Unraveling the complete genome sequence of Advenella mimigardefordensis strain DPN7T and novel...

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Unravelling the complete genome sequence ofAdvenella mimigardefordensis strain DPN7T andnovel insights in the catabolism of the xenobioticpolythioester precursor 3,39-dithiodipropionate

Jan Hendrik Wubbeler,1 Sebastian Hiessl,1 Jorg Schuldes,2

Andrea Thurmer,2 Rolf Daniel2 and Alexander Steinbuchel1,3

Correspondence

Alexander Steinbuchel

[email protected]

Received 17 February 2014

Accepted 14 April 2014

1Institut fur Molekulare Mikrobiologie und Biotechnologie, Westfalische Wilhelms-UniversitatMunster, 48149 Munster, Germany

2Department of Genomic and Applied Microbiology and Gottingen Genomics Laboratory, Institut furMikrobiologie und Genetik, Georg-August-Universitat Gottingen, Gottingen, Germany

3Faculty of Biology, King Abdulaziz University, Jeddah, Saudi Arabia

Advenella mimigardefordensis strain DPN7T is a remarkable betaproteobacterium because of its

extraordinary ability to use the synthetic disulfide 3,39-dithiodipropionic acid (DTDP) as the sole

carbon source and electron donor for aerobic growth. One application of DTDP is as a precursor

substrate for biotechnically synthesized polythioesters (PTEs), which are interesting non-

degradable biopolymers applicable for plastics materials. Metabolic engineering for optimization

of PTE production requires an understanding of DTDP conversion. The genome of A.

mimigardefordensis strain DPN7T was sequenced and annotated. The circular chromosome was

found to be composed of 4 740 516 bp and 4112 predicted ORFs, whereas the circular plasmid

consisted of 23 610 bp and 24 predicted ORFs. The genes participating in DTDP catabolism had

been characterized in detail previously, but knowing the complete genome sequence and with

support of Tn5 : : mob-induced mutants, putatively involved transporter proteins and a

transcriptional regulator were also identified. Most probably, DTDP is transported into the cell by a

specific tripartite tricarboxylate transport system and is then cleaved by the disulfide reductase

LpdA, sulfoxygenated by the 3-mercaptopropionate dioxygenase Mdo, activated by the CoA

ligase SucCD and desulfinated by the acyl-CoA dehydrogenase-like desulfinase AcdA.

Regulation of this pathway is presumably performed by a transcriptional regulator of the xenobiotic

response element family. The excessive sulfate that is inevitably produced is secreted by the cells

by a unique sulfate exporter of the CPA (cation : proton antiporter) superfamily.

INTRODUCTION

Advenella mimigardefordensis strain DPN7T (formerlyTetrathiobacter mimigardefordensis, Wubbeler et al., 2006)is a betaproteobacterium with the extraordinary capacity toutilize the organosulfur compound (OSC) 3,39-dithiodi-propionic acid (DTDP; Fig. 1) as the sole carbon source forgrowth (Wubbeler et al., 2008) and as a precursor for thebiosynthesis of polythioester (PTE; Fig. 1) (Xia et al.,2012).

The genus Advenella was proposed in 2005 (Coenye et al.,2005) and currently comprises four species: the type speciesAdvenella incenata (Coenye et al., 2005), Advenellakashmirensis (Ghosh et al., 2005; Gibello et al., 2009), A.mimigardefordensis (Wubbeler et al., 2006; Gibello et al.,2009) and Advenella faeciporci (Matsuoka et al., 2012).Species of Advenella belong to the family Alcaligenaceae

Abbreviations: 3MP, 3-mercaptopropionic acid; 3SP, 3-sulfinopropionicacid; ABC, ATP-binding cassette; AcdA, acyl-CoA dehydrogenase-likedesulfinase; APS, adenosine 59-phosphosulfate; CPA, cation : protonantiporter; COGs, Clusters of Orthologous Groups; DTDP, 3,39-dithiodipropionic acid; LpdA, dihydrolipoamide dehydrogenase; Mdo,3-mercaptopropionic dioxygenase; MFS, major facilitator superfamily;MIP, major intrinsic protein; OSC, organosulfur compound; PHA,polyhydroxyalkanoate; PHB, polyhydroxybutyrate; PSE, putative sulfateexporter; PTE, polythioester; PTS, phosphotransferase system; SSS,solute : sodium symporter; SucCD, succinate-CoA ligase; T6SS, type VIsecretion system; TAT, twin-arginine translocation; TRAP-T, tripartiteATP-independent periplasmic transporter; TTT, tripartite tricarboxylatetransport; XRE, xenobiotic response element.

The GenBank/EMBL/DDBJ accession numbers for the complete andannotated genome sequence of A. mimigardefordensis strain DPN7T areCP003915 (chromosome MIM_c) and CP003916 (plasmid MIM_24p).

Supplementary material is available with the online version of this paper.

Microbiology (2014), 160, 1401–1416 DOI 10.1099/mic.0.078279-0

078279 G 2014 The Authors Printed in Great Britain 1401


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(De Ley et al., 1986). Strains of this family (Fig. 2) havebeen detected in a variety of habitats, e.g. soil, sewagesludge, and also veterinary and clinical samples. Moreover,Alcaligenaceae perform diverse metabolic reactions andinclude versatile heterotrophs, facultative chemolitho-trophs, sophisticated parasites, as well as pathogens anddegraders of xenobiotics. The most important biochemicaland physiological characteristics of the four speciesaffiliated to the genus Advenella are introduced in thefollowing paragraphs.

A. incenata LMG 22250T was isolated from human sputumand described in 2005, together with some further strains,which were identified in cystic fibrosis patients andveterinary samples (Coenye et al., 2005). Apparently, somestrains of A. incenata are opportunistic pathogens (Coenyeet al., 2005; Vanlaere et al., 2006), but most of them wereisolated worldwide from soil samples of the rhizospherefrom economic plants, such as corn, rice and tobaccoplants (Espinosa-Victoria et al., 2009; Shimoyama et al.,2009; Christiaen et al., 2011; Jin et al., 2011; Shahi et al.,2011). Interestingly, the type strain has the ability todegrade the herbicide terbuthylazine and could be appliedfor bioremediation processes of polluted groundwater(Barra Carracciolo et al., 2010). Unfortunately, no completegenome sequence from any strain of A. incenata is availableat this time; this is also the case for A. faeciporci, which wasisolated from nitrifying/denitrifying activated sludge col-lected from a bioreactor treating piggery wastewater andrepresents the most recently described species of the genusAdvenella (Matsuoka et al., 2012).

The whole-genome of A. kashmirensis WT001T waspublished and is accessible (Ghosh et al., 2011). The typestrain was isolated from a temperate orchard soil (Ghoshet al., 2005), and has been recognized as a thiosulfate- andtetrathionate-oxidizing facultative chemolithotroph (Damet al., 2007). Several isolated soil-dwelling strains of A.

kashmirensis were able to detoxify selenite by reducing it toinsoluble elemental red selenium (Hunter & Manter, 2008).Furthermore, various bacterial isolates, which were affiliatedto A. kashmirensis on the basis of their 16S rRNA genesequence similarity, degrade alkanes or are involved in thebiodegradation of thiodiglycol – the hydrolysis product ofyperite, a highly hazardous derivative of mustard gasemployed in chemical weapons (Dam et al., 2009).

A. mimigardefordensis strain DPN7T was employed initiallyfor research on catabolism of PTE precursor substrates(Wubbeler et al., 2006, 2008) and is now successfullyapplied for the biotechnical production of PTE homo-polymers (Xia et al., 2012). The sulfur-containing homo-PTEs are microbially synthesized, biologically persistentpolymers (Lutke-Eversloh et al., 2002a, b; Kawada et al.,2003; Elbanna et al., 2004; Lutke-Eversloh & Steinbuchel,2004; Kim et al., 2005). The chemical synthesis of PTEs wasreported in 1951 (Marvel & Kotch, 1951), but PTEs were nevertechnically produced for commercialization (Kricheldorf &Schwarz, 2007). The preparation procedures were laboriousand difficult, due to the requirement of toxic reagents, andgave low yields, resulting in high costs (Sanda et al., 2000).Production of PTEs by the methods and principles of greenchemistry was, and still is, desirable; for that reason, the firstreport in 2001 of biotechnically synthesized PTE attractedmuch attention (Lutke-Eversloh et al., 2001). These sulfur-containing polymers possess some unique characteristics,especially auspicious for specialized applications of rarebiomaterials (Steinbuchel, 2005). Until recently, synthesis ofPTE homopolymers was limited, due to the dependency on afew expensive and toxic 3-mercaptoalkanoates (Lutke-Eversloh & Steinbuchel, 2003, 2004; Xia et al., 2012).Alternative economic precursor substrates are essential forfurther enhancement of the large-scale biotechnical pro-duction of established and also novel PTEs (Lutke-Eversloh& Steinbuchel, 2003). Therefore, the catabolic pathway ofthe non-toxic precursor substrate DTDP was studied in A.mimigardefordensis strain DPN7T (Wubbeler et al., 2008,2010; Schurmann et al., 2011, 2013; Fig. 3). An understand-ing of the catabolism, including the regulation of cellularactivities in the presence of DTDP and its degradationintermediates as well as the transport of these sulfurcompounds, would be essential for applications of modernbiochemical and genetic methods to influence the microbialmetabolic networks. Knowledge of the complete genomesequence of A. mimigardefordensis strain DPN7T will bevaluable to further improve the biotechnical production ofPTE homopolymers from economic and distinct com-pounds in the future. Consequently, we sequenced andannotated the genome of A. mimigardefordensis strainDPN7T and performed Tn5 : : mob mutagenesis to elucidatethe entire catabolism of DTDP.

METHODS

Bacterial strains, plasmids and growth conditions. Bacterial

strains and plasmids used in this study are listed in Table S1 (available

S

OR2

yO

R1 O

xz( )

OH SS

O

O

OH

DTDP

PHA PTE

SH OH

O

3MP

Fig. 1. Overview of the chemical structures of 3,39-dithiodipro-pionic acid (DTDP), 3-mercaptopropionic acid (3MP), a typicalpolyhydroxyalkanoate (PHA) and polythioester (PTE). R1, alkylgroup or functionalized alkyl group; R2, hydrogen, methyl or ethylgroup; x/y, number of monomers; z, 1–4 -CH2-.

J. H. Wubbeler and others

1402 Microbiology 160


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in the online Supplementary Material). Escherichia coli strains werecultivated aerobically in lysogeny broth (LB) medium (Sambrook et al.,1989; Berlyn et al., 1996) at 37 uC with the addition of applicableantibiotics, if necessary. E. coli S17-1 harbouring the suicide plasmidpSUP5011 was used for Tn5 : : mob mutagenesis of A. mimigardefordensisstrain DPN7T. Cells of A. mimigardefordensis were cultivated aerobicallyin 0.8 % (w/v) nutrient broth (NB) or in mineral salts medium (MSM;Schlegel et al., 1961) at 30 uC containing the indicated carbon source.Carbon sources were prepared as filter-sterilized 1 M stock solutions andadjusted to pH 6.9–7.4. Solid media contained 1.8–2.0 % (w/v) purifiedagar-agar. Antibiotics were added to growth media at the followingconcentrations: 75 mg ampicillin ml21 and 50 mg kanamycin ml21.

Tn5 : : mob mutagenesis. For Tn5 : : mob mutagenesis, the suicideplasmid technique (Simon et al., 1983; Simon, 1984) was used bytransferring the vector pSUP5011 from E. coli S17-1 to the kanamycin-susceptible A. mimigardefordensis strain DPN7T by conjugationapplying the spot-agar mating technique (Friedrich et al., 1981).Tn5 : : mob-induced mutants were selected on MSM agar platescontaining 50 mg kanamycin ml21 (MSMKm) and 20 mM sodiumpropionate or 20 mM sulfinopropionate (master plates). Putativemutants were transferred in a coordinated pattern on MSMKm agarplates containing 30 mM DTDP (selection plates) and on correspond-ing master plates for further analysis, and were otherwise treated asdescribed previously (Wubbeler et al., 2008; Schurmann et al., 2011).

Thiochemicals. Sulfur-containing chemicals were purchased fromAcros Organics. 3-Sulfinopropionate (3SP) was not available forpurchase; therefore, it was synthesized as the disodium salt accordingto the method defined by Jolles-Bergeret (1974); the procedure wasslightly modified by one repetition of the alkaline cleavage of theintermediate bis-(2-carboxyethyl)sulfone, as described in previousstudies (Wubbeler et al., 2008; Schurmann et al., 2011). Success ofsynthesis and purity of the synthesized compound were confirmed byHPLC and GC-MS analyses.

GC-MS analyses. Purity of 3SP was determined upon methylationafter lyophilization in the presence of 15 % (v/v) sulfuric acid (H2SO4)by GC analyses, as described previously (Schurmann et al., 2013).

HPLC analyses. HPLC analysis was carried out in a LaChrom EliteHPLC apparatus (VWR-Hitachi International) consisting of aMetacarb 67H advanced C column (Varian; Bio-Rad Aminexequivalent) and a 22350 VWR-Hitachi column oven. The column(300 mm66.5 mm) consisted of sulfonated polystyrene resin in theprotonated form. The primary separation mechanism included ligandexchange, ion exclusion and adsorption. The column temperature wasmaintained at 30 uC with a 2350 VWR-Hitachi column oven. An L-2490 VWR-Hitachi refractive index detector was used for detection.Aliquots of 20 ml samples were injected and eluted with 0.005 Nsulfuric acid in double-distilled water at a flow rate of 0.8 ml min21.Online integration and analysis was done with EZ Chrome EliteSoftware (VWR International).

DNA extraction and molecular techniques. Chromosomal DNAof A. mimigardefordensis strain DPN7T and the respective Tn5 : : mob-induced mutants was isolated using the NucleoSpin Tissue kit(Macherey-Nagel) according to the instructions of the manufacturer.To identify the Tn5 : : mob insertion loci, the two-step gene walkingmethod (Pilhofer et al., 2007) was used and subsequent sequenceanalysis was performed by Seqlab. Oligonucleotides used in this studywere synthesized by Eurofins and are listed in Table S1. PCR was doneby applying Biomix (Bioline) and the resulting DNA fragments werepurified using the peqGOLD Gel Extraction Kit I (Peqlab).

Genome sequencing, assembly and gap closure. A combinationof Sanger sequencing and pyrosequencing was used for whole-genome

sequencing of A. mimigardefordensis strain DPN7T. Isolated DNA fromstrain DPN7T was used to generate a 454 shotgun library according tothe GS Rapid Library protocol (454 Life Sciences). The 454 DNA librarywas sequenced with the Genome Sequencer FLX system (454 LifeSciences) using titanium chemistry. A total of 195 897 shotgun readswere generated and assembled de novo into 30 large contigs (.500 bp)using Roche Newbler assembler software 2.0 FLX (454 Life Sciences).Sequence editing was done by using GAP4 as part of the Staden softwarepackage (Staden et al., 2000), and final gap closure was performed byPCR and primer walking using a Bio-X-Act kit (Bioline) and the 5Prime Extender polymerase system (5 Prime) as described by themanufacturers.

Gene prediction, annotation, analysis and comparative geno-

mics. Coding sequences were predicted with YACOP (Tech & Merkl,2003), applying the ORF finders Glimmer, Critica and Z-Curve. Allcoding sequences were curated manually and verified by using criteriasuch as the presence of a ribosome-binding site, GC frame plotanalysis, and comparison with sequences in the publicly availabledatabases Swiss-Prot, Trembl, GenBank, Clusters of OrthologousGroups (COGs), KEGG, ProDom, Pfam, Tigrfam and Prosite,employing the annotation software tools ERGO (Overbeek et al.,2003), IMG/ER (Markowitz et al., 2012) and Artemis (Rutherford et al.,2000). Prediction of TAT (twin-arginine translocation) signalpeptides was carried out using TatP 1.0 (Bendtsen et al., 2005).Comparative genomics was done by implementing software in IMG/ER

using default parameters and inhouse Perl scripts.

RESULTS AND DISCUSSION

General features

Genome. The complete genome of A. mimigardefordensisstrain DPN7T comprises 4 764 126 bp and is distributed ontwo replicons: one circular chromosome consisting of4 740 516 bp and a circular plasmid of 23 610 bp, which arecomposed of 4112 and 24 predicted ORFs, respectively. Aputative function was assigned to 3692 (89.26 %) of allprotein-coding sequences and 3681 (89.00 %) ORFs couldbe assigned to COGs categories (Fig. 4). The mean GC contentwas 54.22 %. The genome harboured seven pseudogenes, 39tRNA genes and two copies of rRNA operons. The generalfeatures are listed in Table 1. Homologues of dnaA, dnaNand gyrB (MIM_c00010–MIM_c00030) are localized adja-cent to each other. Furthermore, single copies of parA andparB, responsible for DNA partitioning, were located on thechromosome (MIM_c40760–MIM_c40750), and a ParA-like encoding gene was also detected on the plasmid(MIM_24p00230). The genome of A. mimigardefordensisstrain DPN7T harbours some ORFs presumably derivedfrom mobile genetic elements or of viral origin. Seven puta-tive integrases and 14 putative transposases were identified(for more details, see Table S2 and the ‘Supplementarysections’ in the online Supplementary Material).

Plasmid. The plasmid pMIM24 harbours 24 ORFs, 18(75 %) of which were assigned to a putative function. Nogenes associated with the utilization of OSCs such as DTDPwere identified on pMIM24. Two ORFs possibly coding foran addiction module consisting of a toxin (MIM_24p00210)and an antidote (MIM_24p00220) were found on this

Genome of A. mimigardefordensis strain DPN7T

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Brackiella oedipodis (AJ277742)

Oligella ureolytica (AJ251912)

Oligella urethralis (AF133538)

Taylorella equigenitalis (X68645)

Taylorella asinigenitalis (AF067729)

Pelistega europaea (Y11890)

1000

Advenella faeciporci (AB567741)

Advenella kashmirensis (AJ864470)

Advenella incenata (AM944734)

Advenella mimigardefordensis (AY880023)

994

926

Kerstersia gyiorum (AY131213)

Achromobacter insolitus (AY170847)

Achromobacter denitrificans (AJ278451)

Achromobacter ruhlandii (AB010840)869

Achromobacter spanius (AY170848)

Achromobacter piechaudii (AB010841)998

Achromobacter xylosoxidans (Y14908)

Bordetella bronchiseptica (U04948)

Bordetella parapertussis (U04949)

Bordetella pertussis (U04950)

Bordetella holmesii (U04820)

890

773

Bordetella hinzii (AF177667)

992

Bordetella trematum (AJ277798)

Bordetella avium (AF177666)

999

Bordetella petrii (AJ249861)

927

Pigmentiphaga kullae (AF282916)

Pigmentiphagada eguensis (EF100696)

Pigmentiphaga litoralis (EU583723)

999

Derxia gummosa (AB089482)

Azohydromonas australica (AB188124)

Azohydromonas lata (AB188125)1000

998

1000

892

700

Pusillimonas ginsengisoli (EF672088)

Pusillimonas soli (GQ241322)

Parapusillimonas granuli (DQ466075)

1000

Pusillimonas noertemannii (AY695828)

857

Castellaniella denitrificans (U82826)

Castellaniella daejeonensis (GQ241321)

Castellaniella defragrans (AJ005447)

931

Castellaniella caeni (AB166879)

Castellaniella ginsengisoli (EU873313)

940

999

Alcaligenes faecalis (D88008)

Alcaligenes faecalis subsp. phenolicus (AY296718)

Alcaligenes faecalis subsp. parafaecalis (AJ242986)

836

Paenalcaligenes hominis (FN391024)

1000

792

996

732

1000

1000

920

0.02




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plasmid. Furthermore, two genes encoding UmuCD (MIM_24p00020–MIM_24p 00030) involved in DNA repair weredetected on the plasmid, but a copy of paralogue umuCDalso exists on the chromosome (MIM_c07720–MIM_c07730). In addition, a transcriptional regulator of theLysR family (MIM_24p00130) and a putative extracyto-plasmic solute-binding receptor of the TTT (tripartitetricarboxylate transporter family) (MIM_24p00110) arelocated on pMIM24. Many paralogues of these two genesexist on the chromosome (Table 2).

A total of five metabolic genes are localized on pMIM24,two of which code for proteins possibly participating intyrosine metabolism: a fumarylacetoacetate hydrolasedomain-containing protein (MIM_24p00140) and a putat-ive gentisate 1,2-dioxygenase (MIM_24p00080). For the

latter, no paralogues were identified on the chromosome.Another ORF on the plasmid encodes a putative 3-hydroxybenzoate 6-hydrolase (MIM_24p00150), whichcould be involved in polycyclic aromatic hydrocarbondegradation. Furthermore, a 3-isopropylmalate dehydra-tase (leuCD, MIM_24p00090–MIM_24p 00100) wasdetected on pMIM24, nonetheless four leuCD paraloguesare also present on the chromosome.

Heterotrophic carbon metabolism

A. mimigardefordensis strain DPN7T was capable of usingseveral amino acids, pentoses, hexoses and organic acids ascarbon and energy sources for heterotrophic growth(Wubbeler et al., 2006; Matsuoka et al., 2012, C. Meinert,

Fig. 2. Phylogenetic tree of type strains belonging to the family Alcaligenaceae. The tree is based on 16S rRNA genesequences, which were obtained from the Ribosomal Database Project (Cole et al., 2009) and the EMBL database (Kanz et al.,2005). Alignment of the sequences was performed with CLUSTAL_X (Thompson et al., 1997) and calculation of the tree was doneusing the neighbour-joining method (Saitou & Nei, 1987). Brackiella oedipodis was used as outgroup. A. mimigardefordensis ishighlighted. Bootstrap values .60 % are shown at the branch points and GenBank accession numbers are given inparentheses. Bar, 0.02 substitutions per nucleotide position.

Centralmetabolism

HO S S

OOH

O

Disulfide reductase LpdAMIM_c19220

NADH+H+

NAD+

NADPH+H+

NADP+

Tripartite tricarboxylatetransport (TTT) systemMIM_c08710 –MIM_c08730

Membrane

ATPCoA

CoACoA

2 x

CoA

ADP + Pi

CoA ligase SucCDMIM_c18280/18290

H2O

H2O H2O H2O

CoA

HS OH

O

OH

O

SHO

O

O

SHO

O

S-CoA

O

S-CoA

O2

3MP

HS CoA

O

CoA

O

CoA

O

3-Mercaptopropionyl-CoA3-Hydroxybutyryl-CoA

Acetoacetyl-CoA

Acetyl-CoA

3SP 3-Sulfinopropionyl-CoA

SO4

SO4

SO3

3MP-dioxygenase MdoMIM_c31400

Desulfinase AcdAMIM_c31390

Sulfite oxidaseMIM_c01600, MIM_c03760,MIM_c12620, MIM_c36020,MIM_c24010

Putative sulfate exporter(PSE) familyMIM_c23530

Propionyl-CoA

Oxaloacetic acid

2-Methylcitric acid2-Methyl-cis-aconitic acid2-Methylisocitric acid

MethylisocitratelyaseMIM_c31320

MethylisocitratedehydrataseMIM_c31340

MethylcitratedehydrataseMIM_c13190

MethylcitratesynthaseMIM_c31350,MIM_c13200

Heterologue Buk and Ptb or native unidentified enzymes

PHA synthasePhaCMIM_c13530

Acteyl-CoA acetyltransferase PhaAMIM_c21300

Acetoacteyl-CoA reductase PhaBMIM_c13540

Homo-PTE synthesisHetero-PTE synthesis

DTDP

HO S S

OOH

O

ATP + CoAADP + P

i

O S

O

O

O

poly(3HB-co-3MP) PMPyx

**

HO OH

O OOHO

OH

HOOH

O

OO

HO OH

O OOHO

HO OH

O OOHO

OH

O

CoA

OOH

S

O

S

O

y

**

Fig. 3. Proposed pathway for utilization of DTDP by A. mimigardefordensis strain DPN7T based on verified enzyme reactions,genome annotation, and Tn5 : : mob-induced and/or deletion mutants. 3HB, 3-hydroxybutyric acid; 3MP, 3-mercaptopropionicacid; 3SP, 3-sulfinopropionic acid; Buk, butyrate kinase from Clostridium acetobutylicum integrated into the genome of A.

mimigardefordensis via homologous recombination; PMP, poly(3-mercaptopropionate); Ptb, phosphotransbutyrylase from C.

acetobutylicum integrated into the genome of A. mimigardefordensis via homologous recombination.




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unpublished). High specific growth rates were obtainedwith TCA cycle intermediates (e.g. succinate or malate),sugar acids (e.g. gluconate) and short-chain fatty acids (e.g.acetate, butyrate or propionate). Furthermore, the strainhad the peculiar ability to utilize DTDP and 3SP as the solecarbon source for growth (see below).

DTDP. DTDP is a xenobiotic and is commonly used indifferent fields of application (Saxena & Gupta, 1984;

Tsutsumi et al., 1998; Lutke-Eversloh & Steinbuchel, 2003;Codognoto et al., 2007; Xia et al., 2012). It is the structuralanalogue of the disulfide amino acid cystine; the onlydifference being the absence of amino groups in DTDP.Formation of DTDP in the natural environment has notyet been described, but it is possible as DTDP is theoxidation product of two molecules of 3-mercapto-propionic acid (3MP; Fig. 1) and 3MP is frequentlydetected in nature (Kiene & Taylor, 1988; Al-Farawati &

04 700 000

470 000

940 000

1 410 000

1 880 000

12 000

22 000 0

2 350 000

2 820 000

3 290 000

3 760 000

4 230 000

Translation, ribosomal structure and biogenesisRNA processing and modification

Cell wall/membrane/envelope biogenesisSignal transduction mechanismsDefence mechanismsCell division and chromosome partitioningReplication, recombination and repairTranscription

Cell motilityCytoskeletonExtracellular structures

Amino acid transport and metabolismCarbohydrate transport and metabolismEnergy production and conversionPost-translational modification, protein turnover and chaperonesIntracellular trafficking, secretion and vesicular transport

Secondary metabolites biosynthesis, transport and catabolismInorganic ion transport and metabolismLipid transport and metabolismCoenzyme transport and metabolismNucleotide transport and metabolism

Function unknownGeneral function prediction only

Fig. 4. Chromosome and plasmid map of A. mimigardefordensis strain DPN7T. Genes encoded by the leading and the laggingstrand (circles 1 and 2, respectively) of the chromosome and plasmid (40-fold enlarged relative size compared with thechromosome) of A. mimigardefordensis strain DPN7T are marked in red and blue, respectively. Circle 3 represents protein-coding regions according to COGs categories. The two inner circles represent the GC content and the GC skew, respectively.




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van den Berg, 2001; Burgmann et al., 2007; Todd et al.,2007). In A. mimigardefordensis strain DPN7T, DTDP ismost probably transported into the cell via the TTT familysystem (Table S3, Fig. S1A; MIM_c08710–MIM_c08730; C.Meinert, unpublished) and cleaved into two molecules of3MP by the disulfide reductase LpdA (Fig. S1B; MIM_

c19220; Wubbeler et al., 2010). 3MP is then oxygenated bythe 3MP dioxygenase Mdo (Fig. S1C; MIM_c31400)yielding 3SP (Bruland et al., 2009), which is activated tothe corresponding CoA thioester by the CoA ligase SucCD(Fig. S1D; MIM_c18280–MIM_c18290; Schurmann et al.,2011). The next step is the abstraction of sulfite from3-sulfinopropionyl-CoA by a reaction of the acyl-CoAdehydrogenase-like desulfinase AcdA (Fig. S1C; MIM_

c31390; Schurmann et al., 2013) and subsequentlypropionyl-CoA enters the central metabolism via themethylcitric acid cycle (Wubbeler et al., 2008). Regula-tion of this pathway is presumably realized by atranscriptional regulator of the XRE (xenobiotic responseelement) family (MIM_c31360), which was located on thechromosome in the vicinity of acd and mdo (Fig. S1C).

The cells of the taxonomically proximate type strain wereincapable of using DTDP or its degradation intermediate3SP as a carbon source (Wubbeler et al., 2006). It is highlyprobable that the reason for this noticeable distinctionbetween A. mimigardefordensis strain DPN7T and A.kashmirensis WT001T is the absence of operating para-logues to mdo (MIM_c31400) and acdA (MIM_c31390) –the two key enzymes in the catabolic pathway of DTDP.The detected genes with the highest sequence similarities inA. kashmirensis WT001T were TKWG_22195 and TKWG_

25370, showing only 49 and 32 % identities to mdo andacdA, respectively. The other two metabolic genes knownto be involved in DTDP catabolism were present in the A.kashmirensis genome: the disulfide reductase (TKWG_

11515; 87 % identical aa to MIM_c19220) and the CoAligase (TKWG_10860–TKWG_10865; .97 % identical aato MIM_c18280–MIM_c18290), which both showed very

high sequence similarities to the orthologues in A. mimi-gardefordensis strain DPN7T.

Furthermore, an operon of the proposed transport systemfor uptake of DTDP could not be predicted for A.kashmirensis. However, the import of DTDP in A.mimigardefordensis strain DPN7T is presumably carriedout by the TTT system (C. Meinert, unpublished) and thesupposed procedure is described in the following (for agraphical overview, see Fig. S2). If DTDP is available, itbinds most probably to the Bug-like extracytoplasmicsolute receptor-binding protein TctC (MIM_c08730).Loaded TctC putatively partitions between the transportpathway via interactions with TctBA (MIM_c08720–MIM_c08710), and the signalling pathway via interactionswith the periplasmic domain of one of the putative sensorkinases TctE (MIM_c39200, MIM_c22990, MIM_c17190),which were located immediately adjacent to three tctAB.Activation of the signal transduction cascade with involve-ment of a putative cytoplasmic response regulator TctD(MIM_c39210, MIM_c23000, MIM_c17200) could resultin an upregulation of the tctCBA transcription. tctC wasdesignated originally as bug (Bordetella uptake gene)because it showed significant sequence similarities to agene family that was found to be strongly over-representedin Bordetella (Antoine et al., 2003). However, Bug-likeextracytoplasmic solute receptor-binding protein encodinggenes (termed tctC) are also abundant in the genomes ofmany other betaproteobacteria (Table 2). The number ofgenes coding for TctC tremendously outnumbers the genescoding for the predicted membrane and sensor compo-nents of the TTT system (Huvent et al., 2006) (Table 2).Therefore, an additional function of this protein insignalling cascades was postulated. Solute receptor proteinsthat participate in such signal transduction cascades couldbe useful for activating transport or metabolic pathways inresponse to the presence of their specific ligands (Antoineet al., 2005). Interestingly, the majority of orphan tctC in A.mimigardefordensis strain DPN7T was often located in thedirect vicinity of genes coding for transcriptional regula-tors, as was also reported for the abundant tctC in thegenome of Cupriavidus necator H16 (Pohlmann et al.,2006), thus supporting the theory of a regulatory role ofthese Bug-like extracytoplasmic solute receptor-bindingproteins. The low number of tctA in the genome of A.kashmirensis is a striking difference to A. mimigardefor-densis strain DPN7T on the genomic level (Table 2). Itshould be noted that the relative number of genes codingfor bacterial main transport systems is comparably high inthe A. mimigardefordensis genome (Table 2). The posses-sion of a convenient number of transporter-encoding genesis an important prerequisite for the ability to utilizexenobiotic compounds like DTDP and 3SP in the firstplace. However, not only is an uptake system necessary, butthe existence of suitable exporters for disposal of toxicintermediates is mandatory for a successful adaptation ofxenobiotics degradation. In addition to the putative sulfateexporter (MIM_c23530), which is responsible for the

Table 1. General features of the genome of A. mimigarde-

fordensis strain DPN7T

Characteristic Count Per cent of

total

Size of the genome 4 764 126 bp 100

chromosome 4 740 516 bp 99.5

plasmid 23 610 bp 0.5

DNA G+C content 2 583 218 bp 54.22

DNA coding sequence 4 113 819 bp 86.35

Predicted protein-coding genes 4091 98.91

with putative function 3692 89.26

with unknown function 399 9.65

RNA genes 45 1.09

rRNA genes 6 0.15

tRNA genes 39 0.94

Pseudogenes 7 0.17




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Table 2. Heat map of abundant genes in the genome of A. mimigardefordensis strain DPN7T and comparison with elected proteobacteria

Colours of the heat map emphasize the comparative quantity of the gene in the particular column: green, most abundant; red, least abundant.

Bacteria Genome

size

(protein)

ABC

(ATPase)

TctC

(TTT)

LysR MFS SDR Acyl-CoA

DH

Aldehyde

DH

DctQ

(TRAP-T)

Thiolase TauE

(exporter)

TctA

(TTT)

n % n % n % n % n % n % n % n % n % n % n %

Advenella mimigardefordensis DPN7 4091 140 3.4 130 3.2 121 3.0 62 1.5 36 0.9 32 0.8 26 0.6 11 0.3 9 0.2 9 0.2 7 0.2

Advenella kashmirensis WT001 3933 88 2.2 122 3.1 102 2.6 50 1.3 32 0.8 21 0.5 15 0.4 11 0.3 10 0.3 6 0.2 1 0.03

Burkholderia xenovorans LB400 8951 204 2.3 13 0.1 186 2.1 151 1.7 101 1.1 56 0.6 62 0.8 3 0.03 17 0.2 7 0.08 5 0.06

Burkholderia mallei ATCC 23344 5414 95 1.8 0 0 82 1.5 78 1.4 32 0.6 20 0.4 24 0.4 0 0 7 0.1 1 0.02 0 0

Bordetella pertussis Tohama I 3761 97 2.6 86 2.3 87 2.3 38 1.0 29 0.8 22 0.6 17 0.5 10 0.3 8 0.2 5 0.1 2 0.05

Ralstonia solanacearum GMI1000 5120 76 1.5 2 0.04 78 1.5 65 1.3 39 0.8 16 0.3 17 0.3 0 0 6 0.1 7 0.1 1 0.02

Roseobacter denitrificans OCh 114 4146 117 2.8 4 0.1 28 0.7 17 0.4 26 0.6 13 0.3 10 0.2 19 0.5 4 0.1 5 0.1 4 0.1

Cupriavidus necator H16 6629 125 1.9 156 2.4 176 2.7 89 1.3 63 1.0 69 1.0 36 0.5 6 0.9 23 0.3 10 0.2 5 0.08

Cupriavidus necator N-1 7832 132 1.7 193 2.5 233 3.0 121 1.5 87 1.1 69 0.9 55 0.7 5 0.6 29 0.4 9 0.1 3 0.04

Variovorax paradoxus B4 6590 190 2.9 172 2.6 190 2.9 67 1.0 66 1.0 33 0.5 30 0.5 17 0.3 14 0.2 9 0.1 7 0.1

Escherichia coli K12 DH1 4160 77 1.9 0 0 53 1.3 62 1.5 14 0.3 4 0.1 14 0.3 1 0.02 5 0.1 1 0.02 0 0

ABC (ATPase), genes coding for the ATP-binding domain-containing protein of the ABC transport system (Pfam: 00005/12848/09821); TctC (TTT), genes coding for the putative Bug (Bordetella uptake gene)-like

extracytoplasmic solute-binding receptor of the TTT family (Pfam: 03401); LysR, genes coding for transcriptional regulators of the LysR family (Pfam: 00126/03466); MFS, genes coding for major facilitator symporter

(Pfam: 07690/13347/05977); SDR, genes coding for short-chain dehydrogenases/reductases (Pfam: 00106); Acyl-CoA DH, genes coding for acyl-CoA dehydrogenase-like proteins (Pfam: 02771/02770/00441/12186/08028/

12418); Aldehyde DH, genes coding for aldehyde dehydrogenases (Pfam: 00171); DctQ (TRAP-T), genes coding for the membrane protein DctQ of the TRAP-T system (Pfam: 04290); thiolase, genes coding for acetyl-CoA

acetyltransferases (thiolases II) and thiolases I (Pfam: 00108/02803); TauE (exporter), genes coding for a putative exporter of sulfite and OSCs (Pfam: 01925); TctA (TTT), genes coding for the large transmembrane protein

of the TTT family (Pfam: 01970).

n, Number of encoding genes in the respective genome; %, per cent of the gene count in the specific genome.

Reasons for selection of bacteria included in this table in addition to A. mimigardefordensis DPN7: A. kashmirensis WT001 is the closest related type strain. Burkholderia xenovorans LB400 is a diverse and versatile non-

pathogenic bacterium that possesses one of the largest annotated bacterial genomes. Burkholderia mallei ATCC 23344, Bordetella pertussis Tohama I and Ralstonia solanacearum GMI1000 are well-studied pathogens with

different specializations. Roseobacter denitrificans OCh 114 belongs to the metabolically diverse purple alphaproteobacteria that are abundant in marine environments. C. necator H16 is able to utilize DTDP and 3MP for the

synthesis of hetero-PTEs. C. necator N-1 can grow with 3SP as the sole carbon source, and Variovorax paradoxus B4 was isolated and annotated due to its ability to use 2-mercaptosuccinic acid for growth as the sole carbon,

energy and sulfur source. E. coli K12 DH1 serves as the best-studied representative of the gammaproteobacteria.

J.H.W

ubbelerand

others

14

08

Micro

bio

logy

16

0


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removal of excessive sulfate, an export system for theharmful intermediate 3MP must also exist in A. mimi-gardefordensis strain DPN7T. During growth with DTDP asthe sole carbon source, 3MP was detectable in highconcentrations in the culture supernatant (Wubbeleret al., 2008) and thus had to be somehow exported.Presumably, the cells exported 3MP to maintain theintracellular osmotic pressure and/or to prevent undesiredside reactions of this thiol. The export system is unknown,but it could be supposed that a TauE exporter is involved,which was described recently as an exporter of sulfite andOSCs (Mayer et al., 2012), and whose correspondingencoding genes are abundant in the genome of A.mimigardefordensis strain DPN7T (Table 2).

Propionate. The catabolism of monocarboxylates such aspropionate is of major importance for bacterial strains,both in their natural habitats as well as during biotechnicalapplications. As only little information is available aboutthe transport of propionate in bacterial cells (Jolkver et al.,2009), it is merely supposed that a transporter of the SSS(solute : sodium symporter) family (TCDB 2.A.21; TableS3) is involved in propionate uptake in A. mimigarde-fordensis strain DPN7T. Inside the cells, propionate ispresumably ligated with CoA by putative acyl-CoA synthe-tases (MIM_c14630, MIM_c23610, MIM_c38630) yieldingpropionyl-CoA. No genes encoding methylmalonyl-CoAmutase, pyruvate ferredoxin oxidoreductase or propionate-CoA transferase, which are the enzymes necessary for themethylmalonyl-CoA pathway, reductive carboxylation and theconversion of lactoyl-CoA into lactate, respectively, weredetected. Nonetheless, all genes involved in the methylcitricacid cycle (Fig. S1C, E) and the b-oxidation of propionyl-CoAvia acryloyl-CoA and 3-hydroxypropionyl-CoA are present inthe genome of A. mimigardefordensis strain DPN7T; therefore,the metabolism of propionyl-CoA is most probably conductedvia these two pathways. Propionyl-CoA formed as an inter-mediate in the DTDP degradation pathway was undoubtedlymetabolized via the methylcitric acid cycle (Wubbeler et al.,2008). This assumption was supported by the finding that aTn5 : : mob insertion in the gene coding for a transcriptionalregulator of the XRE family (MIM_c31360), which waslocated between genes involved in the methylcitric acid cycleand the precedent catabolism of DTDP (Fig. S1C), impairedgrowth of the mutant jhw46 (Table S1) with DTDP orpropionate as the sole carbon source. The utilization of DTDPas a source of sulfur was not impaired in this Tn5 : : mob-induced mutant. It was therefore assumed that the sulfur fromDTDP could still be abstracted by the desulfinase AcdA andused by the cells of A. mimigardefordensis jhw46, but thepropionyl-CoA was not further metabolized to support cellgrowth, most probably due to an inoperable methylcitric acidcycle caused by the disruption of the XRE transcriptionalregulator (MIM_c31360; Fig. S1C).

Glycerol. The simple alcohol glycerol is an abundantsubstance found in nature: It is the structural constituentof several lipids and is frequently produced during

osmoregulation in yeasts (Wang et al., 2001). Thus, manyknown bacteria are able to use glycerol as the sole source ofcarbon and energy, and consequently its application insome industrial processes is a good alternative to tradi-tional carbohydrates (da Silva et al., 2009). In A. mimigar-defordensis strain DPN7T, glycerol was applied successfullyto improve the biotechnical production of PTEs (Xia et al.,2012). The stability of the pH in the cultivation mediumwas the main advantage for employing glycerol as thecarbon source in this process. Transport of glycerol in thecell is usually conducted by passive diffusion through thecytoplasmic membrane and/or via a facilitated diffusionachieved by an integral membrane protein of the MIP(major intrinsic protein) family (Lin, 1976; Romano,1986). A. mimigardefordensis strain DPN7T harbours onegene putatively encoding a MIP (MIM_c22510) and istherefore probably capable of facilitated uptake of glycerol.The catabolic pathway of glycerol in A. mimigardefordensisstrain DPN7T could proceed via two ways. (i) It starts withactivation by a glycerol kinase (MIM_c01080, MIM_c04710)into sn-glycerol 3-phosphate, continuing with conversion indihydroxyacetonephosphate by a glycerol 3-phosphatedehydrogenase (MIM_c01190, MIM_c32210) and leads inthe glycolysis or gluconeogenesis. (ii) Glycerol couldputatively be converted into glyceraldehyde by an aldo/keto reductase (MIM_c13750, MIM_c17820, MIM_c21070).Afterwards, one of the 26 aldehyde dehydrogenases (Table 2)catalyses the transformation into glycerate, followed by theconversion into glycerate 3-phosphate by a glycerate kinase(MIM_c26670). Glycerate 3-phosphate is then furthermetabolized via the Embden–Meyerhof–Parnas pathway.In contrast, the closest related type strain A. kashmirensisWT001T was not able to grow with glycerol as the solecarbon source (Ghosh et al., 2005). The putative glyceroluptake facilitator of the MIP family is present in the A.kashmirensis genome (TKWG_13070), but a frame-shift inits only gene encoding a glycerol 3-phosphate dehydro-genase (TKWG_20235, TKWG_20230) is most probably thecause of the inability to use glycerol for growth. Curiously,the alternative pathway via glyceraldehyde, glycerate andglycerate 3-phosphate could also not be accomplished,because its gene encoding glycerate kinase contains twoframe-shifts (TKWG_08145, TKWG_08150, TKWG_08155).

Sugars and gluconate. Various sugars such as arabinose,D-fucose, D-galactose, D-glucose, D-ribose and D-xylosesupport growth, but A. mimigardefordensis was not able togrow with D-fructose, D-mannose and L-xylose, forexample, as sole carbon sources (Wubbeler et al., 2006;Matsuoka et al., 2012; C. Meinert, unpublished).Interestingly, A. mimigardefordensis strain DPN7T doesnot have a complete phosphotransferase system (PTS);only the genes encoding Enzyme I (EI), EIIA and thephosphocarrier protein HPr were detected (MIM_c21900–MIM_c21920). Transport of glucose by a PTS is the mostcommon uptake system in Gram-negative bacteria,whereas the transport via glucose facilitators [e.g. majorfacilitator superfamily (MFS) type] and ATP-binding




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cassette (ABC) transport systems is the exception (Jahreiset al., 2008). The genome of A. mimigardefordensis strainDPN7T harbours a putative monosaccharide ABC transportsystem (MIM_c39770–MIM_c39800), but the commonlyadjacent sugar kinase regulator is missing and glucosetransport was therefore most probably not conducted in thisway. It is theoretically possible that some sugars could betransported into the cell by MFS-type proteins (Table 2), butrecent data of our laboratory indicated the involvementof the TRAP-T (tripartite ATP-independent periplasmictransporter) family (Table S3; MIM_c39430–MIM_c39450)in the uptake of L-arabinose, D-fucose, D-galactose,D-glucose, D-gluconate and D-xylose (C. Meinert,unpublished). Apparently, there are no genes encodinghexokinases (COG5026), glucokinases (COG0837) orgluconokinases (COG3265) present in the genome, but aribokinase was identified (MIM_c39760). Also, no genecoding for a 6-phosphofructokinase (COG0205) wasdetectable and accordingly glycolysis is not completelyexecutable. However, gluconeogenesis is operable due to tworecognized genes (MIM_c14350, MIM_c23250) encodingfructose 1,6-bisphosphatase. On account of these results andbased on the in silico analyses, it was proposed that glucoseand gluconate are catabolized via the semi-phosphorylatedEntner–Doudoroff pathway (Conway, 1992).

Taurine. The abundant sulfonate taurine (2-aminoethan-sulfonic acid) is frequently used as source of sulfur bymicro-organisms (Cook & Denger, 2002). Taurine is anon-proteinogenic amino acid, and occurs as a majororganic solute in all vertebrates and in a wide range ofmarine invertebrates. In E. coli, the use of taurine as asulfur source was attributed to the tauABCD gene clusterthat encodes a specialized sulfonate–sulfur utilization (ssu)system, which is in most cases exclusively involved inutilization of this substrate (van der Ploeg et al., 1996;Eichhorn et al., 2000; Cook et al., 2006). A. mimigar-defordensis strain DPN7T was able to grow with taurine asthe sole source of carbon and sulfur (Wubbeler et al.,2008). The uptake of taurine is most probably catalysed byABC-type transport systems encoded by tauABC (TableS3). The subsequent release of sulfite is supposedlycatalysed by TauD, an Fe(II) a-ketoglutarate-dependenttaurine dioxygenase (MIM_c03960, MIM_c04000, MIM_c14220, MIM_c38710). This enzyme is absolutely depen-dent on ferrous iron, has a tetrameric structure (Knaueret al., 2012), and converts taurine into sulfite andaminoacetaldehyde. An alternative pathway is conductedby the deamination of taurine into sulfoacetaldehyde,catalysed by the taurine dehydrogenase (MIM_c36670,large subunit; MIM_c36680, small subunit) with theinvolvement of cytochrome c. Sulfoacetaldehyde ispresumably further metabolized by the sulfoacetaldehydeacetyltransferase Xsc (MIM_c36570), resulting in acetylphosphate and sulfite. This desulfonation reaction wasconsidered to be the key reaction of this pathway(Bruggemann et al., 2004). The phosphate acetyltrans-ferase Pta (MIM_c36580) converts acetyl phosphate into

acetyl-CoA, which is then accessible for the centralmetabolism. Thus, the respective gene cluster responsiblefor uptake and degradation of taurine in A. mimigar-defordensis strain DPN7T via aminoacetyldehyde wasidentified as MIM_c38710–MIM_c38740 (Fig. S1F) andvia sulfoacetaldehyde as MIM_c36630–MIM_c36680 (Fig.S1G).

Aliphatic sulfonates. As a fundamental component for alllife, sulfur appears in organisms as both inorganic andorganic compounds. Excluding PTE-producing bacterialstrains, for example, up to 1 % (w/v) of the typical cell dryweight consists of sulfur (Kertesz, 2000), mainly due tocysteine and methionine as components of proteins(Sievert et al., 2007). Additionally, sulfur plays a key rolein iron–sulfur clusters of proteins. Examples of vital OSCsare CoA, biotin (vitamin H or B7), thiamine (vitamin B1),several sulfolipids, various secondary metabolites and theOSCs responsible for the maintenance of cellular redoxpotentials: coenzyme B, ergothiol, glutathione, mycothiol,ovothiol and trypanothione (Hand & Honek, 2005). Thesulfur required for biosynthesis processes is generallyderived from the assimilation of sulfate by plants andbacteria (Kertesz, 2000). In natural environments, onlysmall amounts of sulfur are present as sulfate. Instead,the major amount of sulfur is only accessible as aheterogeneous mixture of diverse OSCs, e.g. sulfateesters, sulfonates and peptides (Mirleau et al., 2005).Therefore, numerous micro-organisms have developed avariety of pathways to make sulfur available for theircentral metabolism. The active inter-conversion of organicand inorganic sulfur forms in soil is due to microbialactivities, and a plant-growth-promoting effect wasdemonstrated for desulfonating bacterial strains (Kertesz& Mirleau, 2004; Schmalenberger & Kertesz, 2007;Schmalenberger et al., 2008). The ssu genes are known tobe responsible for uptake and degradation of aliphaticsulfonates other than taurine (Eichhorn et al., 2000);accordingly, nine putative ssuABC clusters, which code forABC-type aliphatic sulfonate transport systems, wereidentified in the genome of A. mimigardefordensis strainDPN7T (Table S3). Interestingly, the ssuCBA genes with thelocus tag MIM_c03970–MIM_c03990 are encircled by twotauD (MIM_c03960, MIM_c04000). Furthermore, thessuACB gene cluster with the locus tag MIM_c01510–MIM_c01530 (Fig. S1H) is flanked by all the three genes inthe genome that encode alkanesulfonate monooxygenasesSsuD (MIM_c01490, MIM_c01540, MIM_c01550) andanother ssuACB cluster (MIM_c01580–MIM_c01560).SsuD is able to desulfonate a wide range of aliphatic, butnot aromatic sulfonates, with the above-mentionedexception of taurine (Eichhorn et al., 2000). To performthis desulfonation reaction, the two-component SsuDneeds FMNH2 as a substrate, which is provided by theassociated FMN reductase SsuE (MIM_c01500). Inaddition to the nine ssuABC paralogues, another ABC-type nitrate/sulfonate/bicarbonate transport system wasidentified (MIM_c16790–MIM_c16810) and also the




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structurally related nitrate ABC transporters wereidentified (MIM_c09400–MIM_c09420; MIM_c34060–MIM_c34080).

Sulfate metabolism. The import of extracellular sulfateinto cells of A. mimigardefordensis strain DPN7T is per-formed by the specific ABC transport system CysPTWA(Table S3; MIM_c15530–MIM_c 5500), which belongs tothe SulT family (TC 3.A.1.6), and an MFS-type sulfatepermease (Table S3; MIM_c24690) of the SulP family (TC2.A.53) (Aguilar-Barajas et al., 2011). A CysZ-like sulfatepermease (TC 9.B.7) was not identified in the genome of A.mimigardefordensis strain DPN7T. For the subsequentassimilation of sulfur from inorganic sulfate, which seemsto be similar in all prokaryotes, activation by coupling to anucleoside is mandatory and could be accomplished by asulfate adenylyltransferase (cysD/cysN, MIM_c15480/MIM_

c15490) yielding adenosine 59-phosphosulfate (APS). Thenext step is the reduction of APS by the APS reductase (EC1.8.4.10) (MIM_c15470) releasing sulfite (Bick et al., 2000).Apparently, phosphoadenosine 59-phosphosulfate cannot beformed in A. mimigardefordensis strain DPN7T, as no geneencoding adenylylsulfate kinase was identified in thegenome. Sulfite is converted into sulfide by the sulfitereductase (cysI/cysJ, MIM_c29690/MIM_c29700), whichcatalyses a six-electron reduction of sulfite to sulfide(Crane et al., 1995). Sulfide is then metabolized andusually utilized to generate cysteine, either by the cysteinesynthase CysK (MIM_c16980) or CysM (MIM_c22720).

A. mimigardefordensis strain DPN7T was also not able tooxidize sulfane sulfur species directly to sulfate, as iscommon for some alphaproteobacteria (Friedrich et al.,2005). Thus, an entire inorganic sulfur compoundoxidation gene cluster (sox operon) was not detected inthe genome of A. mimigardefordensis strain DPN7T, butpart of the sox operon was reported to exist in A.kashmirensis WT001T (Ghosh et al., 2011), enabling strainWT001T to utilize tetrathionate and thiosulfate chemo-lithotrophically (Ghosh et al., 2005). Paralogues to soxA,soxX, soxW, soxS and soxL were not recognized in thegenome of strain DPN7T. Therefore, A. mimigardefordensisstrain DPN7T was not able to oxidize completely reducedsulfur compounds and subsequently use the resultingelectrons of this process for energy transformation.However, five sulfite oxidases were detected, which allshowed homology to, and could possibly act and beannotated as, soxC (MIM_c01600, MIM_c03760, MIM_

c12620, MIM_c24010, MIM_c36020). Downstream of twoof these soxC-like genes, homologous genes putativelycoding for SoxD were located (MIM_c01590, MIM_

c36010), which are important in the detoxification processof sulfite acting as sulfite oxidases. Furthermore, a putativedimanganese-containing 59-nucleotidase-encoding genesoxB (MIM_c17110), a putative regulator soxR (MIM_

c02390) as well as a fused gene soxYZ (MIM_c36710)coding for the covalently sulfur-binding/chelating proteinwere detectable in A. mimigardefordensis strain DPN7T.

Sulfite and sulfate export. The microbial degradation ofeither naturally occurring or xenobiotic pollutant OSCs suchas dibenzothiophene (McFarland, 1999), di-n-octylsulfide(Kirkwood et al., 2005), dimethylsulfoniopropionic acid(Yoch, 2002) or various C2- (Cook & Denger, 2002) and C3-sulfonates (Cook et al., 2006) has been studied in detail, andthe metabolism of OSCs is an essential part of the globalsulfur cycle (Sievert et al., 2007). During microbialutilization of DTDP as the sole carbon source, the excesssulfur was abstracted from 3SP-CoA as sulfite (Schurmannet al., 2013). Accumulation of charged sulfite species likeHSO3

2 or SO322 leads to reactions with cell constituents,

such as coenzymes, cofactors and nucleic acids, and as aconsequence most likely to irreversible inhibition of cellgrowth (Gunnison, 1981). Despite its toxicity, sulfite is atransient intermediate during the catabolism of cysteineand taurine, as has been known in the degradation ofarylsulfonates and alkylsulfonates for a long time (Kondo &Ishimoto, 1972). However, sulfite is a strong nucleophilethat may convert protein disulfides into sulfonated cysteinederivatives (Bailey & Cole, 1959). As the integrity of disulfidebonds is crucial to the tertiary structure of proteins, it mayhave strong negative effects on growth. If only DTDP, 3SP ortaurine are available as the main carbon source, theoccurrence of excessive sulfite is inevitable. The sulfiteanion is usually not excreted in huge quantities into themedium (Cook et al., 2006) and thus a detoxificationprocess performed by the cells is essential. Commonly, theresulting sulfite is oxidized to sulfate, either by sulfiteoxidases (EC 1.8.3.1) or sulfite dehydrogenases (EC 1.8.2.1)(MIM_c01600, MIM_c03760, MIM_c12620, MIM_c24010,MIM_c36020), which both (COG2041) catalyse the directoxidation of sulfite to sulfate in eukaryotes or prokaryotes,respectively (Feng et al., 2007; Kappler, 2007). Thealternative AMP-dependent pathway with the formation ofadenylyl phosphosulfate is not accomplished by A.mimigardefordensis, because no genes encoding adenylyl-sulfate reductase (EC 1.8.99.2) were detectable in thegenome. Presumably, large amounts of sulfate wereexclusively excreted by a putative membrane protein(Table S3; MIM_c23530; COG2855), which belongs to theCPA (cation : proton antiporter) superfamily, moreprecisely to the PSE (putative sulfate exporter) family(Transporter Classification Database 2.A.98; Bruggemannet al., 2004; Rein et al., 2005; Saier et al., 2009). The encodinggene in A. mimigardefordensis was disrupted by Tn5 : : mobinsertions (Fig. S1I), and the corresponding Tn5 : : mob-induced mutants JG7 and LR32 (Table S1) exhibited a clearphenotype: no growth with DTDP, 3SP or taurine as carbonsource. This very interesting finding confirmed the exigencyof an operable export system for the maintenance of aconstant osmotic pressure in the cell, as also proposedpreviously for bacteria that were growing with an OSC as thesole carbon source (Rein et al., 2005; Cook et al., 2006).Apparently, the gene with the locus tag MIM_c23530 (Fig.S1I) is the only executable sulfate exporter in A.mimigardefordensis, which was verified to be obligatoryduring utilization of DTDP, 3SP and taurine.




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Protein secretion systems and biosynthesis ofstorage compounds

Protein secretion systems. Three complete systems wereidentified from the major translocation systems for thesecretion of proteins across the cytoplasmic membraneknown in bacteria (Desvaux et al., 2009) in A. mimi-gardefordensis strain DPN7T: the well-known TAT pathwayis encoded in one gene cluster (Table S4), the general Secpathway, whose encoding genes are distributed on thechromosome (Table S4), and the type VI secretion system(T6SS; Table S4). The T6SS is versatile and not yetcompletely understood (Silverman et al., 2012). It was firstrecognized, described and later on primarily studied inpathogenic bacteria (Pukatzki et al., 2006; Silverman et al.,2012), and is therefore commonly referred to as a virulencefactor. The peculiar feature is that many pathogens possessat least one T6SS, but most bacteria with a recognized T6SSare not pathogenic (Jani & Cotter, 2010). T6SS playsundoubtedly an important role in virulence, e.g. it isrequired in Burkholderia mallei for the virulence of thispathogen, as demonstrated in a hamster model (Burtnicket al., 2010). Otherwise, the T6SS is involved in interbacterialinteractions (Hood et al., 2010) and can also act as anantipathogenesis factor (Parsons & Heffron, 2005). The twoproteins secreted in a T6SS-dependent manner are basicallythe haemolysin coregulated protein (Hcp) and the valine-glycine repeat protein G (VgrG), which both showed acodependency for export. Interestingly, the genome of A.mimigardefordensis strain DPN7T harboured nine distinctVgr-encoding genes. Three vgrG were identified in the Vibriocholerae O1 genome (Pukatzki et al., 2006) and 10 vgrparalogues were identified in the genome of Pseudomonasaeruginosa PAO1 (Hachani et al., 2011). Thus, the fact thatsome bacteria possess a large number of vgr genes isremarkable, but the reason is still the subject of research.

Polyhydroxyalkanoates (PHAs) and PTE synthesis. Cellsof A. mimigardefordensis accumulated polyhydroxybutyrate(PHB) as an intracellular storage compound (Wubbeleret al., 2006). They were also capable of synthesizing PTE, ifcultivated under the appropriate conditions and treatedwith the necessary genetic modifications (Xia et al., 2012).PHAs (Fig. 1) are polyesters synthesized as storage com-pounds by a wide variety of micro-organisms (Steinbuchel,1991) that can be biotechnically produced by applyingrenewable primary products (Tan, 2004), such as low-costcarbon sources from agriculture. Synthesis and accumu-lation are implemented in bacterial strains if a carbonsource is excessive for growth and another macroelement isrestricted (Potter & Steinbuchel, 2005).

Biosynthesis of PHB depends on three different enzymes(PhaABC) and an enhanced concentration of acetyl-CoAinside the cells. One phaCBR operon was identified in thegenome of A. mimigardefordensis (MIM_c13530–MIM_c13550), but phaA was unexpectedly located elsewhere(MIM_c21300). The genes encoding the PHA synthesisrepressor PhaR (MIM_c13550) and the PHA synthase

PhaC (MIM_c13530) had no paralogues in the genome,whereas phaA and phaB homologues, b-thiolases, andacetoacteyl-CoA reductases were abundant (Table 2). Theabundance of isologs of phaA and phaB is common forPHA-synthesizing bacteria, and was also described, forexample, for C. necator (Pohlmann et al., 2006) – the modelorganism of PHA research (Reinecke & Steinbuchel, 2009).

Another important factor in the biosynthesis of PHB is thesmall non-catalytic amphiphilic phasin proteins (Potter &Steinbuchel, 2005). Phasins (PhaP) are synthesized byPHB-accumulating bacteria, which are localized at, andinteract with, the surface of PHB granules. The genome ofA. mimigardefordensis strain DPN7T harbours two genesencoding PhaP. In silico analyses and comparison withsequence data of C. necator H16 revealed that one of thesephaP (MIM_c10360) is an orthologue of the major phasin-encoding gene phaP1 (H16_A1381; Neumann et al., 2008)and the second phaP (MIM_c36280) is an orthologousgene of phaP5 (H16_B1934). PhaP5 of C. necator is notessential for granule formation (Pfeiffer & Jendrossek,2011), but interacts with the multifunctional proteinPhaM, which is responsible for anchoring PHB granuleswith the cell DNA prior to segmentation (Pfeiffer et al.,2011). The gene in A. mimigardefordensis strain DPN7T

(MIM_c04510) with the highest sequence similarity tophaM (H16_A0141) has only low (33 %) deduced aminoacid identities. Further important PHB granule-associatedproteins involved in homeostasis (Brigham et al., 2012)and degradation are the PHB depolymerases PhaZ and thePHB oligomer hydrolases PhaY. Two phaZ homologueswere detectable in A. mimigardefordensis strain DPN7T, one(MIM_c24170) is an orthologue to phaZ1 from C. necatorand the other (MIM_c30120) to phaZ6. The identified geneencoding PhaY (MIM_c18780) showed the highest sim-ilarities to C. necator phaY2.

In addition to the ability to synthesize polyoxoesters such asPHB, cells of A. mimigardefordensis strain DPN7T are also ableto activate and incorporate 3MP as a building block of PTE,which is accomplished by unknown enzymes and its ownPhaC, if supplied with DTDP as precursor (Wubbeler et al.,2006; Xia et al., 2012). As with C. necator (Lutke-Eversloh &Steinbuchel, 2003), WT A. mimigardefordensis only producesthe heteropolymer poly(3HB-co-3MP). However, after engin-eering of the metabolism when (i) mdo (MIM_c31400) wasdeleted, (ii) the genes from Clostridium acetobutylicum codingfor a butyrate kinase (buk) and a phosphotransbutyrylase(ptb) were introduced into the genome, and (iii) its own phaCwas overexpressed on a suitable vector in addition to the copyon the genome, cells of A. mimigardefordensis producedpoly(3MP) homopolymer up to 25 % (w/w) of the cell dryweight if cultivated in MSM with glycerol as carbon sourceand DTDP as sulfur-containing precursor (Xia et al., 2012).A. mimigardefordensis is at present the best choice for anoptimized production of poly(3MP) applying non-toxicprecursor substrates such as DTDP and/or to establish aPTE synthesis process using only economical alternativeresources as carbon and sulfur sources.




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Conclusions

The detailed information about the genome of A. mimi-gardefordensis strain DPN7T was beneficial for the completeelucidation of the DTDP catabolic pathway (Fig. 3). Inaddition, the three Tn5 : : mob-induced mutants presented inthis study (Table S1) contributed to the identification of theonly functioning exporter of sulfate (MIM_c23530) and ofan important transcriptional regulator (MIM_c31360) inDTDP catabolism. The metabolic genes involved are notclustered in a single operon and are also not localized on theplasmid pMIM24, as presumed at the start of the research onDTDP degradation. Two of the gene products involved, i.e.the CoA ligase (SucCD; MIM_c18280–MIM_c18290) andthe disulfide reductase (LpdA; MIM_c19220), were knownto be involved in other essential metabolic pathways. Thesuccinate-CoA ligase contributed to the TCA cycle and thedisulfide reductase dihydrolipoamide dehydrogenase waspart of the pyruvate dehydrogenase multienzyme complex(MIM_c19200–MIM_c19220). mdo and acdA were in factlocalized adjacent to each other (Fig. S1C), and both werepostulated to be key enzymes of DTDP catabolism. It istherefore supposed that the ability of DTDP degradationevolved due to (i) a broad substrate specificity of pre-existing enzymes and (ii) the duplication of enzyme-codinggenes followed by the subsequent accumulation of pointmutations in the DNA sequence, resulting in a novelassociation with specialized functions of these proteins thatled to the ability to degrade DTDP. It is well-known thatpromiscuous enzymes could be recruited to provide newfunctions when the catalysis of secondary reactions providesa selective advantage to the organism. The so-calledpatchwork combination was often employed in theevolution of degrading pathways for xenobiotic compounds(Copley, 2000; Schmidt et al., 2003; Wagner 2012; Mayeret al., 2012).

As the complete genome sequence of A. mimigardefordensisstrain DPN7T is now available, it will be possible toinvestigate the proteome during growth on DTDP or otherinteresting OSCs by 2D gel electrophoresis and MALDI-TOF. This approach will provide further insights, and willgreatly support and extend our current knowledge ofDTDP catabolism.

ACKNOWLEDGEMENTS

We gratefully acknowledge the assistance of Lena Radamm, JessicaGrote, Madeleine Hamley and Rita Weyer in characterizing mutantsof A. mimigardefordensis.

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