ORIGINAL PAPER

Bacillus pakistanensis sp. nov., a halotolerant bacteriumisolated from salt mines of the Karak Area in Pakistan

Aneela Roohi • Iftikhar Ahmed • Jayoung Paek •

Yeseul Sin • Saira Abbas • Muhammad Jamil •

Young H. Chang

Received: 28 February 2014 / Accepted: 16 April 2014

� Springer International Publishing Switzerland 2014

Abstract A rod shaped, non-motile, endospore form-

ing, Gram-stain positive and moderately halotolerant

strain, designated as NCCP-168T, was isolated from salt

mines sampled in the Karak district of Khyber Pa-

khtunkhwa Province in Pakistan. To delineate its taxo-

nomic position, the strain was subjected to polyphasic

characterization. Cells of strain NCCP-168T can grow at

10–40 sC (optimum at 30–35 sC), in a pH range of

5.0–9.0 (optimum at pH 8.0) and in 0–17 % (w/v) NaCl

on agar medium. The phylogenetic analysis based on the

16S rRNA gene sequence showed that strain NCCP-168T

belongs to the genus Bacillus with the highest similarity

to Bacillus seohaeanensis BH724T (97.1 %), and less

than 97 % similarity with other closely related taxa

(95.6 % with B. subtilis subsp. subtilis NCIB3610T).

DNA–DNA relatedness between strain NCCP-168T and

the type strains of closely related species was lower than

30 %. Chemotaxonomic data (major menaquinone, MK-

7; cell wall peptidoglycan type, A1c [meso-diamino-

pimelic acid]; major fatty acids, iso-C15:0 29.9 %,

anteiso-C15:0 29.3 %, iso-C16:0 11.4 %, iso-C14:0 8.9 %

and anteiso-C17:0 7.0 %; major polar lipids, diphosphat-

idylglycerol, phosphatidylglycerol and phosphatidyleth-

anolamine) support the affiliation of strain NCCP-168T

with genus Bacillus. On the basis of phenotypic,

chemotaxonomic and phylogenetic data, strain NCCP-

168T can be distinguished from the closely related taxa

and thus represents a novel species in the genus Bacillus,

for which the name Bacillus pakistanensis sp. nov. is

proposed, with the type strain NCCP-168T (= KCTC

13786T = DSM 24834T = JCM 18975T).

Keywords Halotolerant bacterium �Karak salt

mines � Bacillus pakistanensis

Introduction

The diversity of moderately halotolerant or halophilic

bacteria has been studied extensively in hypersaline

environments (Ventosa et al. 1998; Oren 1999; Roohi

Aneela Roohi and Iftikhar Ahmed contributed equally to the

experimentation.

Electronic supplementary material The online version ofthis article (doi:10.1007/s10482-014-0177-5) contains supple-mentary material, which is available to authorized users.

A. Roohi � I. Ahmed (&) � S. Abbas

National Culture Collection of Pakistan (NCCP), National

Institute for Genomics and Advanced Biotechnology

(NIGAB), National Agricultural Research Center

(NARC), Park Road, Islamabad 45500, Pakistan

e-mail: iftikharnarc@hotmail.com

A. Roohi � M. Jamil

Department of Biotechnology and Genetic Engineering,

Kohat University of Science and Technology,

Kohat 26000, Pakistan

J. Paek � Y. Sin � Y. H. Chang (&)

Korean Collection for Type Cultures, Biological

Resources Centre, KRIBB, 125 Gwahak-ro, Yuseong-gu,

Daejeon 305-806, Republic of Korea

e-mail: yhchang@kribb.re.kr

123

Antonie van Leeuwenhoek

DOI 10.1007/s10482-014-0177-5

et al. 2012; Quesada et al. 2004, 1982; Arahal and

Ventosa 2002). Many of these halotolerant, Gram-

staining positive, rod-shaped, aerobic, spore-forming

bacteria have been classified or reclassified in the genus

Bacillus, or other related genera, using a polyphasic

taxonomic approach (Garabito et al. 1998; Ventosa et al.

2008; Wainø et al. 1999; Lee et al. 2006; Ahmed et al.

2007a; Ishikawa et al. 2002; Chen et al. 2009; Zhang

et al. 2010). For study of culturable halotolerant

bacterial diversity of salt mines (Roohi et al. 2012,

2014), samples were collected from the Karak region of

the Khyber Pakhtunkhwa Province in Pakistan. During

these studies, a strain designated as NCCP-168T, was

isolated from a salt mine sample on agar media

containing various concentrations (5–20 %, w/v) of

NaCl. The aim of the present study was to determine the

taxonomic status of strain NCCP-168T using a combi-

nation of phenotypic properties, chemotaxonomic and

phylogenetic analyses, and genomic relatedness to meet

the minimal standards for the description of novel taxa

(Logan et al. 2009). On the basis of results, strain NCCP-

168T is considered to represent to be a novel species of

the genus Bacillus, for which the name Bacillus

pakistanensis sp. nov. is proposed.

Materials and methods

Isolation, morphology and phenotypic

characterization

Strain NCCP-168T was isolated on tryptic soya agar

(TSA; Difco) medium containing NaCl salt by the dilution

plate method at 30 �C. The purified strain was maintained

on the agar medium, and also stored as glycerol (35 %,

w/v) stocks at–80 �C, and subjected to phenotypic and

phylogenetic characterization experiments. Strain NCCP-

168T was routinely grown on TSA containing 3 % NaCl

or on marine agar 2216 (MA, Difco) at 30 �C for all the

characterization experiments, except where mentioned

otherwise. The type strains of closely related taxa,

Bacillus seohaeanensis KCTC 3913T, Bacillus marisflavi

KCTC 3906T Bacillus aquimaris KCTC 3903T and the

type species of the genus, Bacillus subtilis subsp. subtilis

KCTC 3135T, were used as reference strains in all of the

experiments (except where mentioned otherwise) under

the same laboratory conditions.

The colony morphology of strain NCCP-168T was

observed after growth on MA or TSA containing 3 %

NaCl (w/v) for 2 days. The cell morphology (and

motility) was observed using phase-contrast micros-

copy with a Nikon Optiphot-2 light microscope and

further detailed using a scanning electron microscope

(S4300 N, Hitachi) according to the previously

described procedure (Jung et al. 2012). Endospores

were observed under a phase contrast microscope

using cells grown on MA and TSA (containing 3 %

NaCl) for 24 h. Gram staining was performed using a

commercial kit according to the manufacturer’s

instructions (bioMerieux, France).

The pH (optimum and range) of medium for growth

was determined in trypticase soy broth (TSB, Difco)

containing 3 % NaCl (w/v) by adjusting pH to a range

of 4.0–10.0 (at increments of 1 pH unit) and by

monitoring the OD600 using a spectrophotometer

(IMPLEN, Germany). The pH values, adjusted by

1 N HCl or 1 N Na2CO3, were verified after auto-

claving. The temperature range for growth was

determined on TSA (pH 7.0) by incubating at different

temperatures (4, 10, 16, 22, 28, 32, 35, 37, 40, 45 and

50 �C) for 6 days. Tolerance to NaCl was determined

using mTGE agar medium (Difco), which contains

(per litre): beef extract (6 g), tryptone (10 g), dextrose

(2 g), agar (15 g) and supplemented with various

concentration of NaCl (0–20 %; w/v), adjusted to pH

7.0, and incubated at 30 �C for 6 days. The relation-

ship to oxygen was determined on TSA containing

3 % NaCl and MA by incubation in an anaerobic

chamber (Mitsubishi Gas Chemicals Co., Inc.) for

10 days.

Physiological and biochemical characteristics were

determined using the API 20E, API 20NE and API

50CH galleries (bioMerieux, France). Resistance to

antibiotics was assessed with an ATB-Vet strip

(bioMerieux, France) and enzyme activities were

determined with an API ZYM strip (bioMerieux,

France). Additional metabolic features of strain

NCCP-168T in comparison to the reference strains

were assessed by using the Biolog GP2 microplate

characterization system (Biolog, USA). Catalase and

oxidase activities were determined by using the API

Color Catalase and API Oxidase Reagent (bio-

Merieux, France), respectively. All commercial API

kits (bioMerieux, France) and Biolog GP2 microplate

(Biolog, USA) were used according to the manufac-

turers’ protocols except that the saline solution

(bioMerieux, France) with NaCl (up to a 3 %, w/v,

final concentration) was used as the inoculation media.

Antonie van Leeuwenhoek

123

16S rRNA gene sequencing and phylogenetic

analyses

A nearly complete 16S rRNA gene was amplified and

sequenced as described previously (Ahmed et al.

2007b; Roohi et al. 2012). The strain was identified

using the sequence of the 16S rRNA gene on the Ez-

Taxon-e Server (http://eztaxon-e.ezbiocloud.net; Kim

et al. (2012) and BLAST search on the DDBJ/NCBI

servers. The phylogenetic analyses were performed

(Roohi et al. 2012) using sequences of closely related

type strains of validly named species retrieved from

the database of the EzTaxon-e Server and the phylo-

genetic trees were constructed with the Kimura

2-parameter model using three algorithms: neighbour-

joining (NJ), maximum parsimony (MP) and maxi-

mum likelihood (MLH). The stability of the relation-

ship was assessed with bootstrap analysis, by

performing 1,000 re-sampling for the tree topology of

the neighbor-joining data.

DNA base composition, DNA–DNA hybridization

For the DNA G?C content analysis and DNA–DNA

hybridization, the DNA of strain NCCP-168T and the

reference strains were isolated according to the

procedure described previously (Chang et al. 2002).

The genomic DNA was digested with P1 nuclease and

alkaline phosphatase. The DNA G?C contents were

analyzed on HPLC (model UFLC, Shimadzu, Japan)

at 270 nm using solvent NH4H2PO4 (0.02 M)-CH3CN

(v/v 20:1) with a Cosmosil 5C18 column (4.6 by

150 mm; Nacalai Tesque; reversed phase silica gel;

C18). DNA–DNA hybridization was performed with

five replications of each sample, at 40 �C with

photobiotin-labelled DNA and microplates as

described by Ezaki et al. (1989), using a Fluoroskan

Ascent Fluorescent plate reader (Thermo Life Sci-

ences, USA) for the fluorescence measurements.

Chemotaxonomic analyses

To quantify whole-cell fatty acids, NCCP-168T and

the reference strains were grown on a TSBA (Difco)

medium (i.e. 1.5 % agar added in TSB) at 30 �C for

24 h. The cellular fatty acid methyl esters were

prepared (Sasser 1990) and were analyzed on a gas

chromatograph (model 6890 N and autosampler 7683;

Agilent, USA) according to the standard protocol of

the Sherlock Microbial Identification System (MIDI

Sherlock version 4.0, MIDI database TSBA40).

Respiratory quinones from strain NCCP-168T and

the reference strains were analyzed from 300 mg of

lyophilized cells grown on marine broth 2216 (MB,

Difco) for 24 h as described by Minnikin et al. (1984).

Isoprenoid quinones were examined by TLC and

HPLC. Polar lipids were extracted from 100 mg of

freeze-dried cells grown on MA and examined by two-

dimensional TLC, as described previously (Tindall

1990). The cell-wall peptidoglycan of NCCP-168T

and the reference strains was analyzed according to the

method of Schleifer and Kandler (1972) using cells

grown on MA for 24 h. The purified cell wall was

analyzed for amino acids using TLC (Merck), as

described elsewhere (Schleifer and Kandler 1972;

Groth et al. 1996; Jung et al. 2012).

Results and discussion

Morphology and phenotypic characterization

The cells of strain NCCP-168T were observed to be

strictly aerobic, Gram-stain positive, non-motile and

rod-shaped (Supplementary Fig. 1). Endospores were

found to be produced in non-swollen sporangia at the

terminal position (Supplementary Fig. 2). The opti-

mum growth of strain NCCP-168T was observed at

30 �C and at pH 8. Strain NCCP-168T, as well as the

reference strains, were positive for catalase and the

hydrolysis of gelatin, but negative for arginine dihy-

drolase, lysine decarboxylase, ornithine decarboxyl-

ase, citrate utilization, H2S production, urease,

tryptophan deaminase, indole production and nitrate

reduction to N2 (the latter positive in B. subtilis subsp.

subtilis KCTC 3135T). The other results of the

phenotypic characterizations are presented in Table 1

and summarised in the species description. In spite of

sharing many common phenotypic characteristics, a

large number of observations differentiated the strain

NCCP-168T from the closely related members and the

type species of the genus Bacillus (Table 1).

Phylogenetic analysis, DNA base composition

and DNA–DNA hybridization

The comparison of the almost complete 16S rRNA

gene sequence (1411 nucleotides; DDBJ/EMBL/

Antonie van Leeuwenhoek

123

Table 1 Differentiating phenotypic and chemotaxonomic characteristics of strain NCCP-168T and type strains of closely related

species

NCCP-168T B. seohaeanensis

KCTC 3913TB. marisflavi

KCTC 3906TB. aquimaris

KCTC 3903TB. subtilis subsp.

subtilis

KCTC 3135T

Growth at:

NaCl range (%) (optimum) 0–17 (2–3) 0–11 (1–2) 0–19 (2–3) 0–16 (2–4) 0–15 (2–3)

Temperature (�C) range (optimum) 10–40 (30–35) 15–50 (35–40)* 10–47 (30–37)* 10–44 (30–37)* (35)*

pH range (optimum) 5.0–9.0 (8) 5.0–8.0* (7.5) 4.5–9.0* (6–8) 5.0–8.5* (6–7) (7)*

Oxidase 2 ? 2 2 ?

Hydrolysis of:

Gelatin 1 1 1 2 1

Esculin 1 2 1 1 1

ONPG (b-galactosidase) 2 2 1 2 1

Voges-Proskauer reaction 2 2 2 2 1

Acid from:

Glycerol 2 w1 1 2 1

L-Arabinose 2 2 2 2 1

D-Ribose 2 w1 1 2 1

D-Xylose 2 w1 1 2 1

D-Mannose 1 2 1 2 1

Inositol 2 2 2 2 1

D-Mannitol 2 w1 1 2 1

D-Sorbitol 2 2 2 2 1

Methyl-aD-mannopyranoside 2 2 1 2 2

Methyl-aD-glucopyranoside 2 2 w1 2 2

N-acetyle glucosamine 1 w1 2 2 2

Amygdalin 1 2 w? 2 1

Arbutin 2 2 2 2 w1

Esculin 1 2 1 w1 1

Salicin 1 2 1 2 w1

D-Celiobiose 1 2 1 2 1

D-Maltose 2 w1 1 w1 1

D-Melibiose 2 2 1 2 1

D-Saccharose (sucrose) 1 2 1 w1 1

Inulin 2 2 2 2 1

D-Rafinose 2 2 2 2 1

Amidon (starch) 2 2 2 w1 1

Glycogen 1 2 2 w1 1

D-Turanose 2 2 2 2 1

D-Tagatose 1 – 2 2 2

D-Arabitol 2 w1 2 – 2

Assimilation of:

Glucose 1 2 1 2 1

Arabinose 2 2 2 2 1

Mannose 1 2 1 2 1

Mannitol 2 2 1 2 1

N-acetyle glucosamine 1 2 2 2 1

Maltose 1 2 1 1 1

Antonie van Leeuwenhoek

123

Table 1 continued

NCCP-168T B. seohaeanensis

KCTC 3913TB. marisflavi

KCTC 3906TB. aquimaris

KCTC 3903TB. subtilis subsp.

subtilis

KCTC 3135T

Potassium gluconate 1 2 2 2 1

Malate 1 2 1 2 1

Trisodium citrate 2 2 1 2 1

Enzyme activity:

Estarase (C 4) ? 1 w? 1 1

Leucine arylamidase 1 2 2 2 2

a-chymotrypsin 1 2 1 2 2

Acid phosphatase 2 2 1 2 1

b-galactosidase w? - 1 2 2

a-glucosidase 1 1 2 1 w?

Resistance to (lg ml-1):

Penicillin (0.25) R S S S R

Oxacillin (2) S S S S Rw

Streptomycin (8) S S R S S

Spectinomycin (64) S S S S Rw

Kanamicin (8) S S S S Rw

Chloramphenicol (8) R S S S Rw

Tetracycline (4) S S S S R

Erythromycin (1) R S S S S

Lincomycin (2) R S R R R

Pristinamycin (2) R S S S R

Tylosin (2) R S S S S

Colistin (4) S S S S R

Sulfamethizol (100) R S R R R

Fusidic Acid (2) Rw S R S S

Metronidazol (4) R S R R R

G?C content, mol %

(as analyzed on HPLC)

39.1 41.0 49.0 38.0 43.2

Cell wall type A1c (m-DAP) A1a (L-Lys direct)* A1c (m-DAP)* A1c (m-DAP)* A1c (m0-DAP)*

All the strains were positive for catalase, hydrolysis of gelatin but negative for arginine dihydrolase, lysine decarboxylase, ornithine

decarboxylase, citrate utilization, H2S production, urease, tryptophane deaminase, indole production, nitrate reduction to N2 (positive in B.

subtilis subsp. subtilis KCTC 3135T). All strains were positive (weakly positive in B. seohaeanensis KCTC 3913T and B. aquimaris KCTC

3903T) for acid production from D-Glucose, D-Fructose and D-Trehalose but negative for erythritol, D-Arabinose, L-Xylose, D-Adonitol,

methyl-bD-xylopyranoside, D-Galactose, L-Sorbose, L-Rhamnose, dulcitol, D-Lactose, D-Melezitose, xylitol, gentibiose, D-Lyxose, D-Fucose,

L-Fucose, L-Arabitol, potassium gluconate, potassium 2-ketogluconate, potassium 5-ketogluconate, (API 50CH). All strains are negative for

fermentation of D-Glucose, and assimilation of capric acid, adipic acid, phenyl acetic acid. (API 20NE). All strains showed strong enzyme

activity of alkaline phosphatase, estarase lipase (C 8), but negative for lipase (C 14), valine arylamidase, cystine arylamidase, trypsin, napthol-

As-BI-phosphohydrolase, a-galactosidase, b-glucoronidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, a-fucosidase. All the

strains are sensitive to the following antibiotics: amoxycilin (4), amox-clav.acid ((4/2), cephalothin (8), cefoperazon (4), gentamicin (4),

apramycin (16), doxycyclin (4), cotrimoxazol (2/38), flumequin (4), oxolinic acid (2), enrofloxacin (0.5), nitrofurantoin (25), rifamcin (4)

? Positive; w 1 weakly positive; – negative; R resistant to the antibiotic; Rw weakly resistant to the antibiotic; S sensitive to the antibiotic.

All data are from this study except if mentioned otherwise

* Data from Lee et al. (2006); Yoon et al. (2003)

Antonie van Leeuwenhoek

123

GenBank accession number AB618147) of strain

NCCP-168T with sequences of the closely related

type strains of validly named species on the Ez-

Taxon Server database showed the highest similarity

as 97.1 % with B. seohaeanensis BH724T (AY667495),

97.0 % with B. aquimaris TF-12T (AF483625), and

less than 97 % with the other species of the genus

Bacillus. A neighbour-joining phylogenetic tree

(Fig. 1) showed that strain NCCP-168T fell within

the radiation of a cluster comprised of B. seoha-

eanensis BH724T (AY667495), B. marisflavi TF-11T

(AF483624), B. aquimaris TF-12T (AF483625) and

B. vietnamensis 15-1T (AB099708), with a relatively

low bootstrap value (\50 %). This cluster joins with

the phylogenetic clade comprised of the type species

of the genus, B. subtilis subsp. subtilis NCIB3610T

(ABQL01000001). A similar tree topology of the

strain NCCP-168T with the closely related species

was observed in analyses performed by at least two

algorithms (i.e. MP/MLH).

The DNA G?C contents of strain NCCP-168T and

the reference strains are given in Table 1. The values

are comparable with previous reports (Lee et al. 2006;

Yoon et al. 2003) and are consistent with the

taxonomic position of the strain within the genus

Bacillus (Shida et al. 1997). The DNA–DNA related-

ness of NCCP-168T was 21 ± 3.2 % with Bacillus

seohaeanensis KCTC 3913T, 29 ± 3.2 % with B.

marisflavi KCTC 3906T, 30 ± 3.5 % with B. aquim-

aris KCTC 3903T and 18 ± 3.0 % with B. subtilis

subsp. subtilis KCTC 3135T. These values are less

than the 70 % threshold recommended to assign the

strain to a novel species (Stackebrandt and Goebel

1994).

Bacillus aquimaris TF-12T

(AF483625)

Bacillus vietnamensis 15-1T

(AB099708)

Bacillus marisflavi TF-11T

(AF483624)

Bacillus seohaeanensis BH724T

(AY667495)

Bacillus pakistanensis NCCP-168T

(AB618147)

Bacillus carboniphilus JCM 9731T

(AB021182)

Bacillus atrophaeus JCM 9070T

(AB021181)

Bacillus subtilis subsp. subtilis NCIB 3610T (ABQL01000001)

Bacillus mojavensis RO-H-1T

(JH600280)

Bacillus shackletonii LMG 18435T (AJ250318)

Bacillus acidicola 105-2T

(AF547209) Bacillus sporothermodurans M215

T (U49079)

Bacillus ginsengihumi Gsoil 114T

(AB245378)

Bacillus methanolicus PB1T

(AFEU01000002)

Bacillus aeolius 4-1T (AJ504797)

Bacillus firmus NCIMB 9366T

(X60616)

Bacillus oceanisediminis H2T

(GQ292772)

Bacillus infantis SMC 4352-1T

(AY904032)

Bacillus gottheilii WCC 4585T (FN995266)

Bacillus circulans ATCC 4513T (AY724690)

Bacillus pocheonensis Gsoil 420T

(AB245377)

Bacillus niacini IFO 15566T

(AB021194)

Bacillus bataviensis LMG 21833T

(AJ542508)

Bacillus soli LMG 21838T

(AJ542513)

Bacillus drentensis LMG 21831T

(AJ542506)

Bacillus novalis LMG 21837T

(AJ542512)

Bacillus vireti LMG 21834T

(AJ542509)

Falsibacillus pallidus CW 7T

(EU364818)

97

61

96

94

57

6475

9194

99

5659

100

89

70

75

0.005

Fig. 1 Neighbour-joining phylogenetic tree showing the inter-

relationships of strain NCCP-168T with the closely related type

strains of the member of genus Bacillus inferred from sequences

of the 16S rRNA gene. Data with gaps and ambiguous

nucleotides were removed during alignment for the construction

of the tree, which is rooted by using Falsibacillus pallidus CW

7T (EU364818) as an out-group. The tree was generated using

the MEGA 5 software package (Tamura et al. 2011) based on a

comparison of 1,300 nucleotides. Bootstrap values (only[50 %

shown), expressed as a percentage of 1,000 replications, are

given at the branching points. Nodes indicated by solid circles

were recovered by three algorithms (NJ, MP and MLH

methods), whereas the nodes with empty circles were recovered

by at least two algorithms. The bar represents the 0.5 %

sequence divergence. The accession number of each type strain

is shown in parentheses

Antonie van Leeuwenhoek

123

Chemotaxonomic analyses

The cellular fatty acid profiles of strain NCCP-168T

and the reference strains are mainly comprised of large

amounts of branched and saturated fatty acids

(Table 2) and were found to contain anteiso-C15:0

and iso-C15:0 as the major cellular fatty acids. This is in

agreement with the profiles of the type species and

other members of the genus Bacillus (Lee et al. 2006;

Yoon et al. 2003). However, strain NCCP-168T

contained some fatty acids such as C16:1 x 11C and

summed feature 4 (iso-C17:1 I or anteiso-C17:1 B, as

defined by the MIDI system) that are not present in the

profiles of the type species and in some other members

of the genus Bacillus studied here under similar

culture conditions. Nevertheless, the cellular fatty acid

profile obtained for strain NCCP-168T is similar to

those of members of the genus Bacillus (Shida et al.,

1997).

Strain NCCP-168T was found to contain MK-7

(98.7 %) as a major component, whilst MK-8 (1.2 %)

was also detected as a minor component. The detec-

tion of MK-7 being the major isoprenoid quinone is in

agreement with the core characteristic described for

the genus Bacillus (Yoon et al. 2003; Kampfer et al.

2006). Strain NCCP-168T shared a similar polar lipids

profile with B. marisflavi KCTC 3906T, B. aquimaris

KCTC 3903T and the type species of the genus, B.

subtilis subsp. subtilis KCTC 3135T, which consisted

predominantly of diphosphatidylglycerol (DPG),

phosphatidylglycerol (PG) and phosphatidylethanol-

amine (PE) (Supplementary Fig. 3; Supplementary

Table 1). However, the presence and patterns of

phospholipids (APL1–APL4), aminophopholipids

(PNI–PN2), glycolipids (GL1), and unknown phos-

pholipids (L1) could differentiate NCCP-168T from

the rest of the type strains analysed. Strain NCCP-168T

was found to contain meso-diaminopimelic acid

(m-DAP) as the diagnostic amino acid, which is also

in agreement with the core characteristics of the genus

Bacillus (Yoon et al. 2003; Kampfer et al. 2006).

According to the recommendations of Kampfer

et al. (2006) for the inclusion of any species in the

genus Bacillus, strain NCCP-168T shares almost all of

the core characteristics (presence of major menaquin-

one as MK-7; dominance of iso and anteiso fatty acids

and profile of polar lipids mainly comprised of

diphosphatidylglycerol, phosphatidylglycerol and

phosphatidylethanolamine) that are present in the type

species of the genus, B. subtilis subsp. subtilis KCTC

3135T. However, NCCP-168T differs from B. subtilis

subsp. subtilis KCTC 3135T in the pattern of polar

Table 2 Cellular fatty acid profiles (%) of strain NCCP-168T in comparison with the closely related taxa

Fatty acid NCCP168T B. seohaeanensis

KCTC 3913TB. aquimaris

KCTC 3903TB. marisflavi

KCTC 3906TB. subtilis subsp.

subtilis KCTC 3135T

iso-C14:0 9.0 7.6 8.1 8.5 5.4

iso-C14:0 3-OH – tr – – 1.1

iso-C15:0 29.9 20.6 48.9 42.4 14.2

anteiso-C15:0 29.3 42.2 17.7 28.4 45.8

iso-C15:0 2-OH – – – tr 2.4

C16:0 1.1 1.4 1.2 1.4 1.8

iso-C16:0 11.5 15.1 9.1 7.9 10.7

C16:1 x 11C 1.6 tr tr – –

C16:1 x 7C alcohol 3.4 1.5 4.0 tr tr

iso-C17:0 2.0 2.3 2.8 2.5 5.3

anteiso-C17:0 7.0 6.4 4.0 6.2 10.7

iso-C17:1 x 10C 1.8 – 1.1 – tr

Summed feature 4� 2.4 tr – – –

All data are obtained in this study. Values are percentages of total fatty acid detected

tr, trace amount (\1.0 %); – not detected� Summed feature 4 contains of iso-C17:1 I/anteiso-C17:1 B that could not be separated by GC using the microbial Identification

System (Microbial ID) software

Antonie van Leeuwenhoek

123

lipids (Supplementary Fig. 3 and Supplementary

Table 1) and cellular fatty acid composition (Table 2).

It also differs from the type species and other closely-

related members of the genus Bacillus in some

phenotypic and chemotaxonomic characteristics

(Table 1).

On the basis of physiological, phylogenetic, che-

motaxonomic and genomic characteristics which we

determined, strain NCCP-168T is considered to be a

new member of the genus Bacillus, and thus its

description is given as follows:

Description of Bacillus pakistanensis sp. nov

Bacillus pakistanensis (pa.kis.tan.en’sis. N.L. masc.

adj. pakistanensis pertaining to Pakistan, where the

organism was isolated).

Cells are Gram-stain positive, strictly aerobic, non-

motile and long rods (1.9–5.6 lm). Sometimes occur

in pairs and/or in chains of up to four cells (end to end).

Occasionally filamentous cells also observed (up to

22 lm long). Endospores are produced in non-swollen

sporangia at a terminal position. Colonies are circular,

smooth, off-white in color and opaque on TSA

supplemented with 3 % (w/v) NaCl. The colony

texture is mucoid and the elevation is flat. Cells can

grow at a temperature range of 10–40 �C (optimum

growth occurs at 30–35 �C). The pH range for growth

is 5.0–9.0 (optimum pH 8.0). Can tolerate 0–17 %

(w/v) NaCl (optimal growth occurs in the presence of

approximately 2–3 % NaCl). Positive for catalase and

the hydrolysis of gelatin and esculin; negative for

oxidase, ONPG (b-galactosidase), Voges-Proskauer

reaction, arginine dihydrolase, lysine & ornithine

decarboxylases, citrate utilization, H2S production,

urease, tryptophan deaminase, indole production and

nitrate reduction. Can assimilate glucose, mannose,

N-acetyl glucosamine, maltose, potassium gluconate

and malate, but cannot assimilate arabinose, mannitol,

capric acid, adipic acid, phenyl acetic acid and

trisodium citrate (API 20NE, bioMerieux, France)

and cannot ferment glucose. Positive for acid produc-

tion (after 48 h to 72 h) from amygdalin, D-Cellobi-

ose, D-Fructose, D-Mannose, D-Glucose, D-Saccharose

(sucrose), D-Tagatose, D-Trehalose, esculin, glycogen,

N-acetyl glucosamine and salicin, but negative for

D-Turanose, D-Arabitol, inulin, D-Raffinose, amidon

(starch), D-Maltose, D-Melibiose, arbutin, inositol,

D-Mannitol, D-Sorbitol, methyl-aD-mannopyranoside,

methyl-aD-glucopyranoside, glycerol, L-Arabinose,

D-Ribose, D-Xylose, erythritol, D-Arabinose, L-Xylose,

D-Adonitol, methyl-bD-xylopyranoside, D-Galactose,

L-Sorbose, L-Rhamnose, dulcitol, D-Lactose, D-Mel-

ezitose, xylitol, gentibiose, D-Lyxose, D-Fucose,

L-Fucose, L-Arabitol, potassium gluconate, potassium

2-ketogluconate, potassium 5-ketogluconate, (API

50CH and 20E galleries, bioMerieux, France). Posi-

tive for the oxidation of dextrin, a-D-Glucose,

D-Mannose, D-Trehalose, stachyose, D-Alanyl-glycine,

glycyl-L-glutamic acid, glycerol and D-Fructose-6-

phosphate; weakly positive for L-Arabinose, L-Fucose,

D-Galacturonic acid, D-Alanine and L-Pyroglutamic

acid; but negative for all other substrates of the Biolog

GP2 plate (Biolog, USA). Strong enzyme activity for

alkaline phosphatase, esterase lipase (C 8), esterase (C

4), leucine arylamidase, a-chymotrypsin, a-glucosi-

dase and weak for b-galactosidase, but negative for

acid phosphatase, lipase (C 14), a-galactosidase,

b-glucuronidase, b-glucosidase, N-acetyl-b-glucosa-

minidase, a-mannosidase, a-fucosidase. valine aryl-

amidase, cysteine arylamidase, naphthol-As–BI–

phosphohydrolase and trypsin (API Zym, bioMerieux,

France). The major polar lipids consist of diphosphat-

idylglycerol, phosphatidylglycerol, phosphatidyletha-

nolamine, three phospholipids, two aminophospholipids,

one glycolipid and one unknown phospholipid. The

cell-wall peptidoglycan type is A1c (contains meso-

diaminopimelic acid as the diagnostic amino acid).

The major cellular fatty acids are iso-C15:0, anteiso-

C15:0, iso-C16:0, iso-C14:0, anteiso-C17:0, followed by

C16:1 x7c alcohol, summed feature 4 (iso-C17:1 I or

anteiso-C17:1 B as defined by MIDI), iso-C17:0, iso-

C17:1 x10c, C16:1 x11c and C16:0. The predominant

respiratory quinone is MK-7. The DNA G?C content

of the type strain is 39.1 mol % (as analyzed using

HPLC).

The type strain, NCCP-168T (= KCTC 13786T =

DSM 24834T = JCM 18975T), was isolated from a

salt mine sample collected from the Karak region of

the Khyber Pakhtunkhwa Province in Pakistan.

The DDBJ/EMBL/GenBank accession number for

the 16S rRNA gene sequence of strain NCCP-168T

(= JCM 18975T = KCTC 13786T = DSM 24834T) is

AB618147.

Acknowledgments This work was partially supported by

PSDP Project ‘‘Research for Agricultural Development Program

Antonie van Leeuwenhoek

123

(RADP)’’ under a sub-project (Grant No. CS-55/RADP/PARC)

entitled ‘‘Establishment of Microbial Bio-Resource Laboratories:

National Culture Collection of Pakistan (NCCP)’’ from Pakistan

Agricultural Research Council (PARC), Islamabad, Pakistan. The

partial financial support of KRIBB to authors, Aneela Roohi and

Saira Abbas in the training course at Korean Collection for Type

Cultures, Biological Resources Centre, KRIBB under the

umbrella of ACM forum is also gratefully acknowledged.

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