ANTIBACTERIAL PEPTIDES FROM MARINE BACILLUS ...

ANTIBACTERIAL PEPTIDES FROM MARINE BACILLUS SPECIES

A thesis submitted in fulfilment of the requirements for the degree

Of

Doctor of Philosophy

Chau Minh Khanh

B.Sc., Ho Chi Minh University of Sciences/Vietnam National University

M.Sc., Ho Chi Minh University of Sciences/Vietnam National University

School of Science

College of Science, Engineering and Health

RMIT University

March 2020


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DECLARATION

I certify that except where due acknowledgement has been made, the work is that of the author

alone; the work has not been submitted previously, in whole or in part, to qualify for any other

academic award; the content of the thesis is the result of work which has been carried out since

the official commencement date of the approved research program; any editorial work, paid or

unpaid, carried out by a third party is acknowledged; and, ethics procedures and guidelines

have been followed.

I acknowledge the support I have received for my research through the provision of an

Australian Government Research Training Program Scholarship.

Signed: ……………………….

Khanh Chau

Date: 27 March 2020


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ACKNOWLEDGEMENTS

I landed in Australia in November 2016 for PhD study and with much curiosity. Since then I

have experienced lots of happiness, also stress, during three years of study. I am so lucky to

have much help from many people that I met in Australia and Vietnam. So, I would like to

sincerely thank them all.

Firstly, I would like to send my deepest gratitude to my supervisor at RMIT, Prof. Robert. J.

Moore for his constant guidance and encouragement. I have always told my friends how lucky

I am to have a chance to be his PhD student. He is not only a knowledgeable supervisor, taking

me into the genetic world of antimicrobial peptides; but also, very patient to improve my

writing skill, because English is my second language. He encouraged and funded me to travel

to Europe to open my horizon on academic research in different continents. I started my

research work in Asia, inhaled the academic education in Australia, and experienced the

research environment during a visit to many research institutions in Europe. It has given me an

overview of academia worldwide. Also, he is very caring to his students with the lots of

celebrating, lab-lunches, and bowling entertainment. Of course, all these events always end

with our smiles.

My second grateful thanks go out to my co-supervisor in Australia, Dr. Hao Van. I felt her

kindness from the first day that I landed in Australia when she took me around the Bundoora

campus to complete administrative works for a newly arrived PhD student. Working with her

in the same lab, she also gave me considerable help and advice, particularly knowledge about

whole-bacterial genome sequencing and data analysis. She always cheered me up with many

invitations to her family for parties and dinners. And I really like her “Bun Bo”- the traditional

food speciality in her hometown and appreciated all the moments to feel at home with her

family members.

I also would like to express my heartfelt gratitude to my co-supervisor in Vietnam, Associate

Prof. Van Quyen Dong. I owe him a special debt of gratitude for the opportunity of the PhD

scholarship that he introduced to me. And he offered me valuable suggestions and advice in

my academic studies. Without Rob’s, Hao’s and Quyen’s consistent instruction, this thesis

could not have reached its present form.

My grateful thanks go to the Moore lab members, Bronwyn Campbell, Ben Vezina, Mian Cheer

Gor, Brydon Davidson, Canh Phung, and Chithralekha Murilidharan. I really enjoyed working


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in the same lab with them, particularly Bronwyn who gave me lots of technical help in the lab.

You were all always very kind and I appreciated all the happy times with you all.

Also, I would like to thank all my Vietnamese friends who I met at RMIT University: Huong

Nguyen, Loc Nguyen, Canh Phung, and Pop. We were working, eating, playing and sharing

during my PhD life. They were always willing to help me with smiles and we travelled together

to explore different cities in Australia.

I am greatly indebted to RMIT University for offering me my PhD scholarship via the RMIT-

VAST cooperative agreement.

I would like to send my great thanks to the Institute of Biotechnology (Vietnam Academy of

Science and Technology), where they set up the scholarship with RMIT University and

prepared a pile of administration works for my scholarship.

I also would like to thank my workplace, “NhaTrang Institute of Technology Research and

Application” (Vietnam Academy of Science and Technology) for supporting me during my

study abroad.

Last but not the least, my acknowledgement also extends to my parents who have given

unconditional assistance; supporting and caring for me all my life. I always love you.

All my best wishes to you.

Khanh Chau


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TABLE OF CONTENT

Chapter 1 INTRODUCTION ..................................................................................................... 3

1.1. The rapid development of antibiotic-resistant bacterial pathogens and reduction in

antibiotic discoveries ................................................................................................................. 3

1.2. The marine environment: an alternative source for antibiotic discoveries ..................... 5

1.3. Marine Bacillus – promising hosts of novel antimicrobial peptides ............................... 6

1.4. Non-ribosomally synthesized peptides (NRPs)- the antimicrobial peptides exhibiting

both antifungal and bactericidal activity. ................................................................................... 6

1.5. The bacteriocins, a promising group of antimicrobial proteins ...................................... 8

1.5.1. Bacteriocins – definition .......................................................................................... 8

1.5.2. Bacteriocin – classification, biosynthesis ................................................................ 9

1.5.2.1. Class I bacteriocins......................................................................................... 10

1.5.2.2. Class II: unmodified bacteriocins ................................................................... 16

1.5.2.3 Class III: large antimicrobial proteins ............................................................ 16

1.5.3. Mode of action and bacteriocin resistance rate surveillance among bacterial

pathogens. ............................................................................................................................. 17

1.6. Current knowledge of marine-derived bacteriocins ...................................................... 18

1.7. Research questions and thesis objectives ...................................................................... 21

1.7.1. Research questions ................................................................................................ 21

1.7.2. Research aims and significance. ............................................................................ 21

1.7.3. Novel contributions ............................................................................................... 22

1.8. Thesis organization ....................................................................................................... 22

Chapter 2 RESEARCH METHODOLOGY ....................................................................... 25

2.1 Sampling and sample preparation for bacterial isolation .............................................. 25

2.2. Isolation of spore-forming bacteria from marine samples ............................................ 25

2.3. Assays to detect the antimicrobial activity. .................................................................. 26

2.3.1. Cross-streak assay for primary screening of marine isolates exhibiting antimicrobial

activity 26


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2.3.2. Well-diffusion assay .............................................................................................. 27

2.3.3. Spot-on-lawn assay ................................................................................................ 29

2.4. Bacterial identification based on 16S rRNA gene sequence and phylogenetic tree

construction .............................................................................................................................. 30

2.4.1. Extraction of genomic DNA by bead beating ....................................................... 30

2.4.2. Amplification of bacterial 16S rRNA gene sequences, Sanger sequencing and

phylogenetic identification ................................................................................................... 30

2.5. The sensitivity of antimicrobial activities to enzyme and heat. .................................... 30

2.6. Analysis of self-depression ability across marine Bacillus/Paenibacillus species ....... 31

2.7. Growth properties, antibiotic susceptibility testing, and enzyme production of isolates

31

2.8. Bacterial whole genome sequencing ............................................................................. 32

2.8.1. Extraction of genomic DNA (gDNA) for whole genome sequencing .................. 32

2.8.2. Qualification of the gDNA .................................................................................... 32

2.8.3. Preparation of DNA library and whole genome sequencing ................................. 33

2.9. Assembly of raw reads generated from whole genome sequencing ............................. 33

2.10. Calculation of Average Nucleotide Identities (ANI) across genomes of terrestrial and

marine Bacillus/Paenibacillus species ..................................................................................... 33

2.11. Estimation of a frequency distribution of CDS across genomes ............................... 34

2.12. Comparison between genomes of marine isolates and genomes of phylogenetically

related terrestrial strains. .......................................................................................................... 35

2.13. In silico prediction of putative antimicrobial peptides within genomes of marine

Bacillus species ........................................................................................................................ 37

2.14. Refinement of media and growth conditions for enhanced bacteriocin production . 39

2.15. Recovery of antimicrobial compounds after fermentation ........................................ 39

2.15.1. Recovery of antimicrobial peptides from the cell-free culture supernatant by

precipitation using ammonium sulphate .............................................................................. 39

2.15.2. Recovery of antimicrobial compounds from cell-free culture supernatants using

Diaion HP-20........................................................................................................................ 40


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2.15.3. Recovery of cell associated antimicrobial compounds from cell pellets by acidic

solvent extraction ................................................................................................................. 40

2.16. Ion exchange chromatography for partial purification of antimicrobial compounds 41

2.16.1. Identification of running buffer and types of resin for ion exchange

chromatography purification of antimicrobial compounds .................................................. 41

2.16.2. Determination of the NaCl concentration used in elution buffer ....................... 42

2.17. Hydrophobic interaction chromatography for partial purification of antimicrobial

compounds ............................................................................................................................... 42

2.18. Reverse phase high-pressure liquid chromatography (RP-HPLC) to obtain pure

antimicrobial compounds ......................................................................................................... 43

2.19. Determination of antimicrobial purity by Matrix-Assisted Laser

Desorption/Ionization time-of-flight mass spectrometry (MALDI-TOF MS) ........................ 43

2.20. Peptide sequencing techniques .................................................................................. 45

2.20.1. Peptide sequencing by de novo peptide sequencing .......................................... 45

2.20.2. Peptide sequencing by N-terminal sequencing (Edman degradation method) .. 45

2.21. Estimation of bacteriocin size by Tricine SDS-PAGE.............................................. 45

2.22. Zymogram assay ....................................................................................................... 47

2.23. Characterisation of the physicochemical properties of antimicrobial compounds ... 47

2.24. Overview of heterologous cloning and expression of bacteriocin ............................ 47

2.25. Extraction of the plasmids from E. coli host ............................................................. 48

2.26. Preparation of the plasmid backbone by enzymatic digestion .................................. 49

2.27. Preparation of the whole sactipeptide genome cluster by PCR amplification. ......... 49

2.28. Preparation of E. coli competent cell. ....................................................................... 49

2.29. Transformation of the fusion plasmid into E. coli by electroporation ...................... 49

2.30. Preparation of Bacillus competent cells .................................................................... 50

2.31. Transformation of the fusion plasmid into Bacillus subtilis BS34A ........................ 50

Chapter 3 BROAD SPECTRUM ANTIMICROBIAL ACTIVITIES OF SPORE-

FORMING BACTERIA FROM THE VIETNAM SEA .................................................... 51


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3.1. Introduction ................................................................................................................... 51

3.2. Results ........................................................................................................................... 52

3.2.1. Thermally resistant, spore-forming, bacteria derived from the marine environment

display antimicrobial activity against important pathogens ................................................. 52

3.2.2. Taxonomic analysis of antimicrobial isolates ....................................................... 57

3.2.3. Proteolytic digestion of antimicrobial culture revealed a dominance of

proteinaceous compounds .................................................................................................... 59

3.2.4. Determination of the growth depression within the short-listed group; showing the

production of potentially novel antimicrobial activities ...................................................... 60

3.2.5. Growth characteristics of antimicrobial-producing isolates .................................. 60

3.3. Summary of results and discussion ............................................................................... 63

Chapter 4 BIOINFORMATIC IDENTIFICATION OF PUTATIVE GENE CLUSTERS

ENCODING ANTIMICROBIAL PEPTIDE PRODUCTION .......................................... 66

4.1. Introduction ................................................................................................................... 66

4.2. Results ........................................................................................................................... 67

4.2.1. Overview of six draft genomes .............................................................................. 67

4.2.2. Calculation of ANI values across the genomes revealed a high degree of similarity

between marine species and terrestrial neighbour strains .................................................... 67

4.2.3. Analysis of the distribution frequency of genes across the species revealed a high

number of strain-specific genes in P. polymyxa genomes ................................................... 70

4.2.4. The pairwise comparison across genomes of B. halotolerans revealed the high

similarity in genome organisation and CDS. ....................................................................... 71

4.2.5. The pairwise comparison across genomes of B. amyloliquefaciens revealed high

similarity in genome organisation and CDS. ....................................................................... 72

4.2.6. The pairwise comparison across genomes of P. polymyxa revealed diversity in

genome organisation and CDS. ............................................................................................ 74

4.2.7. In silico prediction of antimicrobial compounds revealed high numbers of NRPs

and bacteriocins, including novel bacteriocins .................................................................... 76


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4.2.8. Prediction of gene organisation of biosynthetic gene clusters of characterised

bacteriocins........................................................................................................................... 83

4.2.8.1. The LCI gene clusters (found in the genomes of isolates # 06, #08, #11, #13)

83

4.2.8.2. The amylocyclicin gene clusters (found in the genomes of isolates #06, #08,

#11, and #13) .................................................................................................................... 84

4.2.8.3. The mersacidin gene cluster (found in the genome of isolate #08) ............... 85

4.2.8.4. The subtilosin A gene cluster (found in the genome of isolate # 01)............. 87

4.2.8.5. The plantazolicin gene cluster (found in the genome of isolate # 06) ........... 89

4.2.8.6. The paenicidin A gene cluster (found in the genome of isolate #23) ............ 90

4.2.9. Prediction of gene organisation of biosynthetic gene clusters of novel bacteriocins

92

4.2.9.1. The novel lantibiotic gene cluster (found in the genome of isolate #08) ....... 92

4.2.9.2. The novel lantibiotic gene cluster (found in the genome of isolate #23) ....... 94

4.2.9.3. The sactipeptide gene cluster (found in the genome of isolate # 23) ............. 96

4.2.9.4. The lassopeptide gene cluster (found in the genome of isolate #23) ............. 97

4.2.9.5. The thiopeptide gene clusters (found in both isolate #06 and isolate #13) .... 99

4.2.9.6. The thiopeptide gene cluster (found in the genome of isolate #11) ............. 102

4.2.9.7. The two-component lantibiotic gene cluster (found in the genome of isolate

#13) 103

4.3. Summary of results and discussion ............................................................................. 106

Chapter 5 PURIFICATION OF ANTIMICROBIAL PEPTIDES PRODUCED BY

BACILLUS AMYLOLIQUEFACIENS #11 ........................................................................ 111

5.1. Introduction ................................................................................................................. 111

5.2. Result .......................................................................................................................... 113

5.2.1. Analysis of the bacterial growth curve revealed to production of various

antimicrobial compounds ................................................................................................... 113

5.2.2. Purification of antimicrobial compounds from 12 hour-old culture elucidated the

presence of amylocyclicin bacteriocin ............................................................................... 114


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5.2.2.1. Recovery of bacteriocins from cell-free culture and cell pellet. .................. 114

5.2.2.2. Purification of bacteriocins by ion exchange chromatography revealed the

presence of 1 antimicrobial peptide ................................................................................ 116

5.2.2.3. Purification of bacteriocin performed by analytical RP-HPLC elucidated the

presence of amylocyclicin .............................................................................................. 118

5.2.2.4. The amylocyclicin, a heat and pH stable bacteriocin exhibited activity against

only Gram-positive bacteria ........................................................................................... 121

5.2.3. Purification of antimicrobial substances from 36 hours-old culture showed the

presence of three antimicrobial compounds ....................................................................... 123

5.2.3.1. Recovery of antimicrobials compounds from the cell pellet........................ 123

5.2.3.2. RP-HPLC purification of antimicrobial compounds and peptide identification

125

5.3. Summary of results and discussion. ............................................................................ 126

Chapter 6 ANALYSIS OF ANTIMICROBIAL PEPTIDES PRODUCED BY

PAENIBACILLUS POLYMYXA #23 .................................................................................. 130

6.2. Result .......................................................................................................................... 131

6.2.1. Time course analysis of antimicrobial production during bacterial growth revealed

production of multiple antimicrobial substances. .............................................................. 131

6.2.2. Purification of antimicrobial compounds from 24 hour old PB culture revealed the

presence of three antimicrobial compound/s ...................................................................... 133

6.2.2.1. Recovery of antimicrobial compounds from cell-free culture and the cell pellet

133

6.2.2.2. Purification of the antimicrobial peptides in CFS fraction with hydrophobic

exchange chromatography elucidated the presence of two antimicrobial compounds. . 135

6.2.2.3. Purification of 30ACN fraction revealed the presence of polymyxin .......... 136

6.2.2.4. Purification of 40ACN fraction revealed the presence of paenicidin A, tridecaptin

139

6.2.4.1. The paenicidin A, a lantibiotic, displayed antimicrobial activity against Gram-

positive bacteria .............................................................................................................. 140


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6.3. Summary of results and discussion ............................................................................. 141

Chapter 7 CLONING AND HETEROLOGOUS EXPRESSION OF SACTIPEPTIDE, A

NOVEL BACTERIOCIN FROM MARINE P. POLYMYXA #23, IN BACILLUS

SUBTILIS.............................................................................................................................. 145

7.1. Introduction. ................................................................................................................ 145

7.2. Methods and results .................................................................................................... 147

7.2.1. Successful isolation of intact sactipeptide whole gene cluster, and generation of the

expression plasmid (pdLL202/G23sac) ............................................................................. 147

7.2.2. Successful transformation of fusion plasmid into E. coli Top10 ........................ 150

7.2.3. Successful transformation of the fusion-plasmid into E. coli JIR702 for production

of multimeric plasmid. ....................................................................................................... 150

7.2.4. Successful expression of sactipeptide gene cluster in B. subtilis and optimization of

sactipeptide production ...................................................................................................... 152

7.2.5. Two-steps procedure to purify the sactipeptide from 24 hour LB culture .......... 153

7.2.6. Characterisation of sactipeptide revealed the proteinaceous nature and thermal

stability of the sactipeptide ................................................................................................. 155

7.2.7. N-terminal sequencing of the sactipeptide .......................................................... 156

7.3. Summary of result and discussion .............................................................................. 156

Chapter 8 CONCLUSION AND FUTURE WORK ......................................................... 160

8.1. Conclusion .................................................................................................................. 160

8.2. Future work ................................................................................................................. 165

APPENDIX ............................................................................................................................ 183


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ABBREVIATIONS

aa Amino acid

ACN Acetonitrile

AMP(s) Antimicrobial peptide(s)

antiSMASH Antibiotics & secondary metabolite analysis shell

BAGEL Bacteriocin genome mining tool

BLIS Bacteriocin like inhibitory substance

CDS Coding DNA sequence

CFS Cell-free supernatant

CRE Carbapenem- resistant Enterobacteriaceae

CV Colum volume

EDTA Ethylenediaminetetraacetic acid

GES Guanidinium thiocyanate

LAP(s) Linear azol(in)e-containing peptides

LB Luria-Bertani

LPMA Lab prepared marine agar

LPMB Lab prepared marine broth

LP(s) Lipopeptide(s)

MALDI-TOF MS Matrix-assisted laser desorption/ionization mass spectrometry

MHA Muller Hilton Agar

MRKP Multidrug-resistant Klebsiella pneumonia

MRSA Methicillin-resistant Staphylococcus aureus

MSA Multiple sequence alignment

MW Molecular weight

NCBI National Centre for Biotechnology Information

NR Non-redundant

NRS(s) Non-ribosomal synthase(s)

NRSP(s) Non-ribosomally synthesized antimicrobial peptide(s)

ORF Open reading frame

PB Production broth

PBGCs Potential bacteriocin gene clusters


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PCR Polymerase chain reaction

PES Polyethersulfone

PK Polyketide

PKS Polyketide synthase

PTM Post translational modification

RAST Rapid annotation using subsystem technology

RP- HPLC Reverse-phase high-performance liquid chromatography

Sfp 4'-phosphopantetheinyl transferase

SSW Sterile seawater

TEMED Tetramethyl ethylenediamine

TOMM Thiazole/oxazole-modified microcins

TFA Trifluoroacetic acid

TMH Transmembrane helices

TSA Tryptic soy agar

TSB Tryptic soy broth

v/v Volume per volume

w/v Weight per volume

WGS Whole genome sequencing


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LIST OF FIGURES

Figure 1.1. Timeline of antibiotic resistance compared to antibiotic discovery. ....................... 4

Figure 1.2. Minimal domain structure of (A) nonribosomal peptide synthetase (NRPS) and (B)

polyketide synthase (PKS). ........................................................................................................ 7

Figure 1.3. Schematic representation of bacteriocin biosynthesis. ............................................ 9

Figure 1.4. Schematic representation of lantibiotic biosynthesis. ........................................... 11

Figure 1.5. Schematic representation of “head-to-tail linearized bacteriocin”. ....................... 12

Figure 1.6. Schematic presentative of sactipeptide. ................................................................. 13

Figure 1.7. Schematic presentative of LAP biosynthesis. ....................................................... 13

Figure 1.8. Schematic presentative thiopeptide biosynthesis. ................................................. 14

Figure 1.9. Schematic representation of glycocins biosynthesis. ............................................ 15

Figure 1.10. Schematic representation of lassopeptide biosynthesis. ...................................... 16

Figure 1.11.Bacteriocin’s mode of action. ............................................................................... 18

Figure 2.1.Locations of sampling trips within Nhatrang bay (Vietnam sea). .......................... 25

Figure 2.2. Cross-streak assay. ................................................................................................ 26

Figure 2.3. Well-diffusion assay. ............................................................................................. 28

Figure 2.4. Spot-on-lawn assay. ............................................................................................... 29

Figure 2.5. Schematic representation of pairwise genome comparison performed by the

“Sequence-Based Comparison Tool” (A) and MAUVE (B). .................................................. 36

Figure 2.6. Schematic representation of in silico prediction of antimicrobial peptides performed

by BAGEL and AntiSMASH tools. ......................................................................................... 38

Figure 2.7. The process of MALDI-TOF mass spectrometry. ................................................. 44

Figure 3.1. Diversity in the morphology of the marine isolates isolated from NhaTrang bay,

Vietnam Sea. ............................................................................................................................ 53

Figure 3.2. Phylogenetic tree of the 23 short-listed isolates. ................................................... 58

Figure 4.1 The heat map of the average nucleotide identity and genome-based phylogenetic

tree across Bacillus/Paenibacillus genomes ............................................................................ 69

Figure 4.2. Frequency distribution of CDS across the genomes.............................................. 70

Figure 4.3. Genome comparison between marine B. halotolerans #01 and terrestrial neighbour

B. halotolerans F41-3. ............................................................................................................. 71

Figure 4.4. Genome comparison between marine B. amyloliquefaciens and terrestrial B.

amyloliquefaciens .................................................................................................................... 74


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Figure 4.5. Genome comparison between marine P. polymyxa #23 and terrestrial P. polymyxa

species. ..................................................................................................................................... 75

Figure 4.6. Multiple sequence alignment (A) and average distance tree (B) between the

bacteriocins precursors in this study and known precursors .................................................... 82

Figure 4.7 The LCI gene cluster found in marine B. amyloliquefaciens #06, #08, #11 and #13.

.................................................................................................................................................. 83

Figure 4.8. The amylocyclicin gene cluster found in marine B. amyloliquefaciens #06, #08, #11

and #13. .................................................................................................................................... 85

Figure 4.9. The mersacidin gene cluster found in the genome of isolate #08 ......................... 86

Figure 4.10. The subtilosin A gene cluster found in the genome of isolate #01 ..................... 88

Figure 4.11. The plantazolicin gene cluster found in genome of isolate #06 .......................... 89

Figure 4.12. The paenicidin A gene cluster found in the genome of isolate #23 .................... 91

Figure 4.13. The novel lantibiotic found in the genome of B. amyloliquefaciens # 08. .......... 93

Figure 4.14. The novel lantibiotic gene cluster found in the genome of isolate #23. .............. 95

Figure 4.15. The novel sactipeptide gene cluster found in the genome of P. polymyxa #23 and

the precursor sequence. ............................................................................................................ 96

Figure 4.16. The lassopeptide gene cluster found in the genome of isolate #23 ..................... 98

Figure 4.17. The novel thiopeptide gene cluster found in the genomes of B. amyloliquefaciens

#06 and #13. ........................................................................................................................... 100

Figure 4.18.The novel thiopeptide found in the genome of B. amyloliquefaciens #11 ......... 102

Figure 4.19. The two-component lantibiotic gene cluster in the genome of isolate #13. ...... 104

Figure 5.1. The seaweed from which B. amyloliquefaciens #11 isolated (A). Bacterial colony

morphology of #11 on Muller Hilton agar (B) ...................................................................... 112

Figure 5.2. Antimicrobial activity exhibited by marine isolate #11. ..................................... 113

Figure 5.3. Antimicrobial activity exhibited from cell-free supernatants of the cultures collected

at different time-points. .......................................................................................................... 114

Figure 5.4. Antimicrobial activity against L. plantarum exhibited by the fractions collected at

time-point of 12 hours. ........................................................................................................... 115

Figure 5.5. The experiments to select the resin, type of buffer, and NaCl concentration in

elution buffer for ion exchange chromatography ................................................................... 117

Figure 5.6. The MW of antimicrobial peptide presented in 12 hour old culture after partial

purification by cation exchange chromatography. ................................................................. 118

Figure 5.7. RP-HPLC to purify antimicrobial peptide presented in 12 hour old culture. ...... 119

Figure 5.8. Properties of amylocyclicin. ................................................................................ 121


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Figure 5.9. (A) Sequence alignment between amylocyclicin from isolate #11 genome

(11amyA) and FZB42 strains. (B) Maturation of amylocyclicin. ......................................... 122

Figure 5.10. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass

spectrometry (MS) ................................................................................................................. 124

Figure 5.11. RP-HPLC used to separate the freshly dissolved aggregate. ............................ 125

Figure 6.1. Antimicrobial spectrum exhibited by marine P. polymyxa #23. ......................... 131

Figure 6.2. Antimicrobial activity exhibited by P. polymyxa #23 in culture media. ............. 132

Figure 6.3. Recovery of the antimicrobial compounds from the culture supernatant of P.

polymyxa #23 by absorption antimicrobial substances onto Diaion HP-20. ......................... 133

Figure 6.4. Nature of antimicrobial compounds from different fractions obtained during

purification. ............................................................................................................................ 134

Figure 6.5. Hydrophobic interaction chromatography (using Sep-Pak C18 cartridge) to purify

the antimicrobial “cell surface extract” (CFS). ...................................................................... 135

Figure 6.6. Zymogram of polymyxin against S. Enteritidis ................................................... 137

Figure 6.7 Sequencing result of the purified polymyxin. ..................................................... 138

Figure 6.8. The first round of RP-HPLC to separate the 40% ACN fraction.Two fractions

exhibited antimicrobial activity, with peaks at retention times of 23.443 minutes ([m/z] = 1616)

and 23.797 minutes ([m/z] = 3370)........................................................................................ 139

Figure 6.9. MALDI-TOF MS spectra of 40% ACN fraction under detection range of 800 Da –

2000 Da. ................................................................................................................................. 140

Figure 6.10. MW of purified paenicidin A by MALDI-TOF MS with the mass intensity of

[m/z] = 3370. .......................................................................................................................... 140

Figure 7.1. (A) Gene organisation on plasmid pDLL202; (B) The plasmid contains 4 antibiotic

resistance genes and 4 multi cloning sites (MCS); (C) the gene cluster of sactipeptide ....... 146

Figure 7.2. Construction of fusion plasmid. .......................................................................... 149

Figure 7.3. Gene organisation of fusion plasmid pDLL202/G23sac generated after Gibson

assembly. ................................................................................................................................ 150

Figure7.4.Confirmation by PCR for positive transformants after transformation. ................ 151

Figure 7.5. Antimicrobial activity exhibited by some B. subtilis transformants against MRSA

indicator. ................................................................................................................................ 152

Figure7.6. Reverse-phase (RP) high-performance liquid chromatography (HPLC)

chromatograms to separate the AMS fraction. ...................................................................... 154

Figure 7.7. MALDI-TOF MS of purified sactipeptide. ......................................................... 155

Figure 7.8. Properties of sactipeptide. ................................................................................... 155


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LIST OF TABLES

Table 1.1. List of marine bacteriocins published. .................................................................... 19

Table 2.1. List of indicators strains used in antimicrobial screening assays ........................... 27

Table 2.2. Electrode and gel buffers for Tricine–SDS-PAGE, ................................................ 46

Table 2.3. The composition of acrylamide mixtures used to cast the Tricine SDSD gel, ....... 46

Table 2.4. List of strains and plasmids used for cloning /expression ...................................... 48

Table 3.1. Antimicrobial producing bacteria identified from marine samples ........................ 52

Table 3.2. Antimicrobial activity of short-listed isolates against indicators strains. ............... 55

Table 3.3. Closest species of producers, identified by BLASTn of 16S rRNA against 16S rRNA

database (NCBI) (18/06/2019) ................................................................................................. 57

Table 3.4. Enzymatic sensitivity profile of antimicrobial activities. These shortlisted 19 isolates

showed antagonistic activities against C. perfringens. ............................................................ 59

Table 3.5. Antimicrobial activities amongst the short-listed isolates. ..................................... 61

Table 3.6. Characterization of short-listed bacterial isolates. .................................................. 62

Table 4.1.Overview of draft genomes of marine isolates ........................................................ 67

Table 4.2 List of CDS found in genome #01 relating to the biosynthesis of polyketide

compound. These CDS had low similarity with those found in the genome of F41-3 ............ 72

Table 4.3.Low identity CDSs in the genome of isolate #11 relating to the biosynthetic gene

cluster for bacteriocin production. ........................................................................................... 73

Table 4.4. The CDSs relating to unique bacteriocin biosynthetic gene clusters found in the

genome #23 .............................................................................................................................. 76

Table 4.5. List of putative antimicrobial peptides predicted within Bacillus/Paenibacillus draft

genomes. .................................................................................................................................. 78

Table 4.6. Amino acid sequences of precursor bacteriocins predicted within 6 Bacillus genomes

.................................................................................................................................................. 79

Table 4.7. Structural organisation of amylocyclicin gene cluster found in isolates #06, #08, #11

and #13 ..................................................................................................................................... 84

Table 4.8. Structural organisation of mersacidin gene cluster found in isolate #08. ............... 87

Table 4.9. Structural organisation of subtilosin A gene cluster found in the genome of isolate

#01............................................................................................................................................ 88

Table 4.10. Structural organisation of plantazolicin gene cluster found in isolate #06 .......... 90

Table 4.11. Structural organisation of paenicidin A gene cluster found in the genome of isolate

#23............................................................................................................................................ 92


xviii

Table 4.12. Structural organisation of novel lantibiotic gene cluster found in the genome of

isolate #08 ................................................................................................................................ 93

Table 4.13. Structural organisation of novel lantibiotic gene cluster found in the genome of

isolate #23 ................................................................................................................................ 95

Table 4.14. Structural organisation of novel sactipeptide gene cluster found in isolate #23 .. 97

Table 4.15. Structural organization of novel lassopeptide biosynthetic cluster found in isolate

#23............................................................................................................................................ 99

Table 4.16. Structural organization of the novel thiopeptide biosynthetic cluster in isolate #06

and #13 ................................................................................................................................... 101

Table 4.17. Structural organization of the novel thiopeptide biosynthetic cluster found in isolate

#11.......................................................................................................................................... 102

Table 4.18. Structural organization of the novel two-component lantibiotic in isolate #13 .. 105

Table 5.1. The antimicrobial spectrum of amylocyclicin produced by isolate #11 ............... 122

Table 5.2. Antimicrobial spectrum of purified thiopeptide supposed ................................... 126

Table 6.1. Antimicrobial spectra of fractions recovered from a 36-hours old culture ........... 134

Table 6.2. The antimicrobial spectra of 30ACN fraction, and 40ACN fraction. ................... 136

Table 6.3 The antimicrobial spectrum of polymyxin ............................................................. 139

Table 6.4. Antimicrobial activity of compounds present in the 40ACN fraction .................. 141

Table 7.1. List of primers used for cloning and confirmation ............................................... 148

Table 7.2. The growth phase of B. subtilis transformant in LB medium ............................... 153

Table 7.3. Antimicrobial spectrum of sactipeptide ................................................................ 156


1

ABSTRACT

The indiscriminate use of antibiotics in healthcare and agriculture over the past decades has led

to a high incidence of antibiotic-resistant pathogens worldwide. One of the solutions that could

be considered to combat this crisis is the identification of novel antibiotics or other alternatives.

However, the discovery pipeline for novel antimicrobials, identified from organisms in the

terrestrial environment, is sparse. Therefore, we have taken an alternative approach to this issue

by screening marine bacteria to seek novel bacteriocins for further antibiotic development.

Bacteriocins are a family of ribosomally synthesized antimicrobial peptides; some of which are

currently applied as safe food preservatives (nisin) and they are also gaining interest as

promising alternatives to conventional antibiotics. The Vietnam Sea has a variety of marine

ecosystems with diverse marine species, but there has been little exploration of this source for

novel active compounds. Therefore, we undertook a discovery program aimed at identifying

novel bacteriocins from bacterial isolates recovered from the Vietnam Sea.

In stage one, 64 spore-forming bacterial isolates that exhibited antimicrobial activity were

identified after sampling and bacterial isolation (Chapter 3). Inspection of their antimicrobial

spectra resulted in the short-listing of 23 isolates for further analysis. Based on 16S ribosomal

RNA sequences, these 23 isolates were identified as 22 Bacillus species and 1 Paenibacillus

species (Paenibacillus polymyxa), including ubiquitous species (Bacillus subtilis, Bacillus

amyloliquefaciens), and floral species (Bacillus halotolerans, Bacillus safensis, Bacillus

pacificus, Paenibacillus polymyxa, Bacillus licheniformis). They exhibited strong antibacterial

activity against a range of human and veterinary pathogens and food-poisoning bacteria with

dominance of proteinaceous antimicrobial substances. Inspection of the breadth and strength

of antimicrobial activities informed the selection of 6 isolates for whole genome sequencing in

the second stage of the study.

The six isolates subjected to whole genome sequencing were; four B. amyloliquefaciens

isolates (#06, #08, #11, #13), one B. halotolerans (#01) isolate, and one P. polymyxa (#23)

isolate (Chapter 4). Bioinformatic analysis identified genes encoding 61 putative antimicrobial

peptides within the genomes of the six isolates, including 41 mostly characterised non-

ribosomally synthesized peptides (lipopeptides, polyketides and bacilysin) and 20 bacteriocins.

Amongst the 20 putative bacteriocins found, there were 13 different types of bacteriocins. The

putative bacteriocin encoding genes clusters included those for 6 characterised bacteriocins

(mersacidin, paenicidin A, plantazolicin, LCI, amylocyclicin, subtilosin A), and 7


2

uncharacterised/ unique bacteriocins (2 thiopeptides, 1 two-component lantibiotic, 1

sactipeptide, 2 lantibiotic, and 1 lassopeptide).

In the third stage of the study (Chapters 5 and 6); two isolates were selected for antimicrobial

purification. Purification of antimicrobial peptides from isolate #11 resulted in the

identification of amylocyclicin ([m/z]= 6,381), iturin/surfactin ([m/z]= 1,058) and an undefined

peptide ([m/z]= 1,473). The undefined peptide ([m/z]= 1,473), was suspected to be a novel

thiopeptide due to the similarity of MW. However, the amino acid sequence of this peptide

couldn’t be elucidated by an N-terminal sequencing method. Antimicrobial purification from

isolate #23 identified polymyxin ([m/z]= 1168.7), tridecaptin ([m/z]= 1550.8), and paenicidin

A ([m/z]= 3290.4), but no novel bacteriocins.

In the fourth stage of the study, an attempt was made to heterologously produce the novel

sactipeptide which was one of three putative novel bacteriocins predicted from the genome of

isolate #23. The technique employed E. coli/B. subtilis shuttle plasmid, pDLL202, for plasmid

construction and a heterologous B. subtilis host for bacteriocin expression. A 15,487 bp fusion

plasmid (pDLL202/G23sac) was constructed, carrying an 8,556 bp–sactipeptide gene cluster

derived from isolate #23 and the 6,938 bp- plasmid backbone derived from plasmid pDL202.

The Bacilllus transformants successfully expressed the sactipeptide. The sactipeptide had a

molecular weight (MW) of 3,404 Da and depressed growth of Gram-positive bacteria.

Taken together, the marine Bacillus/Paenibacillus are abundant reservoirs of genes coding

novel bacteriocins for further discovery. These antimicrobial compounds produced by marine

Bacillus/Paenibacillus can be developed into therapeutic antimicrobial drugs against

antibiotic-resistant pathogens, particularly against antibiotic-resistant Gram-positive bacteria.


3

Chapter 1 INTRODUCTION

1.1. The rapid development of antibiotic-resistant bacterial pathogens and reduction in

antibiotic discoveries

Antibiotics are natural or synthesized compounds that depress the growth of microorganisms.

The introduction of antibiotics is one of the greatest achievements in pharmacology since

antibiotics now play a vital role in the clinical treatment of many bacterial pathogens.

In 1928, the first antibiotic, Penicillin was discovered from Penicillium notatum by Alexander

Fleming, and in 1934 Penicillin was approved to use then rapidly became an important drug in

clinical treatments, in aquaculture, in veterinary treatments, and agriculture (1). Penicillin

became an important bulwark protecting humankind from numerous pathogenic infections,

decreasing mortality, increasing agricultural profitability, and initiated a wave of antibiotic

discoveries from natural resources [Fig 1.1]. Antibiotics vary greatly in nature, structure, and

mode of action. For example, antibiotics can act as inhibitors of cell wall synthesis

(cephalosporin, carbapenem), DNA synthesis inhibitors (fluoroquinolones), protein synthesis

inhibitors (macrolides, chloramphenicol, rifampin), mycolic acid synthesis inhibitors, and folic

acid synthesis inhibitors (2). Of these, the carbapenem group (meropenem, imipenem) are now

the most widely used antibiotics in the treatment of Gram-negative bacterial pathogens and are

considered as the last line of antibiotics to treat these kinds of pathogens.

However, the indiscriminate overuse of antibiotics for several decades has led to an antibiotic

resistance crisis (3). Bacterial pathogens rapidly form antibiotic-resistance after being exposed

to antibiotic treatment of insufficient dose. Dangerously, these resistance genes are frequently

located on movable genetic elements (transposons, insert sequences, plasmids, prophage), that

can facilitate the transfer of resistance genes to other bacterial recipients. This horizontal and

vertical transfer between pathogens has resulted in the complicated antibiotic resistance crisis

we now face (4-6). For example, in 2009, the first case of carbapenem-resistant Klebsiella

pneumonia, which carried the blaNDM-1 gene encoding a beta-lactamase, was found in India;

and now this resistance gene is found in all nosocomial Gram-negative pathogens

(Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii) in all 5 continents

(7-9). The failure of carbapenem means there is a shortage of antibiotics to control Gram-

negative bacterial pathogens. High resistance rates have also been observed in nosocomial

Gram-positive pathogens. Vancomycin and methicillin have also lost effectiveness against


4

nosocomial Gram-positive bacterial pathogens, since the methicillin-resistant Staphylococcus

aureus and vancomycin-resistant Enterococcus sp. have rapidly spread worldwide (10, 11).

Therefore, antibiotic-resistant pathogens are pushing humans towards an age where there are

no effective antibiotics to fight pathogenic bacterial infections – a frightening and dangerous

situation.

Unfortunately, there is also a lack of research and few new alternative novel antibiotics have

been identified and brought to the market. Very few novel antimicrobial compounds have

recently been discovered from the terrestrial environment. The reason may be due to the

overexploitation of this traditional source for the last 90 years. Thus, the U.S. Food and Drug

Administration reported that the approval rate of new, medically important antibiotics has

decreased by 56% over the last few decades (1998 to 2002 versus 1983 to 1987) without any

evidence for an increase since that study (12). Although much effort has been carried out to

synthesize in vitro novel antibiotics, the natural world still has the potential to provide a rich

array of novel compounds

Figure 1.1. Timeline of antibiotic resistance compared to antibiotic discovery. Figure

from Zaman et al. (2017) (13).


5

1.2. The marine environment: an alternative source for antibiotic discoveries

The marine environment is nowadays gaining interest as an alternative source for antibiotic

discovery. Marine microorganisms potentially serve as reservoirs of novel antimicrobial

compounds (14). However, the current traditional plating method only recovers about 0.001-

1% of total microorganisms from the marine environment (15). Through the recent

development of whole genome sequencing (WGS), numerous genes encoding novel

antimicrobial compounds from these cultivated bacteria have been identified, but the numbers

of such genes are only minor since studies on the marine environment have been limited, and

our knowledge on the marine species’ diversity is significantly less than their terrestrial

counterparts (16). Currently, a majority of marine bacteria are not able to be cultured. This can

lead to difficulties for drug discovery but also emphasizes the high potential of the marine

environment as a rich source of novel antimicrobial compounds for future exploitation.

In contrast to the terrestrial environment, the harsh marine conditions (i.e., limited living space

or nutrient sources; extreme chemo-physical conditions; high competition between marine

microorganisms) may act as a selective pressure driving marine bacteria to evolve increased

abilities for the production of diverse antimicrobial compounds for defence and environmental

adaptation. Notably, bacterial competition is more significant between sponges-associated

microorganism communities. Due to the typical structure with multiple pores, the sponges

provide many ideal ecological niches as sites for bacterial attachment. This leads to a high

density of microbial cells in sponge tissue with a high diversity of bacteria, microalgae, fungi,

and even viruses (17). Some sponges even have 40% of their tissue volumes contributed by

microorganism cells (17). The close cell-by-cell contact in a narrow space and limited nutrient

sources possibly leads to competition, in which bacteria tend to produce antimicrobials as

weapons against other competing bacteria. The plasticity of marine bacterial genomes may be

influenced by uptake of unique external nucleotides derived from the marine environment (18).

Sponges have proven to be a rich source of novel antimicrobials, with about 200 new bioactive

compounds published every year (17). The only way to fully explore the genomic variation of

marine species is by analysing the whole genome sequence in comparison with terrestrial

species.


6

1.3. Marine Bacillus – promising hosts of novel antimicrobial peptides

The Bacillus genus are Gram-positive, spore-forming, facultative anaerobic bacteria, and

mostly non-pathogenic (exceptions include B. cereus and B. anthracis). They are safe for

human consumption as certified by the FDA; are fastidious growers; endogenous spore

formers; and well-known producers of various bioactive compounds including diverse

structural antimicrobial peptides (AMPs). The Bacillus derived AMPs exhibit antimicrobial

activity against a wide range of microorganisms, ranging from viruses, bacteria, and fungi to

parasites. Therefore, Bacillus species are widely used by humans in different applications,

including agriculture (as biocontrol agents, biofertilizers, probiotics), food industries (source

of enzymes, food preservatives, food additives), and pharmaceutical industries (antibiotics)

(19-21). In the marine environment, Bacillus species occupy various ecological niches, like

marine seaweeds, sponges, marine animals, invertebrates, sediments, and seawater. Members

of the marine Bacillus genus include both universal species (B. badius, B. subtilis, B. cereus,

B. licheniformis, B. firmus, B. pumilus, B, amyloliquefaciens, B. mycoides and B. lentus) which

are widely spread in both marine and terrestrial environments; and marine typical Bacillus

species (B. marinus, B. salexigens, B. dipsosauri, Halobacillus sp.). Marine Bacillus species

are considered a likely reservoir of novel AMPs, but they have not been widely studied.

The Bacillus-derived AMPs include both nonribosomal peptides (NRPs) and ribosomally-

synthesized peptides (bacteriocins) (21). They are typically small molecular-weight peptides,

amphipathic, membrane active, and exhibit various pharmaceutical activities, particularly

antimicrobial activity. The machinery to produce bacteriocins and NRPs involves a set of genes

responsible for various synthesis, processing and immunity functions, and are generally

clustered in a DNA region, called a biosynthetic gene cluster.

1.4. Non-ribosomally synthesized peptides (NRPs)- the antimicrobial peptides exhibiting

both antifungal and bactericidal activity.

The NRPs are produced by synthetase enzymes, via non-ribosomal biosynthetic pathways.

They include lipopeptides (LPs), polyketides (PKs), and bacilysin. The LPs are synthesized by

‘non-ribosomal peptide synthetase’ (NRPS) enzymes while PKs are produced by ‘polyketide

synthase’ enzymes (PKs). The NRPs are mostly produced by fungi and bacteria. In the bacterial

kingdom, production of NRPs is frequently observed in bacteria from the Actinobacteria and

Firmicutes phyla (Bacillus, Paenibacillus, Geobacillus, etc) (21-23)


7

The gene clusters of NRPs compounds are typically constituted of genes encoding; i) peptide

synthetase enzymes; ii) enzymes to generate monomers/acyl residues; and iii) enzymes

responsible for modification of the precursor (including processes of N-methylation, acylation,

glycosylation, tailing, and/or heterocyclic ring formation) (24). The synthase enzymes have

very large MW ranging from 100 kDa to more than 1600 kDa (25), and consist of multiple

modules. Each module has multiple domains which are fused covalently (26) [Figure 1.2]. For

details, typical NRPS and PKS enzymes include a “starting module” and multiple “extending

modules”. The starting module of PKS consists of; acyltransferase domain (AT); acyl carrier

protein domain (ACP), while each “extending module” has all above domains, with the

addition of; ketosynthase (KS); and either ketoreductase (KR) or dehydratase (DH) or enoyl

reductase (ER) or thioesterase (ET) domains. While, the NRPS enzymes consist of; peptidyl

carrier protein (PCP), adenylation domain (A), epimerase (E), methyltransferase (MT), 4’-

phosphopantetheine transferase domain (PPT), oxygenation domain (Oxy), and cyclization

domain (Cy). Of these domains, the ACP and PCP domains serve as places where specialized

amino acids/or monomers attach to each module, initiating the formation of chemical bonds

between these monomers to grow the compound chain. The ACP or PCP are usually translated

in the inactive forms and their activations are required for attachment of phosphopantetheine

(4'-PP) moiety derived from coenzyme A (CoA). This process is catalysed by the PPTase

enzyme encoded by the sfp gene. Therefore, the biosynthesis of lipopeptide and polyketide

compounds is dependent on the sfp gene (27).

Figure 1.2. Minimal domain structure of (A) nonribosomal peptide synthetase (NRPS)

and (B) polyketide synthase (PKS). C, condensation domain; PCP, peptidyl carrier

protein; A, adenylation domain; E, epimerase; MT, methyltransferase; PPT, 4’-


8

phosphopantetheine transferase domain; Oxy, oxygenation domain; Cy, cyclization

domain; ACP, acyl carrier protein; AT, acyltransferase domain; KS, ketosynthase

domain; KR, ketoreductase domain; DH, dehydratase domain; ER, enoyl reductase

domain; TE, thioesterase domain. The Figure is from Guzman- Chavez et al. (2018) (28)

The LPs are amphiphilic peptides, consisting of a hydrophilic polypeptide chain and a

hydrophobic acyl chain (lipid). Based on the variation of acyl chain (length, branch, linear and

saturation), LPs are distinguished into various LPs families, including three traditional families

(surfactin, iturin and fengycin), and a newly discovered family (locillomycins) (29, 30). The

LPs mainly exhibit antifungal activity, but some LPs also exhibit antibacterial activity

(paenibacterin and polymyxin) (21). The PKs are compounds that contain the typical carbonyl

methylene residue (-CO-CH2) or are derived from a precursor containing this residue. The

Bacillus-associated PKs include 3 traditional PKs (bacillaene, difficidin and macrolactin) and

newly discovered families (paenimacrolidin, basiliskamide). They mostly exhibit bactericidal

activity (zwittermicin A), but also antifungal activity (paenilamicin and paenilarvins),

antitumor and immunosuppressive activities. In contrast, bacilysin is a dipeptide and its

production is independent of 4′-phosphopantetheinyl transferase (PPTase). The bacilysin

comprises of an l-alanine residue and anonproteinogenic l-anticapsin. Bacilysin exhibits

antibacterial activity (against Erwinia amylovora, M. aeruginosa, A.

flosaquae, Nostoc sp., Anabaena sp), antifungal activity (against yeast) and also anti-algal

activity (against cyanobacteria and microalgae) (31, 32).

1.5. The bacteriocins, a promising group of antimicrobial proteins

1.5.1. Bacteriocins - definition

Bacteriocins are produced on ribosomes and are dependent on messenger RNA transcribed

from bacteriocin encoding genes. They are proteinaceous in nature, fewer than 100 amino acids

in length, and mostly depress the growth of bacterial species that are taxonomical neighbours

with the producing host. Numerous studies have been carried out to screen for bacteriocin

production from terrestrial resources, yielding various bacteriocins produced from various

bacteria (33). Bacteriocins have been most commonly found in Gram-positive bacteria, such

as Lactobacillus, Bacillus, Proteus and Actinobacteria, but some have also been found in

Gram-negative bacteria, such as E. coli, Pseudomonas sp., Vibrio sp. and Shigella sp (34, 35).

In bacterial communities, the lactic acid bacteria (LAB) group and Bacillus are two dominant


9

producers of bacteriocins. Bacillus-derived bacteriocins are more diverse and exhibit broader

spectra of antimicrobial activity (36, 37).

Bacteriocins have been developed and used in a variety of applications, such as natural food

preservatives (nisin), food additives (nosiheptide, duramycin) and antibiotics (thiostrepton)

(19, 20). Bacteriocins are now of interest in pharmacology because they have; (i) strong activity

against antibiotic-resistant pathogens (e.g. MRSA, vancomycin-resistant E. faecalis); (ii) low

resistant rates; (iii) potential to scale up the production via cloning and heterologous

expression; (iv) exhibit a narrow antimicrobial spectrum when compared with conventional

antibiotics, therefore, ensuring the survival of harmless bacteria in human/animal gut.

1.5.2. Bacteriocin – classification, biosynthesis

The machinery to produce bacteriocins involves various genes that are usually located together

in a biosynthetic gene cluster. The gene composition of bacteriocin biosynthetic gene clusters

is significantly diverse, but generally comprises of; structural gene(s) encoding precursor

bacteriocin; gene(s) encoding modification enzyme(s), which are responsible for modify the

precursor (in case of class I bacteriocins); gene(s) encoding transport protein, that is necessary

for bacteriocin secretion; gene(s) encoding immunity protein to protect the host from a suicide-

effect; and regulatory genes to control transcription. The precursor of bacteriocin comprises an

N-terminal leader sequence (or leader sequence) and a C-terminal core sequence. Bacteriocin

maturation frequently involves the eliminating of the signal sequence followed by

modifications of the core sequence [Figure 1.3].

Figure 1.3. Schematic representation of bacteriocin biosynthesis. Firstly, genes within

the bacteriocin gene cluster are transcribed and translated. The modification enzymes


10

recognise the precursor, and subsequently post-translationally modify it (PTMs). The

PTM includes the elimination of signal sequence from a precursor and mediating core

sequence to mature. The matured bacteriocin is then secreted by a transmembrane

transporter. Figure from Ortega et al. (2016) (38).

Many bacteriocins have been described in the scientific literature. They are distinguished based

on structure, mode of action, thermal stability, molecular size and glycosyl tailing (36). The

Bacillus-derived bacteriocins are more diverse than those produced by Gram-negative bacteria

or other Gram-positive bacteria (LAB, Clostridium, etc.). Therefore, many classification

schemes have developed to distinguish Bacillus-derived bacteriocins. The Bacillus-derived

bacteriocins are classified into 3 groups:

Class I bacteriocins are small (<10 kDa), heat stable and are modified compounds.

Class II bacteriocins are small (<10 kDa) and heat stable peptides.

Class III bacteriocins are larger (>10 kDa) and heat sensitive proteins.

1.5.2.1. Class I bacteriocins

Class I bacteriocins are small MW proteins (≤10 kDa). The precursor bacteriocin is firstly

translated from the structural gene and subsequently undergoes post-translational

modifications (PTMs). Based on these PTMs, they are subdivided into 7 subclasses. The

subclass I1: The PTMs generate unusual amino acids (dehydrated amino acids, and cyclised

amino acids) on the core sequence; while for subclasses I2–I7, the precursor bacteriocin

undergoes a single typical modification (head-to-tail peptide, linear azole containing peptide,

lassopeptide, thiopeptide, sactipeptide, and glycosin).

Subclass 1: Lantibiotics

Lantibiotics contain some unusual amino acids residues, such as; 2,3-dehydroalanine (Dha);

2,3-dehydrobutyrine (Dhb); lanthionine (Lan); and methyllanthionine (MeLan) (39). These

amino acids are generated by enzymatic dehydration modification and cyclisation

modification. The hydratase enzyme catalyses the dehydration process by converting all

serine and/or threonine residues on the core sequence into Dha and/or Dhb; the cyclase enzyme

catalyses the cyclisation process by formation of thioether bonds between these dehydrated

amino acids and a nearby thiol group of a cysteine residue to yield Lan (from Dha) and MeLan

(from Dhb). There are two 4 modification enzymes responsible for these modifications,


11

including LanB (dehydratase), LanC (cyclase), LanM (having two domains of LanB and

LanC), LanKC, and LanL (40). Depending on the modification enzymes, lantibiotics are

subclassified into 4 subgroups. Lantibiotic subtype I is modified by LanB and LanC. They are

typical of elongated or screw shape and positively charged compounds. Lantibiotic class II is

modified by LanM. The lantibiotics class II are typical of small MW peptides, globular shape,

or two component peptides, negatively charged or no net charge compounds (mersacidin,

actagardine, and cinnamycin). The dehydration process by LanB and LanM are independent of

phosphorylation. The lantibiotic class III and IV are modified by LanKC, and LanL enzymes

respectively and their dehydration are dependent on phosphorylation. The LanL and LanKC

include 3 domains; an N-terminal lyase, a central kinase and a C-terminal cyclase/putative

cyclase domain. The central kinase domain catalyzes the phosphorylation of the

serine/threonine of the core sequence to generate dehydrated phosphoSer/phorphosphoTher.

Subsequently, the lyase domain removes the phosphate residues to yield Dha and Dhb

[Figure1.4].

The lantibiotics have been reported in B. subtilis, B. thuringinensis, B. cereus, B. megaterium,

B. mycoides, B. clausii, Bacillus sp., Geobacillus thermodenitrificans, Geobacillus

kaustophilus, Paenibacillus polymyxa, P. larvae, P. peoriae and .P. durus (36, 41-43).

Figure 1.4. Schematic representation of lantibiotic biosynthesis. (A) Dehydration and

cyclization to generate the lanthionine, methyl-lanthionine from threonine. B) Four

modification enzymes in lantibiotic biosynthesis. Figure from Yu et al. (2012) (40).


12

Subclass 2: Head to tail cyclized peptides

Head-to-tail cyclized peptides are cyclic peptides, where the cyclization occurs between the N-

terminal amino acid and the C-terminal amino acid [Figure 1.5]. It is modified by a typical

serine protease that cleaves off the leader sequence from the precursor; subsequently an amide

bond forms between the terminal two amino acids on the core sequence strand to generate the

mature cyclic peptide. The head-to-tail cyclized peptides are reported in B. thuringiensis, B.

cereus, B. coagulans, B. pumilus, B. paralicheniformis, B. gobiensis, other Bacillus sp.,

Kyrpidia tusciae, Geobacillus stearothermophilus, Geobacillus kaustophilus, Geobacillus sp.,

Paenibacillus larvae and Paenibacillus mucilaginosus.

Figure 1.5. Schematic representation of “head-to-tail linearized bacteriocin”.The

cyclization formed from 2 amino acid residues at two termini under the catalysis of a

typical protease. Figure from Gabrielsen et al. (2014) (44).

Subclass 3: Sactipeptides

The sactipeptides are cyclic peptides, where cyclization occurs between unusual sulphur and

α-carbon on side chains [Figure 1.6]. This cross-linking bond is catalysed by a typical radical

S-adenosylmethionine (SAM). The sactipetides are known to be produced by B. subtilis, B.

atrophaeus, B. simthii (subtilosin A), B. atrophaeus, B. pumilus, B. subtilis (sporulation killing

factor (SKF)) (45), B. thuringiensis and B. cereus (thuricin H and thuricin CD, thuricin 7,

thuricin S) (46, 47).


13

Figure 1.6. Schematic presentative of sactipeptide. (A) The structure of thuricin CD (as

reference) with sulphur cross-link; (B) Illustration of sulphur cross-link. Figure from

Poorinmohammad et al. (2014) (48).

Subclass 4: Linear azole-containing peptides (LAPs)

The LAPs contain heterocyclic azole/azoline rings, which are generated from enzymatic

modification of serine/threonine and cysteine residues. Three enzymes responsible for

modification include cyclodehydrataseenzyme (protein D), dehydrogenase enzyme (protein B),

and a “docking” enzyme (protein C) [Figure1.7] (49). Protein C and protein D, together, modify

the Cys, Ser, and Thr to generate azoline heterocycles. Subsequently, protein C oxidizes some

of these azolines to azoles. The LAP bacteriocins are mostly found in Lactobacillus, E. coli

(microcin) (50), B. amyloliquefaciens (plantazolicin A and B) (51).

Figure 1.7. Schematic presentative of LAP biosynthesis.(A) LAP biosynthetic gene

cluster comprises a structural bacteriocin gene encoding the precursor of LAP (black), 3

genes encoding 3 modifying enzymes - proteins B, C, D (yellow), a gene encoding

peptidase (brown), an immunity gene (blue), and an export gene (red). The maturation of

LAP involves posttranslational modification (cyclodehydration and dehydration)

catalysed by the BCD complex and proteolysis to remove the leader sequence. (B) The


14

formation of azoline ring is catalysed by protein C and protein D, and azole ring is formed

under catalysis of protein B. Figure from Burkhart et al. (2015) (49).

Subclass 5. Thiopeptides

Thiopeptides contain a nitrogen-containing six-membered ring (piperidine, dehydropiperidine,

or pyridine) which serves as a central ring for the tailing of azole rings and dehydrated amino

acid residues [Figure 1.8]. Thiopeptides are typically low MW (less than 2 kDa), rich in sulphur

residues, and contain heavily modified amino acids. They commonly depress the growth of

Gram-positive bacteria with little or no activity against Gram-negative bacteria. Currently,

there are approximately 100 known thiopeptides, varying in size, charge, and branches of azole

rings. They are found in Actinobacteria, B. cereus ATCC 14579 (thiocillins), B. subtilis,

Bacillus amyloliquefaciens and Lysinibacillus sphaericus. The biosynthesis of thiopeptides

occurs under various posttranslational modification processes, including; (i) dehydration and

cyclization to generate the dehydrated amino acid ;(ii) formation of azole rings; (iii) formation

of 6-membered nitrogen heterocycle; (iv) and elimination of the leader sequence; and variable

tailing processes.

Figure 1.8. Schematic presentative thiopeptide biosynthesis. Gene organisation of the

nosiheptide gene cluster (as an example). (B) Maturation processes of nosiheptile include

the formation of azole rings, dehydration and formation of the 6-membered nitrogen

heterocycle ring. Figure from Ortega et al. (2016) (38).


15

Subclass 6: Glycocins

Glycocins are bacteriocins tailed with a glycosyl residue [Figure 1.9]. The glycocins are found

in B. subtilis (Sublancin 168 containing a β-S-linked glucose moiety), B. thuringiensis , B.

cereus, B. weihenstephanensis, B. lehensis, other Bacillus sp., Geobacillus sp. and

Paenibacillus sp. (52)

Figure 1.9. Schematic representation of glycocins biosynthesis. Sublancin as an example.

The bacteriocin is tailed with sugar moiety. Figure from Hseih et al. (2012) (52).

Subclass 7: Lassopeptides

Lassopeptides contain a macrolactam ring at the N-terminus. Two enzymes; a lasso cyclase

(protein C), and leader peptidase (protein B) are needed to generate the macrolactam ring by

forming a cross-link between either a glycine/alanine/serine or cysteine at the N-terminus and

the carboxylic acid residue of aspartate/glutamate on the side chain to cyclize [Figure 1.10].

The lassopeptides are found in Actinobacteria, Proteobacteria and Firmicutes, such as

Paenibacillus dendritiformis C454, Thermobacillus composti KWC4, Bacillus cereus VD115,

and Paenibacillus polymyxa CR1.


16

Figure 1.10. Schematic representation of lassopeptide biosynthesis.Figure from Zhu et

al. (2016).

1.5.2.2. Class II: unmodified bacteriocins

In contrast to class I bacteriocins, class II bacteriocins undergo no posttranslational

modifications. They typically have an MW of <10 kDa, are heat-stable, and have linear

structure. The biosynthetic gene clusters of class II bacteriocins include a structural gene,

transport gene(s) and immunity gene(s). There are 4 subclasses within the class II bacteriocins,

distinguished based on the shape and ability to kill Listeria sp. Subclass IIa are pediocin-like

bacteriocins and exhibit anti-Listeria activity. The class IIb, class IIc and class IId are non-anti-

Listeria bacteriocins. Subclass IIb are two-component peptides, subclass IIc are circular, and

subclass IId are linear. The class II bacteriocins are found in B. coagulans I-4 (coagulin) (53),

Geobacillus sp. WCH70 (lactobin A, holins), B. subtilis, B. amyloliquefaciens, B. mycoides, B.

pseudomycoides, B. licheniformis, B. pumilus, B. thuringiensis (holin-like peptide BhlA), B.

amyloliquefaciens, Bacillus (LCI) and B. methylotrophicus (aureocin A53) (22, 54).

1.5.2.3 Class III: large antimicrobial proteins

Class III bacteriocins have larger MWs (>10 kDa) and are heat sensitive. The biosynthetic gene

clusters of class III bacteriocins include an immunity gene and a structural gene. The III

bacteriocins are found in B. megaterium ATCC 19213 (megacin), Escherichia coli (Colicin),

Bacillus sp. (M23 metallopeptidase family) (55-57).


17

1.5.3. Mode of action and bacteriocin resistance rate surveillance among bacterial

pathogens.

The bacteriocins are membrane-active peptides, usually cationic-charged; therefore, they bind

to bacterial cell membranes which present mostly negatively charged components, initiating

different modes of action. Class I bacteriocins bind to the lipid II molecules which are main

transporters of cell wall components - the peptidoglycan subunits; therefore, preventing cell

wall formation. The binding results in a ‘bacteriocin-lipid II’ complex, serving as a pore

through the membrane, causing leakage of internal materials of cells. The class II bacteriocins,

which are typically have helical and/or amphiphilic structures, bind to phospholipid heads of

the membrane at hydrophobic regions and subsequently insert the hydrophilic part into the

membrane. The hydrophilic part binds to mannose phosphotransferase systems (PTS-Man)

proteins and together form a pore causing depolarization, resulting in cell death. Class III

bacteriocins can hydrolyse the cell wall components of Gram-positive bacteria, causing loss of

membrane integrity [Figure 1.11] (58).

There are several mechanisms by which bacteriocin-sensitive bacteria can develop resistance,

including; (i) modification of bacteriocins’ receptors on the membrane to prevent bacteriocin

binding; (ii) sequestering of bacteriocins; (iii) formation of efflux pumps to expel the

bacteriocins, or; (iv) production of bacteriocin-degrading enzymes. Different bacteria employ

different strategies, and even the same species can demonstrate different mechanisms.

Bacteriocin resistance frequencies in bacteria are low compared to the resistance rate of

conventional antibiotics, ranging from 10-9 to 10-2 (59, 60).


18

Figure 1.11.Bacteriocin’s mode of action. Figure from Gražina et al. (2012) (58).

1.6. Current knowledge of marine-derived bacteriocins

Marine-derived bacteriocins have been much less studied than terrestrial-derived bacteriocins.

Marine bacteriocins have been mostly reported from lactic acid producing bacteria

(Lactobacillus, Carnobacterium), Bacillus sp. and, to a lesser extent, Enterococcus, Proteus,

Vibrio and Pseudomonas [Table 1.1]. These bacteria encoding bacteriocin production were

isolated mostly from seafood or aquatic animal (fish, prawn) intestines (61, 62), but also

sponges, seaweed and sediment (63, 64). These marine-derived bacteriocins were mostly small

(<10 kDa) and exhibited a variety of antimicrobial activities. Generally, Lactobacillus-

associated bacteriocins exhibited the broadest spectra of activity against both Gram-positive

and Gram-negative bacteria (against Vibrio, Shigella, E. coli, Salmonella sp); while the Vibrio

and Proteus derived bacteriocins commonly exhibited antimicrobial activity against Vibrio sp.

The bacteriocins produced by Carnobacterium or Bacillus possessed antimicrobial activity

mostly against Gram-positive bacteria, but also against Salmonella sp. and Vibrio sp. Most of

the bacteriocins are still not fully characterised, therefore they have been named BLIS

(bacteriocin-like-inhibitory substance) factors. BLIS factors are uncharacterised bacteriocins

that share similar activity and characteristics of typical bacteriocins, but the sequence is still to

be determined.


19

Table 1.1. List of marine bacteriocins published.

Bacteriocins Producers strains MW Killing spectra Isolation source Reference

BLIS Lactobacillus pentosus 39 - A. hydrophila; L. monocytogenes salmon fillets (65)

BLIS Lactobacillus lactis 94 Bacillus, Staphylococcus, Enterococcus, E. coli, Pseudomonas, Shigella

sediment (64)

bacteriocin Lactobacillus fermentum 18 Vibrio, Listeria, Listeria sp., Staphylococcus fish gut, prawn muscle

(66)

SBS001 Lactobacillus fermentum 78 Klebsiella, Pseudomonas, E. coli seawater (67)

AP8 Lactobacillus casei 5 E. coli, Listeria spp., Salmonella sp., Staphylococcus, , Vibrio

fish gut (68)

bacteriocin Lactobacillus acidophilus 2.5 Lactobacillus, Salmonella, Staphylococcus, Bacillus, Salmonella, Escherichia, Klebsiella, Pseudomonas

prawn gut (62)

bacteriocin PSY2 Lactococcus lactis PSY2 Arthrobacter sp., Acinetobacter sp., Bacillus marine perch fish (69)

carnocin U149 Carnobacterium sp. 4.5 Lactobacillus, Pediococcus, Carnobacterium fish gut (70)

divergicin M35 Carnobacterium divergens M35 4,5 Carnobacterium, Listeria smoked mussel (71)

divergicin V41 Carnobacterium divergens V41 4.5 Carnobacterium, Listeria, Enterococcus, fish viscera (72)

carnobacteriocin Carnobacterium piscicola A9b 4.5 Listeria smoked salmon (73)

piscicocin CS526 Carnobacterium piscicola CS526

4.4 Tetragenococcus, Leuconostoc, Listeria, Enterococcus.

frozen surimi (74)

piscicoccin V1a Carnobacterium piscicola V1 4.5 Lactobacillus, Listeria, Enterococcus, Pediococcus. fish (75)

BLIS Enterococcus faecium CHG 2-1 - Enterococcus venus clams (76)

BLIS Enterococcus faecium C-K, C-S - Listeria fish fillet (76)


20

Bacteriocins Producers strains MW Killing spectra Isolation source Reference

enterococinB Enterococcus faeciumALP7 <6.5 Listeria, Staphylococcus, Bacillus, Lactobacillus. fermented shellfish (77)

BLIS Vibrio sp <5 Bacillus sp., Vibrio sp., Pseudomonas sp. marine fish gut (78)

BLIS Vibrio harveyi - - marine fish gut (79)

BLIS Vibrio spp. MMB2 - Aeromonas hydrophila marine microalgae (80)

BLIS Proteus sp.CT1.1, G1 - Vibrio marine fish, lobster (81)

BLIS Bacillus cereus - Vibrio marine fish (81)

BL8 Bacillus licheniformis <3 Staphylococcus, Bacillus sp. sediment (82)

bacteriocin Bacillus sp. NM12 <5 fish pathogens fish gut (78)

bacf3 Bacillus amyloliquefaciens ~3 Bacillus sp., Staphylococcus shark (83)

subtilomycin Bacillus subtilis MMA7 3.2 Bacillus, Listeria, Clostridium, Enterococcus marine sponge (84)

sonorensin Bacillus sonorensis MT93 6.3 Bacillus, Staphylococcus, Listeria, Pseudomonas, E. coli, Vibrio

sediment (85)

lichenicidin Bacillus licheniformis MRSA, Listeria, Enterococcus, Salmonella, E. coli seaweed (86)

pumiviticin Bacillus pumilus DR2 3.9 Salmonella, Proteus, Micrococcus, Lactobacillus seawater (87)

bacteriocin Bacillus sp. - Bacillus, Clostridium, Corynebacterium, Listeria fish gut (88)

bacteriocin Pseudomonas putida FStm2 Bacillus, Staphylococcus, Escherichia, Enterobacter, Serratia, Salmonella sp., Klebsiella, Vibrio, Pseudomonas

shark skin

(89)

BLIS: Bacteriocin-like-inhibitory substance


21

1.7. Research questions and thesis objectives

1.7.1. Research questions

Q1. Can Bacillus strains with a diverse range of antimicrobial activities be isolated from marine

environments?

Q2. Can these antimicrobial activities be harnessed to address a range of pathogenic microbial

threats to humans and animals?

Q3. Are there novel, previously uncharacterised, antimicrobials present within these isolates?

Q4. Can novel bacteriocin(s) be identified?

Q5. Can novel bacteriocin/s in marine isolates be heterologously cloned and expressed?

1.7.2. Research aims and significance.

The overarching aim of this project was to identify and characterise novel antimicrobial

compounds, particularly bacteriocins. This was to be achieved by:

Isolating marine-derived spore-forming bacteria within NhaTrang Bay, Vietnam Sea that

demonstrate inhibition against both Gram-positive and Gram-negative pathogens. The

diversity of antagonistic spore-forming bacteria within this environment was then evaluated.

Characterising isolates that show high antimicrobial activity by:

+ Testing for antimicrobial activity against a range of bacterial pathogens, including food

spoilage bacteria, and human and/or animal pathogens.

+ Bacterial identification based on 16S rRNA sequences.

+ Determining the nature of antimicrobial compounds presented in the media culture.

+ Selecting the isolates that were able to kill other marine antimicrobial producing species.

It likely indicates that these isolates produce novel antimicrobial compounds capable of

attacking other marine bacteria which harbour common immunity systems.

+ Characterising the nature of the antimicrobial products produced by these isolates to provide

evidence of bacteriocin production.

Genomics, via whole genome sequencing for promising isolates which displayed broad

spectra of inhibition. Various bioinformatics approaches were used to identify genes that are


22

likely to encode putative antimicrobials, with aims at understanding the mechanism of

antimicrobial production and identification of novel bacteriocin(s).

Purification of antimicrobial compounds for characterisation, using cation exchange

chromatography, hydrophobic interaction chromatography, reverse-phase high-pressure liquid

chromatography (RP-HPLC), matrix-assisted laser desorption ionization-time of flight mass

spectroscopy (MALDI-TOF MS) and other peptide sequencing techniques (N-terminal

sequencing and de novo sequencing).

Cloning and expression to produce the novel bacteriocins in a heterologous Bacillus subtilis

host.

1.7.3. Novel contributions

Elucidating the diversity of marine antagonistic Bacillus within NhaTrang bay, Vietnam Sea,

which has previously been unexplored

First employment of both genomic and protein approaches for antimicrobial compound

purification to understand the marine Bacillus genome and the potential of antimicrobial

compounds produced by marine Bacillus species.

Providing information about gene organisation of some novel bacteriocin biosynthetic gene

clusters (2 thiopeptides, 1 two-component lantibiotic, 1 sactipeptide and 1 lantibiotic, and 1

lassopeptide), which will be utilised for further cloning and heterologous expression to produce

these bacteriocins for both characterisation and commercialisation.

Successful cloning and expression of the sactipeptide bacteriocin.

1.8. Thesis organization

The project covers the following chapters:

Chapter 1: “Introduction”

Chapter 2: “Research methodology”.

Experimental chapter 3: “Broad antimicrobial spectra from marine spore forming bacteria

isolated from the Vietnam Sea”

This chapter includes the work of; sampling marine samples within the Vietnam Sea; the

isolation of the spore forming bacteria; screening to select candidates exhibiting antimicrobial

activity; selection of the promising candidates which may produce novel bacteriocins.


23

This chapter aimed to understand the diversity of antibacterial marine isolates within NhaTrang

bay and select the isolates producing probable novel antimicrobial compounds. The findings

of this chapter will answer Research Questions 1 and 3.

Experimental Chapter 4: “Bioinformatic identification of biosynthesized gene clusters of

antimicrobial peptides within marine Bacillus genomes”.

Chapter 4 covers the work of; whole genome sequencing, genome annotation, genomic

comparisons between marine isolates and terrestrial neighbour species, to understand the

variation between genomes, and in silico prediction of all putative antimicrobial peptides

within 6 genomes. This chapter aims to understand the marine Bacillus genome characteristics

and select the isolates having biosynthetic genes clusters of novel bacteriocins for antimicrobial

purification. This chapter will answer research question 2.

Experimental chapter 5: “Purification and characterisation of antimicrobial peptides

produced by Bacillus amyloliquefaciens #11”

This chapter covers the purification of antimicrobial compounds produced by this bacterium.

The purification applies various techniques of separation and mass spectrometry.

This chapter aims to purify all the antimicrobial peptides that the bacterium produced to obtain

a novel bacteriocin (thiopeptide) which was predicted from the genome sequence. This chapter

will answer research question 4.

Experimental chapter 6: “Purification and characterisation of antimicrobial peptides

produced by Paenibacillus polymyxa #23”.

This chapter covers the purification of antimicrobial compounds produced by this bacterium.

This chapter aims to purify all the antimicrobial peptides that the bacterium produced to obtain

novel bacteriocins (sactipeptide, lantibiotic, lassopeptide) which were predicted from the

genome sequence. This chapter will answer research question 4.

Experiment chapter 7: “Cloning and heterologous expression of sactipeptide, a novel

bacteriocin from marine Paenibacillus polymyxa #23, in Bacillus subtilis”.

The biosynthetic gene cluster of a novel sactipeptide (8,556 bp) was identified by in silico

analysis of the isolate #23’s genome; however, this bacteriocin was purified (mentioned in

Chapter 6). Therefore, this chapter aims to produce this sactipeptide by a heterologous Bacillus

subtilis host for characterisation.


24

The chapter includes work of; preparation of the sactipeptides gene clusters (8,556 bp) by

amplification from the genome of wild-type P. polymyxa #23 host, preparation of the plasmid

backbone originating from natural plasmid pDLL202, construction of the fusion plasmid from

these two materials by Gibson assembly, a transformation of the fusion plasmid to E. coli

TOP10 to control the plasmid copy number, transformation of fusion plasmid into E. coli

JIR702 to generate the multimeric fusion plasmid, transformation of the multimeric plasmid to

Bacillus subtilis BS34A for expression, optimization of sactipeptide production, purification

of sactipeptide, and characterisation of the sactipeptide. This chapter will answer research

question 2.

Chapter 8. Conclusion and future work.


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Chapter 2 RESEARCH METHODOLOGY

2.1 Sampling and sample preparation for bacterial isolation

Marine samples were collected by scuba-divers from 3 sites in NhaTrang bay (Vietnam Sea),

including Hon Mieu island (12.191837, 109.235086), Hon Mot island (12.173368, 109.271591)

and Hon Rua island (12.289501, 109.242413) [Figure 2.1]. The sites were chosen based on sea

sponge abundance. The sampling included sponges, seaweeds, sediments and seawater

samples. All the samples were transported under refrigerated conditions to the laboratory for

bacterial isolation.

Figure 2.1.Locations of sampling trips within Nhatrang bay (Vietnam sea).Three (X)

symbols indicate the three sites where marine samples were collected.

2.2. Isolation of spore-forming bacteria from marine samples

Solid samples were washed three times with sterile seawater (SSW) to remove externally

attached bacteria, 10 g of material was then homogenised in 90 ml of SSW using a sterile mortar

and pestle. The water samples were concentrated by centrifugation at 2500 x g for 20 minutes

to achieve 10 times concentration (50 mL in initial volume to 5mL). Sediments were dried by

heating at 50°C for 3 hours or air-dried overnight, and 10 g of each were vortexed in 90 mL of

SSW for 5 minutes, followed by centrifugation at 1000 x g for 1 minute. The supernatants were

collected, heated at 80°C for 20 minutes to kill non-spore-forming bacteria, serially diluted 10-


26

fold, and 100 µL aliquots were plated onto laboratory-prepared marine agar (LPMA) to isolate

the spore forming bacteria. Plates were incubated at 30°C for up to 3 days, and colonies were

picked daily based on morphology (size, colour, shape), and re-streaked onto LPMA plates to

obtain pure colonies. All pure isolates were stored at -80°C in LPMB supplemented with 20%

glycerol.

2.3. Assays to detect the antimicrobial activity.

2.3.1. Cross-streak assay for primary screening of marine isolates exhibiting

antimicrobial activity

Each bacterial isolate was streaked vertically onto an LPMA plate and incubated overnight at

30oC. The six indicator strains used in the initial screening were S. aureus, B. cereus, C.

albicans, E. coli, P. aeruginosa, and a methicillin-resistant S. aureus (MRSA). Each indicator

strain was streaked horizontally, from the edge of the plate to the pre-grown isolate streak. The

cross-streak assay plates were then incubated in the growth conditions appropriate for each

indicator strain, as detailed in Table 2.1. The gap between indicator strains and the test isolates’

growth indicated the presence or absence of antimicrobial activity [Figure 2.2]. After inspection

of the strength and spectrum of antimicrobial activity, a short-list of isolates with the strongest

antimicrobial activity was chosen for more detailed analysis (90). Their antagonistic activities

against 14 important pathogens were evaluated. The cross-streak assay was also used to detect

antimicrobial activities produced by the 23 short-listed isolates against the other members of

that group. Each marine isolate was streaked down the middle of LPMA plates, incubated

overnight at 30oC to allow growth and production of antimicrobial compounds, and then the

other 22 isolates were streaked from the edge of the plate to the central streak. The plate was

incubated overnight at 37oC and antimicrobial activity was determined based on the size of the

clear zone between test and indicator bacterial streaks.

Figure 2.2. Cross-streak assay.(A) Plates with antimicrobial-producing marine isolates as

there are zones of no growth between the marine isolate (vertical line), and indicators


27

(horizontal line). (B) Plate with a non-antimicrobial producing isolate without these

zones.

Table 2.1. List of indicators strains used in antimicrobial screening assays

Indicators Origins Media/temp./condition

(A) Gram-positive bacterial indicators

Lactobacillus plantarum Food origin, RMIT MRS/37°C/aerobic

Bacillus cereus ATCC 10876 MH/30°C/aerobic

Streptococcus faecalis ATCC9790 BA /37°C/aerobic

Staphylococcus aureus ATCC25923 BA /37°C/aerobic

MRSA Human pathogen, RMIT BA /37°C/aerobic

Listeria monocytogenes Human pathogen, RMIT BA /37°C/aerobic

Clostridium perfringens Chicken pathogen, RMIT BA /37°C/anaerobic

VRE Human pathogen, RMIT BA /37°C/aerobic

(B) Gram-negative bacterial indicators

Salmonella Enteritidis ATCC 13076 MH/37°C/aerobic

Escherichia coli ATCC 25922 MH/37°C/aerobic

Pseudomonas aeruginosa ATCC 15442 MH/37°C/aerobic

Campylobacter jejuni Chicken pathogen, RMIT BA/37°C/microaerophilic

Multidrug-resistant Klebsiella pneumonia

Human pathogen, RMIT MH/37°C/aerobic

(C) Yeast

Candida albicans ATCC 10231 MH/30°C/aerobic

2.3.2. Well-diffusion assay

The well-diffusion assay (WDA) was used to detect the antimicrobial compounds in a liquid

sample (91). This method relies on the diffusion of antimicrobial compounds through agar


28

plates, which then exhibit antimicrobial activity against sensitive bacteria, reducing or

preventing growth [Figure 2.3]. The diffusion of antimicrobial compounds creates a radial

concentration gradient outward from the well. Indicator strain sensitivity to antimicrobials can

then be quantified based on the measurement of the diameter of inhibition zones. The Muller

Hilton agar (MHA) (Oxoid) was used in the well diffusion assay as it supports the optimal

diffusion of antimicrobial products through the agar. MHA is a non-selective medium allowing

growth of all organisms plated and contains starch which can absorb possible toxins affecting

the bacterial growth, and also serves as a loose agar allowing better diffusion of the

antimicrobial compounds.

The well-diffusion assay was employed at this stage alongside the cross-streak method to

understand if the antimicrobial compounds were secreted into the culture supernatant. To make

the indicator lawn, several fresh colonies of indicator bacteria were suspended in buffered saline

to final turbidity of 0.5 McFarland units (OD60 0~0.08-0.1). A cotton bud was wet in this

bacterial suspension and used to inoculate a lawn of bacteria on MHA. A plug of the agar was

removed using a sterile glass Pasteur pipette (diameter of 6mm) and 100 µL of antimicrobial

samples (or media culture) was added to the wells then allowed to diffuse and incubated at the

indicators strains growth conditions. The diameter of the cell-free zone was measured.

Figure 2.3. Well-diffusion assay.The bacterial culture (or antimicrobial sample) was

added to the well. (+) presence of antimicrobial compounds with no bacterial growth;

(-) no antimicrobial compounds produced


29

2.3.3. Spot-on-lawn assay

The spot-on-lawn assay was also used to detect the presence of antimicrobial compounds in

liquid samples (92). It also relies on the diffusion of antimicrobial compounds across the agar

surface that consequently depresses the growth of indicator bacteria. The antimicrobial sample

is dropped directly onto the agar surface without the requirement of wells, therefore the method

uses less sample than other techniques (10 - 20 µL) and is suitable for enriched samples [Figure

2.4]

In this study, the spot-on-lawn assay was mainly used to detect the antimicrobial fractions

during purification. It was also used to identify marine bacteria exhibiting antimicrobial activity

via detection of inhibitory activity from fermented media culture. In practice, indicator colonies

were suspended in 20 mL of warm liquid soft media agar (0.75% agar) to a final cell density of

108 CFU/ mL, mixed by inversion and poured in a Petri plate and allowed to solidify. Next the

agar surface was dried in the fume hood for 10 minutes. Then 10-20 µL culture supernatant (or

antimicrobial liquid) was spotted directly onto the media surface, allowed to diffuse, and the

plate was incubated at indicators’ growth conditions. A zone of growth inhibition formed

around the bacterial spot indicated the presence of antimicrobial compounds.

Figure 2.4. Spot-on-lawn assay.The spot with the presence of a killing zone indicated the

presence of antimicrobial compounds.


30

2.4. Bacterial identification based on 16S rRNA gene sequence and phylogenetic tree

construction

2.4.1. Extraction of genomic DNA by bead beating

Bacteria were grown in LB broth overnight (until stationary phase with an approximate of 2 x

109 cell/mL). Approximate 2 mL of bacterial cultures were centrifuged to collect the cell pellet.

It was washed twice with TE Buffer low EDTA (Tris-EDTA pH 8.0; 10 mM Tris base, 0.1 mM

EDTA) to remove the remaining culture, then resuspended in 500 µL of TE buffer low EDTA,

and used for DNA extraction by the bead beating method (93). The suspension was transferred

to a screw-cap polypropylene 1.5 ml vial containing 100 µL of 100 μm glass beads. The bead

beating was carried out in a Mini-Beadbeater in two cycles of 1 min at 3,450 oscillations/min

with a 1 min period of cooling on ice between cycles (94). After the bead beating, the cell debris

was removed by centrifugation at 14,000 g for 10 minutes at 4oC; and the supernatant was

collected in a fresh tube for PCR amplification.

2.4.2. Amplification of bacterial 16S rRNA gene sequences, Sanger sequencing and

phylogenetic identification

Bacterial 16S rRNA genes were amplified from the isolated gDNA. PCR was conducted using

primers with the sequences (5’–3’) GGCGTGCCTAATACATGCAA and

TACAAGGCCCGGGAACGT (95, 96). PCR conditions comprised initial denaturation at 98°C

for 30 seconds, 30 cycles of 98°C for 5 seconds, 56°C for 10 seconds and 72°C for 20 seconds,

with a final extension at 72°C for 2 minutes; and then storage at 4°C. PCR products were

checked by electrophoresis in 1% agarose gel, subsequently purified by QIAquick PCR

Purification Kit (Qiagen), and then Sanger-sequenced (Micromon, Monash University,

Australia). The raw reads were trimmed of unclear nucleotides at both 5’ and 3’ terminal ends,

and subsequently BLASTn was used to search for homologies on the Bacterial 16S rDNA

Database (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The highly homologous 16S rDNA

sequences were downloaded for phylogenetic tree construction. All the sequences were aligned

using ClustalW (97) and the phylogenetic tree was subsequently constructed in MEGA7 (98)

using the neighbour joining method with bootstrap tests performed 1000 times and pairwise

detection.

2.5. The sensitivity of antimicrobial activities to enzyme and heat.

Enzymatic treatments of cell-free supernatants (CFSs) were conducted for 3 hours at 37°C with

pronase-E (from Streptomyces griseus), proteinase, trypsin, and lipase, at final concentrations


31

of 2 mg/mL. All enzymes were purchased from Sigma Aldrich. The antimicrobial activities of

the treated CFS preparations were evaluated by well-diffusion assay, against Clostridium

perfringens. To evaluate the thermal stability of antimicrobial substances, the antimicrobial

sample was heated at 60oC, 80 oC, 100 oC for 30 minutes, 60 minutes respectively then chilled

on ice. The antimicrobial activities of the treated samples were evaluated by well-diffusion

assay or spot-on-lawn assays against indicators.

2.6. Analysis of self-depression ability across marine Bacillus/Paenibacillus species

This experiment was designed to demonstrate whether these marine isolates were able to

produce novel antimicrobial compounds. It was set up based on the hypothesis that

antimicrobial-producing bacteria always have an immunity system to protect themselves from

the activity of the antimicrobial compounds that they produce. If a bacterium is killed by another

closely related bacterium (particularly by a strain of same species), it indicates that the bacteria

doing the killing may be producing uncommon antimicrobial compounds. Therefore, the self-

depression observed between strains of the same species may indicate the production of a novel

antimicrobial compound. The experiment was carried out to select isolates for subsequent

whole genome sequencing (WGS), to search novel antimicrobial substances. In this study, the

self-depression experiment employed the cross-streak assay. In practice, each marine Bacillus

isolate was first streaked vertically and the other 22 isolates were streaked horizontally to serve

as indicators. The inhibitory gaps formed between the two biomasses were recorded. The assay

was replicated for all 23 marine isolates.

2.7. Growth properties, antibiotic susceptibility testing, and enzyme production of isolates

The ability of the short-listed isolates to grow on different media was evaluated by spotting 5

µL of bacterial cultures onto several different media, including low nutrition media, such as

marine agar (MA 2216) and LPMA, and rich nutritious media, such as LB agar and Muller

Hilton agar, and incubated at 30oC overnight. Aliquots of cultures were spotted on LB agar

plates, and incubated under microaerophilic, aerobic, and anaerobic conditions at 30°C, and

aerobically incubated at 40°C and 50°C. To measure pH tolerance, bacterial cultures were

spotted onto LB plates in which the media had been adjusted to pH 5.0, 6.0, 7.0, 8.0, and 9.0.

Sodium chloride tolerance was determined on LB plates supplemented with 0%, 1%, 2%, 4%,

6%, 8%, 10%, 12% and 15% (w⁄v) NaCl; while bile salt tolerance was carried out on LB plates

supplemented with bile salt to final concentrations of 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6

M, 0.7 M (Bile Salts Mixture No. 3; Neogen Corporation). For antibiotic susceptibility testing,


32

LB agar plates were prepared supplemented with tetracycline, ampicillin, nalidixic acid, and

kanamycin at a final concentration of 50 µg/mL. The production of proteases, cellulases, and

amylases was evaluated respectively by spotting of 5 µL of bacterial cultures onto skim milk

agar; carbon deficient media (CDM) supplemented with 1% carboxymethylcellulose (CMC),

and CDM supplemented with 1% soluble starch. CDM contained 0.1 g/L yeast extract; 0.5 g/L

peptone; 16.0 g/L agar. Plates were incubated at 30oC overnight, followed by flushing the CMC

plates and starch agar plates with Gram’s iodine solution (Sigma Aldrich) for 1 minute. Positive

reactions, indicating enzymatic activity, were noted via halo zones around the bacterial spots.

2.8. Bacterial whole genome sequencing

2.8.1. Extraction of genomic DNA (gDNA) for whole genome sequencing

Six isolates selected for whole genome sequencing were grown in LB broth overnight (to

stationary phase). The cultures were then centrifuged to collect the cell pellets which were

washed twice with milli-Q water and gDNAs were extracted by the ‘Guanidine thiocyanate

method’ (GES method) (99)

Cell pellets were suspended in 100 µL of 50 mg/mL lysozyme solution in TE buffer and

incubated at 37°C for 30 minutes. Then 500 µL of GES solution was added, gently inverted to

mix. These mixtures were then left at room temperature for 10 minutes. Next, 250 µL of cold

ammonium acetate (7.5 mol/L) was added to cell lysates; mixed by gently inverting the tubes

and left at room temperature for 10 minutes to precipitate proteins and large RNAs. 500 µl of

chloroform:phenol:isoamyl solution (26:24:1) was added; the tubes were inverted 6-8 times to

mix; followed by centrifugation at 25,000 g for 10 minutes. The top aqueous layers were

collected by gently pipetting using plastic transfer pipettes. Then 0.54 volumes of cold

isopropanol were added to these layers, inverted gently 6-8 times to precipitate gDNAs. The

gDNAs were collected by centrifugation at 10,000 g for 2 minutes at 5°C. The supernatant was

decanted and discarded. The DNA pellets were washed 5 times by gently adding 1 mL of cold

70% ethanol to tubes; centrifuging at 8,000 x g for 2 minutes; decanting the supernatants. The

washed DNA pellets were dried using a Speed-Vac at 40oC for 5 minutes and suspended in 100

µl nuclease-free water or 100 µl TE buffer low EDTA.

2.8.2. Qualification of the gDNA

The gDNAs’ quality was assessed with the aid of a NanoDrop One spectrophotometer

(ThermoFisher Scientific) and DNA electrophoresis. Thus, 1 µL of each gDNA was load onto

the NanoDrop; values of DNA concentration, A260/ A280 ratio (wavelengths of 260 nm:


33

wavelengths of 280 nm), and A260/A230 ratio were inspected to estimate the contamination

of RNA and proteins, as well as extraction solvents. The A260/ A280 ratio was expected to be

~1.8 while the A260/ A230 ratio was expected to be within the range of ~1.8-2.2.

Also, 1µL of each gDNA was loaded in an agarose gel (0.8% agarose gel) that was

supplemented with 1X Gelred nucleic acid gel stain (Biotum) during gel casting. The DNA

electrophoresis was performed alongside GeneRuler™ 1kb Plus DNA Ladder (Thermo

Scientific). The electrophoresis conditions were 100V for 40 minutes, followed by visualization

of gDNA bands on the agarose gel with the aid of gelDoc device (BioRad). The samples were

considered to contain gDNAs of suitable quality when there was a single band of DNA which

was higher than the top band of the DNA ladder (10,000bp). The gDNA was then adjusted to a

final concentration of 2.5 µg/mL by the aid of the Qubit dsDNA HS Assay Kit (Thermo Fisher

Scientific) to be ready for DNA library preparation

2.8.3. Preparation of DNA library and whole genome sequencing

The genomic library preparation was performed using the Nextera XT DNA Library

Preparation Kit (Nextera XT DNA Library Prep Reference Guide, Document # 15031942 v05

May 2019) following the manufacturer’s instruction. The libraries were denatured with NaOH,

diluted with hybridization buffer, pooled and sequenced using Illumina MiSeq with 2x300 bp

paired-end reads.

2.9. Assembly of raw reads generated from whole genome sequencing

The raw read data (fastq) were assembled using the A5-miseq pipeline under default

parameters for bacterial species (100). During assembly, the raw reads were automatically

trimmed, filtered by quality, corrected for errors, and finally assembled to generate contigs. The

outcome of the assembly process was draft genomes of bacteria, which contained multiple

contigs.

2.10. Calculation of Average Nucleotide Identities (ANI) across genomes of terrestrial and

marine Bacillus/Paenibacillus species

The genome similarity amongst the marine isolates versus the terrestrial closely related

Bacillus/Paenibacillus strains was estimated using Average Nucleotide Identity (ANI) and pair-

wise BLASTn for similarity search of nucleotide sequences. The terrestrial closely related

Bacillus/Paenibacillus were selected based on the similarity in 16S rDNA sequences with ones

of the marine isolates. All genome sequences were downloaded (from NCBI) as fasta files, and


34

then submitted to web-based ‘Rapid Annotation using Subsystem Technology’ (RAST) for

internal consistency in gene-calling using the Glimmer algorithm and amino acid translation

(101). The majority of the terrestrial genomes had been described and annotated on NCBI, but

their genomes were submitted again to RAST for consistency of annotation. The GENBANK

files generated by RAST were downloaded for calculation of the ANI value across the genomes

by the GET_HOMOLOGUES methodology (102). The distance matrix was visualized as a heat

map to show similarities and differences between genomes within the corresponding

phylogenetic tree.

2.11. Estimation of a frequency distribution of CDS across genomes

The frequency distribution of CDS across genomes were identified by the

GET_HOMOLOGUES tool employing the BLASTp for similarity search of amino acids

sequences (CDS), and the OrthoMCL algorithms (103). The outcome of this calculation was 4

directories; core genes, soft core, shell genes, and cloud genes. The 'core' catalogue contains

genes presented in all genomes. The soft-core catalogue contains genes found in 95% of

genomes. 'Shell' catalogue contains dispensable genes which remain moderately conserved and

present in several genomes. “Cloud’ catalogue contains strain-specific genes.

In practice, three analyses were calculated separately for 3 datasets; one dataset of 6 genomes

of B. amyloliquefaciens species, one dataset for 6 genomes of B. halotolerans species, and one

dataset for 6 genomes of P. polymyxa species. In each calculation, a pangenome matrix was

first obtained by calling ‘get_homologues.pl’ Perl script with option-t 0 reporting all the

computed CDS clusters, OMCL (-M), E < 1e-05 for BLASTp searches; and -C 75% as the cut

off for BLASTp (103). Next the cluster directories were a result of calculation by calling the

‘compare_clusters.pl’ Perl script with ‘pangenomes matrix’ as input generated from the above

script. Next the 'parse_pangenome_matrix.pl' was called to classify genes in these four

compartments of core genes, softcore, shell genes, and cloud.

Ratios between these compartments were compared across datasets (B. halotolerans, B.

amyloliquefaciens, P. polymyxa). This calculation aimed to understand which species having

higher degrees of diversity in genomes, which probably indicates a higher potential of novel

antimicrobial substances for discovery.


35

2.12. Comparison between genomes of marine isolates and genomes of phylogenetically

related terrestrial strains.

The degree of similarity across genomes can be also confirmed by two bioinformatic tools;

“Sequence-based comparisons tool (SBCT)” of RAST annotation tool and MAUVE alignment

were employed to investigate diversity in the genomes across the bacilli species (104, 105).

The SBCT is used to determine the percentage of similar Coding sequences (CDS) found across

the genomes by using pair-wise BLASTp [Figure 2.5A]. The outcome is a circular graphic of

the chromosomes, in which each-by-each CDS was compared for similarity, then colour coded

based on the degree of similarity detected (from red to purple), as well as potentially being

marked as either unique or a unidirectional best hit or a bidirectional best hit [Fig.2.5A]. The

degree of similarity across genomes can be determined via a percentage of similar proteins

shared between genomes identified at a certain similarity threshold. In many similar studies, a

threshold of greater than 70% similarity has been used to evaluate the core proteins (106); while

in a recent publication a threshold of >50% similarity was used (107). In this study, the higher

threshold of greater than 70% similarity was used to obtain the percentage of similar protein

between related isolates. The higher the percentage of similar proteins (with a threshold of

greater than 70%) two bacteria shared the more homologous the two bacteria are.

The MAUVE was employed to compare the genome organisation across the genomes based on

the identification of ‘conserved gene regions’ visualized through ‘Locally Collinear Blocks’

(LCBs) shared between genomes [Figure 2.5B]. In MAUVE figures, two regions on two

genomes are shown as conserved when the LCB of them are the same in colour and are linked

by a thin coloured line. Therefore, the lesser numbers of LCB visualized the more homologous

the two genomes are. In this study, the draft genomes of marine isolates and completed genome

of terrestrial neighbour isolates were submitted to MAUVE software and the progressive-

Mauve mode of analysis was used. The software automatically reorders all contigs of draft

genome based on the gene composition of the completed genome, followed by pair-wise

alignment. The outcome was the organisation of multiple conserved colour blocks on two

genomes [Figure 2.5 B].


36

Figure 2.5. Schematic representation of pairwise genome comparison performed by the

“Sequence-Based Comparison Tool” (A) and MAUVE (B). (A) The output is a schematic of

chromosomes in which CDS are coloured based on the similarity in sequence. (B) Performed

by MAUVE. The output is an organisation of conserved regions on each genome. White colour

within LCB indicates genes loss/insertion caused by mobile DNA elements. The figures were

computed from example genomes provided.


37

2.13. In silico prediction of putative antimicrobial peptides within genomes of marine

Bacillus species

Bacillus species isolates frequently produce antimicrobial peptides (including bacteriocins and

non-ribosomally synthesized antimicrobial peptides) (21, 36); therefore, genes encoding these

kinds of antimicrobial peptides were sought within the selected marine isolates’ genomes by

using two bioinformatic tools; web-based ‘BActeriocin GEnome mining tooL’ (BAGEL) and

web-based the ‘antibiotics and secondary metabolite analysis shell’ (AntiSMASH) (108, 109).

The BAGEL software is the tool of choice to predict putative bacteriocins because it can detect

the small ORFs coding precursors of bacteriocins. These precursors are frequently omitted by

similar software, because of their small size. Draft genomes (in fasta format) of marine isolates

were submitted to the web-based BAGEL tool (http://bagels.molgenrug.nl/) and predictions

were computed. The outcome of the BAGEL prediction was genomic ‘areas of interest’ (AOIs)

that comprises both multiple ORFs which may or may not be indicative of bacteriocin

biosynthesis [Figure 2.6 A]. In this study, manual annotation was subsequently performed to

determine the actual size of bacteriocin biosynthetic gene cluster (BBGC) based on; (i) presence

of proteins required for bacteriocin biosynthesis, including precursor bacteriocin, transporters,

modification enzymes, regulator and immunity proteins; (ii) the biosynthetic gene clusters of

bacteriocins are usually flanked by a promoter and a terminator; (iii) the gene organization of

known similar bacteriocin gene clusters. All ORFs suggested by BAGEL were used as queries

sequences to search against protein databases to attempt to predict function. The protein

databases used included ’uniref 90’ database’, ‘non-redundant’ database (NR) and

BACTIBASE database (for precursor CDS) (110). The BACTIBASE database is a bacteriocin

database which contains large numbers of characterised bacteriocins. Also, SignalP (v5.0) was

employed to predict the cleavage site within the precursor sequence, subsequently resulting in

the prediction of ‘signal sequence’ and ‘core sequence’ (111). The MW of mature bacteriocins

were predicted based on the core sequence using Expasy tool (at

https://web.expasy.org/compute_pi/) (112). The prediction of functional genes required for

bacteriocin processing, transport and immunity were carried out using other software, such as

the TMHMM server (v.3.0) (http://www.cbs.dtu.dk/services/TMHMM/) to predict helical

motifs that are always present in transporters and possibly in immunity proteins (113). The

whole gene cluster encoding bacteriocin production was also predicted based on the evidence

of the flanking promoters and terminators, which were also predicted by BAGEL version 4.


38

The AntiSMASH is a tool to identify non-ribosomal peptides (NRPs) (lipopeptide, polyketide,

hybrids and bacilysin). The tool predicts NRPs compounds via detection of highly conserved

domains of synthase enzymes. In practice, the draft genomes of marine isolates were submitted

to AntiSMASH (at https://AntiSMASH.secondarymetabolites.org/#!/start). The outcome of

AntiSMASH prediction was predicted genomic ‘areas of interest’ (AOIs) encoding production

of NRPs and also bacteriocins, which frequently comprised ORFs with requirement/non-

requirement for biosynthesis [Figure 2.6 B].

Figure 2.6. Schematic representation of in silico prediction of antimicrobial peptides

performed by BAGEL and AntiSMASH tools. (A) The initial the genomic ‘areas of

interest’ containing the bacteriocin gene cluster was determined by the BAGEL. A

precursor of bacteriocin (green), enzymes for posttranslational modification of

bacteriocin (blue). (B) Example of a predicted non-ribosomally- synthesized

antimicrobial peptide encoding region predicted by AntiSMASH. Figure constructed

from the predictions using example genomes provided by the tools.


39

2.14. Refinement of media and growth conditions for enhanced bacteriocin production

Growth curves of bacterial isolates in medium were investigated in association with quantitation

of antimicrobial activity. To determine the kinetics of bacterial growth, a starter culture was

first prepared by inoculating a single colony into 10 mL of selected broth, incubated at 30oC

with a shaking speed of 200 rpm overnight. One mL of this starter culture was inoculated into

100 mL of fresh media, grown at 30oC with a shaking speed of 150 rpm to produce the

antimicrobial compounds. Five mL of culture was removed every 6 hours, up to 36 hours, and

OD600 and pH measurements were taken before being centrifuged at 4,500 g for 20 minutes.

The supernatant was filtered through a 0.45 µm PES membrane to obtain cell-free supernatant.

These cell-free supernatants were utilised in a well-diffusion assay, or spot-on-lawn assay to

detect antimicrobial activity. This analysis was to determine the point in the growth curve at

which the bacteriocin production was maximal.

2.15. Recovery of antimicrobial compounds after fermentation

The antimicrobial compounds not only presented in the bacterial culture but also associated

with the bacterial cell surface. The antimicrobial substances from the bacterial culture

supernatant were recovered by either precipitation using ammonium sulphate or by an

absorbance/elution method using Diaion HP-20 resin. The antimicrobial compounds associated

with the cell surface were recovered by extraction in a polar solvent.

2.15.1. Recovery of antimicrobial peptides from the cell-free culture supernatant by

precipitation using ammonium sulphate

Bacteriocins, being proteinaceous, could be precipitated with the addition of ammonium

sulphate (AMS) to the culture supernatant (114). In principle, a protein’s solubility increases in

the presence of low concentrations of salt (<0.15 M), and decreases in high salt concentrations,

and can precipitate when the salt reaches its saturation concentration. AMS is commonly used

as a salt to precipitate proteins from a liquid suspension. Different proteins can be precipitated

at different saturation concentrations of AMS; therefore, the AMS can be used to partially

purify a protein of interest by judicious selection of AMS concentrations.

In practice, to determine the AMS concentration used to precipitate a bacteriocin, different

amounts of AMS was add to 100 mL samples of cell-free culture supernatant to a final

concentration of AMS of 30%, 40%, 50%, 60%, 70% and 80% at 5oC. The culture supernatants

with added AMS were stirred to dissolve and left at 5oC overnight without shaking. The

mixtures were centrifuged at 17,500 g for 90 minutes and the supernatants were gently


40

decanted. The pellets were dissolved in 2mL of 1X PBS pH7 and 100 µL of dissolved protein

was used in a well-diffusion assay against indicator bacteria of interest. The AMS concentration

at which the precipitated protein exhibited the strongest antimicrobial activity was selected for

large scale precipitation. After precipitation, the dissolved sample was desalted using either a

dialysis membrane (Sigma Aldrich) or centrifugal filter (Millipore) with a protein size cut off

(2 kDa), before ion-exchange chromatography.

2.15.2. Recovery of antimicrobial compounds from cell-free culture supernatants using

Diaion HP-20

The precipitation of bacteriocins with ammonium sulphate followed by dialysis was a time-

consuming process, requiring at least 2 days. Therefore, bacteriocins present in a liquid sample

were also recovered using absorption to Diaion HP-20 resin, as an alternative approach. This

resin is a non-ionic macro reticular; therefore, it can adsorb the amphiphilic antimicrobial

compounds like bacteriocins and NRPs through i) hydrophobic interaction, ii) polar interaction,

iii) pi-pi bonding, and iv) hydrogen bonding. Bacteriocins can be eluted in the presence of polar

solvents (methanol, acetonitrile, and isopropanol).

In practice, the bacterial culture was first adjusted to a pH of 6.5. The Diaion HP-20 resin was

washed with 99.9% HPLC-grade methanol, followed by washing with milli-Q water to

eliminate the organic solvent. Approximate 2% (w/v) of this conditioned resin was added to

cell-free culture supernatant and gently stirred overnight at 5oC. The resin was collected in a

fresh 50mL syringe, washed with milli-Q water (3CV) then with 30% methanol (3CV) and

eluted with 50% methanol (3CV), followed by 70% methanol (3CV). All three elutes which

came from the same methanol concentration were combined. Two elutes (at 50% methanol, and

70% methanol) were concentrated by using speed Vac to eliminate the solvent and used in a

well-diffusion assay or spot-on-lawn assay to determine which elutes exhibiting antimicrobial

activity. After identifying the concentration of methanol to elute the antimicrobial compounds,

this methanol concentration was used in large scale purification.

2.15.3. Recovery of cell associated antimicrobial compounds from cell pellets by acidic

solvent extraction

Antimicrobial peptides are found not only in the bacterial culture supernatant but also

associated with the bacterial cell pellet. This association with cell pellet, probably cell surface,

is based on the ion bonding between positively charged bacteriocins (or NRPs) and negatively

charged bacterial cells; therefore, an organic solvent could be used to release the antimicrobial


41

substances. This step facilitates latter chromatography to purify the antimicrobial substance by

selectively release the antimicrobial compound while remaining most other proteins in the cell

pellet.

In practice, the cell pellets was stirred in 1/20 (v/v) 70% isopropanol pH 2 (using concentrated

HCl to adjust the pH) at 5oC for at least 4 hours. Subsequently, the mixture was centrifuged at

4,500 g for 20 minutes, and the supernatant was then filtered through a syringe membrane

(0.45µm) to obtain a cell-free solvent fraction. The cell-free solvent was concentrated in a rotary

evaporator to 1/100 volume to remove the solvent and adjusted to pH 7 with NaOH (named the

CFS fraction). This fraction was desalted before use in ion exchange chromatography.

2.16. Ion exchange chromatography for partial purification of antimicrobial compounds

Ion exchange chromatography is a separation technique based on the net surface charge of a

protein. It is distinguished into ‘cation exchange chromatography’ and ‘anion exchange

chromatography’. Cation exchange chromatography uses negatively charged resins (SP

sepharose, CM sepharose) which can capture positively charged proteins; while anion exchange

chromatography employs positively charged resins (Q sepharose, DEAE sepharose) to bind to

negatively charged proteins. The protein’s charge varies depending on the protein’s isoelectric

point (pI) and the pH of the buffer that is used to dissolve the protein. The protein’s pI value is

calculated based on amino acid composition. Thus, protein has no charge when dissolved in a

buffer with the same pH as the protein’s pI, or a positive charge when dissolved in a buffer with

a pH higher than the pI value. The proteins (or peptides) that bind to the resin can be released

with elution buffer containing salt (NaCl is commonly used). For an unknown bacteriocin, it is

necessary to select the resin types, running buffer, and elution buffer for a successful separation.

2.16.1. Identification of running buffer and types of resin for ion exchange

chromatography purification of antimicrobial compounds

In practice, five sterile 2 mL-eppendorf tubes were prepared, to 4 of them 500 µL of SP

sepharose added and the fifth tube had 500 µL of Q sepharose. All resins were washed 3 times

with milli-Q water by adding of 1 mL milli-Q water, inverting tubes for 5 minutes, spinning

down, and decanting the milli-Q water. This step aimed to eliminate the ethanol which was used

to store the resin. Next, 1 mL of each of four buffers; 20 mM acetate buffers pH 3.6; 20 mM

acetate buffers pH 4.6; 20 mM acetate buffer pH 5.6; 20 mM phosphate buffer pH 7.0 were

respectively added to one of the tubes containing SP sepharose resin.While 1 mL of 20 mM

Tris HCL pH 8.0 was added to the tube containing Q sepharose. The tubes were inverted for 5


42

minutes, spun down and the buffer was discarded. These steps were replicated 3 times to

equilibrate the resin. One mL of each buffer was added to the corresponding tubes. One hundred

µL of dissolved protein (well-desalted) were aliquoted into the tubes and mixed overnight at

5oC. Next, the tubes were spun down and the supernatants collected (named as GT fraction).

The resins were washed (three times) by adding 1 mL of the same buffer (3 times), inverting

tubes for 5 minutes, and decanting the buffers. One mL of elution buffer (same buffer + 1M

NaCl) wase add to each tube, inverted tubes for 20 minutes, and spun down to collect the

supernatants (EL fraction). One hundred µL of GT fractions and EL fractions were used in a

well-diffusion assay against indicators of interest to detect the presence of antimicrobial

compounds. The most effective resin and buffer combinations were selected for large scale ion

exchange chromatography, based on the antimicrobial activity being concentrated in the EL

fractions.

2.16.2. Determination of the NaCl concentration used in elution buffer

Cation exchange chromatography was performed using the running buffer and resin identified

from the above experiment. The operation of cation exchange included 4 steps, i) equilibrating

resin (3CV) with running buffer, ii) loading the sample onto the resin, iii) washing the resin

with the running buffer (3CV), and iv) eluting the bound antimicrobial compounds with elution

buffer (CV). To determine the concentration of NaCl required for the elution buffer the elution

steps used the elution buffer supplemented with various concentration of NaCl (0.1 M, 0.2 M,

0.3 M, 0.4 M, 0.5 M and 1.0 M). All these elutes were collected and utilized in a well-diffusion

assay against indicators of interest. The elute exhibiting the highest antimicrobial activity with

the lowest concentration of NaCl was selected for large scale ion exchange chromatography.

2.17. Hydrophobic interaction chromatography for partial purification of antimicrobial

compounds

Hydrophobic interaction chromatography is a separation technique based on bacteriocin’s

hydrophobicity. This method employs a resin that is an immobilized hydrophobic ligand. This

ligand can capture hydrophobic compounds. The compounds can be released in the presence of

organic solvents. Therefore, the elution commonly utilises a linear concentration gradient of

organic solvent or a step-gradient process.

In this study hydrophobic interaction chromatography was carried out using a Sep-Pak C18

column (Waters). The Sep-Pak column was first conditioned by equilibrating the resin (3CV)

with 100% acetonitrile (+0.01% TFA) followed by washing (3CV) with 2% acetonitrile in water


43

(+0.01%TFA) to reduce the acetonitrile. Subsequently, the sample was loaded into the column

drop-by-drop and washed (3CV) with 2% acetonitrile in water (+0.01%TFA) to remove the

unbound protein. To determine the concentration of acetonitrile to elute the antimicrobial

compounds, the resin was eluted respectively with different concentration of acetonitrile (10%,

20%, 30%, 40%, 50%, 60%, 70%, 80% and 90%). Each concentration was used with three

column volumes. All elutes were collected, lyophilized, dissolved in milli-Q water and used to

detect the presence of antimicrobial compounds.

2.18. Reverse phase high-pressure liquid chromatography (RP-HPLC) to obtain pure

antimicrobial compounds

HPLC using a reverse-phase column (C4-C18) is used to separate compounds based on the

protein’s hydrophobicity. The sample is injected into the RP-HPLC device, mobile phase (a

mixture of two solvents) carries the sample through the column where the hydrophobic

components in the sample are captured by the ligands immobilized on the matrix. Depending

on the hydrophobicity, these compounds are washed out of the column at different retention

time (RT), corresponding to different concentration of organic solvent. The elution can be

conducted under isocratic conditions or a gradient of organic solvent concentrations. During

operation, the elution of antimicrobial peptide can be visualized by absorbance readings at 280

nm, 230 nm or 214 nm. This chromatography can be used to obtain compounds at high purity

for peptide sequencing.

In this study, RP-HPLC was performed using a Zobax 300SB C18 column (Agilent

Technology).The mobile phase were mixtures of two solutions including solution A (milli-Q

water + 0.1% TFA) and solution B (acetonitrile + 0.1% TFA). The running parameters were

modified after consideration of the compounds’ physicochemical properties. Elutes were

collected every two minutes or based on the peak visualized on the monitor. All elutes were

lyophilized and the antimicrobial activity was evaluated. Some cycles of RP-HPLC were

performed until the elute fractions containing 1 peak visualized on the monitors, and 1 peak

visualized by MALDI-TOF MS.

2.19. Determination of antimicrobial purity by Matrix-Assisted Laser

Desorption/Ionization time-of-flight mass spectrometry (MALDI-TOF MS)

MALDI-TOF MS can be used to detect the presence of antimicrobial peptides in samples,

estimate the purity of antimicrobial samples, and can be utilised for protein identification (115).

The MALDI-TOF MS device consists of a “matrix-assisted laser desorption/ionization


44

(MALDI)” part, a “time-of-flight (TOF)” part containing a mass analyser detector, and a

monitor. A sample is dropped onto a metal plate, allowed to dry and then loaded into the

machine, the MALDI part shoots an ultraviolet laser beam at the sample spot to convert all

molecules present into ions. These ions vapourize into the TOF part where they are separated

according to their velocity under an electrostatic field, due to the charge and mass. The ions are

detected by a mass analyser detector which converts the ions into electrical signals through the

value of mass-to-charge (m/z). These signals are then transmitted to a computer for calculation

[Figure 2.7]. The m/z value of a protein is also the MW of that protein in Dalton.

Figure 2.7. The process of MALDI-TOF mass spectrometry.The sample is mixed with

the matrix which is commonly α-Cyano-4-hydroxycinnamic acid matrix (HACCA). The

mixture is dropped onto the metal plate, dried and the plate is load into the device. The

MALDI part shoots the laser beam to the dry spot of the sample to generate multiple ions

of charged molecules. These ions are separated in the TOF tube and detected by an MS

detector, which is analysed to generate the MS profile. The figure is from Clark et al.

(2013) (116).

In practice, 2 µL of both antimicrobial sample and protein standards were mixed separately

with 2 µL saturated α-Cyano-4-hydroxycinnamic acid matrix (HACCA). In case of

bacteriocins, two types of protein standards kit were selected including “Peptide calibration

standard II” (Burker) which included known peptides with a mass range of ~700 Da – 3,200

Da; and “Protein calibration standard I” (Burker) which consisted of known peptides covering

a mass range of ~4,000 Da - 20,000 Da. Next, 1 µL of each mixture was dropped onto a metal

plate at two different spots and dried at room temperature. This metal plate with sample spots

was load into the Bruker AutoFlex MALDI-TOF MS (Bruker). The operation started with a

calibration process on the protein standard first, followed by the measurement of the sample.

The calibration process was performed by shooting the UV beam at the standard spot multiple

times to obtain the mass range for calculation. After calibration, the sample was measured by


45

shooting at the sample spot until clear protein peaks were obtained. The spectrometry of the

protein sample was visualized through the monitor.

2.20. Peptide sequencing techniques

The bacteriocin consisted of 20 amino acid residues and was sequenced by de novo peptide

sequencing or N-terminal sequencing methods.

2.20.1. Peptide sequencing by de novo peptide sequencing

The HPLC purified bacteriocin samples were first fragmented by proteolytic digestion

(trypsin), which cleaves exclusively at arginine and lysine residues in the amino acid chain.

After desalting to remove residues of enzyme and salt, the pure fragmented bacteriocins were

load into a mass spectrometer, an LC-MS/MS instrument, to obtain the m/z value of all

fragments. The m/z values are searched against the Mascot database

(http://www.matrixscience.com/) to determine the amino acid sequence of each of the

fragments. The method is highly effective for known bacteriocins when the multiple digested

bacteriocins fragments have been assigned into the MASCOT database (117).

2.20.2. Peptide sequencing by N-terminal sequencing (Edman degradation method)

The method is frequently used to detect the amino acid sequence of novel bacteriocins by

identifying each amino acid residue, starting from N-terminus (118). In principle, the pure

peptide is mixed with phenylisothiocyanate (PITC) to form a complex of phenylthiohydantoin

(PTH)-bacteriocin. This mixture is loaded onto the device. Each cycle of the operation removes

an amino acid at the N-terminus until the sequence is fully identified. In this study, the N-

terminal sequencing technique was employed to identify the amino acid residues of

antimicrobial peptides using a Perkin-Elmer Precise Sequencer (Perkin-Elmer).

2.21. Estimation of bacteriocin size by Tricine SDS-PAGE

Tricine SDS-PAGE was used to estimate the bacteriocin size (119). In this study, the

antimicrobial samples were separated on a 16% Tris-Tricine gel. The gel consisted of a 16%

resolving gel layer at the bottom and 4% stacking gel layer at the top (described in table 2.3).


46

Table 2.2. Electrode and gel buffers for Tricine–SDS-PAGE, followed the protocol

suggested by Schagger H, 2006 (119)

Gel components Anode

buffer (10×)

Cathode buffer

(10×)

Gel buffer

(3×)

Tris (M) 1.0 1.0 3.0

Tricine (M) - 1.0 -

HCl (M) 0.225 - 1.0

SDS (%) - 1.0 0.3

pH 8.9 ~8.25 8.45

Table 2.3. The composition of acrylamide mixtures used to cast the Tricine SDSD gel,

followed the protocol suggested by Schagger H, 2006 (119)

Gel component Stacking gel

(mL)

16% resolving gel

(mL)

dH2O 1.10 1.56

40% (w/v) acrylamide mix 0.27 4.00

3X Gel buffer 0.62 3.33

Glycerol (g) - 1.06

10% (w/v) APS 0.01 0.05

TEMED 0.002 0.005

Samples were prepared by adding 32 µL of antimicrobial sample to 8 µL of 4X tricine sample

buffer (Bio-Rad) to give a total of 40 µL of a mixture and boiled at 100 °C for 5 min. Then, 15

µL of the mixture was loaded into two separated wells on a16% Tris-Tricine gel, along with

Dual Xtra Precision Plus Protein™ Prestained Standards (Bio-Rad). The running buffers used

a cathode buffer and an anode buffer (described in table 2.2). The running condition was set at

80 V for 15 minutes to allow the sample in the stacking gel layer to completely migrate into the

resolving gel layer and then increased to 100 V and run for 3.5 hours. The gel was cut into two

halves. One half was stained with Coomassie Blue staining solution or Silver staining solution

(if protein stained by Coomassie Blue was faint), followed by destaining in “destaining buffer”

to visualize the protein bands; the other half of the gel was used to conduct a zymogram assay.


47

Size and location of bacteriocin on the gel was estimated from results of both the stained gel

and zymogram assay.

2.22. Zymogram assay

Half of the Tris-Tricine gel was first fixed in ‘fixing solution’ for 2 hours, was then washed

with milli-Q water for 4 hours (with 4 water changes) and placed on soft media agar (0.75%

agar). This medium agar was initially prepared by mixing 15 mL of molten media agar (at 50

°C and 0.75% agar) with 200μL of an overnight culture of a bacterial indicator, pouring into

the plate to solidify. When the gel was placed on this agar, another 15 mL of seeded molten soft

agar was poured to overlay the gel and allowed to solidify. The plate was incubated at optimal

conditions for the bacterial indicator. Bacteriocin size was then identified by comparing the

position of the killing zone that appeared on zymogram half gel to the stained half gel in

association with the protein ladder (120).

2.23. Characterisation of the physicochemical properties of antimicrobial compounds

To determine enzymatic degradation susceptibility of the antimicrobial compounds, 50 µL of a

sample (or culture) were mixed with various enzymes (proteinase K, pronase E, trypsin,

catalase, and lipase) used at a final concentration of 2 µg/L. The mixtures were incubated at

37oC for 2 hours, followed by heating at 80oC for 5 minutes to inactivate the enzymes. The

antimicrobial activity of the treated samples was evaluated using a well-diffusion assay or spot-

on-lawn assay against indicators of interest. Thermal stability was determined by incubating

purified peptide or culture at various temperatures (40oC, 50oC, 60oC, 80oC, 90oCand 100oC)

for 1 hour, followed by cooling on ice before being used in spot-on-lawn or a well-diffusion

assay.

2.24. Overview of heterologous cloning and expression of bacteriocin

The heterologous cloning and expression employed an E. coli/Bacillus shuttle plasmid,

pDLL202, as the carrier of a whole bacteriocin gene cluster, and B. subtilis BS54A as host for

expression of bacteriocins. This customized plasmid and the bacterium was provided by

Monash University that is designed for cloning a large nucleotide fragment and expression the

gene of interest. The plasmid pDLL202 contains two compatible origins of replication

(colE1rep and repA) enabling replication in both E. coli and B. subtilis hosts, four multi-

cloning-sites (MCS) which include recognition sites of various restriction enzymes, and

antibiotic resistance genes (kanR, ermB, catP, and ampR) encoding resistance to kanamycin,

erythromycin, chloramphenicol and ampicillin. During cloning and transformation, E. coli


48

DH5α cells were used to maintain the native pDLL2002 plasmid. E. coli Top10 served as host

to control the plasmid copy number, E. coli JIR702 served as a host to induce a multimeric

fusion plasmid, described in Table 2.4. The B. subtilis BS54A strain was incubated in Trypton

soy agar (TSA) or broth (TSB) at 30°C while the E. coli strains were propagated aerobically in

Luria−Bertani broth at 37°C. Chloramphenicol was used as a selective antibiotic at a final

concentration of 5 μg/mL for B. subtilis and 30 μg/mL for E. coli strains, while kanamycin was

used at a concentration of 50 μg/mL.

Table 2.4. List of strains and plasmids used for cloning /expression

Strains or plasmids Relevant properties Origins

Strains

E. coli DH5α host for copy control pD202L vectors RMIT university

E. coli Top10 host for copy control pD202L/ insert vectors RMIT university

P. polymyxa #23 Wild type sactipeptide producing strain This study

E. coli JIR702 Host for induction multimeric DNA Monash university

B. subtilis BS34A Host for heterologous expression of sactipeptide Monash university

MRSA indicator strain for an antimicrobial test, Cm5R RMIT university

Plasmids

pDLL202 E. coli and B. subtilis shuttle cloning vector; pCC1BAC origin and pAMbeta1 origin, EmR, CmR, AmpR (10,153 bp)

Monash university

pDLL202/G23sac Fusion plasmid harbouring whole sactipeptide gene cluster (15,479 bp)

This study

2.25. Extraction of the plasmids from E. coli host

To extract the plasmid (pDLL202 and the fusion plasmid), the E. coli host (DH5α, Top10,

JIM702) was sub-cultured in 10mL of selective LB broth. Of that culture,, 2mL was centrifuged

at 5,000 g for 20 minutes and the pellet was collected to extract the plasmid. The plasmid was


49

extracted using the Monarch® Plasmid Miniprep Kit (New England Biolabs). The procedure

followed the manufacturers’ instructions.

2.26. Preparation of the plasmid backbone by enzymatic digestion

The native plasmid pDLL202 was digested by MluI-HF and SpeI-HF (New England Biolabs).

The mixture was then loaded onto a 1% agarose gel and electrophoresed for 40 minutes at 100V.

The band of the plasmid backbone was identified based on the size, excised and purified using

the Monarch® DNA Gel Extraction Kit (NEB).

2.27. Preparation of the whole sactipeptide genome cluster by PCR amplification.

The P. polymyxa #23 genomic DNA was extracted by the GES method and used as a template

in PCR. The whole sactipeptide gene cluster was amplified from P. polymyxa #23’s genome

using the designed primers. Due to the requirement of many optimization steps, the details of

PCR reaction was mentioned in chapter 7.

2.28. Preparation of E. coli competent cell.

To prepare the E. coli competent cells (Top 10, JIR702) for electroporation, a starter culture

was grown by inoculating a single fresh bacterial colony into 10 mL of LB and incubated at

37oC overnight at 200 rpm. All the starter culture was inoculated into 1L of fresh LB media and

incubated at 37oC with shaking until the OD600 reached ~0.35-0.4. The culture was centrifuged

at 1,000 g for 20 minutes at 4oC to collect the bacterial pellet. The pellet was then washed twice

with 250 mL of cold milli-Q water followed with two washes with 100 mL of cold 10% glycerol

before resuspending in 2 mL of 10% glycerol (OD600~200-250). The bacterial suspension (100

µL) was aliquoted to prechilled 1.5 mL Eppendorf tubes and stored at -80 oC.

2.29.Transformation of the fusion plasmid into E. coli by electroporation

The transformation of the fusion plasmid into E. coli (Top10/ JIM702) was carried out by

electroporation. Briefly, the competent E. coli cells (100 μl) were mixed with the given plasmid

vector and held on ice for 5 min. The mixture was transferred to a 0.2 cm electroporation cuvette

(Bio-Rad). After pulsing under specific conditions (field strength 2.1 kV/cm, capacitance 25

μF, resistance 100 Ω), the cell suspension was immediately diluted with 1 ml of pre-warmed

SOC media (see the appendix for recipe), then incubated at 37oC for 1 hour with shaking speed

of 250 rpm. A portion of the cell suspension (100 μl) was placed on selective LB and incubated

at 37°C overnight. The colonies which formed on the plates were patched onto a fresh selective

plate for subsequent confirmation.


50

2.30. Preparation of Bacillus competent cells

The multimeric fusion plasmid was transformed into B. subtilis BS34A via natural

transformation. To prepare the competent Bacillus subtilis BS34A cells, a fresh Bacillus colony

on a TSA plate was suspended in 10 mL of spC media in a 50 mL falcon tube to a final OD600

of~0.2 the suspension was incubated at 37oC at 275-300 rpm until cells reached early stationary

phase. The best indicator for reaching this stage is that the OD increases less than 20% in 30

minutes. Then 2 mL of the culture was diluted by adding it to 10 mL of SpT medium (see the

appendix for recipe) in a fresh flask and incubating at 37oC for 70-90 minutes at 250 rpm and

1 mL of the SpT culture was transferred to a fresh epperdorf tube.

2.31. Transformation of the fusion plasmid into Bacillus subtilis BS34A

To transform B. subtilis, 1-3 μL of plasmid was added to a tube containing the suspension of

Bacillus competent cells. One to three uL of sterile milli-Q water was added to another tube as

a negative control. These tubes were then shaken at 37oC for 30 minutes at 200 rpm. The cells

were harvested by low-speed centrifugation at 4,500 x g for 10 minutes, resuspended in 1-2 mL

of LB broth and incubated with shaking at 37oC for 90 minutes. The bacterial pellet was

centrifuged, resuspended in 0.5 mL of LB, and 0.1 mL of samples were plated onto Tryptone

soya agar (TSA) (Oxoid) plates supplemented with chloramphenicol to the final concentration

of 5 µg/mL. Presence of fusion plasmid in the transformant was confirmed by PCR of the gene

encoding the structural gene from the bacteriocin gene cluster, and by sequencing of the PCR

product.


51

Chapter 3 BROAD SPECTRUM ANTIMICROBIAL ACTIVITIES OF SPORE-

FORMING BACTERIA FROM THE VIETNAM SEA

3.1. Introduction

The emergence and spread of bacterial pathogens resistant to last-line antibiotics, for example,

carbapenem-resistant Gram-negative pathogens, methicillin-resistant Staphylococcus aureus

(MRSA), and vancomycin-resistant Enterococci (VRE), has been of great concern in both

human and veterinary medicine. Antibiotic resistance genes are frequently located on mobile

elements such as conjugative plasmids and transposons which facilitate horizontal and vertical

transmission, leading to increasing numbers of multi-resistant bacteria worldwide. The high

incidence of antibiotic-resistant bacteria has many implications for pathogen control and there

is an urgent need for alternatives to currently available antibiotics to re-control these life-

threating antibiotic-resistant pathogens. The discovery of novel antibiotics from terrestrial

environments over the last few decades has been challenging, due to exhaustion of these

traditional antibiotic sources. According to the FDA, approval of new medically important

antibiotics had decreased by 56% over the last few decades and there has been no evidence for

an increase in discovery rate since that study (12).

The marine environment has been identified as a promising alternative source for antibiotic

discovery, due to the high abundance of antibiotics produced by various members of diverse

marine microbial communities. It has been hypothesized that marine bacteria are under

unusually rigorous selection pressures because of the environmental conditions with which they

must contend. They are typically exposed to low levels of nutrition and rapid changes in

nutrition and physical conditions due to wave and tidal action. These harsh chemo-physical

conditions have selected bacteria that can employ adaptations to out-compete other bacteria.

Diversification of antimicrobial production is one such adaptation that can be essential for

occupying and defending an ecological niche. It is hypothesized that marine environments may

harbour species producing novel antimicrobial compounds which could be effective against a

range of bacteria, including antibiotic-resistant bacteria. Among the diverse marine bacterial

communities, members of the Bacillus genus characterised as spore formers, have been shown

to produce an array of structurally diverse antibiotics, including ribosomally synthesized

peptides (bacteriocins), non-ribosomal secondary metabolites and bacilysin. Bacteriocins are of

interest. These small cationic peptides commonly have a narrow spectrum of activity, affecting

a limited range of bacteria and a low resistance rate; making them attractive antimicrobials for

application against bacteria that have acquired resistance to the current range of available


52

antibiotics. Many terrestrially derived bacteriocins have been characterised, but little is known

of marine-derived bacteriocins. Vietnam covers more than 1 million square kilometres of

coastal areas, incorporating a variety of tropical marine ecosystems with abundant marine

species. These characteristics indicate that the Vietnam Sea is a promising source to explore for

novel antimicrobials.

The objective of this study was to evaluate a diverse collection of spore-forming bacteria from

the Vietnam Sea for antimicrobial molecules with activity against a range of important

pathogens, including multi-antibiotic-resistant pathogens. The bacterial screening was

restricted to spore-forming bacteria because such bacteria have previously proven to be a richer

source of antimicrobials than general bacterial populations, and facilitates for antibiotic

production at the industrial level (121, 122).

3.2. Results

3.2.1. Thermally resistant, spore-forming, bacteria derived from the marine

environment display antimicrobial activity against important pathogens

A total of 389 heat resistant, spore-forming, bacterial isolates were cultured from 50 marine

samples. They demonstrated a range of colony morphologies (Figure 3.1, Table 3.1).

Table 3.1. Antimicrobial producing bacteria identified from marine samples

Number of marine samples collected

Number of isolated spore-forming bacteria

Number of isolates with antimicrobial activity

Percentage of antimicrobial isolates *

Sponges 16 183 28 71.97%

Seaweeds 13 92 15 3.86%

Sediments 13 81 16 4.11%

Seawaters 8 33 6 1.54%

Total 50 389 64 16.45%

* Percentage of antimicrobial isolates was calculated based on the number of isolates

with antimicrobial activity and total number of isolated spore-forming bacteria


53

Figure 3.1. Diversity in the morphology of the marine isolates isolated from NhaTrang

bay, Vietnam Sea.(A) Examples of three of the four kinds of samples collected for

bacterial isolation; sponge, seaweed, sediment. (B) Colony morphologies of some pure

isolates. (C) Plate with colonies isolated from seaweed.

A total of 389 heat resistant, spore-forming, bacterial isolates, were cultured from 50 marine

samples (Table 3.1). They demonstrated a range of colony morphologies (Figure 3.1). Primary

screening against six indicator strains identified 64 isolates (16.4%) with antimicrobial activity

(Figure 3.2.B). The proportion of isolates that exhibited activity against Gram-positive indicator

strains, B. cereus (93.7%), S. aureus (84.3%), and S. faecalis (87.5%), was higher than the

proportion that exhibited activity against the Gram-negative indicators, E. coli (50%) and P.

aeruginosa (4.6%), and the yeast, C. albicans (21.4%).

The spectra of antimicrobial activities, exhibited by a select group of the 23 most potent isolates

from the primary screen, were determined against an expanded panel of 14 indicator strains,

including important multidrug-resistant pathogens, were performed using two assays; a cross-


54

streak assay and a well-diffusion assay. There was considerable variation in the strength and

spectra of antimicrobial activity across the 23 short-listed isolates. The pathogenic indicator

strains most commonly affected by the antimicrobial compounds expressed by the screened

isolates were the Gram-positive species (Table 3.2). Of the bacterial indicators, C. perfringens,

B. cereus and S. aureus were inhibited by 83% (19/23) of the test isolates, whereas the

proportion with activity against the Gram-negative indicators; Campylobacter jejuni, 70%

(16/23), Campylobacter coli, 61% (14/23), was lower. Two antibiotic-resistant Gram-positive

pathogens; MRSA and VRE were inhibited by respectively 83% (19/23) and 57% (13/23) of

isolates, while an antibiotic-resistant Gram-negative pathogen, multidrug-resistant Klebsiella

pneumonia (MRKP), was inhibited by only one isolate, P. polymyxa #23. The Gram-negative

bacteria P.aeruginosa was also inhibited by P. polymyxa #23. The growth of food-borne

pathogens; L. monocytogenes, E. coli and Salmonella Enteritidis were depressed by,

respectively, 78%, 44%, and 57% of the isolates. None of the selected isolates had inhibitory

activity against C. albicans.

The cross-streak assay was found to be more sensitive than the well-diffusion assay. In most

cases, antimicrobial activity was detected in both assays but occasionally the activity was less

or absent in the well-diffusion assay. This difference was particularly apparent when S. aureus,

MRSA and most of the Gram-negative pathogens; C. jejuni, C. coli, E. coli and S. Enteritidis.


55

Table 3.2. Antimicrobial activity of short-listed isolates against indicators strains. The antimicrobial activities were evaluated by well diffusion

assay (WDA) and cross streak assay (CSA) methods

#01 #02 #03 #04 #05 #06 #07 #08 #09 #10 #11 #12

CSA WDA CSA WDA CSA WDA CSA WDA CSA WDA CSA WDA CSA WDA CSA WDA CSA WDA CSA WDA CSA WDA CS WD

C. perfringens ++ ++ ++ + - - + + ++ +++ ++ ++ - - +++

++ - - + + +++ ++ ++ +

S. aureus +++ ++ ++ - + - - - + + +++ - - - +++ - - - ++ ++ ++ - ++ -

B. cereus ++ ++ ++ ++ - - + - + ++ +++ ++ - - +++ +++ ++ - + + +++ ++ + +

L. monocytogenes ++ ++ ++ + - - + - - - +++ ++ + - ++ + - - - - ++ + - -

L. plantarum ++ + + + + + - - - - ++ ++ - - ++ ++ - - + + ++ ++ - -

C. albicans - - - - - - - - - - - - - - - - - - - - - - - -

E. coli + - + - - - - - + - - - - - + + - - - - + + + -

S. Enteritidis + - - - - -

- - - ++ - + - ++ + - - - - ++ + + -

C. jejuni ++ + ++ + - - - - ++ - ++ ++ + - ++ ++ - - - - +++ ++ - -

C. coli + +++ ++ ++ - - ++ +++

+

+ - + +++ + - + +++

+

- - - - ++ +++ ++ ++

P. aeruginosa - - - - - - - - + - - - - - - - - - - - - - - -

MRSA +++ ++ ++ - + - - - + + +++ - - - +++ - - - ++ ++ ++ - ++ -

VRE ++ ++ ++ ++ - - + + - - + - - - +++ +++ - - ++ ++ ++ ++ + ++

MRKP - - - - - - - - - - - - - - - - - - - - - -


56

#13 #14 #15 #16 #17 #18 #19 #20 #21 #22 #23 Percent (%)

CSA WDA CSA WDA CSA WDA CSA WDA CSA WD CSA WDA CSA WDA CSA WDA CSA WDA CSA WDA CSA WDA CSA WDA

C. perfringens +++ +++ ++ + ++ + ++ + ++ ++ ++ + ++ ++ ++ + +++ +++ ++ - ++ + 86.7 82.6

S. aureus +++ - +++ - +++ - +++ - ++ - ++ - ++ +++ +++ - - - +++ - +++ + 82.6 21.7

B. cereus ++ +++ +++ ++ +++ ++ +++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ 82.6 82.6

L. monocytogenes ++ ++ +++ +++ +++ ++ +++ ++ ++ ++ ++ +++ ++ - +++ +++ ++ ++ ++ ++ ++ - 78.2 60.8

L. plantarum ++ +++ - - - - - - - - - - ++ ++ - - ++ ++ - - + + 47.8 47.8

C. albicans - - - - - - - - - - - - - - - - - - - - - - 0 0

E. coli ++ + - - - - - - - - - - ++ - ++ - - - - - ++ ++ 43.5 17.3

S. Enteritidis ++ - - - + - + - + - + - ++ - ++ - +++ + + - +++ ++ 56.5 17.3

C. jejuni + - ++ - + - + - ++ - ++ - - - - - + - ++ - ++ ++ 69.5 26

C. coli + ++++ - - - - - - ++ - + - ++ - + - ++ ++++ + - +++ ++ 60.8 43.5

P. aeruginosa - - - - - - - - - - - - - - - - - - - - ++ ++ 4.3 4.3

MRSA +++ - +++ ++ +++ ++ +++ ++ ++ ++ ++ +++ ++ + +++ ++ - - +++ ++ +++ ++ 82.6 21.7

VRE ++ +++ - - - - - - - - + + + ++ - - ++ ++ - - + + 56.5 52.2

MRKP - - - - - - - - - - - - - - - - - - - - ++ ++ 4.3 4.3

+: zone of inhibition observed with a clear halo of growth inhibition in at least one time point; ++ and +++: increased activity as assessed visually by the

increased diameter of the inhibition zone; -: no inhibition. The percentage values were calculated based on the ratio between the number of isolates with

antimicrobial activity against the indicator strain and the total 23 isolates tested. MRKP: Multidrug-resistant Klebsiella pneumonia; VRE: Vancomycin-

resistant Enterococcus faecalis; MRSA: Methicillin-resistant Staphylococcus aureus.


57

3.2.2. Taxonomic analysis of antimicrobial isolates

Of the 23 isolates selected as expressing the most antimicrobial activity, there were 22 Bacillus

species isolates and 1 Paenibacillus polymyxa isolate. Their 16S rRNA sequences shared 99%

- 100% identity with, mostly, terrestrially derived species (Table 3.3, Figure 3.2). The most

commonly identified species amongst the short-listed isolates were B. subtilis (10 isolates) and

other members of the B. subtilis group, such as B. amyloliquefaciens (5 isolates), B.

licheniformis (1 isolate), and B. safensis (1 isolate). Besides, members of other Bacillus groups

were also identified including B. pacificus (2 isolates), belonging to the B. cereus group; B.

halotolerans (3 isolates), and P. polymyxa (1 isolate)

Table 3.3. Closest species of producers, identified by BLASTn of 16S rRNA against 16S

rRNA database (NCBI) (18/06/2019)

Isolates Closed species Degree of identity (%)

#01 Bacillus halotolerans / Bacillus mojavensis/ Bacillus subtilis subsp. 100 #02 Bacillus subtilis 100 #03 Bacillus licheniformis/ Bacillus haynesii 99.92 #04 Bacillus subtilis 100 #05 Bacillus subtilis 100 #06 Bacillus amyloliquefaciens 99.92 #07 Bacillus pacificus/ Bacillus paranthracis / Bacillus cereus 100 #08 Bacillus amyloliquefaciens 99.92 #09 Bacillus pacificus/ Bacillus paranthracis / Bacillus cereus 100 #10 Bacillus safensis/ Bacillusaustralimaris/ Bacillus pumilus 100 #11 Bacillus amyloliquefaciens 99.92 #12 Bacillus subtilis 100 #13 Bacillus amyloliquefaciens 99.92 #14 Bacillus subtilis 100 #15 Bacillus subtilis 99.92 #16 Bacillus subtilis 99.87 #17 Bacillus subtilis 99.92 #18 Bacillus subtilis 99.76 #19 Bacillus halotolerans/ Bacillus mojavensis 100 #20 Bacillus tequilensis/ Bacillus subtilis 100 #21 Bacillus amyloliquefaciens subsp. plantarum/ Bacillus siamensis 99.84 #22 Bacillus subtilis 100 #23 Paenibacillus polymyxa 99.45


58

Figure 3.2. Phylogenetic tree of the 23 short-listed isolates.The neighbour-joining

phylogenetic tree was constructed using the maximum composite likelihood method,

bootstrap method of 1000 replication and pairwise deletion by MEGA &. In this tree, the

23 marine isolates were displayed by assigned number, while all reference strains bacteria

were closely related bacteria identified and downloaded from NCBI.


59

3.2.3. Proteolytic digestion of antimicrobial culture revealed a dominance of

proteinaceous compounds

The proteinaceous nature of the anti-C. perfringens activity of the isolates was determined by

digesting cell culture supernatants used in the well-diffusion assay with proteases. In 16 of the

19 isolates tested the antimicrobial activities against C. perfringens were reduced or lost after

treatment with at least one proteolytic enzyme (Table 3.4). The antimicrobial activity of each

preparation, treated with each of the enzymes, were directly compared to the untreated control

on the same plate and therefore the zones of clearing are directly comparable and small

differences in zone diameter are informative. The pronase-E enzyme eliminated anti-C.

perfringens activity from 10 isolates, while proteinase-K removed the activity from 4 isolates.

Some antimicrobials (produced by isolates #06, #11, and #21) were affected by both proteinases

and a lipase. Other isolates showed reduced, but not eliminated, antimicrobial activity following

protease treatment (#01, #08, #20, #21). The antimicrobial activities produced by isolates #5,

#18, #19 were not affected by any of the enzymes used. Of the isolates in which antimicrobial

activity was not affected by proteases, isolates #05 and #19 were also resistant to heat treatment

whereas anti- C. perfringens activity was abolished when #18 supernatant was heated.

Table 3.4. Enzymatic sensitivity profile of antimicrobial activities. These shortlisted 19

isolates showed antagonistic activities against C. perfringens.

Isolates Killing

zone (mm)

Proteinase

K

Pronase

E

Trypsin Lipase Sensitive to

#01 14 11 11 14 14 Pronase, proteinase K #02 11 9 - 11 11 Pronase, proteinase K #04 9 0 - - 9 Pronase, proteinase K trypsin #05 24 24 24 24 24 - #06 15 13 11 - - Pronase, trypsin, lipase #08 14 12 12 14 14 Pronase, proteinase K #10 9 - - - 9 Pronase, proteinase K trypsin #11 17 15 - 15 - Pronase, proteinase K lipase #12 12 9 - - 12 Pronase, proteinase K, trypsin #13 16 16 16 13 18 Trypsin #14 9 9 - - - Pronase, proteinase K trypsin, #15 9 0 - 9 9 Pronase, trypsin #16 12 0 - - 12 Pronase, proteinase K, trypsin #17 12 10 - - 9 Pronase, proteinase K, trypsin #18 10 10 10 10 10 - #19 14 14 14 14 13 - #20 11 11 9 11 11 Pronase #21 15 11 12 15 12 Pronase, proteinase K, lipase #23 13 13 - 13 13 Pronase


60

(*): The diameter of the killing zone (in millimetres). The value included the diameter of

wells (~6mm)

3.2.4. Determination of the growth depression within the short-listed group;

showing the production of potentially novel antimicrobial activities

It is hypothesised that bacteria produce antimicrobial compounds to compete within an

ecological niche. We, therefore, investigated whether this group of isolates exhibited any

antimicrobial activities against each other [Table 3.5]. Growth inhibition was detected, using

the cross-streak assay, between strains of the same species for isolates B. amyloliquefaciens

#06, #08, and B. subtilis #05. Cross-species antimicrobial activities were detected for many of

the isolates, including, B. amyloliquefaciens #06, #08, #11, #13, B. licheniformis #03; B.

safensis #10; B. halotolerans #01, and P. polymyxa #23. Particularly, P. polymyxa #23

depressed growth of the entire marine Bacillus species.

3.2.5. Growth characteristics of antimicrobial-producing isolates

The basic growth parameters of the 23 isolates were qualitatively evaluated (Table 3.6). All

isolates could grow on various kinds of media. Growth was better in rich nutritious medias such

as Luria-Bertani (LB), brain heart infusion (BHI), Muller Hinton agar (MH), and blood agar

(BA), than growth in less nutritious media such as; lab-prepared marine agar (LPMA) and

marine broth (Difco 2216). All 23 isolates grew vigorously under both aerobic and

microaerophilic conditions but not in anaerobic condition, under incubations temperatures of

30°C, 37oC, 40°C, 50°C, and under pHs ranging from 6.0 to 9.0. Some isolates had reduced

growth at pH5. All isolates could grow in media supplemented with 5% NaCl, and all but isolate

#23 grew in 7% NaCl. No isolate could tolerate 12% NaCl. The marine environment, from

which the isolates were derived, typically has a salt content of 3.5%. No isolates grew in LB

media supplemented with bile salts, even at the lowest concentration tested (0.1M). All isolates

were sensitive to 3 antibiotics; nalidixic acid (50 µg/mL), kanamycin (50 µg/mL) and

tetracycline (50 µg/mL), but 14 of the 23 isolates were resistant to ampicillin (50 µg/mL). All

the isolates had detectable levels of cellulolytic, proteolytic or amylolytic activity


61

Table 3.5. Antimicrobial activities amongst the short-listed isolates. The analysis was conducted by cross streak method. Columns with highlight

colour were results from growth depression by isolates of the same species

Marine isolates

#02 #04 #05 #12 #14 #15 #16 #17 #18 #20 #22 #06 #08 #11 #13 #21 #07 #09 #03 #10 #19 #01 #23 #02 B. subtilis - - - - - - - - - - - - + - - - - + - - - - - #04 B. subtilis - - - - - - - - - - - - + - - - - - - - - - - #05 B. subtilis + + - + - + + + + + + + + + + + + + + + - - + #12 B. subtilis - - - - - - - - - - - - - + - - - - - - - - #14 B. subtilis - - - - - - - - - - - - - - - - - - - - - - - #15 B. subtilis - - - - - - - - - - - - - - - - - - - + - - - #16 B. subtilis - - - - - - - - - - - + + - + ++ - - - ++ + + + #17 B. subtilis - - - - - - - - - - - + + - ++ ++ - - - ++ ++ - + #18 B. subtilis - - - - - - - - - - - + + - ++ ++ - + - + ++ - ++ #20 B. subtilis - - - - - - - - - - - - - - ++ + - - - - + - - #22 B. subtilis - - - - - - - - - - - - - - ++ ++ - + - - ++ - ++ #06 B. amyloliquefaciens + + + + - - ++ ++ ++ ++ ++ - + + + ++ + + + + ++ + ++ #08 B. amyloliquefaciens + + + ++ + + + + + ++ ++ + - + ++ + + - + - + + + #11 B. amyloliquefaciens - - - - - - - - - - - - - - - - - - - + - - - #13 B. amyloliquefaciens - - - ++ - - + + + ++ ++ - - - - - ++ ++ - + + - ++ #21 B. amyloliquefaciens - - - + - + - + + + + - - - - - + + - - ++ - - #07 B. pacificus - - - - - - - - - - - - - - - - - - - + - - - #09 B. pacificus + + + + + + - + + + + + + + + + - - + + + + + #03 B. licheniformis + + + + ++ + ++ ++ ++ ++ ++ + + + ++ ++ + + - + ++ + + #10 B. safensis - - - - - - - - - - - + + - + + - - - - + - + #01 B. halotolerans + + - + + + + + + + + + + + + + + + ++ + - - + #19 B. halotolerans + + - + ++ ++ + + ++ + + + + + + + + + ++ + - - + #23 P. polymyxa ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ -

+: zone of inhibition observed with a clear halo of growth inhibition; -: no inhibition. The isolates’ names were temporarily identified based on the name

of first species mentioned 16S rRNA analysis in Table 3. The isolates are arranged to group the isolates of the same species together.


62

Table 3.6. Characterization of short-listed bacterial isolates.

Isolates # #01 #02 #03 #04 #05 #06 #07 #08 #09 #10 #11 #12 #13 #14 #15 #16 #17 #18 #19 #20 #21 #22 #23 Salinity tolerance (NaCl) 3%-5% ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ 6%-7% ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ - 8% ++ ++ + ++ ++ + - + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + - 9% + ++ - ++ ++ - - - ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ - - 10% - + - + + - - - + + + + + + + + + + ++ + + - - 12% - - - - - - - - - - - - - - - - - - - - - - - Bile salt tolerance (mole/L) 0.1M -0.7M - - - - - - - - - - - - - - - - - - - - - - - Antibiotic susceptibility Kanamycin - - - - - - - - - - - - - - - - - - - - - - - Tetracycline - - - - - - - - - - - - - - - - - - - - - - - Ampicillin + + + - + + + + + - - - - + + + + - - - + + - Nalidixic acid - - - - - - - - - - - - - - - - - - - - - - - pH tolerance 5 ++ ++ + + + + + + + + + + + + + + ++ ++ ++ ++ ++ ++ + 6, 7, 8, 9 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ Thermal tolerance 30°C, 40oC 50oC ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ oxygen requirement for growth

Aerobic, Microaerophillic ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ Anaerobic - - - - - - - - - - - - - - - - - - - - - - - Enzymes production Protease +++ + - + - +++ + ++ ++ + + + ++ - + + ++ ++ +++ - ++ + ++ cellulose degradation ++ ++ ++ ++ ++ ++ + ++ + - ++ ++ ++ ++ ++ ++ ++ ++ ++ - - ++ ++ starch degradation + + - + + + + + + - + + + + + + + + + + + + + Haemolysis β β α β β α ϓ α ϓ β β β α β β β β β β β ϓ β ϓ Growth on media LPMA, MA + + + + + + + + + + + + + + + + + + + ++ ++ ++ ++ LBA, MHA, BHI ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++


63

3.3. Summary of results and discussion

There is growing interest in the marine environment as a potential source of bacteria that

produce novel antimicrobial compounds, particularly bacteriocins. The antimicrobial peptides

have been gaining interest as potential drug candidates for clinical treatment of antibiotic-

resistant pathogens (19). In this study, spore-forming bacteria isolated from the coastal marine

environment of Nha Trang (Vietnam Sea) were screened to identify Bacillus isolates producing

antimicrobial compounds. Members of the Bacillus genus were targeted because they are well-

recognized as producers of structurally diverse. Of the various types of marine samples

collected, the antimicrobial producing isolates were most frequently recovered from sponges,

followed by sediments, seaweeds and sea-water samples. Strains with pigments were regularly

detected with low/no antimicrobial activity. Marine sponges have multi-porous structures that

may trap and maintain high bacterial densities, leading to higher recovery rates of antimicrobial

producers. The recovery rate in marine sponges, at 7.6%, was at the lower end of the range

reported in previous studies (5.5% to 50.0%) (123). A lower recovery rate of antimicrobial

producing isolates was also seen from seaweeds, at 3.6%, whereas previous studies had

identified them from seaweeds at 11.0%–16.0% (63). The differences between this study and

previous studies could be due to geographical differences, marine conditions, or heat treatment

to select the spore-formers that may eliminate the metabolically active vegetative cells.

The methods used to detect antimicrobial activity had different levels of sensitivity. The well-

diffusion assay was less sensitive than the cross-streak assay. This effect was most obvious with

the failure to detect activity against Gram-negative bacteria, S. aureus and MRSA when the

latter assay was used. This may occur because of the production of multiple antimicrobial

compounds, with variable relative expression levels in liquid and solid media-based cultivation.

Many studies have reported the influence of various factors on bacteriocin production in liquid

culture such as type of culture media, pH, temperature, growth phase, and quorum sensing

regulation (124). Therefore, the production conditions for some bacteriocins may require

optimization, like varing the concentrations of medium components, screening of incubation

condition (temperature, shaking speed).

The most commonly identified species amongst the 23 shortlisted isolates that exhibited the

strongest antimicrobial activities, were members of the B. subtilis group. These species have

been widely reported in both marine environments and terrestrial environments and can tolerate

the broad environmental conditions (nutrients, pHs, chemo-physical conditions) that are

typically found in marine environments. For example, a study by Ivanova et al. (1999) reported


64

that 55.0% (11/ 20) of endospore-forming bacteria isolated from different areas of the Pacific

Ocean were B. subtilis species (125). While B. subtilis, B. amyloliquefaciens, B. pumilus and

P. polymyxa were all identified amongst aerobic spore-forming isolates from marine sources

from the Gulf of Mexico (126), or isolated from seaweed samples collected from the Irish Sea

(86). Other typical marine species, for example, B. aquimaris, B. algicola and B. hwajinpoensis

may require additional special nutrients or salts to recover, or could have been eliminated during

screening and shortlisting based on the strength of antimicrobial activity.

The marine Bacillus/Paenibacillus isolates that we have characterised had broad antimicrobial

activity against a range of human, veterinary and foodborne pathogens, including three

antibiotic-resistant pathogens (MRKP, MRSA and VRE). B. amyloliquefaciens #06, #08, #11,

#13, B. halotolerans #01, #19, B. licheniformis #03, B. safensis #10, P. polymyxa #23 had

broad antimicrobial activity against both Gram-positive and Gram-negative pathogen indicator

strains and other marine Bacillus of different species. This indicated production of either

multiple antimicrobial compounds by a single Bacillus, or a broad-spectrum antimicrobial

compound. Members of the Bacillus genus are known to produce various types of antimicrobial

compounds including polyketides, lipopeptides, bacteriocins, bacilysin, and volatile

compounds. The synergistic effects of these antimicrobials could result in a broad spectrum of

antimicrobial activity, such as noted for a number of the isolates in this study. Also, production

of broad-spectrum bacteriocin was recently reported for a marine Bacillus; sonorensin,

identified from a marine B. sonorensis isolate, exhibited broad-spectrum antibacterial activity

towards both Gram-positive and Gram-negative bacteria (85). Amongst Gram-positive bacteria,

such as Bacillus sp., the expression of bacteriocins and other antimicrobial compounds with

activity against other Gram-positive bacteria is widespread and extensively studied. However,

the production of compounds with activity against Gram-negative bacteria is less common,

therefore, the isolates that have antimicrobial activity against Gram-negative bacteria are of

particular interest. Rarely observed antimicrobial activities such as that observed against

Campylobacter, P. aeruginosa, and even multidrug-resistant K. pneumonia, represent

potentially novel compounds. The P. polymyxa #23 isolate had the strongest and broadest

activity and the activity against Gram-negative bacteria likely results from the expression of

polymyxin and tridecaptin, which has long been known to have such activity (42, 127). The

other isolates with activity against some of the Gram-negative bacteria tested, for example, B.

amyloliquefaciens #11, which had significant activity against both Campylobacter species but

lesser activity against E. coli and Salmonella, appears to indicate a spectrum of activity that has


65

not previously been reported and so the compound responsible may be novel and hence warrants

further investigation.

The finding that many of the compounds that had activity against C. perfringens were

inactivated by proteases, indicated that the antimicrobial compounds produced probably

included bacteriocins. Interestingly, the production of large quantities of these antimicrobial

compounds for drug development in the future could likely be achieved as it was demonstrated

that most of the isolates were well adapted to a broad range of growth conditions (various kinds

of nutrients, pHs, salt concentration, temperature, etc). These characteristics of the isolates

represent advantages that could facilitate manufacturing processes, products storage, and the

potential harnessing of these isolates for in vivo use in animal or food applications. The culture

conditions may affect the level of bacteriocin production. Besides, these marine

Bacillus/Paenibacillus isolates were also shown to have proteolytic, cellulolytic, and

amylolytic activity and hence may represent a promising source of important industrial enzymes

such as proteases, cellulases, and amylases. Marine-derived enzymes have been noted to have

significant advantages in manufacturing because they commonly have high adaptability to high-

salt concentration, and fluctuating temperature, pH, organic solvents, and ions (128).

Based on the results of the chapter, it can be confirmed that the Vietnam Sea region harbors an

abundance of marine Bacillus isolates expressing antimicrobial activities against a wide range

of bacterial pathogens, particularly antimicrobial-resistant bacterial pathogens. Some of the

antimicrobials may represent novel compounds that warrant further studies. This supports the

hypothesis raised in the introduction.


66

Chapter 4 BIOINFORMATIC IDENTIFICATION OF PUTATIVE GENE CLUSTERS

ENCODING ANTIMICROBIAL PEPTIDE PRODUCTION

4.1. Introduction

The recent development of affordable whole genome sequencing (WGS) and various

bioinformatic tools for genome analysis are speeding up antibiotic discovery from natural

resources. Nowadays, the elucidation of antimicrobial potential of bacteria is facilitated by in

silico prediction of genes encoding all putative antimicrobial substances. The method allows

the identification of candidates which harbour genes of novel substances for latter purification,

therefore, i) omitting the unnecessary purification processes of replicated compounds. In

addition, ii) the prediction of antimicrobial peptides from genomes can provide information on

gene components of bacteriocins biosynthetic gene cluster for further cloning/ expression for

mass production; and iii) can provide information on properties of antimicrobial substance

which are key factors to determine appropriate purification procedures. Therefore, genome

analysis is a powerful tool that can be applied before the direct purification, to save time and

reduce the range of purifications that need to be trialed.

In this study 6 marine isolates including 4 B. amyloliquefaciens (#06, #08, #11 and #13), 1 B.

halotolerans (#01), and 1 P. polymyxa (#23) were selected for genome sequencing using the

Illumina MiSeq sequencing platform. They were selected because they exhibited strong

antimicrobial activity against a range of important hospital-acquired pathogens and foodborne

pathogens such as Gram-negative bacteria (Salmonella, E. coli, Campylobacter coli,

Campylobacter jejuni, Pseudomonas aeruginosa), (129) and a majority of the Gram-positive

bacteria tested. In practice, the genome analysis included the steps of (i) generation of raw

sequence data, (ii) assembly the raw reads using the A5-miseq pipeline to generate draft

genomes; (iii) annotation of the draft genomes using web-based RAST for gene calling and

amino acid translation; iv) calculation of ANI values to estimate the genome similarity within

the marine species and versus the genomes of terrestrial species; (v) confirmation of the genome

similarity by pairwise comparison using the SBCT tool (of RAST) and MAUVE, to visualize

the percentage of similar CDS shared between genomes, and genome patterns; (vi) in silico

prediction of putative antimicrobial peptide encoding genes, including bacteriocins and NRPs,

by BAGEL and AntiSMASH; and vii) determination of gene components of bacteriocin gene

clusters. The final aim is to identify candidate biosynthetic gene clusters of putative novel

bacteriocins and to select strains for purification of bacteriocins


67

4.2. Results

4.2.1. Overview of six draft genomes

After assembly, the size of the draft genomes varied based on the species [Table 4.1]. Thus,

five Bacillus species had genome sizes ranging from 3.87 Mbps to 4.14 Mbps, and the genome

of P. polymyxa #23 was ~6.01 Mbps. The G+C contents of all genomes were similar, around

43.8% - 46.5%. There were no plasmids found. The draft genome of P. polymyxa #23 consisted

of 68 contigs while the number of contigs in genomes of Bacillus species ranged from 17 to 33

contigs.

Table 4.1.Overview of draft genomes of marine isolates

Marine isolates

#01 # 06 #08 # 11 #13 #23

Identification B. halotolerans

B.

amyloliquefaciens

P. polymyxa

Source Sediment sponge sponge seaweed sponge sponge

Numbers of contigs 31 17 23 33 19 68

Genome size 4.14 3.87 3.95 4.01 3.90 6.01

RNAs 119 114 102 118 102 187

No. CDSs 4033 3720 3839 3900 3776 5236

Subsystem coverage 48% 49% 48% 49% 48% 42%

N50 1077099 988071 991768 339244 1024489 469875

G+C (%) 43.8 46.5 46.3 46.2 46.5 45.2

Subsystems quantity 467 462 459 469 465 452

4.2.2. Calculation of ANI values across the genomes revealed a high degree of similarity

between marine species and terrestrial neighbour strains

To evaluate the genetic similarity of genomes between marine strains and closely related

bacteria isolated from terrestrial sources, an average nucleotide identity (ANI) plot (Figure 4.1)

was generated using GET-HOMOLOGOUS. The Bacillus/Paenibacillus species used for this

calculation included 6 B. amyloliquefaciens subsp. plantarum, 4 of which were isolated in this

study and 2 were from the NCBI genome database (#06, #08, #11, #13, FZB42, CAUB419),; 6

P. polymyxa (#23, CR1, E681, SC2, SQR21, HY962); 6 B. halotolerans (#01, ZB201701,


68

F413, NRRLB41617, ZB201702, ATC25096). These terrestrial strains were selected because

they had high similarity in 16S rRNA sequences with those of the marine isolates.

The phylogenetic tree included three distinct clades; clades A (comprising 6 B.

amyloliquefaciens species); clade B (comprising 6 B. halotolerans), and clade C (comprising 6

P. polymyxa species) [Figure 4.1]. The ANI values across genomes ranged from 50.27% to

96.03% where the lowest ANI value was observed between the genomes of B.

amyloliquefaciens #08 and P. polymyxa CR1; the highest ANI value was seen between genomes

of B. amyloliquefaciens #6 and B. amyloliquefaciens #08. The ANI was highest across genomes

of B. amyloliquefaciens species (94.08%-96.03%), followed by B. halotolerans (77.79% -

78.83%), and lowest ANI was observed across P. polymyxa (50.02% - 51.17%). Within the B.

amyloliquefaciens species, B. amyloliquefaciens #11 was closer to B. amyloliquefaciens FZB42

(ANI=95%) while B. amyloliquefaciens #06, #08 #13 were closer to B. amyloliquefaciens

CAUB419 (95.73%, 95.71%, 95.65%).


69

Figure 4.1 The heat map of the average nucleotide identity and genome-based phylogenetic tree across Bacillus/Paenibacillus genomes. These

genomes included 6 B. amyloliquefaciens (Ba) (#06, #08, #11, #13, FZB42, CAUB41, 6 P. polymyxa (Pp) (#23, CR1, E681, SC2, SQR21) and 6

B. halotolerans (Bh) (#01, ZB201701, F413, NRRLB41617). The graphic was generated by calculation of GET-HOMOLOGOUS software using

BLASTn for gene clustering and determination of similarity across the genomes dataset. The highest similarity across genomes was observed at B.

amyloliquefaciens, followed by B. halotolerans, and the P. polymyxa genomes displayed the lowest similarity.


70

4.2.3. Analysis of the distribution frequency of genes across the species revealed a high

number of strain-specific genes in P. polymyxa genomes

The frequency distribution of orthologous coding sequence (CDS) across the genomes of 6 B.

amyloliquefaciens, 6 P. polymyxa, and 6 B. halotolerans genomes were calculated separately

and then compared. The result showed that 6 B. amyloliquefaciens genomes contained 5172

orthologous proteins comprising 1337 cloud CDS (25.85%), 312 CDS in the shell (6.03%) and

3523 CDS in the cloud (68.12%), 3320 in the core (64.19%). The B. halotolerans contained

6063 orthologous proteins including 2071 CDS in clouds (34.16%), 467 CDS in shells (7.70%),

3525 CDS in soft-core (58.14%), and 2395 CDS in the core (39.50%). The 6 P. polymyxa

genomes contained 9206 orthologous CDS, comprising of 4444 CDS in the cloud (48.27%),

and 707 in the shell (7.68%), 4055 soft-core (44.05%), and 3549 genes in the core (38.55%).

Therefore, numbers of CDS in the cloud catalogue was highest at P. polymyxa, followed by B.

halotolerans and B. amyloliquefaciens. The ratio between the number of CDS presented in 1

genome (‘strict cloud’) and the number of genes presented in 6 genomes ('strict' cores') was ~1

in P. polymyxa, higher than that in B. halotolerans, and B. amyloliquefaciens (about ~1/2 X).

This indicated a sightly enrichment of strains-specific genes in P. polymyxa genomes [Table

4.2]

Figure 4.2. Frequency distribution of CDS across the genomes.. (A) Of 6 B. halotolerans

genomes; (B) 6 B. amyloliquefaciens genomes; (C) 6 P. polymyxa. Bars from the left to

right illustrated a number of CDS presented in 1 genome (named as 'strict' cloud); 2

genomes, 3 genomes, 4 genomes 5 genomes, and 6 genomes (called as ‘strict’ core)

respectively. The ratio of 'strict' cloud: 'strict' core were ~1/2 in B. halotolerans and B.


71

amyloliquefaciens, but there was a slight enrichment of unique CDS in P. polymyxa when

this ratio was ~1.

4.2.4. The pairwise comparison across genomes of B. halotolerans revealed the high

similarity in genome organisation and CDS.

The similarity across the genomes was confirmed by MAUVE and SBCT tools. Among the

isolates of B. halotolerans, the genome of #01 was closer to B. halotolerans F41-3 (ANI =

94.85%). Genomes alignment performed by MAUVE showed that the genome patterns between

B. halotolerans #01 and B. halotolerans F41-3 had 31 LCBs, most of the genomes were

collinear, no translocations, no inversions, but the presence of some deleted block, indicating

the integration of mobile nucleotide elements (probably phage, transposons and/or integrases)

into the genomes [Figure 4.3 A]

Pairwise comparison between genomes of the isolate #01 and F41-3 resulted in 90.7% similar

CDS (3822/ 4210) (amino acid similarity of ≥70%) [Figure 4.3 B]. There were approximated

2.5% CDS (106/4210) with low similarity found in the only genome of isolate #01 but not in

genome of B. halotolerans F41-3, which were then annotated as; transporters responsible to

transport maltose/maltodextrin and relating to multidrug resistance, transcription regulators,

phage related proteins, hypothetical proteins, and a biosynthetic gene cluster of unknown

polyketide [Table 4.2].

Figure 4.3. Genome comparison between marine B. halotolerans #01 and terrestrial

neighbour B. halotolerans F41-3. (A) Performed by MAUVE to visualize the


72

organisation of conserved DNA regions on genome. (B) And by SBCT to identify the

similar proteins. The comparison was conducted between B. halotolerans F41-3 (bottom)

and B. halotolerans #01 as reference (top).

Table 4.2 List of CDS found in genome #01 relating to the biosynthesis of polyketide

compound. These CDS had low similarity with those found in the genome of F41-3

Gene ID Function

fig|6666666.473256.peg.3879 Polyketide synthase modules and related proteins



fig|6666666.473256.peg.3711 Polyketide biosynthesis 3-hydroxy-3-methylglutaryl-ACP

synthase PksG

fig|6666666.473256.peg.3704 Malonyl CoA-acyl carrier protein transacylase (EC 2.3.1.39)

fig|6666666.473256.peg.3710 Polyketide biosynthesis malonyl-ACP decarboxylase PksF

fig|6666666.473256.peg.3717 Modular polyketide synthase

fig|6666666.473256.peg.1975 Uncharacterized transcriptional response regulator YcbL

fig|6666666.473256.peg.1978 Bacitracin transport ATP-binding protein bcrA

fig|6666666.473256.peg.1673 Two-component system sensor histidine kinase

fig|6666666.473256.peg.3712 Probable polyketide biosynthesis enoyl-CoA hydratase

PksH

fig|6666666.473256.peg.3713 Modular polyketide synthase

fig|6666666.473256.peg.3709 Polyketide biosynthesis acyl-carrier-protein AcpK

fig|6666666.473256.peg.3710 Polyketide biosynthesis malonyl-ACP decarboxylase PksF

fig|6666666.473256.peg.3020 3-oxoacyl-[acyl-carrier-protein] synthase, KASII (EC

2.3.1.179)

fig|6666666.473256.peg.2003 ABC-type Na+ transport system not coupled with H+ or K+

uptake, ATPase component NatA

4.2.5. The pairwise comparison across genomes of B. amyloliquefaciens revealed high

similarity in genome organisation and CDS.

Both MAUVE and SBCT concluded a high degree of similarity between the genomes of marine

B. amyloliquefaciens species and those of terrestrial B. amyloliquefaciens. Of these species,

isolate #11 was closer to B. amyloliquefaciens FZB42 while isolates #06, #08 and #13 were

closer to B. amyloliquefaciens CAUB946.


73

The MAUVE arrangement showed that there were only 3 LCBs between isolate #11 and FZB42

without genes inversion; 11 LCBs between genomes of #13 and FZB42 with some inversions

and translocations [Figure 4.4]. The high similarity in genomes between #11 and FZB42 was

again confirmed by SBCT when the tool detected that the two genomes had 97.51% similar

proteins, which was significantly higher than the 89.33% similarity between FZB42 and isolate

#13 [Figure 4.4B]. The genome of #13 was closer to B. amyloliquefaciens CAUB946; they

shared 93.83% similar proteins. The comparison performed between marine species indicated

the genome of #13 was similar to that of isolate #08 (had 100% similar proteins), followed by

#06 (had 93.19% similar proteins). Besides, all these marine B. amyloliquefaciens were

‘amyloliquefaciens’ sub-species, as the proportion of similar CDS sharing between marine B.

amyloliquefaciens and strains of ‘plantarum’ subspecies (B. amyloliquefaciens FZB42 or B.

amyloliquefaciens CAUB946) was 90.64%, therefore a higher degree of similarity than that

shared between marine isolates and strain of ‘amyloliquefaciens’ subspecies (B.

amyloliquefaciens DSM 07); 86.67%. There were 4.19% (166/3961) CDS found in the genome

of isolate #11 but presented as low similarity CDS or non-present in the genome of FZB42

[Table 4.3]. These CDS were annotated as hypothetical proteins (65.06%) with unknown

function, phage-relating proteins, and a gene cluster responsible for the biosynthesis of

thiopeptide, a class I bacteriocin.

Table 4.3.Low identity CDSs in the genome of isolate #11 relating to the biosynthetic

gene cluster for bacteriocin production.

Gene code Annotation

fig|6666666.242001.peg.370 Bacitracin ABC transporter, permease protein, putative

fig|6666666.242001.peg.372 Sensory box histidine kinase

fig|6666666.242001.peg.373 Positive regulation of late competence genes and surfactin production (comS, srfA, rapA)

fig|6666666.242001.peg.374 Hypothetical protein

fig|6666666.242001.peg.375 Lanthionine biosynthesis protein, LanM

fig|6666666.242001.peg.376 methionine ABC transporter ATP-binding protein

fig|6666666.242001.peg.377 hypothetical protein

fig|6666666.242001.peg.378 lanthionine biosynthesis protein, LanM


74

Figure 4.4. Genome comparison between marine B. amyloliquefaciens and terrestrial B.

amyloliquefaciens (A) Performance by MAUVE between the genome of isolate #11 and

genome FZB42 (A1), and between the genome of isolate #13 and FZB42 (A2). (B)

Performance by SBCT. From the outer ring to inner ring were genomes of FZB42, #08,

#06, DSM7, CAUB946, CBMB205, #11. The comparison was performed against the

genome of isolate #13 as reference.

4.2.6. The pairwise comparison across genomes of P. polymyxa revealed diversity in

genome organisation and CDS.

Both MAUVE and RAST revealed an average degree of similarity between genomes of marine

P. polymyxa #23 and terrestrial neighbour P. polymyxa [Figure 4.3]. The genome of isolate #23

shared 87.20% similar CDS with P. polymyxa SQR21, 81.72% with P. polymyxa SC2, and

78.47% CDS with P. polymyxa CR1 [Table 4.5]. Therefore, the genome of #23 was closer to

the genome of P. polymyxa SQR21. The MAUVE analysis confirmed this similarity between


75

these two isolates when there was less number of LCBs between the genome of isolate #23 and

SQR21 than that observed between isolate #23 and P. polymyxa CR1.

Figure 4.5. Genome comparison between marine P. polymyxa #23 and terrestrial P.

polymyxa species. (A) The performance was carried out by MAUVE analysis between

genomes of isolate #23 and the genome of terrestrial P. polymyxa SQR21 (A1); and

between isolate # 23 and terrestrial P. polymyxa CR1 (A2). (B) The performance was

carried out by SBCT between marine P. polymyxa # 23 (used as reference) and genomes

of P. polymyxa SQR21, P. polymyxa CR1, P. polymyxa SC2 (from the inner ring to outer

ring).


76

There were 12.71% (680/ 5352) CDSs found in the genome of isolate #23 which had low

similarity to those found in the genome of SQR21. These low similarity CDSs were located

mostly in contigs #33 and #34. They were annotated as hypothetical proteins (64.85%),

transcriptional regulators (4.41%) including HXlR, XRE, ADA, AraC, HcpR, MerR, TetR,

LysR, ArsR, AsnC, LrpA, AbrB, PadR which were known to regulate carbon-storage, sugar

metabolism, stress response; transmembrane transporters (2.05%) which including multidrug-

efflux transporter, bacitracin ABC transport, multidrug resistant transporter, Bcr/cflA family,

methionine transporters which may be responsible for antibiotic resistance; and proteins

relating a bacteriocin biosynthetic gene cluster [Table 4.4].

Table 4.4. The CDSs relating to unique bacteriocin biosynthetic gene clusters found in

the genome #23 but had low similarity to those found in the genome of isolate CAUB419

Gene code Functions suggested by RAST

Bacteriocin gene cluster

fig|6666666.242000.peg.4718 Methionine ABC transporter ATP-binding protein

fig|6666666.242000.peg.4719 Lipid A export ATP-binding/permease protein MsbA

fig|6666666.242000.peg.4720 Glycosyl transferase, family 8

fig|6666666.242000.peg.4721 Glycosyltransferase, group 2 family protein


fig|6666666.242000.peg.4723 UDP-4-amino-4-deoxy-L-arabinose--oxoglutarate aminotransferase (EC 2.6.1.-)


fig|6666666.242000.peg.4725 Radical SAM


4.2.7. In silico prediction of antimicrobial compounds revealed high numbers of NRPs

and bacteriocins, including novel bacteriocins

In silico prediction of putative antimicrobial peptides from these six genomes by both BAGEL

and antiSMASH bioinformatic tools resulted in the detection of a total of 61antimicrobial

peptides (AMPs) including 20 bacteriocins and 41 NRPs compounds (lipopeptides, polyketides,

hybrids, bacilysin) [Table 4.5]. The B. amyloliquefaciens and P. polymyxa harboured higher

numbers of AMPs than those found in the genome of B. halotolerans #01. Types of NRPs

predicted from genomes from the Bacillus taxon were different to the types predicted to be


77

present in the P. polymyxa taxon, which included lipopeptides (tridecaptin, polymyxin,

paenibacterin, and an unknown LP), polyketides (fusaricidin and an unknown polyketide).

Although 41 NRSPs were predicted, there were only 14 different types of substances. This was

because many Bacillus derived polyketides (bacillaene and macrolactin), lipopeptides

(surfactin, fengycin, and bacillomycin D), siderophores (bacillibactin) and a dipeptide bacilysin

were presented in all members of the Bacillus taxon. However, the genes encoding the difficidin

were found in all B. amyloliquefaciens species but were not found in the genome of B.

halotolerans #01. The NRPs gene clusters were large in size (commonly larger than 12 kDa),

including multiple replicated modules of synthetases enzymes (28), which would give many

difficulties for further cloning/ expression to characterise. Most of the NRPs gene clusters were

well characterised, the gene determinants of some gene clusters of undefined compounds were

separated on different contigs due to multiple gaps presented within the incompleted

chromosome after miseq sequencing, therefore the identification of full gene clusters would

have many challenges, therefore gene organisation of the NRPs gene cluster was not further

annotated in this study. Also, they were not types that were of interest for this study, because

they were predicted with less novelty of substances.

Although 20 bacteriocins were predicted within six genomes there were only 13 different types

[Table 4.5, Table 4.6]. This was because of the replication of several bacteriocins (LCI,

amylocyclicin) in all the B. amyloliquefaciens genomes; and 1 novel thiopeptide cluster

presented in genomes of both isolates #06 and #13. Of these 13 bacteriocins, 6 bacteriocins

were found to be characterised from earlier studies including subtilosin A (isolate # 01),

plantazolicin (# 06), mersacidin (# 08), paenicidin A (#23), LCI and amylocyclicin (all #06,

#08, #11, #13); and 7 uncharacterised bacteriocins including 2 thiopeptides (one presented in

both #08 and #13, and one found in #11), 1 two-component lantibiotic (#13), 1 sactipeptide

(#23), 1 lantibiotic (#23), 1 lantibiotic (#08) and 1 uncharacterised lassopeptide (#23). A

bacteriocin, in this study, was considered as a unique or uncharacterised bacteriocin when the

precursor’s amino acid sequence had a low identity (< 90% of similarity), or was unmatched to

any characterised bacteriocins listed from the bacteriocin database (BACTIBASE), and uniref

90 database (UNIPROT).


78

Table 4.5. List of putative antimicrobial peptides predicted within Bacillus/Paenibacillus

draft genomes.

Similar known peptides

Class Similarity Marine isolates

#01 #06 #08 #11 #13 #23

Bacteriocins

LCI class II 100% • • • • •

amylocyclicin head to tail cyclized peptide

100% • • • • •

subtilosin A sactipeptide 100% •

paenicidin A lantibiotic type 1 - •

mersacidin lantibiotic type 1 100% •

plantazolicin LAPs 100% •

unknown thiopeptide - • •

unknown thiopeptide - •

unknown sactipeptide - •

paeninodin lassopeptide 60% •

unknown lantibiotic type 1 - •

unknown lantibiotic type 2 - •

fengycin lipopeptide 100% • • • • •

bacillibactin lipopeptide 100% • • • • •

surfactin lipopeptide 100% • • • • •

fusaricidin lipopeptide 100% •

penibacterin lipopeptide 60% •

tridecaptin lipopeptide 100% •

polymyxin lipopeptide 100% •

unknown lipopeptide - •

bacillaene polyketide 100% • • • • •

macrolactin polyketide 100% • • • • •

difficidin polyketide 100% • • • •

unknown polyketide •

bacilysin other 100% • • • • •


79

Table 4.6. Amino acid sequences of precursor bacteriocins predicted within 6 Bacillus genomes

. Hosts

Class Amino acid sequence MW (kDa)

Similar known bacteriocin

Similarity (%)

Characterized bacteriocins

1 #01 sactipeptide MKKAVIVENKGCATCSIGAACLVDGPIPDFEIAGATGLFGLWG

3.44 subtilosin A 100%

2 #06; #08; #11; #13

unmodified MKFKKVLTGSALSLALLMSAAPAFAASPTASASVENSPISTKADAGINAIKLVQSPNGNFAASFVLDGTKWIFKSKYYDSSKGYWVGIYESVDK

5 LCI 100%

3 #06 LAPs MNSLSINFMEEVTIMTQIKVPTALIASVHGEGQHLFEPMAARCTCTTIISSSSTF

1.34 plantazolicin 100%

4 #06; #08; #11; #13

head to tail cyclized peptide

MMNLVKSNKKSFILFGAALAAATLVYALLLTGTELNVAAAHAFSANAELASTLGISTAAAKKAIDIIDAASTIASIISLIGIVTGAGAISYAIVATAKTMIKKYGKKYAAAW

6.38 amylocyclicin 100%

5 #08 lantibiotic MSQEAIIRSWKDPFSRENSTQNPAGNPFSELKEAQMDKLVGAGDMEAACTFTLPGGGGVCTLTSECIC

1.98 mersacidine 100%

6 #23 lantibiotic MAENLFDLDIQVNKSQGSVEPQVLSIVACSSGCGSGKTAASCVETCGNRCFTNVGSLC

3.38 paenicidin A 100%

7 #23 lassopeptide MAAINPAKDHYRITYGEEVYVSDMDGEKVMMSIHTGKYYNLGFTGGRIWELAEFSPSIEDIVTVLTNEYEVDEEQCRQQVHTFVAELEREGLLRLLRELD

paeninodine 60%

Uncharacterized bacteriocins

8 #06, #13 thiopeptide MKKEKNDLLNNLEITGLDVTEITDSISIPETGATSGSGGHSTCGSSSCCSSCCS

1.88 unique -


80

. Hosts

Class Amino acid sequence MW (kDa)

Similar known bacteriocin

Similarity (%)

9 #08 lantibiotic MSEKQIERTIRAWKDPEFRKTLSDVADHPSGSIEARQLSMLGSEDNVNPMTTPVSPAPTVVIRVSLKVCPTFKIKCKV

lichenicidinVK21A2

37%

10 #11 thiopeptide MEKMMSQNVKEKEDSLDLTSILDEIESIDIMEVSESMLLSETGATSGSSSSSSTSCCGSCSCASCGACTSCG

- unique -

11 #13 lantibiotic MKKDFQALTPMTEEELKNLAGGSDATPMTVTPTTITIPISLAGCPTTKCASIVSPCND

3.61 haloduracin β 41%

12 #23 sactipeptide MRKLVKRSTNVGDTIEAFGCGCSCYCPCSCYCAGSLTRSSNTSRESDGSYRRDNGTGIGNY

4.4.6 unique -

13 #23 lantibiotic MNSPELVFFEQEDTLDLDLQINDLTLKQAKNPCTSTVTCSVSRCLGTHVTCECWC

2.82 unique -

The bold letters indicated the position of core sequence on precursor, normal letters denoted the leader sequence. It was predicted by SingalP.v5.0

prediction (http://www.cbs.dtu.dk/services/SignalP/). The MW was predicted based on the core sequence using expasy tool

(https://web.expasy.org/compute_pi/).


81

A1 A2

A3

0BAC014_|_mersacidin 4008lan 2423lan1 913lan 1023lan2 2BAC215_|_Paenicidin_A 2BAC197_|_Lichenicidin_A2 0BAC196_|_Lichenicidin_A1

Consensus

.M......

.S......

.Q......

.E......

.A......

.I......

.I......

.R......

.S......

.W......

.K......

.D......

.P......

.F......

.S......

.R......

.EM.....

.NA.....

.SE.....

.TN.....

.QL.....

.NF.....

.PD.....

.AL.....

.GD.....

.NI.....

.PQ.....

.FV.....

.SN.....

.EK.....

.LS.M...

.KQMN...

.EGKS...

.ASKP...

.QVDE...

.MEFL...

.DPQV...

.KQAF...

.LVLFVT.

.VLTELT.


Consensus

.GSPQSP.

.AIMEIAT

.GVTDVTI

.DAETATT

.MCELCSLs

.ESEDSSS

.ASLLSWT

.AGKDGTCc

CCCNLCCA

TTGLQGII

FFSAISTL

TTGGNGAS

LLKGDKGK

PP.SL..P

.

.

.DT..L

.

.

.A....

.

.

.T....

.

.

.P....

.

.

.M....

.

.

.T....

.

.

.V....

.

.

.TL...

.

.

.PK...

.

.

.TQ...

.

.

.TA...

.

.

.IK.V.t

GGTTNTTG

GGAIPAVN

GGAPCASN

GGSITSAG

.

.CSSCSYv

VVVLTVLL

CCEAVE.Ct

TTTGTT.Tc

LLCCCCCV

TTGPSGPT

SSNTVNTK

EERTSRTEc

CCCKRCKC

IIFCCFCM


Consensus

.

.TALTTP

.

.NSGN..

.

.VITV..

.

.GVHG..s

.

.SSVSS.

.

.LPTLRSc

CCCCCCCC

.

.

.NE..N

.

.

.DC...

.

.

.

.W...

3101sac 4023sac 31BAC098_|_subtilosin 22BAC217_|_Thuricin_CD_beta 20BAC201_|_Thurincin_H 31BAC166_|_Propionicin-F

Consensus

.M....

.R....

.K....

.L....

.V....

.K....

.R....

.S...W

MTM..F

KNK..Y

KVK..Q

AGA..G

VDV..M

ITI..N

VIV..Ie

EEE..A

NANGDI

KFKWWYg

GGGVTAc

CCCACN

.G....

ACACWI

TSTVSGc

CCCGCG

SYSALV

ICICVAg

GPGGCNa

ACATAIa

ASAVAIc

CCCCCGl

LYLLSYv

VCVAVT

DADSEEg

GGGG.A

PSP..A

ILIGLV

PTPVLA

DRDGNT

FSFTLLe

ESEE..

4301sac 6023sac 43BAC098_|_subtilosin 30BAC217_|_Thuricin_CD_beta 31BAC201_|_Thurincin_H 43BAC166_|_Propionicin-F

Consensus

INIFVLa

ATAATGa

GSGAAAa

ARAAAVt

TETSTVg

GSGYGA

LDLFAV

FGFLSA

GSG.TP

LYL.AV

WRW.SV

GRG..P

.D....

.N....

.G....

.T....

.G....

.I....

.G....

.N....

26nosM 25nocM 33tsrH 36sioH 29cltA 21tpdA 22tpaA 2913thioA 4011thioA

Consensus

.

.

.

.

.

.

.

.M

.

.

.

.

.

.

.

.E

.

.

.

.

.

.

.

.K

.

.MM....M

.

.SS....M

.

.NT....S

.

.AA....Q

.

.AA....N

.

.LI....V

.

.

.V....K

.

.

.GM..ME

.

.

.QE..KK

.

.EEK..KE

M.IIE..ED

DMGGL..KS

ASVVV..NL

AAEDLM.DD

HDGGDDMLL

LLLLLLNLT

SSTTASKNS

DAGGDDDNI

LLLLLLILL

DNDDSPDED

.

.

.

.

.

.

.

.E

IIVVVMLII

DDDDDDSTE

ASTAEVAGS

LLLLLFILI

EEEEDEEDD

IIIIVLIVI

SSSSLASTM

EEDDPDDEE

FFYYT.LIV

LLMMS.ITS

DDDDPDSDE

EDEEGGESS

SSTTAVTIM

RRLLGAESL

LLLLLVQIL

EEDDEESPS

50nosM 49nocM 58tsrH 61sioH 60cltA 49tpdA 48tpaA 5413thioA 7111thioA

Consensus

DDGGSSDEE

SSEEILDTT

EEDDNTAGG

VVLLVALAA

VVTSGGSTT

AAVVHHQSS

KKTTAGVGG

VVMMMMMSS

MMIVVTAGS

SSASEEAGS

.

.SSIVSHS

.

.

.

.GG..S

.

.

.

.

.

.

.

.S

.

.

.

.A...T

AAAASACSS

SSSSNSTTC

CCCCCCTCC

TTTTTNTGG

TTTTS.GSS

CCCCTCCSC

EEIIGFASS

CCCCTCCCC

CSTTPYSCA

.

.

.

.AI..S

.

.

.

.S....

CCCCCCSSC

.

.

.

.CC..G

SSSSSSSSA

CCCCCCSCC

SSSSCSSCT

SSSSCSSSS

.

.

.

.CAT.C


82

B3

B2

B1

Figure 4.6. Multiple sequence alignment (A) and average distance tree (B) between the bacteriocins precursors in this study and known

precursors . The calculations were performed on DNAman software using Clustal W (130). (1) lantibiotic precursors (13lan1, 13lan2 were

novel precursors), (2) lassopeptide precursors (23sacA was novel precursor); (3) thiopeptides precursors (06thioA, 13thioA were novel

precursor). The letters in black (100% of identity), pink (≥70%), and blue (50%). The thiopeptides for comparison were acquired from

THIOBASE (131) while sactipeptides and lantibiotic were downloaded from BACTIBASE database (110).

06thioA

13thio

11thioA

ZP_00237834.1

ZP_00237835.1

ZP_00237837.1

ZP_00237830.1

ZP_00237832.1

NP_834755.1

NP_834756.1

NP_834757.1

NP_834758.1

nosM

sioH

YP_003974670.1

YP_003974672.1

cltA

100%

100%

100%

100%

100%

100%

100%

95%

61%

53%

45%

33%

26%

20%

17%

11%

100% 0%80% 60% 40% 20%

01sac

BAC098_|_subtilosin

BAC201_|_Thurincin_H

23sac

BAC217_|_Thuricin_CD_beta

BAC166_|_Propionicin-F

100%

32%

17%

15%

4%

100% 0%80% 60% 40% 20%

BAC014_|_mersacidin

08lan

BAC196_|_Lichenicidin_A1

23lan1

BAC215_|_Paenicidin_A

13lan

BAC197_|_Lichenicidin_A2

23lan2

100%

100%

59%

35%

24%

16%

11%

100% 0%80% 60% 40% 20%


83

4.2.8. Prediction of gene organisation of biosynthetic gene clusters of characterised

bacteriocins

4.2.8.1. The LCI gene clusters (found in the genomes of isolates # 06, #08, #11, #13)

The biosynthetic gene clusters of LCI were found in all 4 B. amyloliquefaciens genomes (#06,

#08, #11, #13) [Figure 4.7]. The gene clusters were the same in all 4 species, and also had

100% identity to the LCI gene cluster found ubiquitously in the B. subtilis/B.

amyloliquefaciens group. The LCI is an unmodified bacteriocin (class II), pI of 9.85,

molecular weight (MW) of 5 kDa and exhibits antimicrobial activity against Gram-negative

bacteria (Xanthomonas sp., P. solanacearum) but is not active against E. coli (54).

The gene organisation of the LCI biosynthetic gene clusters found in these marine isolate

genomes were the same as the well-characterised ones (54, 132). It consisted of a structural

gene encoding a precursor bacteriocin and an ABC transport gene that was responsible for

immunity/transportation. The precursor LCI sequence had 94 amino acids consisting of a

leader sequence and a 46 aa core sequence. The core sequence had a molecular mass of 5.46

kDa and a pI of 10.25.

Figure 4.7 The LCI gene cluster found in marine B. amyloliquefaciens #06, #08, #11

and #13. (A) The gene organisation of the LCI gene cluster. Green colour shows

structural gene. (B) The multiple sequence alignment (MSA) between LCI of #11, #06,

#08, #13 and B. amyloliquefaciens FZB42.


84

4.2.8.2. The amylocyclicin gene clusters (found in genomes of #06, #08, #11 and#13)

The biosynthetic gene clusters of amylocyclicin were also found in all marine B.

amyloliquefaciens species (#06, #08, #11 and #13) (named 11amy) [Figure 4.8]. The gene

clusters were the same in all 4 species, and also had 100% identity to the amylocyclicin gene

cluster found in B. amyloliquefaciens FZB42 (133). This bacteriocin is a head-to-tail cyclised

bacteriocin (class Ib) with a pI of 9.8, MW of 6.38 kDa and is active against Gram-positive

bacteria (Bacillus, Staphylococcus, MRSA, Lactobacillus Listeria and Enterococcus).

The gene organisation of these amylocyclicin biosynthetic gene clusters were the same as the

well characterised one found in B. amyloliquefaciens FZB42 genome (133). It was 4,490 bp

in length, flanked by 2 terminators and consists of 6 ORFs encoding 6 proteins, 11amyA-F

[Figure 4.8, Table 4.7]. The 111 aa precursor bacteriocin (11amyA) had a 47 aa leader

sequence at the C-terminus and a 64 aa core sequence at the N-terminus. The maturation of

head-to-tail bacteriocin commonly relates to three steps: enzymatic cleavage of the leader

sequence and circularization (44). In this gene cluster, the 11amyC was 100% identical to

acnC (found in the one of FZB42) which were responsible to cleavage of the leader sequence

and cyclise two amino acids at two terminal ends of core sequence (133). Two ORFs; 11amyD

(ATP binding domain) and 11amyE (permease domain), which were homology to acnD and

acnE, formed an ATP-dependant two-component ABC transport for secreting the bacteriocin

(134). The 11amyF was an immunity protein for protecting the host from the bacteriocin

effect, and the 11amyB was a hypothetical protein with unknown function

Table 4.7. Structural organisation of amylocyclicin gene cluster found in isolates #06,

#08, #11 and #13

Protein Description Function Similar proteins*

11amyB hypothetical protein unknown function acnB

11amy A precursor precursor acnA

11amy C FIG01241261: hypothetical protein modification enzyme acnC

11amy D ABC transporter, ATP-binding protein ABC Transport

ABC permease

acnD

11amy E uncharacterized protein MJ0793 acnE

11amy F hypothetical protein immunity acnF


85

* The similar proteins were observed from the amylocyclicin gene cluster of Bacillus

amyloliquefaciens FZB42 (133).

Figure 4.8. The amylocyclicin gene cluster found in marine B. amyloliquefaciens #06,

#08, #11 and #13. (A) The gene organisation of the gene cluster. (C) The multiple

sequence alignment (MSA) between precursors of #11, #06, #08, #13 and B.

amyloliquefaciens FZB42. The precursor sequences were 100% identical.

4.2.8.3. The mersacidin gene cluster (found in the genome of isolate #08)

The biosynthetic gene cluster of mersacidin was found within scaffold #01 of B.

amyloliquefaciens #08’s draft genome [Figure 4.9]. The mersacidin gene cluster was 100%

similar to that found in Bacillus sp. HIL-Y85/54728 (GenBank AJ250862.2, or BGC0000527)

(135). The mersacidin has an MW of 1,825 Da, globular structure, pI of 4.2, and displays

antimicrobial activity against Gram-positive bacteria including MRSA (41).

The genome organisation of the mersacidin biosynthetic gene cluster in the genome of isolate

08 (named as 08mrs) was the same as that of Bacillus sp. HIL-Y85/54728 (135). It was 12,300

bp in length and consisted of 10 ORFs encoding 10 proteins, 08mrsA-K [Figure 4.9, Table

4.8]. The precursor bacteriocin (08mrsA) comprised a deduced 48 aa leader sequence at the


86

C-terminal end and a 20 aa core sequence at the N-terminal end. The lantibiotics commonly

mature via two modification steps; i) removal of the signal sequence, ii) enzymatic

dehydration and cyclisation to convert serine and/or threonine residues to yield unusual amino

acids that are typical for lantibiotic (Dha, Dhb, Lan, MeLan) (39). The 08mrsD was annotated

as a protease, therefore, it was responsible to cleave off the leader sequence. The 08mrsM was

annotated as a lanM-type enzyme which was a typical modification enzyme to modify the

class Ib lantibiotic (39, 40, 136). The matured form of bacteriocin was secreted by 08mrsT

(lanT-type transporter). Three ORFs, mrsF, mrsG, and mrsH formed a group II type ABC

transporter system serving as an immune system to protect host (136-138). Besides, the gene

cluster also harbours a two-component regulatory system formed from 08mrsI and 08mrsJ

(139).

Figure 4.9. The mersacidin gene cluster found in the genome of isolate #08 . (A) Gene

organisation of the gene cluster. (B) MSA between the mersacidin precursors of #08

(08mrsA) and Bacillus sp. HIL-Y85/54728. (C) Multiple sequence alignment at the

gene cluster level (MSAG) between the mersacidin gene cluster of isolate #08 and

Bacillus sp. HIL- Y85/ 54728 (135). The multiple sequences alignment at gene level

(MSAG) was performed by ‘MultiGeneBlast’ tool indicated 100 % similarity between

two gene clusters (140).


87

Table 4.8. Structural organisation of mersacidin gene cluster found in isolate #08.

Proteins Description Functions Similar protein*

08mrsJ protein putative histidine-kinase regulatory mrsK2

08mrsI protein putative response-regulator regulatory mrsR2

08mrsH ABC-transporter immunity mrsF

08mrsG ABC-transporter integral membrane protein immunity mrsG

08mrsF ABC-transporter integral membrane protein immunity mrsE

08mrsA mersacidin precursor precursor mrsA

08mrsE protein putative response-regulator regulator mrsR1

08mrsD peptidase maturation mrsD

08mrsC lanM- lantibiotic modification enzyme maturation mrsM

08mrsB lanT transportation protein transportation mrsT

* The similar proteins were observed from the mersacidin gene cluster of Bacillus sp. HIL-

Y85/54728.

4.2.8.4. The subtilosin A gene cluster (found in the genome of isolate # 01)

The biosynthetic gene cluster of subtilosin A was found in the genome of isolate #01 [Figure

4.10]. The whole gene cluster had a high degree of similarity (100%) with that of B. subtilis

subsp. spizizenii ATCC 6633 (AJ430547.1, or BGC0000602) (141). The subtilosin A is a

sactipeptide with an MW of 3398.9 Da, pI of 3.88, and exhibits antimicrobial activity against

Gram-positive bacteria (Bacillus sp., Enterococcus sp. and Listeria sp.) as well as Gram-

negative bacteria at high concentration (142).

The biosynthetic gene cluster of subtilosin A found in #01’s genome (named 01sbt) was 8,250

bp in length, flanked by two terminators, and consisted of 8 genes encoding proteins, 01sblA-

H [Figure 4.10, Table 4.9]. The bacteriocin precursor (01sbtA) contained 42 amino acids. The

sactipeptide commonly matures via modification steps; removal of the leader sequence and

the core sequence is modified by a radical SAM that catalyses to form a cross-linking bond

between two amino acid residues (143). In this gene cluster, the putative zinc protease

(01sbtG) and peptidase (01sbtH) were responsible for the elimination of the leader peptide,

modification on core peptide under the catalysis of SAM-radical-enzyme (01sbtB) to mature.

Next, a two-component transport system (clustered by 01stbE and 01stbG) was responsible

for secreting the bacteriocin. The immunity protein (01stbD) was found within the cluster that


88

protected the host from bacteriocin activity. The role of two hypothetical proteins; 01slbI,

01sbtB were unknown.

Figure 4.10. The subtilosin A gene cluster found in the genome of isolate #01. (A) Gene

organisation of the subtilosin A gene cluster. (B) MSA of the mersacidin precursors of

#01 ((01-sbtA), and Bacillus sp. HIL-Y85/54728 indicated 100% of similarity. (C) The

MSAG between the subtilosin A gene cluster of isolate #01 and B. subtilis subsp.

spizizenii ATCC 6633. Two gene clusters shared 100% sequence similarity.

Table 4.9. Structural organisation of subtilosin A gene cluster found in the genome of

isolate #01

Protein Description Functions Similar proteins*

01sbtA precursor pepetide precursor sboA

01sbtB unknown unknown sboX

01sbC radical SAM maturation Alba

01sbtD immunity immunity albB

01sbtE ABC transport (ATP binding) transportation albC

01sbtG ABC transport permease transportation albD

01sbtG putative zinc protease maturation able

01sbtH peptidases maturation albF

01sblI unknown unknown albG

* The proteins were observed from the subtilosin A gene cluster of B. subtilis subsp. spizizenii

ATCC 6633.


89

4.2.8.5. The plantazolicin gene cluster (found in the genome of isolate # 06)

The plantazolicin gene cluster was found in the genome of B. amyloliquefaciens #06. The

gene cluster shared 91% similarity to that of B. amyloliquefaciens FZB42 [Figure 4.11] (51).

The plantazolicin is a ‘linearized-azole-peptide’ bacteriocin. It has MW of 1,336 Da and

displays antimicrobial activity only against Gram-positive bacteria.

The plantazolicin gene cluster (06plz) was 13.3 kb in length and comprised 10 ORFs encoding

10 proteins, 06plzA-N [Figure 4.11, Table 4.10]. The 40aa precursor bacteriocin (06plzA)

was composed of a 26 aa leader sequence at the C-terminus and a 14 aa core sequence at the

N-terminus. The maturation of linearized azole peptides (LAPs) are common via typical

modification steps; enzymatic removal of the leader sequence and modification on core

peptide under the catalysis of 3 enzymes; cyclodehydratase, dehydrogenase, a

docking/scaffolding protein (51, 144). Therefore, these genes were searched in this gene

cluster. Four ORFs were annotated as modification enzymes found including zinc protease

(06plzK) for the elimination of the signal peptides, and cyclodehydratase (06plzH),

dehydrogenase (06plzJ) and a docking/scaffolding protein (06plzI) for modification of the

core sequence (51, 144). Besides, a methyltransferase (06plzL) was found in the gene cluster

that may carry and attach additional methyl moieties to the side chain (51, 145). The

bacteriocin was secreted by a two-component ABC transporter (clustered by 06plzD and

06plzE). The hypothetical protein (06plzF) was also included within the gene cluster but the

function was unknown. A transmembrane protein (06plzB) served an immunity function for

protection.

Figure 4.11. The plantazolicin gene cluster found in genome of isolate #06. (A) Gene

organisation of the plantazolicin gene cluster. (B) MSA of the mersacidin precursors of


90

#06 (06plzA) and B. amyloliquefaciens FZB42 indicated 100% of similarity. (C) The

MSAG between the subtilosin A gene cluster of isolate #01 and the one of B. subtilis

subsp. spizizenii ATCC 6633. Two gene clusters shared 91% sequence similarity.

Table 4.10. Structural organisation of plantazolicin gene cluster found in isolate #06


06plzB hypothetical protein unknown plzF

06plzC transcriptional regulators, marR/emrR family regulation plzK

06plzD TOMM export ABC transporter, ATP-binding transportation plzG

06plzE TOMM export ABC transporter, permease protein transportation plzH

06plzF Similar to Uncharacterized low-complexity proteins

unknown plzI

06plzA precursor bacteriocins precursor plzA

06plzG hypothetical protein (FIG01245785) maturation plzJ

06plzH TOMM biosynthesis cyclodehydratase (protein C) maturation plzC

06plzI TOMM biosynthesis docking scaffold (protein D) maturation plzD

06plzJ TOMM biosynthesis dehydrogenase (protein B) maturation plzB

06plzK TOMM biosynthesis zinc protease removal of leader sequence

plzE

06plzL SAM-dependent methyltransferase (EC 2.1.1) tailing plzL

* The proteins were from the plantazolicin gene cluster of B. amyloliquefaciens FZB42.

4.2.8.6. The paenicidin A gene cluster (found in the genome of isolate #23)

The biosynthetic gene cluster of paenicidin A was found in the genome of P. polymyxa #23

[Figure 4.12]. The whole genome cluster was similar to that of P. polymyxa NRRL B-30509

(146). The paenicidin A has an MW of 3376.5 Da, and displays antibacterial activity against

Gram-positive bacteria (Bacillus sp., Lactobacillus lactis, L. monocytogenes and E. faecium).

The paenicidin A gene cluster (23pae) was 9,667 bp in length and consisted of 6 ORFs

encoding 6 proteins, 23paeA-23paeF [Figure 4.12, Table 4.11]. The precursor (23paeA) was

57 amino acids, consisting of a 21 aa leader sequence at the C- terminus and a 36 aa core

sequence at the N-terminus. The sequence was 100% identical to the paenicidin A precursor


91

of P. polymyxa NRRL B-30509. The maturation of paenicidin A occurred under catalysis of

3 enzymes; LanB-type enzyme (23paeB); LanC-type enzyme (23paeD); and LanT-type ABC

transporter (23paeC). The LanB and lanC were well known as two typical modification

enzymes of subclass Ia which catalysed the dehydration and cyclisation of amino acid residues

to convert them to Dha, Dhb amino acids (40, 147). The lanT- type ABC transport had

conserved domains (peptidase, ATPase, permease), therefore it had both functions of removal

of the leader sequence and secretion of the matured peptide (39, 138, 148). A cluster of 3

proteins (23PaeF, 23PaeG, 23PaeH) formed an ABC transport type II that was an immunity

system (137).

Figure 4.12. The paenicidin A gene cluster found in the genome of isolate #23. (A) Gene

organisation of the gene cluster. (B) MSA of the mersacidin precursors of #23 (23paeA)

and P. polymyxa NRRL B-30509. Two precursors were 100% identical. (C) The MSAG

between the paenicidin A gene cluster of isolate #23 and P. polymyxa NRRL B-30509.

Two gene clusters shared 100% sequence similarity.


92

Table 4.11. Structural organisation of paenicidin A gene cluster found in the genome of

isolate #23


23paeA paenicidin precursor precursor paeA

23paeH transporter ATP binding protein immunity paeF

23pae G ABC transporter membrane-bound subunit

transportation paeE

23pae F ABC transporter membrane-bound subunit

transportation paeG

23pae D lantibiotic dehydratase modification enzymes

paeB

23paeC lantibiotic ABC transporter transport paeT

23paeB lantibiotic cyclase modification enzyme

paeC

* The proteins were from the paenicidin A gene cluster of P. polymyxa NRRL B-30509.

4.2.9. Prediction of gene organisation of biosynthetic gene clusters of novel bacteriocins

4.2.9.1. The novel lantibiotic gene cluster (found in the genome of isolate #08)

The biosynthetic gene cluster of a novel lantibiotic (named 08lan) was found within the isolate

#08’s genome [Figure 4.13]. Presence of the lanM-type enzyme within the gene cluster

indicated that this bacteriocin was a lantibiotic subclass Ib (40).

The 08lan lantibiotic gene cluster was 5,977 bp in length, flanked by 2 terminators and

consisted of 4 ORFs encoding 4 proteins 08lanA-D [Figure 4.13, Table 4.12]. The precursor

bacteriocin (08lanA) had 78 aa, comprising a 48 aa leader sequence and a 35 aa core sequence

at the N-terminal. The amino acid sequence was similar to lichenidin A1 (37% similarity) and

lichenidin VK21A2 (43% similarity), but had 100% identity to an uncharacterized

mersacidin/lichenicidin family type 2 lantibiotic (WP_052587026.1) in the NR database

[Figure 4.6]. This novel lantibiotic possibly matured under the modifications processes under

the catalysis of a protease (08lanD) for cleavage of the signal sequence and a lanM-type

enzyme (08lanB) to modify the core sequence. The 08lanC shared 42.42% similarity to a

multidrug resistance ABC transporter ATP-binding/ permease protein, BmrA, and possessed


93

5 transmembrane helices in the structure suggesting a possibility of either secretion and/or an

immunity function (149, 150)

Figure 4.13. The novel lantibiotic found in the genome of B. amyloliquefaciens # 08.(A)

Gene organisation of the gene cluster. (B) Precursor bacteriocin coding sequence and

amino acid translation. The amino acid sequence of precursor bacteriocin was

highlighted in green colour. The grey box indicated the presence of the ribosomal

binding site (RBS) and start codon (methionine).

Table 4.12. Structural organisation of novel lantibiotic gene cluster found in the genome

of isolate #08

Proteins TMH* Description Function Similar proteins in uniref90 database

08lanD 0 serine protease removal of leader sequence

28.22%

08lanC 5 ABC transporter ATP-binding/permease protein

secretion 42.42%.

08lanB 0 mersacidin modifying enzyme

modification 35.32%

08lanA 0 bacteriocin precursor precursor -

* TMH, number of transmembrane helices predicted by the TMHMM server, v.2.0

(http://www.cbs.dtu.dk/services/TMHMM/).


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4.2.9.2. The novel lantibiotic gene cluster (found in the genome of isolate #23)

The biosynthetic gene cluster of a novel lantibiotic (named as 23lan) was found within

scaffold #1 of P. polymyxa #23’s genome. The whole gene cluster included two parts; short

DNA regions containing 3 ORFs that inserted in-between the S-layer glycan biosynthetic gene

cluster, and a part containing the ORFs borrowed from S-glycan gene cluster [Figure 4.14].

The S-layer glycan gene cluster is responsible to produce transmembrane glycans that serve

as antigens on the bacterial surface and exhibits no antimicrobial activity (151). The presence

of two typical lantibiotic modification enzymes; lanB and lanC within the gene cluster

indicated that this bacteriocin was a lantibiotic of subclass Ia (40)

The insert region including 3 ORFs; a precursor bacteriocin (23lanA), a lanB-like enzyme

(23lanB) and a lanC-like enzyme (23lanC), was flanked by 2 terminators. The precursor

(23lanA) was unable to be detected by the BAGEL, only elucidated by manual annotation by

reducing the threshold value (cut off at least 50 amino acids) for gene calling. The precursor

bacteriocin had 53 amino acids, consisting of a 29 aa signal sequence at the C-terminus and

24 aa core sequence at the N-terminus with a theoretical MW of 2375.2 Da. The sequence

was unique on the BACTIBASE database and shared low identity (45%) with a hypothetical

protein, PAC_2c01280, found in Paenibacillus alvei DSM23 (EJW18364.1). Two proteins;

23lanC, 23lanB were annotated as lanB-like dehydratase and lanC-like cyclase which

modified the core sequence.

Other functional proteins were found on the S-layer gene cluster. Two ORFs (23lanE and

23lanF) located at upstream of the insert region formed a two-component ABC transport for

bacteriocin secretion. The 23lanD containing 15 transmembrane helicases was possibly an

immunity protein (149, 152). However, it contained two conserved domains (Wzy_C

(IPR007016) and TPR-like helical_dom_sf (IPR011990)) which were annotated without the

evidence of immunity function. There was no ORF at the surrounding of the insert regions

functioning in the removal of the leader sequence.

The gene cluster was flanked by two terminators; one was at upstream of the ABC transport

and one was at downstream of 23lanA. Based on these terminators, the gene cluster was

temporarily predicted with a length of 12,147 bp, consisting of 6 ORFs encoding 6 proteins

(23lanA-F) [Table 4.13].


95

Figure 4.14. The novel lantibiotic gene cluster found in the genome of isolate #23.(A)

Gene organisation of the gene cluster. (B) The MSAG between the lantibiotic gene

cluster of isolate #23 and S-layer glycan. It indicated the insert regions in-between of

S-layer glycan gene cluster (in the box). (C) Precursor coding sequence and amino acid

translation after manual prediction on Snapgene. The grey box indicates the presence of

the ribosomal binding site (RBS) and start codon (methionine).

Table 4.13. Structural organisation of novel lantibiotic gene cluster found in the genome

of isolate #23

Proteins TMH Description Function Similar proteins in uniref90 database

23lanA 0 precursor bacteriocin precursor -

23lanB 0 lanB-type enzyme (PF04738, PF14028)

maturation V9W9P1_9BACL (65.4%); P. larvae DSM 2543

E3EGY4_PAEPS (60.2%); - P. polymyxa (strain SC2)

23lanC 0 lantibiotic biosynthesis protein (PF05147)

maturation V9WAJ5_9BACL (63.1% ); P. larvae DSM 25430

23lanD 15 polymerase immunity E3EAL6_PAEPS (88.4%); P. polymyxa (strain SC2)

23lanE 6 transport permease protein (PF1061)

transportation A0A0M0WD78_9BACI (85.3%); Bacillus sp. FJAT-21945

23lanF 0 ABC transporter, ATP-binding protein (PF00005; PF14524)

transportation C6J2R6_9BACL (68.15%) Paenibacillus sp. oral 786 str. D14


96

4.2.9.3. The sactipeptide gene cluster (found in the genome of isolate # 23)

The biosynthetic gene cluster of a novel sactipeptide (23sac) was found within scaffold #7 of

#23’s genome [Figure 4.15]. The presence of a radical SAM enzyme within the gene cluster

indicated that this gene cluster related to a sactipeptide (143).

The 23sac gene cluster was 8556 bp in length, consisting of 8 ORFs encoding 8 proteins,

23sacA-H [Figure 4.15, Table 4.14]. It was flanked by 2 terminators, one was downstream of

23sacG and one was at upstream of 23sacH. The precursor bacteriocin (23sacA) had 61 aa,

comprising a 19 aa leader sequence and a 42 aa core sequence. The precursor sactipeptide

was 100% identical to an uncharacterized putative bacteriocin, CLI_3235 family

(A0A378XQ17-P. polymyxa) but unique on BACTISBASE database. The 42 aa core

sequence was cysteine-rich with a theoretical MW of 4,455 Da and a pI of 7.73.

The maturation of the sactipeptide was probably initiated by a subtilase-family serine protease

(23sacH) which was responsible for cleavage of the signal sequence (153), followed by

modification of the core sequence by 2 enzymes; the radical SAM protein (23sacD) and an

‘acyl carrier protein homolog’ (TIGR04069) (23sacF) (154-156). The matured peptide was

then secreted by a putative ABC transporter (23sacC). The 23sacE was a hypothetical protein

belonging to IGR04066 family. This protein family contained subunits of an H+-transporting

two-sector ATPase, suggesting a function in immunity. The 23sacG was annotated as ‘muscle

M-line assembly protein unc-89 uncoordinated protein with unknown function for bacteriocin

biosynthesis.

Figure 4.15. The novel sactipeptide gene cluster found in the genome of P. polymyxa #23 and

the precursor sequence.


97

Table 4.14. Structural organisation of novel sactipeptide gene cluster found in the

genome of isolate #23

Proteins TMH*

Description Function Similar proteins in uniref90 database

23sacA Precursor bacteriocin precursor -

23sacB ECF subfamily RNA polymerase sigma-24 subunit

unknown A0A378XN73 (99.3%); P. polymyxa

23sacC 6 Multidrug ABC transporter protein

transportation A0A378XMM5 (100%); P. polymyxa

23sacD 0 Cys-rich peptide radical SAM maturase TIGR04068

modification A0A2S6NY22 (95.4%); P. peoriae

23sacE 0 Protein with domain TIGR04066

immunity UPI000D2F5CC2 (10%); P. polymyxa

23sacF 0 Peptide maturation system acyl carrier-related protein (TIGR04069)

modification A0A378XN50 (100%); P. polymyxa

23sacG 0 Muscle M-line assembly protein unc-89 Uncoordinated protein 89

unknown A0A378XQ26 (100%); P. polymyxa

23sacH 0 Subtilin-like serine protease removal of leader sequence

A0A378XQL5 (100%); P. polymyxa



4.2.9.4. The lassopeptide gene cluster (found in the genome of isolate #23).

This gene cluster of a lassopeptide (named as 23las) was located within scaffold 0 of #23’s

draft genome (namely 23las) [Figure 4.16]. Bioinformatic analysis using whole 23las amino

acid sequence 40% identical to the paeninodin gene cluster found in P. dendritiformis C454

(157). This biosynthetic gene cluster was 9233 bp in length and consisted of 8 ORFs encoding

8 proteins; 23lasA-H [Figure 4.16, Table 4.15]. The gene cluster was flanked by a promoter

locating upstream of 23lasI and a terminator at downstream of 23lasB. The precursor

lassopeptide (23lasA) comprised a deduced 21 aa signal sequence and a 21 aa core sequence

that has a predicted MW of 2413.1 Da. The 23lasA precursor bacteriocin was found to be


98

unique on the BACTIBASE database, but highly identical (difference in 1 amino acid) to an

uncharacterized lassopeptide ‘papoA’ of P. polymyxa CR1 (158, 159).

The lassopeptide maturation frequently relates to the modification steps; removal of leader

sequence by an ‘ATP-dependent cysteine protease’ (named as B protein), and a lassopeptide

cyclase (C protein) fold the amino acid residue at N-terminus of the core sequence and

attached to an Asp/Glu residue on the side chain to form a macrolactam ring (38, 160, 161).

The protein B can present at a “split” form; B1, B2 (162). In this gene cluster, B1-protein

(23lasE); B2 protein (23lasD) and C protein (23lasG) were found. The 23lasF was an HPr

kinase/phosphorylase that was responsible for the regulation (159). It regulated the operon by

first recognition of the precursor, binding and then activating the modification processes.

Additionally, a sulfotransferase protein (23lasH) transferred and attached sulphur moieties to

the side chain (159). The lassopeptide was secreted by an ABC transport (23lasB) which also

served as an immunity protein. A kinase (23lasD) was responsible to regulate the operon's

transcription.

Figure 4.16. The lassopeptide gene cluster found in the genome of isolate #23. (A) Gene

organisation of the gene cluster. (B) MSA of the lassopeptide precusor of #23 (23paeA)

and known lassopeptide precursors (baceA from B. cereus VD115, thcoA from

Thermobacillus composti KWC4 (163), padeA from P. dendritiformis C454, papoA

from P. polymyxa CR1(157)). (C) The homology tree across the precursor sequences.


99

Table 4.15. Structural organization of novel lassopeptide biosynthetic cluster found in

isolate #23

Proteins TMH*


23lasA Precursor bacteriocin precursor -

23lasB 5 ABC transporter ATP-binding protein/ Permease (PF00005.27; PF00664.23)

Transportation, immunity.

A0A378XVV8 (97.7%); P. polymyxa

23lasC 0 Uncharacterized nucleotidyltransferase

tailing A0A378XWG9 (43.1%); Paenibacillus sp. BC26

23lasD 0 Lasso peptide biosynthesis B2 protein

maturation A0A3G8R0N7 (95.7%); P. polymyxa 152

23lasE 0 Coenzyme PQQ synthesis protein D (PqqD) (protein B1)

maturation A0A378XW98 (98.0%); P. polymyxa

23lasF 0 HPr kinase/phosphorylase regulation A0A378XTM6 (98.5%); P. polymyxa

23lasG 0 Asparagine synthase (protein C)

maturation A0A378XUA7 (98.7%) P. polymyxa

23lasH 0 Protein-tyrosine sulfotransferase (PF09037.5 )

tailing A0A378XVA8 (97.2%); P. polymyxa



4.2.9.5. The thiopeptide gene clusters (found in the genomes of isolates #06 and #13).

The thiopeptide gene cluster was located within the genomes of both #06 and #13 [Figure

4.17; Table 4.16]. The thiopeptide gene clusters in #06 and #13 were the same. Therefore it

was named as 13thio gene cluster.

This 13thio gene cluster was 12 kbp in length and was comprised of 13 ORFs encoding 13

proteins, 13thioA-K [Figure 4.17, Table 4.16]. The precursor (13thioA) had 51 amino acids,

comprising a 31 aa leader sequence at the C-terminus and a 21 aa core sequence at the N-

terminus. The sequence was unique on BACTIBASE database but 100% identical with an


100

“uncharacterized thiopeptide" of Bacillus species” on the non-redundant (NR) database. The

core sequence had a theoretical MW of 1884.97 Da and theoretical pI of 6.39.

Thiopeptide commonly matures via heavy modification processes; i) cyclization, dehydration,

or oxidation of Ser, Thr, and Cys residues on the precursor to form oxazole ring; ii) conversion

of the Ser and Thr to Dha, Dhb, Lan and MeLan residues; iii) formation of the central six-

membered ring along with the elimination of the signal sequence; iv) additional modifications

on side chain like oxidations, cyclizations, methylations, tailing of indolic or quinaldic acid

moieties (164). To find these highly conserved proteins in the genome of isolate #13, these

proteins were acquired from the ‘nosiheptide genes clusters’ (165), then searched against the

genome of isolate #13 to locate homology CDS. The proteins included a pair of NosG (SpaB

C-terminal domain, PF14028) and nosF (Lant_dehyd_C, PF04738), a pair of NosD (YcaO

domain, PF02624) and nosE (PF00881), and the pair of nosO and nosH. After searching, two

ORFs (13thio G, and 13thioF) were found to be homology with NosG and nosF which were

responsible to convert amino acid to lantibiotic-typical amino acids through dehydration and

cyclisation. A pair of (11thioD and 11thiE) were homology to (NosD and nosE) catalysing to

form the azole rings; and (13thioO, 13thioH) were homology to (nosO and nosH) that

functioned in folding the peptide chain to form a six membered ring and also elimination of

the leader sequence. The thiopeptide was secreted by an ABC transport system (formed from

13thioI, and 13thioJ). There was a transcriptional regulator (13thioK) locating at end the of

the gene cluster. However, there was no immunity gene found so it was unclear if this cluster

had the potential to produce an antimicrobial.

Figure 4.17. The novel thiopeptide gene cluster found in the genomes of B. amyloliquefaciens

#06 and #13.


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Table 4.16. Structural organization of the novel thiopeptide biosynthetic cluster in

isolate #06 and #13

Proteins TMH*


13thioA Precursor bacteriocin precursor -

13thioB 0 Lantibiotic dehydratase domain protein (protein B) PF04738

dehydration of

aa residue

UPI0006246BFE (99.7%); B. amyloliquefaciens

13thioC 0 Lantibiotic biosynthesis dehydratase C-term (PF14028)

cyclisation of

aa residue

A0 A1G6Y285; (24.8%); Aquimonas voraii

13thioD 0 Hypothetical protein unknown UPI00084A0EF5 (100.0%); B. amyloliquefacines

13thioE 0 TOMM precursor leader sequence-binding protein (PF02624)

formation of azole

UPI0006246BFE; (99.7%); B. amyloliquefacines

13thioF 0 Lantibiotic biosynthesis dehydratase C-term (PF14028)

formation of azole

J8HJV6 (51.3%); B. mycoides

13thioG 0 SagB/ThcOx family dehydrogenase (PF00881); putative nitroreductase MJ1384

formation of azole

UPI00052AFFE7 (100%); B. amyloliquefacines

13thioH 0 TOMM precursor leader sequence-binding protein (TGR02603)

formation of pyrine ring.

UPI00104D7783 (25.7%); Tumebacillus sp

13thioI 0 ABC transporter ATP-binding protein

transportation UPI000653513B (100%); Bacillus sp.

13thioJ 6 ABC transporter permease transportation UPI00052AA050 (100%); Bacillus sp.

13thioK 0 Transcriptional regulator regulator I2C213 (93.8%); B. amyloliquefaciens




102

4.2.9.6. The thiopeptide gene cluster (found in the genome of isolate #11).

This novel thiopeptide gene cluster (namely 11thio) was located within scaffold 8 of isolate

#11’s draft genome [Figure 4.18]. The gene organization was like the thiopeptide gene cluster

found in #13 and #06 but with some variance.

Figure 4.18.The novel thiopeptide found in the genome of B. amyloliquefaciens #11

Table 4.17. Structural organization of the novel thiopeptide biosynthetic cluster found

in isolate #11

Proteins TMH*


11thioA precursor bacteriocin precursor

11thioB 0 lantibiotic dehydratase protein (PF04738)

maturation A0A369C8Z1 (100%); B. amyloliquefaciens

11thioC 0 unknown unknown -

11thioD 0 unknown unknown -

11thioE 0 bacteriocin biosynthesis docking scaffold, SagD family (PF02624)

maturation J8I0L0 ( 34.5%); B. mycoides

11thioF 0 sagB/ThcOx family dehydrogenase (PF00881)

maturation A0A418UV33; Actinomyces sp. 2119

11thioG 0 lantibiotic biosynthesis dehydratase C-term (PF14028)

maturation A0A1M5UR24 (39.9%) Caloranaerobacter azorensis DSM 13643

11thioH 0 sagB-type dehydrogenase enzyme

maturation A0A369CBC6 (100%); B. amyloliquefaciens

11thioI 0 hypothetical protein unknown

11thioJ 6 ABC-2 type transport system ATP-binding protein (PF00005)

transportation A0A369C905 (100%); B. amyloliquefaciens

11thioK 0 ABC transporter permease transportation A0A2I5JX77 (100%); B. amyloliquefaciens


103



The 11thio gene cluster was 13,386 bp in length and consisted of 11 ORFs, encoding 11

proteins 11thioA-K [Figure 4.18, Table 4.17]. The precursor (11thioA) had 72 aa, comprised

of a signal sequence at the C-terminus and a 16aa core sequence at the N-terminus. The core

sequence had a theoretical MW of 1777.98 Da and pI of 5.23, was unique on BACTIBASE

database but identical with an “uncharacterized thiopeptide-like bacteriocin” found in B.

amyloliquefaciens (H57 Ga0082361_104/ KACC 18228) on the NR database.

Similar annotation like the 13thioeptpide gene cluster, the pair (11thio G, and 11thioF) were

found to be homology with (Nos G and nosF), pair of (11thioD and 11thiE) were homology

to (NosD and nosE); and (11thioO, 11thioH) were homology to (nosO and nosH). A two-

component ABC transport (clustered from 11thioJ and11thioK) was responsible to secrete the

bacteriocin (150). Two hypothetical proteins (11thioC, 11thioI) were unknown functions,

possibly serving as immunity proteins because this kind of functional protein was still not

found within the gene cluster.

The 11thio gene cluster was flanked by a promoter upstream of 11thioA and a terminator

located downstream of 11thioK. Within the gene cluster, 11thioA itself was flanked by a

promoter and Rho-independent terminator; there was a second promoter detected upstream of

11thioB. Therefore, it suggested that the 11thioA was first transcribed, subsequently serving

as an enhancer to promote the expression of the whole operon using the second promoter.

4.2.9.7. The two-component lantibiotic gene cluster (found in genome of isolate #13).

This lantibiotic gene cluster was found in #13’s draft genome (namely 13lan) [Figure 4.19].

It was 15,526 bp in length, was comprised of 11 ORFs; 13lanA-J [Figure 4.19, Table 4.18].

There were 2 precursors (13lanA1; 13lanA2) within the gene cluster. The 13lanA1 precursor

was found to be 100% identical to ‘Plantaricin C family lantibiotic’ from Bacillus species

(WP_003154891.1). The 13lanA2 had low identical (27%) to ‘lichenicidinVK21A2’ (B.

licheniformis strain DSM 13). The 13lanA2 had 58 aa, comprising a leader sequence at the

C-terminus and a core sequence at the N-terminus via a conserved processing site of two

glycine residues (166, 167).


104

There were two lanM-like enzymes (13lanH, 13lanJ) that were responsible to modify the

precursor bacteriocins; therefore, each of the enzymes may modify one precursor. This kind

of gene distribution was also typical in the biosynthetic gene cluster of two-component

lantibiotics (168). A LanT-type ABC transport (13lanI), which contained a C39 peptidase

domain (PF03412) and ABC_tran (PF00005) functioned in cleaving off the leader sequence

and secreting the mature bacteriocin. Another ABC transport (clustered from 13lanD and

13lanE) may serve as an immunity system that protected the host from bacteriocin activity

(169). Besides, a lanKR-type two-component signal transduction system (clustered from

13lanB and 13lanC) regulated the transcription of the bacteriocin gene cluster (170). Another

two-component regulatory system (13lanF, 13lanG) was also found that also played a role in

the regulation of bacteriocin production.

Figure 4.19. The two-component lantibiotic gene cluster found in the genome of isolate

#13. (A) Gene organisation of the gene cluster. (B) MSA of the two precursors of #23

(23lanA1, and 23lanA2) and lichenicidine A2. (C) The homology tree across the

precursor sequences. Tree and MSA were calculated by DNAman software.


105

Table 4.18. Structural organization of the novel two-component lantibiotic in isolate

#13

Protein TMH*

Description Function Similar proteins (on Uniref90 database)

13lanB 0 Transcriptional regulatory protein YvrH (PF00486, PF00072)

transcription P94504 (36.56%); Bacilli

13lanC 2 Sensor histidine kinase YbdK (PF00512)

transcription O31433 (25.42%); Bacillii

13lanD 0 Lantibiotic ABC transporter ATP-binding protein (PF00005)

transportation Q5WIQ7_BACSK (46.01%); B. clausii KSM-K16

13lanE 6 Lantibiotic ABC transporter/ permease (PF12730.7)

modification A0A1W6HGX2 (97.1%); B. venezensis

13lanF 2 Sensor histidine kinase ComP (PF02518)

transcription Q99027 (29.49%); Bacillaceae

13lanG 0 Transcriptional regulatory protein ComA (PF00072)

transcription P14204 (41.23%); Bacilli

13lanA1 Precursor precursor -

13lanH 0 Lantibiotic modifying enzyme (PF05147)

modification Q65DC3_BACLD; (32.07%); B. licheniformis DSM 13

13lanI 5 MrsT protein (PF00005; PF03412)

transportation and cleaving off the leader sequence

Q9RC21_BACSY (35.49%); Bacillus sp. strain HIL-Y85/54728

13lanA2 Structural bacteriocin (PF04604, PF16934)

precursor -

13lanA3 Precursor precursor -

13lanJ 0 Lantibiotic modifying enzyme (PF05147.13, PF13575.6)

modification Q62NU6_BACLD (27.32%); B. licheniformis DSM 13




106


There is an urgent need for novel antimicrobial substances to be identified for new antibiotic

development when the majority of antibiotics currently used in the treatment of hospital-

acquired infection are found to be less effective with the development of resistance. Much

effort has been made to synthesize in vitro novel antimicrobial agents; nevertheless, nature is

still a rich and cheap source to search for novel antimicrobials substances. In this

investigation, 6 marine Bacillus/Paenibacillus isolates were selected to search for novel

bacteriocins, including; 1 P. polymyxa #23; 4 B. amyloliquefaciens (#06, #08, #11, #13); and

1 B. halotolerans (#01). These bacteria were chosen because they displayed antimicrobial

activity against a wide range of problematic pathogens, including Gram-positive bacteria, and

some serious hospital-acquired Gram-negative bacteria such as Campylobacter, Klebsiella,

and Pseudomonas.

First, genomes were sequenced, assembled and annotated and then comparisons were

performed to visualize the difference in genomes within marine isolates versus closely related

bacteria derived from terrestrial sources. All genome comparison calculations (first

employing calculation of ANI across the genomes and later confirming the finding from ANI

calculation by SBCT tool and MAUVE) were in agreement that genomes of B.

amyloliquefaciens were significantly homologous, followed by B. halotolerans, and the P.

polymyxa displayed the lowest similarity in genomes. Also, analysis of the frequency

distribution of CDS across the genomes of each of species (including both marine strains and

terrestrial strains), verified by GET-HOMOLOGOUS, indicated the highest diversity of the

gene repertoire of P. polymyxa when compared with B. amyloliquefaciens, and B.

halotolerans, emphasizing the potential of this species in the discovery of novel antimicrobial

agents. The high numbers of strains-specific CDS in P. polymyxa genomes can be derived

from prophage sequences or external movable DNA that infected to the host by horizontal

transfer event (171, 172). The infection can be observed via the presence of the deleted blocks

within LCBs of the genome from the MAUVE analysis. Due to the larger chromosome, P.

polymyxa can carry high numbers of prophages sequences that often possess unique genes

and genes relating biosynthetic gene clusters of ribosomally synthesized peptides (microcins,

lantibiotic) (141, 172).

In silico prediction of antimicrobial peptide encoding genes from these 6 genomes, by both

BAGEL and antiSMASH, elucidated the presence of 61 gene clusters relating to the


107

biosynthesis of lipopeptides (LPs), polyketides (PKs), bacilysin and bacteriocins. The number

of predicted lipopeptides encoding regions was twice the number of polyketide, hybrid, or

bacteriocin encoding regions. This finding is in agreement with earlier studies on drug

discoveries from bacilli. It was reported that 31% of the Firmicutes had gene clusters predicted

to encode antimicrobial peptides, with 70% of the total predictions being for NRPs synthesis

and 30% for hybrid LPs/PKS or PKS (173). The presence of both NRPs and bacteriocin agents

mark a difference between the antimicrobial potential of Bacillus and Lactobacillus isolates,

with the Lactobacillus hosts only carrying genes for bacteriocin synthesis (37)

There were 41 NRPs gene clusters predicted. They related to the biosynthesis of 14 different

types of NRPs compounds. This is because some NRPs were replicated in all Bacillus species,

including LPs (bacillaene, and macrolactin), lipopeptides (surfactin, fengycin, and

bacillomycin D), and siderophores (bacillibactin). These NRPs types of Bacillus were

different from those of Paenibacillus genomes (tridecaptin, polymyxin, putative

paenibacterin, fusaricidin), suggesting that the NRPs are taxon associated. During searches

for the presence of these derived NRPs from other species, we found that these NRPs were

also found in other members of B. subtilis groups (174-176) but not in other members of the

Bacillus group, probably suggesting that they are associated with the B. subtilis group only.

This may indicate that these NRPs gene clusters were acquired in a common ancestor, and

then distributed and maintained within the members of the group as it diversified. However,

the lipopeptide difficidin was only found in genomes of B. amyloliquefaciens but not in the

B. halotolerans genome, suggesting that this lipopeptide is only associated with B.

amyloliquefaciens. This observation is similar to earlier studies which also reported that the

distribution of the fengycin gene cluster was in a narrow subset of the B. subtilis group, or

bacitracin cluster presented in some species of B. subtilis group (such as B. licheniformis, B.

subtilis) (172, 177). The potential novelty of completely new NRPs compounds is less

reported, although the variants of traditional agents are seldom mentioned due to addition/loss

of acyl residue attached on main chains (127). To conclude, the presence of these NRPs

compounds in marine Bacillus/Paenibacillus emphasizes the potential application of these

marine species in pharmaceuticals and agriculture. Several Bacillus-derived NRPs are

approved for use in various treatment, such as immunosuppressive (rapamycin), anti-cancer

(bleomycin, daunorubicin, mitomycin) and even antibiotics (amphotericin B, erythromycin,

vancomycin, polymyxin, tridecaptin) (24, 56). In agriculture, AMPs genes are widely used as

markers to evaluate the application ability of a bacterium as biocontrol. For example, bmyB


108

(coding bacillomycin D synthetase B), and fenD genes (fengycin synthetaseD) were used as

markers to evaluate the B. amyloliquefaciens FZB42 (178); or bmyB, fenD, and ituC (iturin

synthetase D) are used to evaluate strains QST713, RGAF51, UMAF6639, RLID 12.1 (179)

The study has shown that marine Bacillus/Paenibacillus are a rich resource for the discovery

of novel bacteriocins, with 53.8% of all the predicted bacteriocins identified as novel or

uncharacterised. The six genomes investigated contained 20 bacteriocin gene clusters, with

each genome encoding at least 3 types of bacteriocins, excepting the genome of B.

halotolerans that contained only a single subtilosin A encoding region. This emphasizes the

high potential of marine B. amyloliquefaciens, and Paenibacillus as abundant pools of

bacteriocins, including novel bacteriocins. Although 20 bacteriocin encoding gene clusters

were found, they were predicted to only encode 13 different types of bacteriocins and these

belonged mostly to class I bacteriocins (lantibiotics, lassopeptide, thiopeptide, sactipeptide,

LAP). There was 1 predicted class II bacteriocin, and no class III. Lantibiotics were the most

common type (4/12) (including mersacidin, paenicidin A, two unknown lantibiotics).

The P. polymyxa #23 derived bacteriocins were more diverse; including 4 bacteriocins

belonging to 3 different subclasses of class I bacteriocins (sactipeptide, lassopeptide, 2

lantibiotics). Three of 4 bacteriocins were novel/uncharacterised and one bacteriocin that has

been previously characterised (paenicidin A), marking this species as the one with the most

potential for seeking novel bacteriocins in the marine environment (at least in this

investigation). This is an unsurprising result as the genome of this species was found to be

highly variable, indicating great flexibility in adapting to environmental changes (last chapter,

mentioned in the study of comparative genomics). Of three novel bacteriocins, the predicted

lassopeptide encoding gene cluster had 40% similarity to a paeninodin encoding gene cluster

found in the genome of P. dendritiformis C454, and the precursor sequence had 1 amino acid

differing to the lassopeptide precursor in P. polymyxa CR1; however, this predicted peptide

is yet to be characterised.

Amongst the 5 sequenced Bacillus isolates, there were 8 gene clusters of bacteriocins found,

including 5 previously characterised ones (mersacidin, subtilosin A, plantazolicin, LCI, and

amylocyclicin). The bacteriocins found in the Bacillus isolates were also different from those

of P. polymyxa. Three bacteriocins (LCI, amylocyclicin and thiopeptide) were found in all B.

amyloliquefaciens isolates, but not in the sequenced B. halotolerans isolate. The LCI

bacteriocin has been also reported in many studies, not only associated with B.


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amyloliquefaciens but also found in B. subtilis, suggesting that the presence of this bacteriocin

is associated with the related species of the B. subtilis group. Both amylocyclicin and

thiopeptides are found only in genomes of marine B. amyloliquefaciens, indicating strain-

specific bacteriocins. Therefore, it appears that some of the Bacillus derived bacteriocins may

be specific/restricted to certain species. There were 3 novel bacteriocins (two thiopeptides,

one two-component lantibiotic). All the predicted thiopeptides found in this investigation are

novel. The thiopeptides are highly modified peptides of low MW (< 2 kDa), and their

biosynthesis uses both non-ribosomal synthesis, and ribosomal synthesis. The thiopeptides

are recognised as a promising bacteriocin group for drug development; some of them are

already in clinical trials for the treatment of human infections. Besides, thiopeptides also

exhibit anti-cancer, anti-plasmodial, immunosuppressive, antifungal, and even anti-malaria

activity which address important areas of therapeutic application.

It is interesting to review a number of the performance of the bioinformatic tools that were

used to predict the antimicrobial peptide encoding regions; BAGEL and AntiSMASH.

BAGEL is currently the tool of choice for prediction of putative bacteriocins. It has been

designed to better perform the task when compared with other similar software because it can

detect small ORFs of structural genes which are frequently omitted by other software, and it

predicts the bacteriocins cluster by also interrogating surrounding genes to identify other

genes that may participate in bacteriocin biosynthesis, processing, and export. In this

investigation, it was found that BAGEL was unable to detect the thiopeptide gene clusters,

but AntiSMASH could. AntiSMASH is the tool of choice for prediction of NRPs compounds

in bacterial genomes. However, both software was unable to reveal in detail the gene

organisation of thiopeptide gene clusters and locate the structural gene within the gene cluster.

Therefore, the attempt to locate gene components was supplemented by manual annotation

by i) reducing the minimum peptide length cut off to 50 amino acids for calling genes

encoding precursor. The sequence of precursor peptide comprises of a core sequence of 12 to

17 residues at C- terminus, and a leader sequence of 34 to 55 residues at the N-terminus (164).

ii) using highly conserved sequences of modification enzymes typical for thiopeptide

biosynthesis (like nosF, nosG, nosE, nosD, nosF in nosiheptide cluster) as reference (or driver

sequences) to locate homologous CDS within the cluster. Therefore, prediction of

antimicrobial peptides from Bacillus/Paenibacillus may require a combination of BAGEL,

AntiSMASH, and manual annotation, to fully explore all the gene clusters. However, due to

incompleteness of genomes (draft genomes, with gaps) generated from Illumina sequencing,


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some clusters lack the context of some surrounding gene-determinants, particularly

loss/division of gene components across multiple gaps, into different contigs, is commonly

observed from large gene clusters such as those used to produce non-ribosomally synthesized

peptides. This can cause overprediction of the number of antimicrobial peptide encoding

regions. To avoid this it would be desirable to close genomes and generate complete

assembled genome sequences (e.g., by adding data from long sequence read technologies such

as Pacbio or MinIon) (180, 181), to give a conclusive picture of antimicrobial potential from

marine Bacillus/Paenibacillus. Taken together, the results emphasized that the marine

Bacillus/Paenibacillus from the Vietnam Sea are abundant sources of non-ribosomally

synthesized antimicrobial peptides and bacteriocins, and novel bacteriocins. Although the list

of the compounds and their biosynthetic gene clusters were well-analysed in this study, the

overall picture of antimicrobial potential of these marine Bacillus/Paenibacillu is still unclear

until in vitro characterisation is carried out to validate the findings. Therefore, two of the

bacterial isolates were used to purify the antimicrobial substances produced. That work is

detailed in the next chapters.


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Chapter 5 PURIFICATION OF ANTIMICROBIAL PEPTIDES PRODUCED BY

Bacillus amyloliquefaciens #11

5.1. Introduction

Bacillus amyloliquefaciens is a Gram-positive, spore-forming non-pathogenic bacterium. It is

ubiquitous in both the terrestrial and marine environment. This species has two subspecies;

plantarum (commonly associated with plants) and amyloliquefaciens (non-plant associated).

Terrestrially derived B. amyloliquefaciens are well-known producers of enzymes, and

antimicrobial peptides (particularly bacteriocins) (36, 182, 183). The potential of bacteriocins

produced by marine-derived B. amyloliquefaciens is still yet to be exploited in any detail and

therefore they may prove to be a promising reservoir for the discovery of novel bacteriocins.

In the last Chapter 4, B. amyloliquefaciens #11 was isolated from a seaweed sample and

exhibited strong antibacterial activity. The antibacterial activity exhibited by isolate #11

encompassed both Gram-positive pathogens (Staphylococcus sp., Enterococcus sp., Bacillus

sp., Streptococcus sp, Clostridium sp., MRSA) and Gram-negative bacteria (E. coli, S.

Enteritidis, C. coli). Therefore, it was selected for whole genome sequencing to search for

novel bacteriocins. The bacterium was identified based on the 16S rDNA sequence sharing

99.9% of similarity to one of B. amyloliquefaciens FZB42. The FZB42 which is a plant

growth-promoting rhizobacteria, and serves as a Gram-positive bacterium model for

biocontrol in agriculture (184). The comparison of the in silico predicted antimicrobials

peptides from the genome of #11, with the prediction of those encoded by FZB42, indicates

significant similarity in antimicrobial potential between these two bacteria. Both bacteria have

gene clusters predicted to encode the same 8 - 10 types of antimicrobial peptides with high

homology, including non-ribosomally synthesized peptides (siderophore bacillibactin,

difficidin, macrolactin, surfactin, fengycin, bacillomycin D) and two bacteriocins

(amylocyclicin, and LCI). They are only different due to the presence of plantazolicin

bacteriocin and an uncharacterised LPs (nrs gene cluster), which are found only in FZB42,

and a novel thiopeptide which is found only in the genome of #11. The plantazolicin belongs

to the linear azol(in)e peptides (LAP) subclass; both LAP and thiopeptide are two of members

of the TOMM class (thiazole/oxazole-modified microcins). This group is typically small

bacteriocins (MW of less than 2 kDa), highly modified structures containing azoline rings

generated from enzymatic modification of serine/threonine and cysteine residues. By

comparing with LAP bacteriocins, the thiopeptides have the addition of both Dha/Dhb


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residues and a nitrogen-containing six-membered ring; therefore, the gene cluster of

thiopeptide comprised more genes than LAP. Therefore, the heterologous cloning and

expression of a thiopeptide gene cluster is likely to be more challenging than cloning and

expression of other classes of bacteriocins. Thiopeptides have applications in

pharmaceuticals; with some of them used in the treatment of vaginal infections (185).

This chapter aimed to purify the thiopeptide from #11, for characterisation in vitro. To

achieved this aim, a purification procedure was developed including; i) the growth curve

analysis of the bacterium in LPMA to identify at which time-point the maximal antimicrobial

activity against two indicators, L. plantarum and B. cereus, was present; ii) chromatography

(ion exchange chromatography, hydrophobic interaction chromatography, reverse phase-

HPLC) to partially purify the compound, and iii) mass spectrometry (MALDI-TOF MS, N-

Terminal sequencing) to identify the composition. The work presented in this chapter

describes the bacterium, to underpin its use for pharmaceutical, industrial and agricultural

applications.

Figure 5.1. The seaweed from which B. amyloliquefaciens #11 isolated (A). Bacterial

colony morphology of #11 on Muller Hilton agar (B).


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Figure 5.2. Antimicrobial activity exhibited by marine isolate #11. (A) Image

demonstrated the antimicrobial activities against some Gram-positive bacteria including

S. aureus (1), MRSA (2), S. faecalis (3), B. cereus (4). (B) Image demonstrated the

activities against some Gram-negative bacteria such as S. Enteritidis (1), E. coli (2), L.

monocytogenes (3); and P. aeruginosa (4).

5.2. Result

5.2.1. Analysis of the bacterial growth curve revealed to production of various

antimicrobial compounds

The growth of B. amyloliquefaciens in LPMB broth was monitored up to 48 hours (30oC, 150

rpm) and sampled over that period. The samples were tested in an antimicrobial assay against

two indicators; L. plantarum and B. cereus [Figure 5.3]. The first indication of antimicrobial

activity in the culture was observed at 8 hours, with inhibition of L. plantarum. The strongest

activity was noted at 12 hours and activity was lost after 24 hours. Activity against B. cereus

was first observed after 12 hours and continued to be observed over all time points, up to 48

hours. It was obvious from these results that the bacterium produced at least two antimicrobial

compounds that peaked in activity, within the culture, after different fermentation times. To

further explore the antimicrobial peptides produced, 12 hour old culture and 24 hour old

culture were collected and purified separately.


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Figure 5.3. Antimicrobial activity exhibited from cell-free supernatants of the cultures

collected at different time-points. (A) The spot-on lawn assay was used to detect

antimicrobial activity against L. plantarum on MRS agar; (B) the well diffusion assay

was used to detect the antimicrobial activity against B. cereus on Muller Hilton agar.

cultures at 12 hours (4), at 18 hours (5); at 24 hours (6), 36 hours (7); 42 hours (8), 48

hours (9); (-) uninoculated media was used as a negative control. The activity against L.

plantarum was strongly observed at 12 hours old culture and was lost after 24 hours,

while the activity against B. cereus was first observed at 12 hour-old culture and from

then on continuously observed.

5.2.2. Purification of antimicrobial compounds from 12 hour-old culture elucidated the

presence of amylocyclicin bacteriocin

5.2.2.1. Recovery of bacteriocins from cell-free culture and cell pellet.

The culture lost the antimicrobial activity following proteolytic treatment (by proteinase K or

pronase E), indicating the presence of a proteinaceous antimicrobial compound, probably a

bacteriocin. During purification, we found that both cell-free culture and extract from cell

pellet exhibited similar antimicrobial activity [Figure 5.4]. Therefore, two separate procedures

were set up to recover the antimicrobial compounds.

The bacteriocin present in the cell-free culture was precipitated using ammonium sulphate.

An experiment was first carried out to determine the concentration of ammonium sulphate to

precipitate this bacteriocin. It was found that the bacteriocin precipitated when the ammonium

sulphate was at 40% (w/v) at 5oC. After centrifugation, the protein pellet was collected. The

protein pellet was dissolved in 1X phosphate-buffered saline (PBS) and desalted against milli-


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Q water using a dialysis tubing membrane (protein cut off 2 kDa) (Sigma Aldrich), to yield a

desalted ‘AMS fraction’. The remaining supernatant was also retained, adjusted to pH 2 by

concentrated HCl, and left overnight at 5oC without stirring. The protein precipitate was

collected by centrifugation at 17,500 g for 90 minutes, dissolved in 1X PBS to yield ‘LP

fraction’.

The cell pellet was also collected, then stirred in 70% isopropanol pH 2 at 5oC overnight

(acidified by concentrated HCl). The volume of isopropanol used was 20% of the volume of

bacterial culture. The cell-free solvent was collected by; centrifugation at 4,500 g for 20

minutes; filtered through a syringe membrane (0.45 µm, PES membrane), and then

concentrated in a rotary evaporator to 1/100 volume, to remove the solvent. The concentrate

was adjusted to pH 7 using NaOH, desalted against milli-Q water overnight at 5oC to yield a

desalted ‘fraction CFS’.

All three fractions exhibited antimicrobial activity against L. plantarum [Figure 5.4]. Of them,

the AMP yielded larger inhibitory diameter than LP or CFS. The antimicrobial compounds

presented in LP faction was considered due to the remaining of protein pellet during decanting

of the supernatant after centrifugation

Figure 5.4. Antimicrobial activity against L. plantarum exhibited by the fractions

collected at time-point of 12 hours. AMP: AMP fraction; LP: LP fractions, SF: CFS

fraction; CR: crude bacterial culture; (-) 1X PBS as negative control.


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To estimate the size of the antimicrobial peptides, two of the fractions; AMP and CFS, were

loaded on a 16% Tricine-SDS-PAGE and zymography were then performed. After

electrophoresis, one half of the gel was stained with Coomassie blue staining solution, but the

protein bands on stained gel appeared too faint to visualize; although antimicrobial band

appeared clearly on zymogram gel [Figure 5.6 B]. Therefore, the half gel was stained in silver

staining solution to increase the sensitivity of detection. This showed there was only a single

protein band on the gel. This single protein band, visualised by silver staining was at the same

position as the region of activity identified by zymogram. The band and activity derived from

AMP and CFS fractions were similar, with an estimated MW of ~5-10 kDa [Figure 5.6 A].

However, the AMP fraction contained lots of contaminating proteins when compared with

the CFS fraction. Therefore, CFS would be more suitable for use in future experiments for

quick purification of bacteriocin. The CFS fraction was then desalted by either dialysis (cut

off of 2 kDa) (Sigma Aldrich) or Amicon® Ultra Centrifugal Filters with MW cut off of

approximately 3 kDa (Merck Millipore). The desalted sample was then further purified by ion

exchange chromatography.

5.2.2.2. Purification of bacteriocins by ion exchange chromatography revealed the

presence of 1 antimicrobial peptide

To purify the unknown bacteriocin by ion exchange chromatography, two experiments were

first carried out to select the most suitable types of running buffer, types of resin and

concentration of NaCl for elution.

The first experiment showed that the antimicrobial substance was absorbed onto SP sepharose

in either of 20 mM acetate buffer pH 3.6 or pH 4.6 or pH 5.6 or 20 mM phosphate buffer pH

6.6. In contrast, it was not absorbed onto Q sepharose in 20 mM Tris-HCl buffer pH 8 [Figure

5.5A]. This indicated cation exchange chromatography was suitable to purify the bacteriocin,

and this bacteriocin had a high pI value, which may be more than 9. Subsequently, the second

experiment was carried out to determine the concentration of NaCl in the elution buffer. In

this stage, the cation exchange chromatography using SP sepharose was set up using 20 mM

acetate buffer pH 3.6 as running buffer. The result showed that the bacteriocin was eluted in

the presence of 0.2 M NaCl [Figure 5.5B]. Three elutes with 0.2 M NaCl were combined,

desalted by using Amicon® Ultra Centrifugal Filters (cut off of approximately 3 kDa).

MALDI-TOF MS identified two peaks; one with [m/z] = 6.390; one with [m/z]= 3.056 [Figure

5.6 C]. The desalted sample was then separated by RP-HPLC.


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Figure 5.5. The experiments to select the resin, type of buffer, and NaCl concentration

in elution buffer for ion exchange chromatography (A) The experiments to select the

resin and buffer. Well #1 to well # 4: SP sepharose and running buffer at pH 3.6, 4.6,

5.6, 6.6 respectively; well # 5: Q sepharose and buffer pH 8. (a) Antimicrobial sample

collected after mixing with the resin for 20 minutes. (b) Elutes of resin by elution buffer

supplemented with 0.5M NaCl. (B) The experiment to determine the concentration of

NaCl in the elution buffer. The cation exchange chromatography was performed in this

experiment using SP-sepharose and 20 mM acetate buffer pH 3.2. (S) The sample

collected after running through the resin, (w) the buffers collected after washing the

resin (twice), and antimicrobial fractions after eluting the resin using buffer

supplemented with 0.1 M (0.1), 0.2 M (0.2), 0.3 M (0.3) to 1M NaCl respectively. The

experiment indicated the antimicrobial was able to be eluted by elution buffer

supplemented with 0.2M NaCl.


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Figure 5.6. The MW of antimicrobial peptide presented in 12 hour old culture after

partial purification by cation exchange chromatography. (A) Estimated by Tricine-SDS

PAGE from AMP fraction (1), CFS fraction (2), purified bacteriocin (3) in the

visualization of (B) zymogram gel against L. plantarum (the same antimicrobial bands

presented in AMP and CFS samples indicated the presence of the same peptide). (C)

MALDI-TOF MS detected two protein peaks from the desalted sample obtained from

cation exchange chromatography. The peptide had mass intensity at m/z 6,399.27.

5.2.2.3. Purification of bacteriocin performed by analytical RP-HPLC elucidated the

presence of amylocyclicin

The desalted sample was injected in a C18 column and was operated under a 60 min-

acetonitrile-water gradient. The operation consisted of; 2% buffer B for 5 minutes; 2%-100%

solution B from minute 6-55; 100% solution B from minutes 56-60. Solution A was milli-Q

water + 0.1% trifluoroacetic acid (TFA) and solution B was 100% HPLC-grade acetonitrile

+ 0.1% TFA. The operation was monitored under light wavelengths of 230 nm and 280 nm

[Figure 5.7 A]. The active fraction was eluted at a retention time of 43.9 minutes (against L.


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plantarum) and was well-visualized under the wavelength of 230nm. Mass spectrometry of

antimicrobial compounds was [m/z] = 6380.853 [Figure 5.7 b]. The differences in the mass

peak between HPLC- based peptide and desalted- peptide from cation exchange

chromatography was possible due to the loss of 1 H2O atom (18 in MW) in HPLC-based

peptide when the peptide was in the acetonitrile solvent.

Figure 5.7. RP-HPLC to purify antimicrobial peptide presented in 12 hour old culture.

(A) The RP-HPLC to separate the desalted sample after cation exchange

chromatography. The result showed elution of amylocyclicin occurred at a retention

time of 43.9 minutes, (B) MALDI TOF-MS measured the purified bacteriocin with a

major peak at [m/z]=6,380.85.

Taken together, a full purification procedure for amylocyclicin could be proposed:

- Fermentation step: One percent of overnight bacterial culture is inoculated to fresh LPMA

culture. The culture is shaken at 120 rpm for 12 hours, centrifuges at 4,500 g for 20 minutes


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to collect both bacterial pellet and supernatant. The bacterial pellet is washed twice with milli-

Q water. The supernatant is filtered through a filter membrane (cut off 0.45 um, PES) to obtain

cell-free supernatant.

- Recovery step: bacteriocin can be recovered from either cell-free supernatant or cell pellet.

However, the cell pellet source contains less contaminating proteins and required less time

for purification than recovery from cell-free supernatant. Precipitating the bacteriocin from

cell-free supernatant can be achieved by using 40% saturated ammonium sulphate (at 5oC

overnight), subsequently centrifuging (17,500 x g, 90 minutes, 5oC) to collect the protein

pellet which is dissolved in 20 mM acetate buffer pH 3.6 (volume of the buffer: volume of

supernatant = 1: 50). Stirring the bacterial cell pellet in 1/5 volume of isopropanol (adjusted

to pH 2 by concentrated HCl) at 5oC overnight, subsequently centrifuging (4500 x g, 20

minutes) followed by filtering of supernatant through a syringe membrane (0.45um, PES) and

concentrating to 1/10 volume. These two fractions can be combined and dialysed against 20

mM acetate buffer pH 3.6 overnight at 5oC using dialysis tubing (cut off 2 kDa).

- Purification step: The desalted sample is passed through SP sepharose column for a cation

exchange chromatography, using 20 mM acetate buffer pH 3.6 as running buffer. The

chromatography can be operated automatically or manually. The automatic chromatography

uses prepacked HiTrap SP HP cation exchange chromatography column (GE Healthcare Life)

and with the aid of ÄKTA pure chromatography system (GE Healthcare Life). The operation

is under a gradient of solution A (20 mM acetate buffer pH 3.6) and solution B (20 mM acetate

buffer pH 3.6 + 1 M NaCl), visualized under the wavelength of 230 nm. The manual

chromatography used SP Sepharose Fast Flow cation exchange chromatography resin (Sigma

Aldrich). The operation included some steps; (i) packing the SP sepharose into an empty

column; (ii) washing the resin by adding milli-Q water (3CV) to remove ethanol; (iii)

equilibrating the resin by adding 20 mM acetate buffer pH 3.6 to column (3 CV); (iv) loading

the sample by dripping the sample into the resin; (v) washing resin (3CV) with 20 mM acetate

buffer pH 3.6; (vi) washing resin (3CV) with 20 mM acetate buffer pH 3.6 supplemented with

0.1 M NaCl to eliminate the weakly- bound proteins; and (iv) eluting the bacteriocin by adding

20 mM acetate buffer pH 3.6 supplemented with 0.2 M NaCl (3CV).

- All antimicrobial fractions are combined, desalted and concentrated using Amicon® Ultra

Centrifugal Filters (MW cut off of approximately 3 kDa)


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- Injecting desalted sample into an RP-HPLC C18 column. Operation is carried out using a

C18 Column with solution A (milli-Q water +0.1% TFA) and solution B (100% HPLC-grade

acetonitrile + 0.1%TFA). Operation condition includes 2% solution B for 5 minutes; 2%-

100% solution B from minute 6-55 minutes; 100% solution B from minutes 56–60 minutes

with visualization under a wavelength of 230 nm. The pure bacteriocin is eluted at a retention

time of 43.9 minutes.

5.2.2.4. The amylocyclicin, a heat and pH stable bacteriocin exhibited activity against

only Gram-positive bacteria

The purified bacteriocin was used to determine antimicrobial activity against a panel of

bacterial pathogens. The result showed that it could depress the growth of only Gram-positive

bacteria [Figure 5.8 C, Table 5.1]. It lost activity after proteolytic treatment with protease E,

proteinase K, but was resistant to trypsin, lipase and catalase treatment. The bacteriocin was

thermo-stable to 80oC for 1 hour, only losing activity at 100oC [Figure 5.8 A, B]. From these

finding, along with inspection of the bacteriocins predicted from the genomic data, it was

determined that this bacteriocin was amylocyclicin (133).

Figure 5.8. Properties of amylocyclicin. Thermal stability; activity remained after being heat

at 80oC for 1 hour. (B) Enzymatic degradability against pronase E (proE) and proteinase K

(proK). (C) antimicrobial activity of amylocyclicin against some indicators; from left to

right and from top to bottom was C. perfringens, S. aureus, MRSA, L. plantarum, E.

faecalis, E. coli, C. jejuni, S. Enteritidis, P. aeruginosa.


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Figure 5.9. (A) Sequence alignment between amylocyclicin from isolate #11 genome

(11amyA) and FZB42 strains. (B) Maturation of amylocyclicin. The amylocyclicin

precursor included a 48 aa N-terminal leader sequence, the core sequence. The

maturation related to the cleavage of the signal sequence at the cleavage site between

E−1 and L1 (vertical arrow), and circulation of core sequence between W64 and L1.

Six tryptic fragments and their mass were detected after digestion by trypsin including

the cyclization site WL (bold letters). Image B was obtained from Scholz et al. (2014)

(133)

Table 5.1. The antimicrobial spectrum of amylocyclicin produced by isolate #11

Indicators Medium used Antagonistic activity

S. aureus MHA ++

MRSA MHA ++

B. cereus MHA +

L. monocytogenes MHA +

C. perfringens MHA ++

L. plantarum MRS +++

E. faecalis (VRE) MRS ++


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Indicators Medium used Antagonistic activity

S. Enteritidis MHA -

E. coli MHA -

C. jejuni MHA -

C. albicans MHA -

5.2.3. Purification of antimicrobial substances from 36 hours-old culture showed the

presence of three antimicrobial compounds

5.2.3.1. Recovery of antimicrobials compounds from the cell pellet

The novel thiopeptide was not found from 12 hour culture; therefore, the 36 hour culture was

investigated alternatively. The antimicrobial activity against B. cereus in the culture

supernatant remained after proteolytic digestion by trypsin, pronase E, proteinase K, but no

antimicrobial band(s) could be detected on zymogram plates against B. cereus.

The antimicrobial substances were collected from 36 hour cell pellet. The cell pellet collected

from 36 hours old culture was stirred in acetonitrile (70% pH 2 (acidified by TFA)) overnight.

Acetonitrile and TFA were used instead of isopropanol and acid HCl because they were

volatile solvents, therefore, easily eliminated during rotary evaporation. The mixture was

centrifuged at 5,000 g for 20 minutes and the supernatant was retained. A cell-free solvent

fraction was obtained by filtering the supernatant through a PES syringe membrane (0.45µm).

MALDI-TOF was used to identify peptides presented in the cell-free solvent fraction,

detecting three peak clusters from this sample [Figure 5.9 A]. Based on the mass identity of

NRPs compounds mentioned in the literature (186), one peak cluster with mass of m/z 1002

-1068 indicated the presence of an iturin/surfactin with its iso-protons; a peak with a mass

intensity of m/z 800 was an unknown compound; and one cluster of peaks at a mass range of

m/z 1,500 -1,543 was consistent with fengycin or possibly thiopeptide as both fengycin and

thiopeptide have similar MW.


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Figure 5.10. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)

mass spectrometry (MS) of (A) CFS fraction from 24-hour culture, showing compounds

in the MW range of 800 – 2000 Da. The m/z 1002 -1068 peaks related to iturin, while

the peaks at m/z 1500 -1543 indicated the presence of an unidentified peptide, with an

MW similar to fengycin and thiopeptide; (B) MALDI-TOF trace obtained from the

purified compound with a mass of m/z 1500 -1543.

A

B


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5.2.3.2. RP-HPLC purification of antimicrobial compounds and peptide identification

The cell-free solvent fraction was concentrated to 1/20 volume under reduced pressure in a

rotary evaporator. During concentration, a precipitate appeared. This precipitate was

separated from the supernatant by centrifugation, washed twice with milli-Q water and

dissolved in 50% acetonitrile (it was insoluble in water or PBS buffer). This dissolve material

had antimicrobial activity against Gram-positive bacteria (but not Staphylococcus species).

During storage at 5oC or -20oC this dissolve formed aggregated again and was insoluble in

50% acetonitrile. Therefore, the freshly-dissolved aggregate was injected into an RP-HPLC

(C18 column) and the separation was carried out under a 60 minutes acetonitrile-water

gradient cycle including; 30% buffer B for 5 minutes, 30%-50% of buffer B from minute 6 to

minute 0; 50%-100% buffer B from minute 11–minute 55; 100% buffer B for minutes 55-60.

The chromatography elutes were visualized under wavelengths of 230 nm, of 214 nm and 280

nm. The antimicrobial compound was collected at a retention time of 44.618 minutes,

corresponding to approximately 80% acetonitrile. The MALDI-TOF MS revealed a major

peak cluster at an intensity of m/z 1417-1663; indicating the MW of the compound and its

isoprotons [Figure 5.9 B]. This peptide was only detected under UV wavelength of 214nm

and 230 nm but not under the wavelength of 280 nm, indicating a peptide [Figure 5.10]

Taken together these findings suggest that this antimicrobial peptide was highly hydrophobic

and exhibited an antimicrobial spectrum against Gram-positive bacteria. We failed to

determine the peptide sequence using Edman degradation N-terminal sequencing.

Figure 5.11. RP-HPLC used to separate the freshly dissolved aggregate.The antimicrobial

was eluted at a retention time of 44.6 minutes


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Table 5.2. Antimicrobial spectrum of purified thiopeptide supposed

Indicators Media Antagonistic activity

Staphylococcus aureus MHA -

MRSA MHA -

Bacillus cereus MHA +

Listeria monocytogenes MHA -

Clostridium perfringens MHA +

Lactobacillus plantarum MRS -

Enterococcus faecalis (VRE) MRS -

Salmonella Enteritidis MHA +

Escherichia coli MHA +

Campylobacter jejuni Blood agar +

Candida albicans MHA -

5.3. Summary of results and discussion.

The B. amyloliquefaciens #11 was isolated from seaweed Sargasum which was collected in

NhaTrang bay, Vietnam Sea. Genome analysis revealed it possessed gene clusters encoding

synthesis of non-ribosomally synthesized lipopeptides (macrolactin, surfactin, fengycin,

bacillomycin D), two polyketides (bacillibactin), dipeptide bacilysin, and three bacteriocins

(amylocyclicin, LCI, and 1 thiopeptide). Of three bacteriocins, thiopeptide bacteriocin was

identified as novel bacteriocin as its sequence was unmatched to any bacteriocin previously

characterised. The questions arose as to which antimicrobial substances were produced in

vitro, and if the production of the novel thiopeptide occurs. The answer is only clear when all

the antimicrobial compounds produced by this isolate are characterised. Therefore, different

culture fractions and different purification methods were employed to purify all the

antimicrobial substances produced by this bacterium.

First, the growth curve of bacteria in LPMA was observed in association with the ability of

antimicrobial exhibition against two indicators, L. plantarum and B. cereus. From the studies

carried out during the course of this whole research program it was found that L plantarum is

consistently one of the most sensitive indicators for detection of bacteriocin activity, while B.


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cereus is more sensitive to NRPs, and also bacteriocins. One of the key factors possibly

contributing to the high sensitivity of L. plantarum to the Bacillus-derived bacteriocins is

probably due to usage of the MRS medium for well-diffusion assay or spot-on-lawn assay.

This media contains the detergent, Tween 80, which is known as an agent that stabilizes highly

modified compounds like bacteriocins, and consequently can enhance bacteriocin activity

(187). This media is known as the best media for production of Lactobacillus-derived

bacteriocins (188, 189). Besides, B. cereus may require higher killing dosages of bacteriocins

(high Minimum inhibitory concentration’ (MIC)) than L. plantarum. Therefore, selection of

appropriate indicators for bacteriocin detection is also important.

Harvesting cultures grown in LMPB, at different points in culture growth, demonstrated that

the antimicrobial compounds produced by B. amyloliquefaciens #11 varied, such that one type

of activity peaked at about 12 hours of growth whereas other activity peaked after 36 hours

of growth, identified by assessment of antimicrobial activity against B. cereus, and L.

plantarum. This indicated the production of multiple antimicrobial substances. Thus, the

culture broth during log-phase growth (< 12 hours of cultivation) was able to kill L.plantarum,

but not B. cereus. While the broths collected during the latter growth phase inhibited the

growth of B. cereus but not L. plantarum. The 12 hour old broth lost antimicrobial activity

when it was incubated with protease enzymes but was resistant to lipase, indicating that the

antimicrobial peptide contains no lipid moiety for its biological activity, therefore, it could be

a bacteriocin. The 36 hours-culture still retained antimicrobial activity when it was incubated

with proteolytic enzymes and also lipase, indicating the unknown nature of the antimicrobial

substances. The growth curve analysis indicated the possible production of different

antimicrobial peptides with different properties at the two time points of 12 hours and 36

hours. Therefore, these broths were collected to purify the antimicrobial peptides.

Purification of the antimicrobial peptides from the 12 hour culture was successful via a three-

step procedure, including precipitating the antimicrobial compounds with ammonium

sulphate, then partial purification via cation exchange chromatography, followed by

purification on an RP-HPLC C18 column. The purity and MW of compound were monitored

by MALDI-TOF MS. Purification resulted in the isolation of an antimicrobial substance

having MW of 6,381 Da; It displayed strong activity against Gram-positive bacteria,

particularly against MSRA, but no activity against Gram-negative bacteria; it was sensitive to

proteinase K and pronase E but resistant to trypsin and lipase, it was very heat-stable, still

retaining activity after being heated to 80°C for 60 min. Based on the properties of this


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compound and properties of the bacteriocins predicted from genome, this antimicrobial

substance is likely to be amylocyclicin. It has the same physiochemical properties as

amylocyclicin, isolated from Bacillus amyloliquefaciens FZB42 (133). In the literature, heat

stable, pH stable bacteriocins are also reported for other Bacillus bacteriocins, like pumilicin

and laterosporulin (190, 191)

The putative novel thiopeptide was not seen in the 12 hour culture, therefore it was searched

in 36 hour cultures. Antimicrobial peptides were recovered from the bacterial cell pellet of 36

hour cultures using a solvent extraction method. A 2-step process was used; i) the bacterial

cells were extracted by stirring in 70% acetonitrile (adjusted to pH 2 by TFA) to release the

antimicrobial peptides into the solvent, and the cell-free solvent was then collected after

concentration of cell-free solvent under reduced pressure (Speed-Vac, or Rotary evaporator);

ii) chromatography on an RP-HPLC C18 column to separate and purify the antimicrobial

peptides from the concentrated solvent extract. After extraction from cell pellets, the cell-free

solvent presented 3 peptide peaks detected using MALDI-TOF under a detective range of

800-2,000 Da. They were (([m/z] = 1,058 Da, (([m/z] = 800), ([m/z] = 1,473) with isoforms.

In the literature, the Bacillus-derived antimicrobial peptides derived from liquid culture-

grown supernatants are reported as mostly falling into three mass ranges of; 850–950 m/z

(kurstakins); 1000–1100 m/z (surfactins, iturins); and 1450–1550 m/z (fengycins,

polymyxins, and bacitracins) (186). Based on this data, the 1058 Da substance could be an

iturin/surfactin. The 800Da-compounds is of similar MW as kurstakins, but this LPs was not

predicted from the genome and therefore, it is an unknown peptide. The 1,473 Da-substance

may be a fengycin or possibly a thiopeptide, as both peptides have similar MWs and

physicochemical properties (high hydrophobicity). To elucidate, this solvent extract was

concentrated in a rotary evaporator for separation using RP-HPLC. During concentration

under low pressure of rotary evaporator, the 1,473 Da compound was precipitated, collected

by centrifugation, and washed several times with milli-Q water, and dissolved in 50%

acetonitrile, as it is unable to dissolve in water or aqueous buffer. The material exhibited

antimicrobial activity against B. cereus. However, when it was stored at chilled temperatures

(5oC or -20oC), the antimicrobial peptide aggregated again and did not redissolve in water or

acetonitrile. Purification of freshly prepared aggregate, using an RP-HPLC C18 column,

resulted in an antimicrobial peptide at a retention time of 44.618 minutes, indicating elution

at approximately 85% acetonitrile. This suggested that this antimicrobial peptide was highly

hydrophobic. However, attempts to elucidate the amino acid sequence of this peptide by N-


129

terminal sequencing failed, suggesting its structure may be blocked at N and/or C terminus,

either by some form of chemical modification/protection or cyclization.

Some bacteriocin and NRPs compounds which were predicted from the genome analysis were

not found in any of the culture fraction. These included LCI and difficin. There are some

possible reasons why these compounds were not identified, including i) limitation in the

extraction procedures, such as usage of bacterial indicators that were not sensitive; ii) loss of

peptides during dialysis (using a membrane with cut off 3 kDa), or iii) the media used for

fermentation is inappropriate. This last hypothesis is raised because bacteriocin production

is known to be highly affected by media components, and other tress response factors.

Alternatively, the gene clusters predicted to encode synthesis of these undetected compounds

may be inactive due to repression or mutations. Activation of expression may require stress-

responding factors (nutrients stress, or factors for quorum sensing).

To conclude, the study has identified two antimicrobial compounds that appear to be novel,

one with an MW of 800 Da and the other, which may be a thiopeptide, with an MW of 1,473

Da. The 1,473 Da compound is suspected to be a novel thiopeptide because such a molecule

was predicted as a novel bacteriocin from the genome. However, a full structural analysis of

the peptide is required to finally resolve its identity. Therefore, the therapeutic application of

this bacterium is still unclear, but the B. amyloliquefaciens #11 isolate clearly has extensive

antimicrobial activity and could potentially be used as a biocontrol agent in various

applications, including in agriculture for control of plant and animal pathogens.


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Chapter 6 ANALYSIS OF ANTIMICROBIAL PEPTIDES PRODUCED BY

PAENIBACILLUS POLYMYXA #23

6.1. Introduction

Paenibacillus polymyxa is a non-pathogenic, spore-forming, Gram-positive bacterium. It is

ubiquitous in both terrestrial and marine environments (192). Terrestrial P. polymyxa is

known to exhibit various bioactivities like; nitrogen fixation, leading its use as a biofertilizer

in agriculture (193); production of phytohormones (194), and hence used as a biocontrol agent

in agriculture and aquaculture application (193, 195); lipid, cellulose, and starch degradation

which gives it value for the production of important enzymes (protease, lipase, cellulase,

amylases) (196). These enzymes are widely used in food and waste treatment industries.

Isolates of P. polymyxa also, typically produce a range of antimicrobial compounds with

activity against a wide range of fungi and bacteria. Such isolates have the potential to be

developed as probiotics or the purified antimicrobials used as pharmaceuticals. There are still

only a few limited studies conducted on marine-derived isolates of this species, to understand

the antimicrobial potential of this species.

In this study, P. polymyxa #23, isolated from a marine sponge collected in the NhaTrang Sea,

Vietnam, was examined. The bacterium exhibited antimicrobial activity against a wide range

of bacterial indicator strains, including Gram-positive bacteria (S. aureus, C. perfringens, B.

cereus, L. plantarum, L. monocytogenes, MRSA and VRE), Gram-negative bacteria (C.

jejuni, P. aeruginosa, E. coli, S. Enteritidis and even multidrug-resistant K. pneumonia) and

all of the marine antimicrobial producing Bacillus [Figure 6.1, Table 6.1]. It indicates that the

bacterium may produce multiple antimicrobial compounds. Therefore, it was selected for

sequencing and analysis to predict the putative antimicrobial peptides encoded within the

genome. In Chapter 4, in silico prediction of antimicrobial peptides potentially produced by

this isolate uncovered the presence of LPs (1 paenibacterin, 1 tridecaptin, 1 polymyxin, 1

unknown LPs), PKs (1 unknown polyketide), and 4 bacteriocins (paenicilin A, 1 novel

lantibiotic, 1 novel sactipeptide, 1 uncharacterised lassopetide). The work presented in this

chapter aimed to purify some of these novel bacteriocins for in vitro characterisation. The

bacterium was fermented and the antimicrobial peptides present in the culture were purified,

employing various separation techniques and in aid of mass spectrometry for identification of

peptides. The procedures included; i) selection of media for enhancement of antimicrobial

production, and identification of the time-point during bacterial growth for best yield of


131

antimicrobial products; ii) extraction of antimicrobial peptides from the cell surface using

acidic polar solvent; iii) RP-HPLC. The result in this chapter describes the antimicrobial

potential to underpin applications in pharmaceuticals.

Figure 6.1. Antimicrobial spectrum exhibited by marine P. polymyxa #23. (A) Several

representative Gram-positive bacteria inhibited. (1) S. aureus, (2) MRSA, (3) S.

faecalis, (4) B. cereus. (B) Several representative Gram-negative bacteria and yeast

inhibited such as P. aeruginosa (Ps), S. Enteriditis (Sal), E. coli (E), C. albicans (Ca).

The test was performed by cross streak assay with a duplicate of indictors.

6.2. Result

6.2.1. Time course analysis of antimicrobial production during bacterial growth

revealed production of multiple antimicrobial substances.

To compare the effect of media composition on bacteriocin production, six different media

formulations were tested, including production media (PB) (51); marine broth (Difco 2216)

(MB); lab-prepared marine broth (LPMB); nutrient broth (NB) (Oxoid); tryptone soy broth

(TSB) (Oxoid), and TSB supplemented with 0.6% yeast extract (TSBY). See the appendix for

the recipes of all defined media. An overnight starter bacterial culture, grown in LB broth was

added (1 mL) to 100 mL of each of the above media. All inoculated media were incubated at

30oC under a shaking speed of 150 rpm. Five mL aliquots of each culture were collected after

12, 18, 24, 36, 42, 48, 60, and 66 hours of incubation, filtered through a 0.45µm PES

membrane and the antimicrobial activity in the cell-free supernatant was evaluated against S.

Enteritidis and B. cereus as indicators.


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The results showed that growth in PB medium yielded the highest levels of antimicrobial

production, compared with other media [Figure 6.2 A]. In the PB grown culture, the

antimicrobial activity against S. Enteritidis was first observed after 6 hours of fermentation,

and the strongest inhibitory effect was seen from the 24 hour culture, and activity remained

at later times. The killing zones (from the well-diffusion assay) against Salmonella were

characterised by sharp boundaries at the edge of the zone in the majority of time-points, but

only 48 hours of culture, resulted in a more diffuse, blurred edge of the killing zone. The

activity against B. cereus was first observed after 18 hours of cultivation, reached peak

activity after 24 hours of cultivation, and started reducing after 36 hours [Figure 6.2 B]. Taken

together, it was clear that the antimicrobial activity against both indicator strains peaked in 24

hour culture; therefore, the 24 hour culture was collected for and used for antimicrobial

purification.

Figure 6.2. Antimicrobial activity exhibited by P. polymyxa #23 in culture media. (A)

Media selection for antimicrobial production. Six media were selected for antimicrobial

production including PB (1), LPMB (2), MB (3), NB (4), TSB (5). The cultures were

collected after 12 hours (a), 24 hours (b) and 36 hours (c) for testing against Salmonella

(left) and B. cereus (right). (B) The antimicrobial activity exhibited across the growth

phases in PB medium. Culture aliquots were collected after 6 hours to 66 hours and


133

tested against two indicators. Resulted showed that the activity against Salmonella was

started being observed from 6 hours culture. Activity against B. cereus was started being

observed from 18 hours old culture. The blurred edge of killing zone from 48 hours old

culture indicated the production of additional antimicrobial substances at this time

point.

6.2.2. Purification of antimicrobial compounds from 24 hour old PB culture revealed

the presence of three antimicrobial compound/s

6.2.2.1. Recovery of antimicrobial compounds from cell-free culture and the cell pellet

In the 24 hour old culture, the antimicrobial activity was observed from both cell-free culture

supernatant and cell pellet extracts. Therefore, two separate procedures were designed to

recover the antimicrobial compounds from these two sources. The antimicrobial presented in

the cell-free culture supernatant was absorbed onto Diaion HP-20, and the antimicrobial

substance was eluted with 70% acetonitrile [Figure 6.3]

Figure 6.3. Recovery of the antimicrobial compounds from the culture supernatant of

P. polymyxa #23 by absorption antimicrobial substances onto Diaion HP-20. Elutions

were carried out with; 30%, 50%,70%, and 100% ACN. The antimicrobial compound

could be eluted at least 70% acetonitrile.

The elute was then centrifuged at 5,000 g to collect the supernatant, filtered through a syringe

membrane (0.45µm PES) to obtain the cell-free solvent containing the antimicrobial

compound. The cell-free solvent was concentrated by a rotary evaporator under reduced

pressure. During concentration, a precipitate appeared which was separated from clear

supernatant (fraction CFS) by centrifugation. The precipitate was washed (twice) with milli-

Q water and dissolved in 40% acetonitrile (named SFP). Both SFP and CFS exhibited


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antimicrobial activity [Table 6.1, Figure 6.4 B]. The antimicrobial activities from both

fractions were resistant to proteolytic enzymatic digestion [Figure 6.4 A]

Table 6.1. Antimicrobial spectra of fractions recovered from a 36 hours old culture

Indicators Supernatant

(24 hour)

CFS SFP

P. aeruginosa + +++ -

E. coli ++ ++++ +++

S. Enteritidis ++ ++++ +++

C. jejuni ++ +++ +

C. coli ++ +++ ++

L. monocytogenes ++ ++++ ++

C. perfringens ++ + -

E. faecalis - - -

S. aureus ++ +++ +

MRSA ++ +++ +

L. plantarum - + -

Figure 6.4. Nature of antimicrobial compounds from different fractions obtained during

purification. CFS fraction and SPF fraction. The enzymes were used including


135

proteinase K (1), pronase E (2), trypsin (3), catalase (4), lipase (5), and fraction without

treatment as a positive control (6).

6.2.2.2. Purification of the antimicrobial peptides in CFS fraction with hydrophobic

exchange chromatography elucidated the presence of two antimicrobial

compounds.

The CFS sample was loaded into a C18 cartridge (Waters), and the column was then eluted

with a linear concentration gradient, ranging from 30% to 100% ACN (+0.01%TFA) to

recover the antimicrobial compounds. All elutes were collected and evaluated for

antimicrobial activity against both Bacillus and Salmonella. Antimicrobial activity was

observed in two fractions; at 30% acetonitrile (named as 30ACN elute) and at 40% acetonitrile

(named as 40ACN elute). The 30ACN elute displayed activity against S. Enteritidis while the

40ACN exhibited activity against both S. Enteritidis and B. cereus [Figure 6.5, Table 6.2].

Therefore, these two elutes were lyophilized, redissolved in milli-Q water to inject separately

into an RP-HPLC using C18 column.

Figure 6.5. Hydrophobic interaction chromatography (using Sep-Pak C18 cartridge) to

purify the antimicrobial “cell surface extract” (CFS).Two elutes with antimicrobial

activity were detected with elution at 30% acetonitrile (30ACN) and 40% acetonitrile

(40%ACN).


136

Table 6.2. The antimicrobial spectra of 30ACN fraction, and 40ACN fraction.

Indicators 30%ACN 40%ACN

B. cereus - +

L. monocytogenes - +

S. aureus - +

MRSA - +

L. plantarum - +

E. faecalis ++ +

E. coli ++ +

S. Enteritidis - +

C. jejuni ++ -

K. pneumonia ++ -

6.2.2.3. Purification of 30ACN fraction revealed the presence of polymyxin

Separating this 30ACN elute using RP-HPLC (C18 column) resulted in an antimicrobial

fraction at 25.6 minutes. MALDI-TOF MS showed that this fraction had a single peak at [m/z]

=1,168.772. It was thermally stable, as its activity was unaffected after being heated at 100oC

for 1 hour; and it was stable under various enzymatic digestions (proteinase K, protein E,

trypsin, lipase, catalase and lysozyme). It exhibited antimicrobial activity against most Gram-

negative bacteria (but not against C. jejuni) and displayed a circle of inhibition on a zymogram

gel at a range of 2-10 kDa against S. Enteritidis [Figure 6.6].


137

Figure 6.6. Zymogram of polymyxin against S. Enteritidis

The determination of the amino acid sequence of this compound failed with N-terminal

sequencing (Edman degradation technique). This indicated that the peptide’s N-terminal was

blocked. As an alternatively, tandem MS was employed. During the cycle, the compound

presented in three charged states. The single charged version also demonstrated some gas-

phase dimerization, most likely due to the high intensity [Figure 6.7]. Although strong tandem

MS spectra were acquired for all three charge states, no primary sequence was forthcoming

from automated de novo sequencing from the PEAKS software. Additionally, wildcard

searches in Byonic, of Bacillus proteins (197), where unknown masses may be added to

peptides, did not yield reasonable sequences. Therefore, manual de novo sequencing was

performed. Numerous water losses were observed, and most mass differences did not

correspond to proteinogenic amino acid residues, though there was potentially some evidence

for threonine and isoleucine/leucine. Several instances of mass differences that could

correspond to non-proteinogenic amino acids occurred in the spectra, including;

diaminopropanoic acid, diaminobutanoic acid (like shorter chain lysines) and

hydroxynorvaline. Using the most intense ions and water losses as a reference, a potential

sequence based on the doubly charged fragment spectrum was vLLvTvvTvu, where v is 2,4-

diaminobutanoic acid (or equivalent, C4H10N2O2) and u is something akin to Amino(1H-

pyrrol-2-YL) acetic acid (C6H8N2O2).


138

Figure 6.7 Sequencing result of the purified polymyxin. (A) Peptide sequencing of the

purified polymyxin by modified tandem mass spectrometry. (B) Structure of known

polymyxin. The image was obtained from Poirel et al, 2017 (198).


139

Table 6.3 The antimicrobial spectrum of polymyxin

Indicators organisms Activity

B. cereus -

L. monocytogenes -

S. aureus -

MRSA -

L. plantarum -

E. faecalis ++

E. coli ++

S. Enteritidis +

C. jejuni -

K. pneumonia ++

+ and ++ indicate the relative strength of antimicrobial activity.

6.2.2.4. Purification of 40ACN fraction revealed the presence of paenicidin A,

tridecaptin

Purification of antimicrobial peptides from the 40ACN elute was performed using an RP-

HPLC using a C18 column, yielding two fractions exhibiting the antimicrobial activity at

retention times of 23.443 minutes ([m/z] = 1616) and 23.797 minutes ([m/z] = 3370) [Figure

6.9]. The 3,370 Da antimicrobial peptide displayed antagonistic activity against only B.

cereus, while the 1,616 Da compound was able to kill both Salmonella and B. cereus.

Figure 6.8. The first round of RP-HPLC to separate the 40% ACN fraction.Two

fractions exhibited antimicrobial activity, with peaks at retention times of 23.443

minutes ([m/z] = 1616) and 23.797 minutes ([m/z] = 3370)


140

Figure 6.9. MALDI-TOF MS spectra of 40% ACN fraction under detection range of

800 Da – 2000 Da. The high mass intensity in the m/z range of 1,400–1,700 indicated

the presence of tridecaptin.

6.2.4.1. The paenicidin A, a lantibiotic, displayed antimicrobial activity against Gram-

positive bacteria

The 3,370 Da antimicrobial compound was sensitive to proteolytic treatment (pronase E and

proteinase K) and demonstrated antimicrobial activity against only Gram-positive bacteria

(but not against L. plantarum and E. faecalis) [Table 6.8]. Based on the antimicrobial

spectrum, MW, and list of antimicrobial peptides predicted by in silico analysis of the

genomic sequence, this antimicrobial peptide is likely to be paenicidin A [Figure 6.10; Table

6.4]

Figure 6.10. MW of purified paenicidin A by MALDI-TOF MS with the mass intensity

of [m/z] = 3370.

6.2.4.2. The tridecaptin, a lipopeptide with anti-Campylobacter activity

The 1,616Da-compound was resistant to proteolytic treatment and exhibited antimicrobial

activity against only Gram-negative bacteria (C. jejuni, S. Enteritidis and E. coli but not P.

0940.383 1614.894

977.096

983.091

1660.637

1668.588

1570.349

1683.232

1690.817

1004.225

1150.014

1706.565

1483.229 1789.3721848.7771038.312

1010.264

1729.125

1544.438

1529.0891114.570 1251.774

Native23_60%eluteACN_22min 0:D5 MS Raw

0.5

1.0

1.5

5x10In

ten

s. [a

.u.]

900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900m/z


141

aeruginosa and K. pneumonia) [Table 6.4]. Based on the antimicrobial spectrum, MW, and

list of antimicrobial peptides predicted from the genome, this compound was identified as

tridecaptin.

Table 6.4. Antimicrobial activity of compounds present in the 40ACN fraction

Indicators PaenicidinA Tridecaptin

B. cereus ++ -

L. monocytogenes ++ -

S. aureus + -

MRSA + -

L. plantarum + -

E. faecalis + -

E. coli - ++

S. Enteritidis - ++

C. jejuni - +

K. pneumonia - -

P. aeruginosa - -


Attempt to extract the predicted novel thiopeptide bacteriocin from B. amyloliquefaciens #11

culture failed. Therefore, as an alternative approach, P. polymyxa #23 was used to attempt

isolation of novel bacteriocins. The P. polymyxa #23 genome had 3 novel bacteriocins gene

clusters (1 lassopeptide, 1 lantibiotic, 1 sactippetide) along with paenicidin A and various

NRPs substances (tridecaptin A, polymyxin, and fusaricidin, paenibacterin). The

antimicrobial potential of #23 is similar to that of P. polymyxa NRRL B-30509, which is

known as a producer of polymyxin, tridecaptin, and paenicidin A (146). To determine if the

predicted novel bacteriocins were produced in vitro, #23 was grown in liquid culture, and

various purification methods were applied to obtain these novel bacteriocins for

characterisation.

First, six media types (PB, MB, LMPB, NA, TSB, and TSBY) were selected to culture the

bacterium, to evaluate the yield of antimicrobial compounds. These media were selected

because they were used in earlier studies to ferment for bacteriocin production (51, 84, 199,


142

200). The selection of culture conditions for enhancement of bacteriocin production is a

complex process, which medium formulations and growth conditions are two factors highly

influencing bacterial growth and bacteriocin production. The result showed that cultivation of

bacterium in PB media resulted in the strongest antimicrobial activity against two indicators

(S. Enteritidis, and B. cereus) across growth-phases. The PB is a completely defined media

which is formulated with rich nutrients (carbon and nitrogens source) and minerals and can

produce high cell densities and in consequence high bacteriocin production. This observation

is in agreement with earlier studies which reported that completely defined medium frequently

increased the yield of bacteriocin production (201).

The bacterium was cultured in PB medium, and samples were collected every 6 hours and

antimicrobial activities were evaluated against two indicators; S. Enteritidis and B. cereus.

The two indicators were killed by cultures collected at different time-points, indicating the

production of multiple antimicrobial substances. Thus, activity against Salmonella was

observed starting at 6 hours of cultivation, reached a maximum at 24 hours, and was

maintained throughout the rest of the culture monitoring period (66 hours). Interestingly, the

culture collected at 60 hours exhibited a strange diffuse edge to the killing zone, suggesting

the production of additional antimicrobial substances. The activity against B. cereus was first

observed from the 12 hour sample and steadily reduced from 36 hours. Because the 24 hour

broth displayed strong activity against both Salmonella and Bacillus, the culture at this time-

point was collected for antimicrobial purification, to characterise all the putative antimicrobial

peptides produced

The purification of antimicrobial compounds from 24hour old PB culture resulted in the

presence of 3 antimicrobial peptides, which were; polymyxin ([m/z]= 1168.7), paenicidin A

([m/z]= 3290.4), and tridecaptin ([m/z]= = 1550.8). It is assumed that the activities against

Gram-negative bacteria are due to polymyxin and tridecaptin. Of them, the activity against C.

jejuni is mainly by tridecaptin, and ability to inhibit P. aeruginosa and K. pneumonia is a

result of polymyxin activity. The activity against Gram-positive bacteria is due to paenicidin

A, which is commonly found in Paenibacillus species (43, 146). In the literature, polymyxin

is found and extracted from various terrestrial Paenibacillus species (202, 203). Eight variants

of polymyxin (A to E, M, S, and T) are described in works of literature, but only polymyxin

B and E are widely used in the treatment of serious Gram-negative bacterial infections with

another name as colistin (colistin E, colistin B) (204, 205). The tridecaptin is a lipopeptide

previously extracted from P. jamilae M-152, P. terrae NRRL B-30644 and P. polymyxa


143

NRRL B-30509, with the presence of some variants (tridecaptin A1, newly tridecaptin M)

(127, 146). Polymyxin, and tridecaptin display different killing modes against the targeted

bacteria, therefore they can be used in combination to cure various infectious diseases caused

by Gram-negative bacteria, even carbapenem-resistant Enterobacteriaceae (8, 206). Due to

the difference in the mode of action, tridecaptin may be a good compound for further

development in treatment against colistin-resistant pathogens. The identification of

tridecaptin and polymyxin as antimicrobials produced from isolate #23 emphasizes the

potential therapeutical value of this bacterium.

Some other NRPs compounds and novel bacteriocins were not observed during purification.

There are several possible explanations for the inability to purify these antimicrobials; i) the

novel bacteriocins were not produced under the culture conditions used, or ii) loss of the novel

bacteriocin during purification.

Production of bacteriocin by bacteria is known to vary greatly, depending on types of

bacteriocin, type of host, and types of media used for fermentation, and the growth phase

(124, 207). Refining media for production is a complicated process, but it is necessary for the

enhancement of compounds of interest. Thus, some bacteriocin production was enhanced

when media was supplemented with surfactant (like Tween), NaCl, or ethanol (187, 207-210).

Of them, tween may stabilize the bacteriocin structure by preventing the aggregation of

bacteriocin molecules. Tween 80 is a common supplement in MRS medium, which is reported

as the best medium for high yield of bacteriocins deriving from Lactobacillus (211).

Supplementation of NaCl and ethanol can also increase bacteriocin yield, but in some cases,

high concentrations of NaCl and ethanol can also result in inhibition of bacteriocin

production, such as curvacin A (210). Besides, media with fewer nutrients is reported to

enhance bacteriocin yield; such conditions may activate bacteriocin transcription in response

to nutrient stress (212). However, some bacteriocin productions are only maximized in media

with which supply appropriate levels of sugars, vitamins, and nitrogen (207). High nutrient

media can yield a high density of cells, therefore improving bacteriocin yield; however, an

oversupply of these nutrient sources can lead to inhibition of both bacterial growth and

bacteriocin production.

While, loss of novel bacteriocins during the purification process is also possible, probably due

to limitation of this purification procedure or inactivation during the processing steps used.

Thus, the bacteriocin may be produced at low concentration in an unsuitable media, which


144

may lower the ability to detect the antimicrobial activity against indicator strains. This may

result in an inability to detect fractions containing these bacteriocins, during purification.

Solutions that could be considered to overcome this failure include heterologous cloning and

expression of the bacteriocin gene cluster to produce the antimicrobial in an engineered

system, or inactivation the genes predicted to be required for NRPs production (sfp genes) to

stop NRPs production and identify the change in the antimicrobial activity of the mutated

strain (27).

Three solutions can be suggested to overcome purification failure based on the above reasons.

Firstly, selecting an appropriate bacterial indicator may be a critical factor in determining

success in the detection of novel bacteriocins production. For instance, use of mutant strain

HB0042 of B. subtilis as an indicator. This bacterium was used as an indicator to detect the

presence of novel plantazolicin and amylocyclicin produced by Bacillus amyloliquefaciens

FZB42 (51, 133). This strain contains the gene encoding an extracytoplasmic sigma factor

(SigW) that provides the resistance against the NRPs produced by bacilli (213). Alternatively,

the use of a marine Bacillus species among the shortlisting of 23 marine isolates in this study

as an indicator. The marine strain selected has a known genome sequence which contains the

genes responsible for the biosynthesis of NRPs compounds, but deficient genes of novel

bacteriocins. For some novel bacteriocins that their productions appear to be inactive with

unknown reasons, dead indicator cells can be added to the fermentation culture. The cells can

serve as inducing molecules to activate the “silent” bacteriocin production via a quorum

sensing mechanism. Secondly, deficient in the genes relating to NRPs of wide-type bacteria

by genetic modifications, then fermentation of this mutant for production of novel bacteriocin.

Thirdly, heterologous cloning and expression of the gene clusters of novel peptides

Taken together, the main aim of this chapter, to purify the putative novel antimicrobials

predicted to be produced by P. polymyxin, was not achieved. However, the study has provided

information to understand the isolate, particularly the antimicrobial activities that could be

developed to address issues in therapeutics and agriculture.


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Chapter 7 CLONING AND HETEROLOGOUS EXPRESSION OF SACTIPEPTIDE,

A NOVEL BACTERIOCIN FROM MARINE P. POLYMYXA #23, IN BACILLUS

SUBTILIS

7.1. Introduction.

Genomic analysis of P polymyxa #23 identified 13 biosynthetic gene clusters that were

predicted to direct the synthesis of antimicrobial compounds. Three were predicted to encode

uncharacterised bacteriocins; 1sactipeptide, 1 lantibiotic, 1 lassopeptide. However, these three

novel bacteriocins were not observed during the purification procedures outlined in Chapter

6, even though the gene clusters appear to have full complements of all the accessory genes

required for biosynthesis. Therefore, heterologous cloning and expression of sactipeptide

were attempted, to provide an alternative source from which to purify the bacteriocin for in

vitro characterisation. This technique was expected to enable sactipeptide production by using

strong promoters that are unlikely to be responsive to suppression by inhibitors which may

have interfered with biosynthesis from the native gene cluster.

Cloning and expression techniques employ a variety of host-vector systems which are selected

depending on the type of gene(s) to be expressed and the size of gene clusters used. For

bacteriocin expression, it is often necessary to clone the whole gene cluster carrying the

structural gene for bacteriocin and all the accessory genes. Therefore, the bacterial host for

expression must be able to receive large vectors, be deficient in other bacteriocin genes, and

contains accessories genes supporting expression. Examples of early studies to heterologous

express bacteriocins include the production of mersacidin (214), pediocin PA-1 (215),

plantaricin ZJ5 (216), and nisin (217). In the literature, the majority of reported studies used

E. coli as the host for expression and variable vector types.

In this study, the cloning and expression of sactipeptide were processed using plasmid

pDLL202 (kindly provided by Monash University) and B. subtilis cells for expression. The

plasmid pDLL202 contains two compatible origins of replication (colE1 rep and repA),

enabling replication in both E. coli and B. subtilis hosts [Figure 7.1 A]. The plasmid contains

four multi-cloning-sites (MCS) which include recognition sites of various restriction

enzymes, for genetically engineering, and 4 antibiotic resistance genes; kanR, ermB, catP,

and ampR, which confer on the host resistance to kanamycin, erythromycin, chloramphenicol,

and ampicillin. The sactipeptide biosynthetic gene cluster that was cloned is 8,556 bp in length

and consisted of a precursor bacteriocin, 3 modification enzymes, a transporter for bacteriocin


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secretion, and a putative immunity protein [Figure 7.1B]. The precursor bacteriocin is 61 aa,

comprising of a 19 aa leader sequence and a 42 aa core sequence. The core sequence was

cysteine-rich, and was predicted with a theoretical MW of 4,455.81 Da, and a pI of 7.73. The

MW of sactipeptide was estimated from core sequence, which was predicted by SignalP

software.

Figure 7.1. (A) Gene organisation on plasmid pDLL202; (B) The plasmid contains 4

antibiotic resistance genes and 4 multi cloning sites (MCS), and (C) the gene cluster of

sactipeptide;. The figure of plasmid was provided from Monash University. The gene

organisation of the sactipeptide gene cluster is derived from the annotation of the P.

polymyxa #23 genome sequence.


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Transformation into B. subtilis cells is more successful with plasmids in multimeric form

rather than ones in monomeric form (218, 219); therefore, the procedure included an

intermediate step of transformation into E. coli JIR702 cells that produce fusion plasmids to

generate the multimeric plasmid. In practice, the whole procedure consisted of the following

steps; (i) amplification of the sactipeptide gene cluster from the genome of P. polymyxa #23

using designed primers and high fidelity polymerase; ii) preparation of the plasmid backbone

by double restriction enzyme digestion of plasmid pDLL202, to accept the amplified gene

cluster; (iii) construction to generate the fusion plasmid, pDLL202/G23sac, by Gibson

assembly (220) ; (iv) transformation of the fusion plasmid into E. coli Top 10 cell to recover

the constructed clone; (v) extraction of plasmid DNA from E. coli Top 10 cells and

transformed into the E. coli JIR702 to generate the multimeric form of the plasmid; (vi)

extraction and transformation of this multimeric fusion plasmid into B. subtilis BS34A for

expression of sactipeptide; (vii) selection of culture media for bacteriocin expression by B.

subtilis transformants; and (vii) purification of the sactipeptide for characterisation.

7.2. Methods and results

7.2.1. Successful isolation of intact sactipeptide whole gene cluster, and generation of

the expression plasmid (pdLL202/G23sac)

The whole sactipeptide gene cluster was amplified from P. polymyxa #23’s genome using the

designed primers [Table 7.1]. The P. polymyxa #23 genomic DNA was extracted by the GES

method and used as a template in PCR. The 50 μL PCR reaction included 2.5 μL of each 10

mM forward and reverse primer (G23sac_F/ G23sac_R), 1.5 μL of DMSO, 25 μL of

Phusion® High-Fidelity PCR Master Mix (Thermo Fisher Scientific), 1 μL of HF buffer, 2

μL of intact gDNA (100 ng) and 15.5 μL of water. The PCR conditions were; 98oC for 30

seconds, 35 cycles of; 98oC for 10 seconds, 56oC for 30 seconds, 72oC for 4.5 minutes, and a

final extension at 72oC for 10 minutes. The PCR product was purified using the Monarch PCR

clean kit (NEB). The primers (G23sac_F/ G23sac_R) were designed to generate a PCR

product containing the sactipeptide gene cluster flanked by ends regions of 20 nucleotides,

which overlapped with the terminal regions on the plasmid vector backbone.


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Table 7.1. List of primers used for cloning and confirmation

Primers 5’-3’ sequence Nucleotide Tm

(to)

Purpose Source

G23sac_F CAGCGAGATCTGATCACGCGCAGTTTACCAGTAGCTGCTC

40 58 amplification of 8,556 bp- sactipeptide gene cluster from wide-type P. polymyxa #23

This study

G23sac_R GCTAGCGACGTCTAGACTAGCGGGAATTAAGTAGGGGTATAGG

43 59

23Sac-test_F

ATAATTACCAATTCCCGTTGC

21 51 amplification of 249 bp- DNA region inbetween sactipeptide gene cluster for confirmation

This study

23Sac-test_R

GTAAGGAATTAGTATGGGTGAAC

23 51

Both the purified plasmid vector and the purified whole sactipeptide gene cluster were

checked to be of the expected approximate size by electrophoresis on a 0.8% agarose gel. The

1 kbp DNA ladder (Thermo Scientific) was used as a standard to estimate size [Figure 7.2].

The plasmid and amplicon bands were of the expected size. The PCR product of sactipeptide

gene cluster was further confirmed by PCR amplification using a second pair of primers

(23Sac-test_F/ 23sac_test_R). The 20 μL PCR reaction included; 0.8 µL of each 10 mM

primers (23sac-test_F and 23sac_test_R), 10 µL of MyTaq Red HS master mix (Bioline), 1

µL of purified sactipeptide gene cluster, and 8.4 µL of molecular water. The PCR conditions

were 95oC for 1 minute, 30 cycles of; 95oC for 15 seconds; 49oC for 20 seconds; 72oC for 10

seconds, and a final extension at 72oC for 10 minutes, and then held at 5oC. This primer pair

was designed to amplify a 249 bp- DNA region locating within the sactipeptide gene cluster

but did not amplify any DNA regions within the plasmid pDLL202 or genome of the B.

subtilis BS34A host.


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Figure 7.2. Construction of fusion plasmid. Band of 8,556 bp gene cluster of

sactipeptide amplified from #23 genome (A); the band of 6,938 bp of the plasmid vector,

from restriction enzyme digestion of plasmid pdLL202 (B); the band of 15,497 bp

fusion plasmid generated after Gibson assembly (C); 1 kbp DNA ladder (M)

The fusion plasmid was generated from the purified plasmid, pDLL202, and the purified PCR

product of the sactipeptide gene cluster using the Gibson assembly method. Briefly, a 20 µL

reaction was prepared including; 10 µL of NEBuilder HiFi DNA Assembly Master Mix

(NEB); ~0.15 pmol of insert and plasmid backbone (ratio of 1: 3); and water. The Gibson

reaction was incubated at 50oC for 60 minutes and immediately used for transformation or

stored at -20oC until usage. The gene content of the fusion plasmid shown in Figure 7.3.


150

Figure 7.3. Gene organisation of fusion plasmid pDLL202/G23sac generated after

Gibson assembly. The figure was prepared in Snap-gene software.

7.2.2. Successful transformation of fusion plasmid into E. coli Top10

After assembling the mixture was transformed into E. coli Top10 by electroporation. The

transformation mix was plated on selective LB (chloramphenicol) and all colonies forming

on the plate were picked and purified by streaking onto another fresh selective plate.

Successful cloning was confirmed by both PCR amplification and restriction enzyme

digestion of the plasmid. The colony PCR was carried out on all the transformant colonies

using the primer pair (sacti-test_F/ sacti-test_R). Results showed the PCR product presented

from some colonies when compared with positive control [Figure 7.4]. The plasmid DNA

from the PCR-positive transformants was isolated using a Monarch® DNA Gel Extraction

Kit (NEB) and confirmed again by restriction enzyme digestion, analysed on an agarose gel.

7.2.3. Successful transformation of the fusion-plasmid into E. coli JIR702 for

production of multimeric plasmid.


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The fusion plasmid DNA was extracted from an overnight culture of E. coli Top10

transformants using the Monarch® DNA Gel Extraction Kit (NEB). It was transformed into

E. coli JIR702 by electroporation to induce the multimeric form of this plasmid. The

multimeric plasmid was required for transformation into the B. subtilis BS34A host. The

transformation procedure was similar to that performed into the E. coli Top10. The positive

transformants were also confirmed by PCR primer pair G23sac_test_F/ G23sac_test_R via

size confirmation and enzymatic digestion. The multimeric form of the pDLL202/G23sac

plasmid was extracted by the Monarch® DNA Gel Extraction Kit (NEB) and subsequently

transformed into B. subtilis BS34A cells under the natural transformation method. The outline

of the method was mentioned in the methodology of chapter 2.

Figure7.4. Confirmation by PCR for positive transformants after transformation.(A) On

E. coli JIR702 transformants. #01 to #14: used transformants’ DNA as templates, well

#15: positive control using genomic DNA of P. polymyxa #23 as a template, #16:

negative control (water as a template). (B) For B. subtilis BS34A transformants; using

transformants’ DNA as templates, #14: positive control using genomic DNA of P.

polymyxa #23 as a template.


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7.2.4. Successful expression of sactipeptide gene cluster in B. subtilis and optimization

of sactipeptide production

The Bacillus transformants which were confirmed for the presence of fusion plasmid were

fermented in two types of culture media including Production broth (PB); and LB broth, to

observe the antimicrobial production. The production of sactipeptide was only seen in LB

broth [Figure 7.5]. During purification of the sactipeptide, all cultures and solid media were

supplemented with 5 µg/mL chloramphenicol to maintain the fusion plasmid. The methicillin-

resistant S. aureus (MRSA), which was resistant to chloramphenicol, was used as an indicator

in antimicrobial detection assays (the strain was provided by Microlab-RMIT University)

Analysis on bacterial growth in LB medium in association with a strength of antimicrobial

exhibition showed that the sactipeptide was first produced after 6 hours of fermentation, and

exhibited the strongest activity against MRSA at 24 hours of incubation (corresponding to an

of OD600 of ~1.4) and reduced and lost activity after 32-hours of incubation [Figure 7.5 B,

Table 7.2]. Therefore, the 24hour-old culture was collected for purification.

Figure 7.5. Antimicrobial activity exhibited by some B. subtilis transformants against

MRSA indicator.(A) Cultivation of 3 B. subtilis transformants in Production media (PB)

and Lueria media (LB). The antimicrobial activity was only seen in LB media. Culture

of the B. subtilis harbouring native pDLL202 was used as a negative control. (B)


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Antimicrobial activity exhibited by LB cultures collected at different growth phases.

The production of sactipeptide was initially observed after 6 hours of cultivation.

Table 7.2. The growth phase of B. subtilis transformant in LB medium

Time-points OD600 Killing diameter

against MRSA(cm)

2 hours 0.020 0

4 hours 0.056 0

6 hours 0.134 11

8 hours 0.306 13

10 hours 0.802 12

12 hours 0.872 12

24 hours 1.405 16

26 hours 1.306 14

28 hours 1.738 12

30 hours 1.955 13

32 hours 2.01 10

7.2.5. Two-steps procedure to purify the sactipeptide from 24 hour LB culture

The purification procedure included two steps; precipitation of sactipeptide in the culture at

70% saturated concentration of ammonium sulphate; and separation under two cycles of

HPLC using a reverse-phase column (C18). In practice, the ammonium sulphate was added

to the cell-free LB supernatant to final 70% saturation concentration. The mixture was

centrifuged at 17,500 g for 90 minutes, and the protein pellet was dissolved in a minimum

volume of milli-Q water. Next, the material was purified by RP-HPLC (C18 column) with

two cycles, until the pure compound could be collected. During purification, MRSA was used

as an indicator to detect the fractions exhibiting the antimicrobial activity using the spot-on-

lawn method. In the first round of RP-HPLC, the chromatography was operated with a

solution A (milli-Q water + 0.1% (TFA)) and solution B (100% HPLC-grade acetonitrile +

0.1% TFA). The cycle was operated at a flow rate of 1 mL per minute under 55 minutes cycles

of; 2% B in 5 minutes, a gradient of 2%- 95% B in minute #6 to #45, and isocratic elution of

95% eluent B for 10 min. The elution was visualized under UV wavelengths of 214 nm, 230


154

nm, and 280 nm. Elutes were collected every 2 minutes and used in the antimicrobial testing

assay. The first RP-HPLC cycle resulted in an antimicrobial fraction at a retention time of 30–

32 minutes [Figure7.6 A]. This fraction was concentrated by SpeedVac (at 37oC) and loaded

into the RP-HPLC as input for the 2nd operation. The second round of RP-HPLC resulted in

the elution of an antimicrobial compound at a retention time of 32.7 minutes [Figure7.6 B].

The MALDI-TOF-MS revealed a major peak with m/z 3404 Da [Figure 7.7].

Figure7.6. Reverse-phase (RP) high-performance liquid chromatography (HPLC)

chromatograms to separate the AMS fraction.(A) The first round of RP-HPLC resulted

in antimicrobial elution at tR of 30–32 minutes. (B) The second round of RP-HPLC

resulted in antimicrobial elution at tR of 32.77 minutes.


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Figure 7.7. MALDI-TOF MS of purified sactipeptide.The measurement showed a major

peak of the sactipeptide at m/z 3404.

7.2.6. Characterisation of sactipeptide revealed the proteinaceous nature and thermal

stability of the sactipeptide

The thermal and enzymatic stability of the antimicrobial substance was identified.

Consequently, it displayed thermal stability, because the antimicrobial activity remained after

heat-treatment of 100 °C for 1 hour, although the activity was observed to be reduced. It was

sensitive to pronase E, proteinase K, and trypsin but was resistant to lipase and catalase. The

antimicrobial spectrum of sactipeptide was assayed using the HPLC-based, purified material,

resulting in activity against only Gram-positive bacteria. It strongly depressed the growth of

MRSA [Figure 7.8, Table 7.3].

Figure 7.8. Properties of sactipeptide. Enzymatic degradability: catalase (C), lipase (L),

pronase E (E), proteinase K (K) and trypsin (T). The result showed that the activity was

reduced after digestion with trypsin, pronase E and proteinase K. The thermal stability


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determined by heating peptide at 40oC, 50oC, 60oC, 70oC, 80oC, 90oC; 100oC for 1 hour.

The result showed that activity remained after thermal treatment at 100oC

Table 7.3. Antimicrobial spectrum of sactipeptide

Indicators Media Antagonistic activity

S. aureus MHA +

MRSA MHA ++

B. subtilis MHA +

B. cereus MHA +

L. monocytogenes MHA -

C. perfringens MHA -

L. plantarum MRS -

E. faecalis (VRE) MRS +

S. Enteritidis MHA -

E. coli MHA -

C. jejuni MHA+ 5% blood -

C. albicans MHA -

7.2.7. N-terminal sequencing of the sactipeptide

The bacteriocin sequence was unable to be determined by N-terminal sequencing, indicating

a blocked N-terminus.

7.3. Summary of result and discussion

The sactipeptide is one of three uncharacterised bacteriocins predicted from the genome of P.

polymyxa #23 with all of the genes expected to be required for biosynthesis, but it was not

observed during purification. Therefore, heterologous cloning and expression were employed

to enhance sactipeptide production.

In this study, the whole sactipeptide gene cluster was isolated from P. polymyxa #23 and

cloned into the pDLL202 plasmid vector, and B. subtilis BS34A was used as the expression

host. It is an E. coli/Bacillus shuttle plasmid enabling the transformation into both E. coli and

B. subtilis. The heterologous cloning and expression of sactipeptide were successfully

performed but each step had to be refined and modified to achieve success. (a) The PCR to


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amplify the large sactipeptide gene cluster had to meet some demands such as; intact PCR

product with few or no nonspecific bands; high yield; high purity; no mutations during

amplification. These requirements were achieved through; i) selection of a gDNA extraction

method that gave long genomic DNA fragments from P. polymyxa #23; ii) selection of a high

fidelity polymerase enzyme, iii) optimisation of PCR mixture components (e.g. primer

concentration), iv) testing of a variety of PCR running conditions to reduce the nonspecific

bands, v) selection of appropriate kits for the tasks of DNA plasmid extraction, gel

purification, cleaning-up for high efficiency of recovery. The GES DNA extraction method

was shown to be suitable to extract intact genomic DNA from P. polymyxa. It may be due to

the activity of guanidine thiocyanate in extraction buffer, which served as an inhibitory agent

of endonuclease abundantly released from P. polymyxa cells after cell breakage. Also, all kits

(plasmid extraction kit, gel purification, PCR product purification) purchased from NEB

(Monarch kit) always gave good DNA yields compared to kits purchased from several other

manufacturers). The Phusion ® High-Fidelity polymerase (NEB) was the most suitable for

amplification of large DNA fragments and resulted in intact and high yields of PCR product

(when compared with Q5 polymerase), and HF buffer supplemented into the PCR mixture

enhanced PCR efficiency.

- (b) There was no success when standard ligation conditions were used to attempt to construct

the fusion expression plasmid. Construction was only successful when Gibson assembly was

used. For Gibson assembly, the PCR amplification primers were redesigned to generate

nucleotide fragments which contained the sactipeptide gene cluster flanked by two regions of

20-nucleotide sequences at the termini. These two regions were designed to be

complementary to sequences at the ends of the linearized plasmid vector.

- (c) Transformation was difficult because of the large size of the fusion plasmid (>10 kbp).

It was achieved by selection of appropriate E. coli types, and transformation methods

(electroporation or natural transformation).

- (d) Purification of sactipeptide from Bacillus transformants was achieved by selection of a

medium in which expression occurred and by the use of sensitive indicator cells for detection

of antimicrobial activity during purification. In our investigation, we found that the

sactipeptide was only produced in LB media, not PB media. And an MRSA strain which can

resistant to chloramphenicol was used as the indicator strain.


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The analysis of the growth curve found that the antimicrobial substance was produced at a

stationary growth phase, similar to earlier studies on bacteriocin production (84, 221).

Purification of the sactipeptide from LB liquid culture was achieved via a 2 steps-procedure;

(i) precipitation of the peptide by adding ammonium sulphate to 70% saturation concentration

at 5oC; (ii) purification of the sactipeptide by RP-HPLC employing a C18 column. The pure

sactipeptide was identified with an MW of 3.4 kDa; was heat-stable; sensitive to proteinase

K, pronase E and trypsin; and displayed antimicrobial activity against only Gram-positive

bacteria. However, the amino acid sequence of sactipeptide was not elucidated because N-

terminal sequencing was not successful, suggesting that the N-terminal of the protein is

blocked. The failure of N-terminal sequencing has been frequently reported for highly

modified bacteriocins such as Enterocin AS-48 (222), or subtilomyxin (84), subtilosin A

(223), circularin A and closticin 574 (224)

The MW of the purified sactipeptide was 3.4 kDa, while the MW predicted, based on the

amino acid sequence of the core sequence, was 4,455.81 Da. Based on multiple sequence

alignment between this sactipeptide and previously characterised sactipeptides (obtained from

BACTIBASE database), the #23 sactipeptide sequence is closest to subtilosin A. Both these

two bacteriocins have the same numbers of amino acids residues and have similar MWs of

~3-4 kDa. Therefore, this discrepancy in MW may be a result of a misprediction of the SignalP

software for identifying the signal sequence cleavage position on the precursor, subsequently

causing misprediction of MW of core sequence. To confirm, we submitted sequences of well-

characterised bacteriocin precursors (like amylocyclicin, mersacidin and paenicidin A) to this

software for calculation of cleavage site, but the software suggested incorrect recognition

sites, leading to an incorrect prediction of the MW of core sequence. Although SignalP is

widely used for this purpose, it is clear that the software is sometimes ineffective in the case

of small peptides like bacteriocins. The #23 sactipeptide only depressed growth of Gram-

positive bacteria, while subtilosin A has antimicrobial activity extending to both Gram-

positive bacteria and Gram-negative bacteria (142). This indicates the diversity in

antimicrobial spectra of sactipeptide-type bacteriocins.

The use of a synthetic biology approach to isolate operons from uncultivable strains for

improvement on already isolated operons may be interesting. Although there is no example

of this sort of approach in the literature it may have the potential to greatly expand access to

novel antimicrobial operons from uncultivable bacteria. Metagenomic analysis of

environmental samples could be used as a tool to achieve this purpose. However, for smany


159

antimicrobial compound, the bioactive antimicrobial compounds need the expression of many

genes for accessory protein products that participate in the various functions of immunity,

secretion, posttranslational modification, and precursor processing. These genes are often

located together in one operon but are sometimes dispersed and hence difficult to identify.

Due to the ineffectiveness of current genetic systems, metagenomics may be a difficult task

to manipulate large size antimicrobial gene cluster. Currently, many studies mainly

concentrate on developing different heterologous expression systems to enhance the

efficiency to capture the antimicrobial gene clusters derived from cultivable bacteria, such as

TAR System, IR System, Red/ET recombineering (225-227). Alternatively, the studies are

performed to improve heterologous cloning/expression by evaluating the impact of different

media and media components on bacteriocin production, i.e., Garvicin KS (228).

Taken together, the findings in this chapter confirms the usefulness of plasmid pDLL202 for

cloning of the bacteriocin from bacilli and facilitated successful expression to produce the

recombinant bacteriocin. The pure novel #23 sactipeptide can now be further characterised,

both in terms of structural analysis and antimicrobial activity.


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Chapter 8 CONCLUSION AND FUTURE WORK

8.1. Conclusion

In the circumstance of emerging antibiotics resistance of current pathogens, humankind is

seeking insights outside of the traditional bacterial reservoirs and increased investment in

other classes of antimicrobial peptides for antibiotic development, such as bacteriocins.

Hypothesizing that there is an abundance of novel bacteriocins unidentified in marine Bacillus

since marine Bacillus/Paenibacillus species have been under-utilised, we therefore selected

an unexplored sea region, the NhaTrang bay (Vietnam Sea) to seek novel bacteriocins, for

application in pharmaceuticals, aquaculture, agriculture and food industries.

From the results of all the experimental chapters, five research questions raised in Chapter 1

have been clarified. Bacillus bacteria with diverse antimicrobial activities could be isolated

from marine environments. The sampling and bacterial isolation work detailed in Chapter 3

resulted in 16.4% of the total spore-forming bacteria isolated from the Vietnam showing clear

antimicrobial activity. Of the isolates, the Bacillus and Paenibacillus, with a diversity of

species, were found, including members of the B. subtilis group (including B. licheniformis,

B. safensis and ubiquitous isolates like B. subtilis, B. amyloliquefaciens), members of the B.

cereus group (B. pacificus) and Paenibacillus (P. polymyxa). The activities against Gram-

positive bacterial indicators were most commonly observed, including against antibiotic-

resistant pathogens (VRE, MRSA); and activities against Gram-negative bacteria, although

limited, were found in the isolate collection. Six isolates (B. halotolerans 01, B.

amyloliquefaciens #06, #08, #11, #13 and P. polymyxa #23) killed 11 or more of the 14

indicators strains, covering a range of bacterial pathogens including food spoilage bacteria,

and human and/or animal pathogens. A high percentage of the antimicrobial substances were

found to be sensitive to proteolytic enzymes and the dominance of antimicrobial activity

against Gram-positive bacteria indicated production of bacteriocins. Therefore, antimicrobial

compounds derived from marine Bacillus/Paenibacillus could potentially be used to address

a range of pathogenic microbial threats to humans and animals. The aims of Chapter 3, to

understand the antimicrobial spectra of spore-forming bacteria in Vietnam Sea, and to select

the promising candidates for genome sequencings, were achieved.

Discovery of novel bacteriocins (and other antimicrobial peptides) is now facilitated by

analysis of bacterial genomes to identify genes encoding these novel compounds. Therefore,

6 marine isolates (4 B. amyloliquefaciens, 1 B. halotolerans #01, 1 P. polymyxa #23), that


161

were selected in Chapter 3 were sequenced and the genomic information was used to all the

encoded putative antimicrobial peptides. The in silico screen resulted in the identification of

an abundance of known and novel antimicrobials (bacteriocins, secondary metabolites) from

the 6 genomes. A total of 61 gene clusters of various AMPs were identified, including 41

NRPs compounds (mostly LPs), 20 bacteriocins that comprised 13 different types of

bacteriocins. Of these 13 bacteriocins, 54% (7/13) were novel and belonged to different

subclasses of bacteriocin class I (1 sactipeptide, 1 lassopeptide, 2 thiopeptides, and 3

lantibiotics). The gene organisation of every bacteriocin gene cluster found in this study was

analysed to understand the mechanism of bacteriocin production. Therefore, the aims of the

chapter, to understand the mechanism of antimicrobial production and identification of novel

bacteriocin(s) and select the candidates harbouring novel bacteriocins for in vitro

characterisation, were achieved. Seven gene clusters of novel bacteriocins were identified;

two potentially novel compounds, produced by isolates #23 and isolate #11, were targeted for

purification. The results of the chapter showed that marine B. amyloliquefaciens and P.

polymyxa contained a rich source of novel bacteriocins gene clusters for further in vitro

characterisation

In addition, in this chapter genome comparisons were performed within these marine species

and versus the terrestrially derived closely related species. The findings demonstrated the high

degree of similarity across the genomes, as judged by, ANI values, and the similarity of the

coding sequence (CDS) potential shared between the genomes. Of the three species

investigated, B. amyloliquefaciens had a significantly higher degree of similarity in genomes,

followed by B. halotolerans, but genomes of P. polymyxa isolates were found to be highly

variable and P. polymyxa had a significantly high diversity of gene repertoire, which may

indicate that future studies could efficiently focus on these sorts of isolates to increase the

discovery rate of novel antimicrobial compounds. The high potential for discovery of novel

bacteriocins from P. polymyxa was confirmed by the in silico prediction of putative

antimicrobial peptides encoded by P. polymyxa #23. The analysis predicted the presence of

many gene clusters of various antimicrobial peptides, including two gene clusters relating to

novel NRPs compounds and three clusters of uncharacterised bacteriocins (1 sactipeptide, 1

lassopeptide, 1 lantibiotic), alongside the presence of gene clusters for NRPs and bacteriocin

characterised previously (tridecaptin, polymyxin, fursacidin, paenicidin A). The marine B.

amyloliquefaciens also serves as a second promising pool of novel bacteriocins, with 3 gene

clusters of novel bacteriocins found in 4 genomes (2 thiopeptides, and 1 two component


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lantibiotic). The B. halotolerans contained genes for production of 1 subtilosin A, therefore,

the potential of novel bacteriocin production from this species is limited.

The purification of antimicrobial peptides was carried out for two bacteria; isolate #11 which

contained genes of novel thiopeptide, and isolate #23 which contained genes of 3

uncharacterised bacteriocins (lasso peptide, 1 lantibiotic, and 1 sactipeptide). The aim was to

obtain the novel bacteriocins for in vitro characterisation; however, the novel bacteriocins are

yet to be identified. The purification of antimicrobial peptides produced by isolate #11 in

LPMA elucidated the presence of amylocyclicin bacteriocin ([m/z] = 6,381], a surfactin

([m/z] =1,058 Da], and [m/z= 1,473]) in the culture. The 1,473 Da substance was suspected

to be a novel thiopeptide, or it could also be a fengycin, because these two compounds have

similar MW to this peptide mass. However, the N-terminal sequencing failed to determine the

amino acid sequence of this compound, therefore, production of novel thiopeptide in culture

is yet to be fully confirmed. Purification of antimicrobial peptides from culture of P. polymyxa

identified the presence of polymyxin ([m/z] = 1168.7), paenicidin A ([m/z]= 3290.4), and

tridecaptin ([m/z]= 1550.8). Attempts to purify novel bacteriocins directly from the isolates

was difficult because various lipopeptides and polyketides were co-produced, and these

exhibited strong antimicrobial activities which hide the presence of other antimicrobial

compounds. The production of NRPs (fengycin, tridecaptin, surfactin), excepting polymyxin,

were frequently observed during the latter phase of culture growth. Bacteriocins

(amylocyclicin, paenicidin) production tended to occur at earlier stages of growth. Therefore,

it is necessary to select the correct time-point for harvesting cultures for purification of

bacteriocins. All the Bacillus/Paenibacillus-derived antimicrobial peptides that were purified

could not be analysed by N-terminal sequencing. This probably indicated that the

Bacillus/Paenibacillus derived AMPs were highly modified peptides which contained either

blocked N-terminus, unusual amino acid residues, and/or various cross-linkages resulting in

complex tertiary structure. Therefore, characterisation and sequencing of Bacillus-derived

AMPs were more challenging compared to that generally found with the characterisation of

Lactobacillus bacteriocins. As an alternative, the peptides could be identified by nuclear

magnetic resonance (NMR) techniques, which have been widely used to identify bacteriocins

(229, 230).

The absence of some of the novel bacteriocins and other NRPs compounds, which were

predicted in silico from genomes, during purification could be due to many reasons.


163

i) Gene clusters relating to these compounds are not transcribed in the conditions produced

by growth in LPMA, or PB medium. It may be activated in other media or requires the

supplement of some factors that can induce expression. For example, the presence of dead

cells of indicators in the culture can activate the production of some bacteriocin via the

quorum sensing system (231, 232). The selection of media for enhancement of bacteriocin

production is a complex process requiring a balance between the most efficient

macronutrients (carbon, nitrogen, phosphate) and the growth-essential micronutrients (zinc,

iron, and manganese) (233, 234). Apart from nutrient effects, the bacteriocin production is

also dependent on the pH, dissolved oxygen level, growth phase (235, 236). The production

of novel sactipeptide only in LB media but not LPMA or PB media can confirm it the media

dependence of sactipeptide production.

ii) The loss of these compounds during purification is possible due to the low detection of

novel bacteriocin. This can happen if the novel bacteriocins are produced at low

concentration; lower than MICs required to kill the indicators. The MIC values of bacteriocins

against indicators are variable depending on types of bacteriocin and types of indicator cells.

And due to the coproduction of non-ribosomally synthesized peptides that commonly exhibit

strong antimicrobial activities B. cereus, the weaker antimicrobial activities of novel

bacteriocin are possibly omitted during purification by accident, particularly when

inappropriate indicators selected.

iii) Loss of compounds during purification is also possible due to the limitation of the

extraction procedure. Bacteriocin (with MWs of less than 2 kDa) can be lost during the

dialysis using 3 kDa cut-off membrane. Therefore, it would be advisable to apply MALDI-

TOF MS analysis at the beginning of the procedure to check the presence/loss of all peptides

during purification.

However, some approaches could be modified to obtain the pure novel bacteriocins for

characterisation; including, a) generation of a mutant which is deficient in NRPs production,

which can mask the presence of other AMPs, and fermentation of this mutant for bacteriocin

production, and; b) heterologous cloning and expression of the gene clusters of novel peptides.

a) The first approach could be achieved by deleting the sfp gene and bacA from the wild-type

isolates. The sfp gene encodes the sfp - 4'-phosphopantetheinyl transferase, required for

activation of synthase enzymes in the biosynthesis of lipopeptide and polyketides. The bacA


164

gene is a structural gene encoding bacilysin. This technique was used previously to

characterise the antimicrobial peptides produced by B. amyloliquefaciens FZB42 (237)

b) The second approach can be an ideal method to produce, heterologously, the novel

bacteriocins, when the gene clusters of these bacteriocins were well annotated, as in the last

chapter. However, the selection of specific expression vectors and host for successful

bacteriocin production must be carefully considered. Successful expression of bacteriocin

requires cloning of the whole bacteriocin biosynthetic gene cluster into a vector to generate

the large expression plasmid. The larger plasmid can be challenging to construct, manipulate

and transform into different hosts for expression analysis. Particularly, in the case of

thiopeptide, of which the biosynthetic gene clusters are large (>12 bps), and required a large

number of optimization steps and trials

In chapter 07, the heterologous cloning and expression methods were employed to produce

one of three uncharacterised bacteriocins found in the genome of P. polymyxa #23. This

bacteriocin was predicted in silico from the genome, and its biosynthesis was predicted to be

encoded by an 8.5 kbp gene cluster containing all accessories genes required for biosynthesis;

however, it was not observed during purification from the wild-type isolate. The heterologous

cloning and expression of the sactipeptide gene cluster employed a shuttle E. coli/Bacillus

pDLL202 plasmid vector and was expressed in B. subtilis BS54B. Attempts with lots of

optimization steps were made and finally led to the successful production of sactipeptide

(question 5). Optimization included i) Selection of an appropriate method to extract the intact

gDNA from wild-type P. polymyxa #23. In this investigation, the genomic DNA of wild-type

P. polymyxa was extracted using a guanidine thiocyanate method (GES). ii) Amplification of

the sactipeptide gene cluster from intact gDNA needs a specific pair of primers, Phusion High-

Fidelity Master-Mix (Thermo Scientific), and HF buffer as a supplement to improve amplicon

yield. iii) Preparation of plasmid backbone from native pDLL202 plasmid by enzymatic

digestion of native plasmid. iv) Joining the gene cluster and plasmid vector DNA molecules

were only achieved with the aid of the Gibson assembly method. v) Transformation of fusion

plasmid into B. subtilis cell required intermediate transformations employing E. coli hosts (E.

coli Top10 and E. coli JIR 702 cells), which can receive a large size fusion plasmid to control

the plasmid copies as well as produce the multimeric form of the fusion plasmid. vi) Finally,

the expression of a novel sactipeptide in Bacillus transformant cells was successful in LB

medium, and MRSA (resistant to chloramphenicol) was used as an indicator. The sactipeptide

had MW of 3,404 Da, was sensitive to protease, proteianase K, trypsin, but not lipase. It had


165

antimicrobial activity against most Gram-positive bacteria. Unfortunately, the amino acid

sequence of the sactipeptide could not be confirmed by N-terminal sequencing, suggesting

the presence of a block at the N-terminus.

The therapeutic potential of isolate #11 is unclear as the production of the predicted

thiopeptide in vitro could not be confirmed. However, the potential to use isolate #23 in

therapeutic applications is clear, with tridecaptin and polymyxin both produced by in vitro

grown cultures. These antimicrobial compounds are currently proven as useful AMPs to cure

hospital-acquired Gram-negative bacteria, particularly against carbapenem-resistant

Enterobacteriaceae. Also, the novel sactipeptide produced heterologously exhibits activity

against MRSA and VRE, therefore, it displayed potential as an antimicrobial for the treatment

of antibiotic-resistant pathogens. Nevertheless, this potential requires more studies in future

to characterise the molecules and properties such as crystallisation of molecules,

determination of toxicity to human cells, stability analysis under different physical condition.

They could be used to protect plants against the invasion and growth of various phyto-fungi

and/or pathogenic bacteria or to maintain the microbiota in soils and water without usage of

antibiotics or pesticides. These isolates could be effective solutions to promote

environmentally friendly, and sustainable, agriculture and aquaculture. In addition, the

isolates were observed to have the ability to degrade different organic substances, like starch,

cellulose, and protein, indicating that these bacteria can produce enzymes like amylase,

cellullase and protease. These enzymes are currently used in many manufacturing processes

and the waste treatment industry. The marine-derive enzymes identified in this study are

effective enzymes with some unusual properties, that have potential for a wide range of

application. This may be, because of the highly variable environment from which they are

derived. A further analysis of the collection of marine isolates established at the start of this

study can be expected to yield additional candidates for application in health and industry

8.2. Future work

There are many avenues for future research, including:

Sequencing the #23’s novel sactipeptide and an un-identified antimicrobial peptide

produced by isolate #11 that was considered a thiopeptide. Attempts could be made using de

novo sequencing techniques or NMR methods.


166

The deletion of the structural genes of the thiopeptide from the genome to confirm the above

antimicrobial peptide as a thiopeptide.

Characterisation of novel bacteriocins, including; bacteriocin crystallization for structural

analysis, mode of action, MIC against selected pathogens, and optimizing media culture for

optimal novel bacteriocin production.

Testing the marine isolates to develop into probiotic products for animal-food supplements

or for aquaculture since the marine Bacillus can tolerate high salt concentration.

Testing the marine bacteriocins to develop into food preservatives or antibiotics.


167

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APPENDIX

General media, solutions, and buffers

Acrylamide- bisacrylamide mixture (49.5% T, 3% C): 48% (w/v) acrylamide, 1.5% (w/v)

bisacrylamide

Coomassie blue stain for SDS-PAGE: 40% (v/v) methanol, 10% (v/v) acetic acid, 0.05%

Coomassie brilliant blue R-250 in Milli-Q water

Destaining solution for SDS-PAGE: 40% (v/v) methanol and 10% (v/v) glacial acetic acid.

Electrophoresis buffers for Tricine-SDS-PAGE:

+ Cathode buffer (inside the cassette): 0.1 M Tris, 0.1 M Tricine, 0.1% SDS

+ Anode buffer (outside the cassette, inside the chamber): 0.2 M Tris-HCl, pH 8.9

Fixing solution: 20% (v/v) isopropanol; 10% (v/v) acetic acid

GES solution: 0.5% v/v Sarkosyl (GES reagent), 5 mol/l Guanidium thiocyanate, and 100mM

EDTA)

LPMB medium: 2.5 g/L yeast extract, 5 g/L peptone, 1 g/L dextrose, 0,2 g/L K2HPO4, 0.05

g/L MgSO4.7H2O, 750 mL/L aged sea water, 250 mL/L water, pH= 7.5). The aged seawater

was prepared by storing the fresh seawater in dark for 2-4 weeks, aiming to stabilize/ or

neutralize the chemicals (heavy metals, toxic compounds)

PB medium: 40 g soy peptone, 40 g dextrin 10, 1.8 g KH2PO4, 4.5 g K2HPO4, 0.3 g

MgSO4·7H2O, and 0.2 ml KellyT trace metal solution per litter was used. KellyT trace metal

solution contains 25 mg EDTA disodium salt dihydrate, 0.5 g ZnSO4·7H2O, 3.67 g

CaCl2·2H2O, 1.25 g MnCl2·4H2O, 0.25 g CoCl2·6 H2O, 0.25 g ammonium molybdate, 2.5 g

FeSO4·7H2O, and 0.1 g CuSO4·5H2O adjusted to pH 6 with NaOH, 500 mL H2O (51)

Saline water: 0.85% (w/v) sodium chloride (NaCl) in Milli-Q water.

SpC medium: 0.1 ml /L 50% glucose; 0.1 ml/L 2% MgSO4; 0.25 ml/L 1% casamino acids;

0.2 mL /L 10% nutrient broth; 0.05 ml /L 1% required amino acid*

SpT medium: 0.1 mL /L 50% glucose; 0.41mL/L 2% MgSO4; 0.1 mL /L 1% casamino acids;

0.1 ml /L 10% nutrient broth; 0.05mL/L 1% required amino acid*


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* Because these are defined media, cultures of auxotrophs should be supplemented with any

required nutrients (amino acids, vitamins, purines, etc.). The Bacillus subtilisBS34A growth

requires addition of filter-sterilised L-Tryptophan (1% in dH20 – should be clear solution)

made fresh on day of experiment, which may need to be shaken at 37°C for 30 min to dissolve

TE buffer (10mmol Tris-HCl; 1mmol/L EDTA, pH 8) for 30 minutes at 37°C. 500µL of GES

solution (0.5% v/v Sarkosyl (GES reagent), 5 mol/l Guanidium thiocyanate, and 100mM

EDTA)

T-base media: 2 g/L (NH4)2SO4; 14 g/L K2HPO4; 6 g/L KH2PO4; 1g/L Na3-citrate-6H2O;

dH2O to 1L

TAE buffer (50×): Tris-base (24.2% w/v), glacial acetic acid (5.17% w/v) and EDTA (1.86%

w/v) were dissolved in Milli-Q water. This was diluted to 1× with Milli-Q water before use.

ANTIBACTERIAL PEPTIDES FROM MARINE BACILLUS ...

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