Open Access

Permanent draft genome sequence of Comamonas testosteroni KF-1

  • Michael Weiss,
  • , Anna I. Kesberg
  • , Kurt M. LaButti
  • , Sam Pitluck
  • , David Bruce
  • , Loren Hauser
  • , Alex Copeland
  • , Tanja Woyke
  • , Stephen Lowry
  • , Susan Lucas
  • , Miriam Land
  • , Lynne Goodwin,
  • , Staffan Kjelleberg
  • , Alasdair M. Cook,
  • , Matthias Buhmann
  • , Torsten Thomas
  • and David Schleheck,
Corresponding author

DOI: 10.4056/sigs.3847890

Received: 30 May 2013

Accepted: 30 May 2013

Published: 15 June 2013

Abstract

Comamonas testosteroni KF-1 is a model organism for the elucidation of the novel biochemical degradation pathways for xenobiotic 4-sulfophenylcarboxylates (SPC) formed during biodegradation of synthetic 4-sulfophenylalkane surfactants (linear alkylbenzenesulfonates, LAS) by bacterial communities. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 6,026,527 bp long chromosome (one sequencing gap) exhibits an average G+C content of 61.79% and is predicted to encode 5,492 protein-coding genes and 114 RNA genes.

Keywords:

Comamonas testosteroni KF-1aerobicGram-negativeComamonadaceaexenobiotic surfactant biodegradation

Introduction

Comamonas testosteroni strain KF-1 (DSM14576) was isolated for its ability to degrade xenobiotic sulfophenylcarboxylates (SPC), which are degradation intermediates of the synthetic laundry surfactants linear alkylbenzenesulfonates (LAS) [1]. LAS is in use worldwide (appr. 3 × 106 tons per year [2]) and consists of a complex mixture of linear alkanes (C10-C13) sub-terminally substituted by 4-sulfophenyl rings (i.e., 38 different compounds) [2]. Commercial LAS is completely biodegradable, as known for more than 50 years [3], e.g., in sewage treatment plants, and its degradation is catalyzed by heterotrophic aerobic bacterial communities in two steps. First, an initial degradation step is catalyzed by bacteria such as Parvibaculum lavamentivorans DS-1T [4] through activation and shortening of the alkyl-chains of LAS, and many short-chain degradation intermediates are excreted by these organisms, i.e., approximately 50 different SPCs and related compounds [1,5-8]. Secondly, the ultimate degradation step, i.e., mineralization of all SPCs, is catalyzed by other bacteria in the community, and one representative of these is Comamonas testosteroni KF-1. In particular, strain KF-1 was isolated from a laboratory trickling filter that had been used to enrich a bacterial community from sewage sludge that completely degraded commercial LAS and SPCs [1,6]. Strain KF-1 is able to utilize four individual SPCs (both enantiomers), namely R/S-3-(4-sulfopenyl)butyrate (3-C4-SPC), enoyl-3-C4-SPC, R/S-3-(4-sulfopenyl)pentanoate (3-C5-SPC), and enoyl-3-C5-SPC (see therefore also below), as novel carbon an energy sources for its heterotrophic aerobic growth [1,9,10].

The first Comamonas testosteroni (formerly Pseudomonas testosteroni [11]) strain, type-strain ATCC 11996, was enriched from soil and isolated in 1952 for its ability to degrade testosterone [12,13]. Since then, the physiology, biochemistry, genetics, and regulation of steroid degradation in this and in other C. testosteroni strains have been elucidated in great detail [e.g., 14-21]. Most recently, the genome of C. testosteroni ATCC 11996T has been sequenced in order to further improve the understanding of the molecular basis for the degradation of steroids [22].

In the environment, members of the genus Comamonas may also be important degraders of aromatic compounds other than steroids, especially of xenobiotic pollutants, since they have frequently been enriched and isolated for their ability to utilize (xenobiotic) aromatic compounds. For example, Comamonas sp. strain JS46 is able to grow with 3-nitrobenzoate [23], Comamonas sp. strain CNB-1 with 4-chloronitrobenzene [24], C. testosteroni T-2 with 4-toluenesulfonate and 4-sulfobenzoate [25], C. testosteroni WDL7 with chloroaniline [26], Comamonas sp. strain JS765 with nitrobenzene [27], Comamonas sp. strain B-9 with lignin-polymer fragments [28], C. testosteroni B-356 with biphenyl and 4-chlorobiphenyl [29], Comamonas sp. strain KD-7 with dibenzofuran [30], Comamonas sp. strain 4BC with naphthalene-2-sulfonate [31], or C. testosteroni SPB-2 (as well as strain KF-1) with 4‑sulfophenylcarboxylates [1]. In several C. testosteroni strains, the physiology, biochemistry, genetics, and/or regulation of the utilization of aromatic compounds have been elucidated [e.g., 10,23,25,27,29,32-48]. Furthermore, the genome sequence of (plasmid-cured) C. testosteroni CNB-2 has been published [24], and the sequence of its plasmid pCNB1 (of C. testosteroni CNB-1) [49], in order to further improve the understanding of the molecular basis for the ability of C. testosteroni to degrade such a large array of aromatic compounds.

Members of the genus Comamonas are able to cope with harsh environmental conditions such as high concentrations of arsenate [50,51], zinc [52], cobalt and nickel [53], or phenol [54], and can exhibit increased resistance to oxidative stress [55] or antibiotics [56]. Another C. testosteroni genome sequence, of strain S44, has recently been established in order to improve the understanding of the molecular basis for its resistance to increased concentrations of zinc [52]. Notably, an increased antibiotic resistance (and enhanced insecticide catabolism) as a consequence of induction of the steroid degradation pathway has been shown for C. testosteroni ATCC 11996T [56].

Here, we present a summary classification and a set of features for another C. testosteroni strain, strain KF-1, which has been genome-sequenced in order to improve the understanding of the molecular basis for its ability to degrade xenobiotic compounds, particularly xenobiotic, chiral 3-C4-SPC, and how this novel degradation pathway has been assembled in this organism, together with the description of its draft genome sequence and annotation. The genome sequence and its annotation have been established as part of the Microbial Genomics Program 2006 of the DOE Joint Genome Institute, and are accessible via the IMG platform [57].

Classifications and features

Morphology and growth conditions

C. testosteroni KF-1 is a rod-shaped (size, appr. 0.5 x 2 µm, Figure 1) Gram-negative bacterium that can be motile and grows strictly aerobically with complex medium (e.g., in LB- or peptone medium) or in a prototrophic manner when cultivated in mineral-salts medium [58] with a single carbon source (e.g., acetate). Strain KF-1 grows overnight on LB-agar plates and forms whitish-beige colonies [Table 1]. The strain grew with all amino acids tested (D-alanine, L-alanine, L-aspartate, L-phenylalanine, L-valine, glycine, L-histidine, L-methionine), but not with any of the sugars tested (D-glucose, D-fructose, D-galactose, D-arabinose, and D-maltose). Strain KF-1 utilized the following alcohols and carboxylic acids when tested (in this study): ethanol, acetate, glycerol, glycolate, glyoxylate, butanol, butyrate, isobutyrate, succinate, meso-tartaric acid, D- and L-malate, mesaconate, and nicotinate. Furthermore, strain KF-1 was positive for growth with poly-beta-hydroxybutyrate (this study). Strain KF-1 is able to utilize the steroids testosterone and progesterone (confirmed in this study), as well as taurocholate and cholate (and taurine and N-methyl taurine) [19], and taurodeoxycholate; strain KF-1 was tested negative for growth with cholesterol, ergosterol, 17β-estradiol and ethinylestradiol (this study), correlating with the findings for C. testosteroni strain TA441 [20].

Figure 1

Scanning electron micrograph of Comamonas testosteroni KF-1 . Cells derived from a liquid culture that grew in LB medium.

Table 1

Classification and general features of Comamonas testosteroni KF-1 according to the MIGS recommendations [59].

MIGS ID

    Property

     Term

    Evidence codea

    Current classification

     Domain Bacteria

    TAS [60]

     Phylum Proteobacteria

    TAS [61]

     Class Betaproteobacteria

    TAS [62,63]

     Order Burkholderiales

    TAS [62,64]

     Family Comamonadaceae

    TAS [65]

     Genus Comamonas

    TAS [11,66-69]

     Species Comamonas testosteroni

    TAS [11,68]

     Strain KF-1

    TAS [1]

    Gram stain

     Negative

    Cell shape

     small rod

    Motility

     Motile

    Sporulation

     non-sporulating

    Temperature range

     Mesophile

    TAS [1]

    Optimum temperature

     30ºC

    TAS [1]

    Carbon source

     3-(4-sulfophenyl)butyrate (3-C4-SPC) and other SPCs [see text],     4-sulfoacetophenone, 4-sulfophenyl acetate, 4-sulfophenol, testosterone,     progesterone, taurocholate, cholate, taurine, benzoate, 4-hydroxybenzoate, vanillate, isovanillate

    IDA,TAS [1,19]

    Energy source

     Chemoorganotroph

    TAS [1,6]

    Terminal electron receptor

     molecular oxygen

    TAS [1,6]

MIGS-6

    Habitat

     aerobic habitat

    TAS [1,6]

MIGS-22

    Oxygen requirement

     Aerobic

    TAS [1,6]

MIGS-15

    Biotic relationship

     free-living

    TAS [1,6]

MIGS-14

    Pathogenicity

     nonpathogenic, Risk group 1 (classification according to German TRBA)

MIGS-4

    Geographic location

     isolated from a LAS surfactant-degrading laboratory trickling filter (University of Konstanz, Germany)     that had been inoculated with sludge from a communal     sewage treatment plant (Herisau, Switzerland).

    TAS [1,6]

MIGS-5

    Collection date

     1999

    TAS [1,6]

MIGS-4.1

    Latitude

     47° 41' 27.24"

    TAS [1,6]

MIGS-4.2

    Longitude

     9° 11' 16.25"

    TAS [1,6]

MIGS-4.4

    Altitude

     440 m

    TAS [1,6]

a Evidence codes - IDA: Inferred from Direct Assay; TAS: Traceable Author Statement; NAS: Non-traceable Author Statement. These evidence codes are from the Gene Ontology project [70]. If the evidence is IDA, then the property was directly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements.

In respect to other aromatic compounds, strain KF-1 is known to utilize benzoate, 3- and 4-hydroxybenzoate, protocatechuate (3,4-dihydroxybenzoate), gentisate (2,5-dihydroxybenzoate), phthalate, terephthalate, vanillate, isovanillate, veratrate, 2- and 3-hydroxyphenylacetate (tested in this study, and ref. 1). Xenobiotic aromatic substrates for strain KF-1 known are the 4-sulfophenylcarboxylates R/S-3-(4-sulfophenyl)butyrate (R/S-3-C4-SPC), 3-(4-sulfophenyl)-∆2-enoylbutyrate (enoyl-3-C4-SPC), R/S-3-(4-sulfophenyl)pentanoate (R/S-3-C5-SPC), 3-(4-sulfophenyl)-∆2-enoylpentanoate (enoyl-3-C5-SPC), as well as the three xenobiotic metabolites in the 3-C4-SPC-pathway, 4-sulfoacetophenone (4-acetylbenzenesulfonate), 4-sulfophenol acetate, and 4-sulfophenol [1,9]. Finally, strain KF-1 did not utilize the following, other carbon sources tested (this study and refs. 1,9): n-alkanes (C6-C12), cycloalkanes (C8-C12), secondary-4-sulfophenylalkanes (LAS surfactants), secondary alkanesulfonates (SAS surfactants), dodecylsulfate (SDS surfactant), benzene sulfonate, 4-toluenesulfonate, 4-sulfobenzoate, phenylacetate, 3-phenylpropionate, 3- and 4-phenylbutyrate, 4-sulfostyrene, 4-sulfobenzoate, 4-sulfocatechol, cyclohexanone, 4-aminoacetophenone, gallic acid (3,4,5-trihydroxybenzoic acid) and gallotannic acid, pentanesulfonate, isethionate, sulfoacetate, D-tartaric acid, acetamide, gamma-aminobutyrate, oxalate, methanol, methylamine, methanesulfonate or formate, and not 2-C4-SPC (2-[4-sulfophenyl]butyrate), 4-C5-SPC, 4-C6-SPC, 5-C6-SPC, or any of the C7 – C9 SPCs generated during commercial LAS surfactant degradation.

C. testosteroni KF-1 has been recognized for its poor ability to form structured biofilms on surfaces [71] [see also ref. 72], or micro- or macroscopic cellular aggregates in liquid cultures [73], in direct comparison to ‘good’ biofilm forming organisms such as Delftia acidovorans SPH-1 [71], Pseudomonas aeruginosa PAO1 [73], or C. testosteroni SPB-2 [1].

No significant production of siderophores could be observed for C. testosteroni KF-1 when grown in presence of non-inhibitory levels of iron chelator 2,2'-dipyridyl [see 74], in comparison to siderophore-producing Delftia acidovorans SPH-1, Pseudomonas aeruginosa PAO1, and Pseudoalteromonas tunicata D2 [75] (reported in this study, data not shown).

Finally, strain KF-1 is able to grow in the presence of up to 500 µg/ml ampicillin or 600 µg/ml kanamycin in liquid cultures, as tested in this study.

Phylogeny

Based on its 16S rRNA gene sequence, strain KF-1 is a member of the genus Comamonas, which is placed in the family Comamonadaceae within the order Burkholderiales of Betaproteobacteria, as illustrated by a phylogenetic tree shown in Figure 2. Currently, 686 genome sequences of members of the order Burkholderiales of Betaproteobacteria, and 147 genome sequences within the family Comamonadaceae, have been, currently are, or are targeted to be established (GOLD database; May 2013).

Figure 2

Illustration of the phylogenetic position of Comamonas testosteroni KF-1 within the order Burkholderiales of Betaproteobacteria. The 16S rRNA gene alignment included the three other C. testosteroni strains whose genome sequences have been published, strain S44 [52], strain CNB-2 [24], and type-strain ATCC 11996 [22], and some of other genome-sequenced representatives of the family Comamonadaceae or of other families within the order Burkholderiales. The corresponding genome-project accession numbers, or 16S rRNA gene accession numbers, are indicated. “T” indicates a type strain. The sequences were aligned using the RDP tree builder [76] and displayed using MEGA4 [77]. Bootstrap values are indicated; bar, 0.02 substitutions per nucleotide position.

Genome sequencing information

Genome project history

The genome was selected for sequencing as part of the U.S. Department of Energy - Microbial Genomics Program 2006. The DNA sample was submitted in February 2006 and the initial sequencing phase was completed in July 2006. After the finishing and assembly phase the genome was presented for public access on January 2009; a modified version was presented (IMG) in August 2011. Table 2 presents the project information and its association with MIGS version 2.0 compliance [78].

Table 2

Project information

MIGS ID

     Property

    Term

MIGS-31.1

     Sequencing status

    Complete

MIGS-28

     Libraries used

    3.5 kb, 9 kb and 37 kb DNA libraries

MIGS-29

     Sequencing platforms

    Sanger

MIGS-31.2

     Sequencing depth

    12.8×

MIGS-30

     Assemblers

    Phred/Phrap/Consed

MIGS-32

     Gene calling method

    Prodigal

     Genbank ID

    17465

     Genbank Date of Release

    January 14, 2009

     GOLD ID

    Gi01330

MIGS-13

     Source material identifier

    DSM 14576

     Project relevance

    Biotechnological

Growth conditions and DNA isolation

Comamonas testosteroni KF-1, obtained from the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM14576), was grown on LB agar plates and transferred into selective medium (6 mM 4-sulfophenol/mineral-salts medium) in the 3-ml scale, and this culture was sub-cultivated in larger scale; cell pellets were stored frozen until DNA preparation. DNA was prepared following the JGI’s DNA Isolation Bacterial CTAB Protocol.

Genome sequencing and assembly

The genome of Comamonas testosteroni KF-1 was sequenced at the Joint Genome Institute (JGI) using a combination of 3.5 kb, 9 kb and 37 kb DNA libraries. All general aspects of library construction and sequencing performed at the JGI can be found at JGI website [79]. In total, 66.91 Mbp of Sanger sequence data were generated for the assembly from all three libraries, which provided for a 12.8-fold coverage of the genome. The Phred/Phrap/Consed software package was used for sequence assembly and quality assessment [80-82]. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher [83], PCR amplification, or transposon bombing of bridging clones (Epicentre Biotechnologies, Madison, WI, USA). Gaps between contigs were closed by editing in Consed, custom primer walk or PCR amplification (Roche Applied Science, Indianapolis, IN, USA). The genome could not be closed due to clone viability issues, however, several clones circularized the contig, and a PCR product was obtained that spanned the ends, but all attempts at primer walking and transforming the amplicon were unsuccessful. At this time no additional work is planned for this project (labeled as Permanent Draft; one linear contig).

Genome annotation

Genes were identified using Prodigal [84] as part of the genome annotation pipeline at Oak Ridge National Laboratory (ORNL), Oak Ridge, TN, USA, followed by a round of manual curation using the JGI GenePRIMP pipeline [85]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Non-coding genes and miscellaneous features were predicted using tRNAscan-SE [86], RNAMMer [87], Rfam [88], TMHMM [89], and signalP [90]. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes (IMG) platform [91] developed by the Joint Genome Institute, Walnut Creek, CA, USA [92].

Genome properties

The genome of C. testosteroni KF-1 comprises a chromosome of 6,026,527 bp (61.76% GC content) (Table 3), for which a total number of 5,606 genes were predicted. Of these predicted genes, 5,492 are protein-coding genes, and 4,009 of the protein-coding genes were assigned to a putative function and the remaining annotated as hypothetical proteins. Genome analysis predicted 114 RNA genes and six rRNA operons. The properties and the statistics of the genome are summarized in Table 3, the distribution of genes into COGs functional categories is presented in Table 4, and the chromosome map of the genome of C. testosteroni KF-1 is illustrated in Figure 3.

Table 3

Nucleotide and gene count levels of the genome of C. testosteroni KF-1

Attribute

    Value

     % of totala

Genome size (bp)

    6,026,527

     100

DNA coding region (bp)

    5,275,818

     87.54

DNA G+C content (bp)

    3,723,913

     61.79

Number of replicons

    1

Extrachromosomal elements

    0

Genes total number

    5,606

     100

Protein-coding genes

    5,492

     97.97

RNA genes

    114

     2.03

rRNA operon count

    6

Genes with function prediction

    4,009

     71.51

Genes in paralog clusters

    1314

     23.44

Genes assigned to COGs

    4,131

     73.69

Genes assigned to Pfam domains

    4,375

     78.04

Genes connected to KEGG pathways

    1,502

     26.79

Genes with transmembrane helices

    1,265

     22.57

Genes with signal peptides

    1,410

     25.15

a) The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome.

Table 4

Number of genes associated with the general COG functional categories in C. testosteroni KF-1

Code

     Value

     %age

    Description

J

     187

        4.02

    Translation, ribosomal structure and biogenesis

A

     2

        0.04

    RNA processing and modification

K

     407

        8.75

    Transcription

L

     212

        4.56

    Replication, recombination and repair

B

     2

        0.04

    Chromatin structure and dynamics

D

     32

        0.69

    Cell cycle control, cell division, chromosome partitioning

Y

     -

     -

    Nuclear structure

V

     53

        1.14

    Defense mechanisms

T

     263

        5.65

    Signal transduction mechanisms

M

     239

        5.14

    Cell wall/membrane/envelope biogenesis

N

     102

        2.19

    Cell motility

Z

     -

     -

    Cytoskeleton

W

    Extracellular structures

U

     159

        3.42

    Intracellular trafficking, secretion, and vesicular transport

O

     154

        3.31

    Posttranslational modification, protein turnover, chaperones

C

     304

        6.54

    Energy production and conversion

G

     170

        3.66

    Carbohydrate transport and metabolism

E

     361

        7.76

    Amino acid transport and metabolism

F

     90

        1.94

    Nucleotide transport and metabolism

H

     164

        3.53

    Coenzyme transport and metabolism

I

     283

        6.08

    Lipid transport and metabolism

P

     303

        6.51

    Inorganic ion transport and metabolism

Q

     154

        3.31

    Secondary metabolites biosynthesis, transport and catabolism

R

     546

        11.74

    General function prediction only

S

     464

        9.98

    Function unknown

NA

     1475

        26.31

    Not in COGs

a) The total is based on the total number of protein coding genes in the annotated genome.

Figure 3

Chromosome map of the genome of C. testosteroni KF-1. From bottom to top: Genes on forward strand (colour by COG categories), genes on reverse strand (color by COG categories), RNA genes (tRNA, green; rRNA, red; other RNAs, black), GC content.

The chromosome of C. testosteroni KF-1 (6.03 Mb) is larger in comparison to these of the three other C. testosteroni strains whose sequences have been published, of strain S44 [52] (5.53 Mb), strain CNB-2 [24] (5.46 Mb), and strain ATCC 11996 [22] (5.41 Mb), and in comparison to that of C. testosteroni NBRC 100989 (5.59 Mb) whose draft sequence has not yet been published (BioProject ID PRJNA70139). Upon genomic BLAST comparison however, the strain NBRC 100989 chromosome showed the highest similarity to the chromosome of C. testosteroni KF-1.

For the three C. testosteroni genomes accessible within the IMG platform for direct comparison [57], strains KF-1, S44 and CNB-2, the gene abundance profile indicated, most strikingly, a much higher abundance of transposases (COG2801, COG2826 and COG4644) in strain KF-1 (42 total) in comparison to strains S44 (4 total) and CNB-2 (9 total); retroviral integrases (pfam00665) are more abundant in strain KF-1 (36 total) in comparison to strains S44 (none) and CNB-2 (13 total), and hemagluttinin repeat proteins (pfam05594) implicated in cell aggregation are more abundant (10 total) in comparison to strains S44 (none) and CNB-2 (none).

In respect to candidate genes encoding the metabolic features of C. testosteroni KF-1 (see above), almost identical (syntenic) gene clusters were found for the main steroid degradation genes characterized in C. testosteroni TA441 [16,17,20], including the genes characterized in C. testosteroni ATCC 11996 [18,21,52,93-95]; the strain KF-1 genes are up to 98% identical in their amino-acid sequences. Candidate genes for the degradation of the acyl-sidechain of cholate in Pseudomonas sp. strain Chol1 [96,97] were also found (thiolase, locus tag CtesDRAFT_PD3654; acyl-CoA dehydrogenase, PD3666), and the genes for inversion of the cholate-stereochemistry in Comamonas testosteroni TA441 [98] (PD3740-44). In respect to the complete degradation of taurocholate [19], several candidate genes for bile-salts hydrolase (taurocholate hydrolase) and candidate genes for the complete degradation of the taurine-moiety (2-aminoethanesulfonate) [19], e.g., for sulfoacetaldehyde acetyltransferase (Xsc, PD0776), were found.

Strain KF-1 has acquired the ability to utilize xenobiotic 3-C4-SPC, 3-C4-SPC-2H, 3-C5-SPC and 3-C5-SPC-2H, 4-sulfoacetophenone (SAP), and 4-sulfophenol (SP) (see above) [1,9]. The 3-C4-SPC is converted to SAP [9] and further to 4-sulfophenol acetate (SPAc) by a recently identified Baeyer-Villiger monooxygenase (‘SAPMO’, PD5437), and SPAc hydrolyzed by a recently identified carboxylester hydrolase encoded by the next gene in the genome (PD5438), to yield acetate and SP [10]. The two identified genes, together with other (predicted) catabolic genes, are framed by IS1071 insertion sequence elements (Tn3-family transposase genes), which suggests that these genes have only recently been acquired, possibly in the form of a ‘catabolic composite transposon’ through horizontal gene transfer [10]. Genes for other sections of the proposed 3-C4-SPC degradation pathway in strain KF-1, i.e., the ‘upper’ and ‘lower’ pathway, from 3-C4-SPC to SAP and from SP further to central metabolites, respectively [9], are examined in our present work (unpublished).

C. testosteroni KF-1 encodes a wealth of genes for aromatic ring-cleavage oxygenases and aromatic-ring hydroxylating oxygenase (systems), as commonly observed for members of the order Burkholderiales [99]. Firstly, the complete protocatechuate 4,5-cleavage (meta) degradation operon (pmd-operon) characterized in C. testosteroni strain BR6020 [35,43], strain E6 [47] and CNB-1 [48] involved in the degradation pathways for vanillate, isovanillate and 3- and 4-hydroxybenzoate, was found in strain KF-1 (pmdB, PD1898) (and two pmdB paralogs, PD1614 and 1810). An ortholog of the 3-hydroxybenzoate monooxygenase characterized in C. testosteroni GZ39 [100] was found in strain KF-1 (PD1242), as were the genes for conversion of vanillate and isovanillate (vanA/ivaA: PD0400/PD0403) [43].

Gene clusters of the meta-pathway enzymes for degradation of phenol as characterized in C. testosteroni TA441, i.e., aphCEFGHJI [101] and aphKLMNOPQB [102]), were not found in strain KF-1, but in strains S44 and CNB-2. However, homologs for all meta-pathway enzymes (corresponding to aphCEFGHJI) seem to be distributed at different locations in the strain KF-1 genome, but a valid candidate gene cluster of the phenol hydroxylase components (aph- [102] or phcKLMNOP [34] genes) and catechol 2,3-dioxygenase (aphB) could not be found in the strain KF-1 genome. Also the gene cluster for the 3-(3-hydroxyphenyl) propionic acid degradation pathway (mhp-operon) characterized in Comamonas testosteroni TA441 [103] was not found in the genome of strain KF-1, nor in strain CNB-2, but was found in strain S44; homologs for all pathway enzymes (corresponding to mhpABDFE) seem to be distributed at different locations in the strain KF-1 genome.

An almost identical gene cluster for the terepthalate (benzene-1,4-dicarboxylic acid) pathway (tph-cluster) as characterized in C. testosteroni YZW-D [104] and strain E6 [44,46] was found in strain KF-1 (tphA, PD2130). The gene cluster for the isophthalate (benzene-1,3-dicarboxylic acid) pathway of C. testosteroni YZW-D [104] and strain E6 [45] was also found in strain KF-1 (iphA, PD2139), encoded directly upstream of the tph-cluster. Notably, at least nine other Rieske-domain ring-hydroxylating oxygenase component genes (COG4638) similar to tpaA/iphA (PD2130/PD2139) and vanA/ivaA (see above, PD0400/PD0403), seem to be encoded in strain KF-1 (PD2042, 1888, 4205, 2022, 0968, 3693, 1612, 2032, 5293).

No ortholog of the catechol 2,3-ring cleavage dioxygenase (non-heme Fe2+) of the phenol-pathway gene cluster (aphB) [102] was found in strain KF-1, but two other class I/II extradiol ring-cleavage dioxygenase candidates (PD0021, 5290) in addition to a (decarboxylating) 4-hydroxyphenylpyruvate dioxygenase candidate (PD0347) (also in CNB-2 and S44), tesB of the steroid gene cluster (PD3739), and the class-III type extradiol ring-cleavage dioxygenases mentioned above (PmdAB) were found.

In respect to intradiol ring-cleavage dioxygenases, three candidates for (non-heme Fe3+) catechol 1,2-dioxygenase/protocatechuate 3,4-dioxygenase beta subunit/hydroxyquinol 1,2-dioxygenase were found in strain KF-1, i.e., PD0424, 5469, and 5471; notably, the latter two candidates are not represented in strains CNB-2 and S44.

Also not represented in the C. testosteroni KF-1 genome is the nitrobenzene (nbz) degradation gene cluster of Comamonas sp. JS765 [38], the 3-nitrobenzoate (mnb) degradation cluster of C. testosteroni BR6020 [23], the 4-chlorobenzoate uptake and degradation cluster of Comamonas sp. strain DJ-12 [51,105], and not the 4-chloronitrobenzene (cnb) cluster on plasmid pCNB1 in C. testosteroni CNB-1 [49] and the upper-pathway chloroaniline (dca) cluster on plasmid pWDL7 in C. testosteroni WDL7 [26]. Finally, an ortholog of the aliphatic nitrilase/cyanide hydratase (NitA) characterized in a C. testosteroni soil isolate [106] was also not found in the genome of strain KF-1, nor in those of CNB-2 or S44.

Strain KF-1 utilized none of the sugars tested (see above), and this observation is reflected by an absence of appropriate candidate genes in strain KF-1 for hexokinase and glucokinase in glycolysis, as well as of genes of the oxidative branch of the pentose phosphate pathway, as reported also for C. testosteroni CNB-2 [24].

Strain KF-1 is able to utilize nicotinate for growth and encodes an orthologous set of genes for the nicotinate dehydrogenase /hydroxylase complex (PD0815-13) characterized in C. testosteroni JA1 [107].

The poly(3-hydroxybutyrate) (PHB) biosynthesis and utilization operon of Comamonas sp. EB172 [108] is also encoded in strain KF-1 (e.g., PD2272). Furthermore, strain KF-1 tested positive for growth with extracellular poly(3-hydroxybutyrate) (this study), and strain KF-1 encodes an ortholog (PD3795) of the characterized poly(3-hydroxybutyrate) depolymerase precursor (PhaZ) of Comamonas sp. strain 31A [109]; notably, the ortholog was also found in C. testosteroni ATCC 11996T, but not in strains S44 and CNB-2.

In respect to the ampicillin (beta-lactam) antibiotic resistance of strain KF-1, the genome encodes at least two beta-lactamase class A (PD2722, 4357) and one beta-lactamase class B (PD0340) candidates, and with respect to kanamycin (aminoglycoside) resistance, two aminoglycoside phosphotransferase candidates (PD3717, 1418); notably, the latter two are not represented in strains CNB-2 and S44.

All four heavy metal exporter ATPase genes (zntA) and five CzcA-family exporter gene clusters described for highly zinc-resistant C. testosteroni S44 [52] were found in strain KF‑1, and in total eight zntA and 11 cntA candidates. Two arsenical resistance gene clusters (PD1708-06 and 3544-42), each with candidates for arsenical pump (ArsB), arsenate reductase (ArsC), NADPH:FMN oxidoreductases (ArsH), and transcriptional regulator (ArsR), and a third arsC candidate (PD0567), were found in strain KF-1.

Declarations

Acknowledgements

We thank Joachim Hentschel for SEM operation, and several students of our practical classes for testing growth substrates. The work was financially supported by the University of Konstanz and the Konstanz Research School Chemical Biology, the University of New South Wales and the Centre for Marine Bio-Innovation, and by the Deutsche Forschungsgemeinschaft (DFG grant SCHL 1936/1-1 to D.S.). The work conducted by the U.S. Department of Energy Joint Genome Institute was supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231, and by the University of California, Lawrence Berkeley National Laboratory under contract No. W-7405-Eng-48, Lawrence Berkeley National Laboratory under contract No. DE-AC03-76SF00098 and Los Alamos National Laboratory under contract No. W-7405-ENG-36.


This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

References

  1. Schleheck D, Knepper TP, Fischer K and Cook AM. Mineralization of individual congeners of linear alkylbenzenesulfonate by defined pairs of heterotrophic bacteria. Appl Environ Microbiol. 2004; 70:4053-4063 View ArticlePubMed
  2. Knepper TP, Barceló D, deVoogt P. Analysis and fate of surfactants in the quatic environment. Amsterdam: Elsevier; 2003.
  3. Swisher RD. Surfactant biodegradation. New York: Marcel Dekker; 1970.
  4. Schleheck D, Weiss M, Pitluck S, Bruce D, Land ML, Han S, Saunders E, Tapia R, Detter C and Brettin T. Complete genome sequence of Parvibaculum lavamentivorans type strain (DS-1T). Stand Genomic Sci. 2011; 5:298-310 View ArticlePubMed
  5. Schleheck D, Dong W, Denger K, Heinzle E and Cook AM. An alpha-proteobacterium converts linear alkylbenzenesulfonate surfactants into sulfophenylcarboxylates and linear alkyldiphenyletherdisulfonate surfactants into sulfodiphenylethercarboxylates. Appl Environ Microbiol. 2000; 66:1911-1916 View ArticlePubMed
  6. Dong W, Eichhorn P, Radajewski S, Schleheck D, Denger K, Knepper TP, Murrell JC and Cook AM. Parvibaculum lavamentivorans converts linear alkylbenzenesulphonate surfactant to sulphophenylcarboxylates, alpha,beta-unsaturated sulphophenylcarboxylates and sulphophenyldicarboxylates, which are degraded in communities. J Appl Microbiol. 2004; 96:630-640 View ArticlePubMed
  7. Schleheck D, Tindall BJ, Rossello-Mora R and Cook AM. Parvibaculum lavamentivorans gen. nov., sp. nov., a novel heterotroph that initiates catabolism of linear alkylbenzenesulfonate. Int J Syst Evol Microbiol. 2004; 54:1489-1497 View ArticlePubMed
  8. Schleheck D, Knepper TP, Eichhorn P and Cook AM. Parvibaculum lavamentivorans DS-1T degrades centrally substituted congeners of commercial linear alkylbenzenesulfonate to sulfophenyl carboxylates and sulfophenyl dicarboxylates. Appl Environ Microbiol. 2007; 73:4725-4732 View ArticlePubMed
  9. Schleheck D, von Netzer F, Fleischmann T, Rentsch D, Huhn T, Cook AM and Kohler HP. The missing link in linear alkylbenzenesulfonate surfactant degradation: 4-sulfoacetophenone as a transient intermediate in the degradation of 3-(4-sulfophenyl)butyrate by Comamonas testosteroni KF-1. Appl Environ Microbiol. 2010; 76:196-202 View ArticlePubMed
  10. Weiss M, Denger K, Huhn T and Schleheck D. Two enzymes of a complete degradation pathway for linear alkylbenzenesulfonate (LAS) surfactants: 4-sulfoacetophenone Baeyer-Villiger monooxygenase and 4-sulfophenylacetate esterase in Comamonas testosteroni KF-1. Appl Environ Microbiol. 2012; 78:8254-8263 View ArticlePubMed
  11. Tamaoka J, Ha DM and Komagata K. Reclassification of Pseudomonas acidovorans Den Dooren De Jong 1926 and Pseudomonas testosteroni Marcus and Talalay 1956 as Comamonas acidovorans comb. nov. and Comamonas testosteroni comb. nov., with an emended description of the genus Comamonas. Int J Syst Bacteriol. 1987; 37:52-59 View Article
  12. Talalay P, Dobson MM and Tapley DF. Oxidative degradation of testosterone by adaptive enzymes. Nature. 1952; 170:620-621 View ArticlePubMed
  13. Talalay P. A fascination with enzymes: The journey not the arrival matters. J Biol Chem. 2005; 280:28829-28847 View ArticlePubMed
  14. Shaw DA, Borkenhagen LF and Talalay P. Enzymatic oxidation of steroids by cell-free extracts of Pseudomonas testosteroni: isolation of cleavage products of ring A. Proc Natl Acad Sci USA. 1965; 54:837-844 View ArticlePubMed
  15. Marcus PI and Talalay P. Induction and purification of alpha- and beta-hydroxysteroid dehydrogenases. J Biol Chem. 1956; 218:661-674PubMed
  16. Horinouchi M, Yamamoto T, Taguchi K, Arai H and Kudo T. Meta-cleavage enzyme gene tesB is necessary for testosterone degradation in Comamonas testosteroni TA441. Microbiology. 2001; 147:3367-3375PubMed
  17. Horinouchi M, Hayashi T, Yamamoto T and Kudo T. A new bacterial steroid degradation gene cluster in Comamonas testosteroni TA441 which consists of aromatic-compound degradation genes for seco-steroids and 3-ketosteroid dehydrogenase genes. Appl Environ Microbiol. 2003; 69:4421-4430 View ArticlePubMed
  18. Xiong G, Martin HJ and Maser E. Identification and characterization of a novel translational repressor of the steroid-inducible 3 alpha-hydroxysteroid dehydrogenase/carbonyl reductase gene in Comamonas testosteroni. J Biol Chem. 2003; 278:47400-47407 View ArticlePubMed
  19. Rösch V, Denger K, Schleheck D, Smits TH and Cook AM. Different bacterial strategies to degrade taurocholate. Arch Microbiol. 2008; 190:11-18 View ArticlePubMed
  20. Horinouchi M, Kurita T, Hayashi T and Kudo T. Steroid degradation genes in Comamonas testosteroni TA441: Isolation of genes encoding a delta 4(5)-isomerase and 3 alpha- and 3 beta-dehydrogenases and evidence for a 100 kb steroid degradation gene hot spot. J Steroid Biochem Mol Biol. 2010; 122:253-263 View ArticlePubMed
  21. Gong W, Xiong G and Maser E. Identification and characterization of the LysR-type transcriptional regulator HsdR for steroid-inducible expression of the 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase gene in Comamonas testosteroni. Appl Environ Microbiol. 2012; 78:941-950 View ArticlePubMed
  22. Gong W, Kisiela M, Schilhabel MB, Xiong G and Maser E. Genome sequence of Comamonas testosteroni ATCC 11996, a representative strain involved in steroid degradation. J Bacteriol. 2012; 194:1633-1634 View ArticlePubMed
  23. Providenti MA, Shaye RE, Lynes KD, McKenna NT, O'Brien JM, Rosolen S, Wyndham RC and Lambert LB. The locus coding for the 3-nitrobenzoate dioxygenase of Comamonas sp. strain JS46 is flanked by IS1071 elements and is subject to deletion and inversion events. Appl Environ Microbiol. 2006; 72:2651-2660 View ArticlePubMed
  24. Ma YF, Zhang Y, Zhang JY, Chen DW, Zhu YQ, Zheng HJ, Wang SY, Jiang CY, Zhao GP and Liu SJ. The complete genome of Comamonas testosteroni reveals its genetic adaptations to changing environments. Appl Environ Microbiol. 2009; 75:6812-6819 View ArticlePubMed
  25. Locher HH and Leisinger TAMC. Degradation of p-toluenesulphonic acid via sidechain oxidation, desulphonation and meta ring cleavage in Pseudomonas (Comamonas) testosteroni T-2. J Gen Microbiol. 1989; 135:1969-1978PubMed
  26. Krol JE, Penrod JT, McCaslin H, Rogers LM, Yano H, Stancik AD, Dejonghe W, Brown CJ, Parales RE and Wuertz S. Role of IncP-1beta plasmids pWDL7:rfp and pNB8c in chloroaniline catabolism as determined by genomic and functional analyses. Appl Environ Microbiol. 2012; 78:828-838 View ArticlePubMed
  27. Nishino SF and Spain JC. Oxidative pathway for the biodegradation of nitrobenzene by Comamonas sp. strain JS765. Appl Environ Microbiol. 1995; 61:2308-2313PubMed
  28. Chen YH, Chai LY, Zhu YH, Yang ZH, Zheng Y and Zhang H. Biodegradation of kraft lignin by a bacterial strain Comamonas sp. B-9 isolated from eroded bamboo slips. J Appl Microbiol. 2012; 112:900-906 View ArticlePubMed
  29. Ahmad D, Massé R and Sylvestre M. Cloning and expression of genes involved in 4-chlorobiphenyl transformation by Pseudomonas testosteroni: homology to polychlorobiphenyl-degrading genes in other bacteria. Gene. 1990; 86:53-61 View ArticlePubMed
  30. Wang Y, Yamazoe A, Suzuki S, Liu CT, Aono T and Oyaizu H. Isolation and characterization of dibenzofuran-degrading Comamonas sp. strains isolated from white clover roots. Curr Microbiol. 2004; 49:288-294 View ArticlePubMed
  31. Song Z, Edwards SR and Burns RG. Biodegradation of naphthalene-2-sulfonic acid present in tannery wastewater by bacterial isolates Arthrobacter sp. 2AC and Comamonas sp. 4BC. Biodegradation. 2005; •••:237-252 View ArticlePubMed
  32. Ornston MK and Ornston LN. Regulation of beta-ketoadipate pathway in Pseudomonas acidovorans and Pseudomonas testosteroni. J Gen Microbiol. 1972; 73:455-464 View ArticlePubMed
  33. Schläfli HR, Weiss MA, Leisinger T and Cook AM. Terephthalate 1,2-dioxygenase system from Comamonas testosteroni T-2 - Purification and some properties of the oxygenase component. J Bacteriol. 1994; 176:6644-6652PubMed
  34. Teramoto M, Futamata H, Harayama S and Watanabe K. Characterization of a high-affinity phenol hydroxylase from Comamonas testosteroni R5 by gene cloning, and expression in Pseudomonas aeruginosa PAO1c. Mol Gen Genet. 1999; 262:552-558 View ArticlePubMed
  35. Providenti MA, Mampel J, MacSween S, Cook AM and Wyndham RC. Comamonas testosteroni BR6020 possesses a single genetic locus for extradiol cleavage of protocatechuate. Microbiology-Sgm. 2001; 147:2157-2167PubMed
  36. Sylvestre M, Sirois M, Hurtubise Y, Bergeron J, Ahmad D, Shareck F, Barriault D, Guillemette I and Juteau JM. Sequencing of Comamonas testosteroni strain B-356-biphenyl/chlorobiphenyl dioxygenase genes: evolutionary relationships among Gram-negative bacterial biphenyl dioxygenases. Gene. 1996; 174:195-202 View ArticlePubMed
  37. Tralau T, Cook AM and Ruff J. Map of the IncP1beta plasmid pTSA encoding the widespread genes (tsa) for p-toluenesulfonate degradation in Comamonas testosteroni T-2. Appl Environ Microbiol. 2001; 67:1508-1516 View ArticlePubMed
  38. Lessner DJ, Johnson GR, Parales RE, Spain JC and Gibson DT. Molecular characterization and substrate specificity of nitrobenzene dioxygenase from Comamonas sp. strain JS765. Appl Environ Microbiol. 2002; 68:634-641 View ArticlePubMed
  39. Tralau T, Cook AM and Ruff J. An additional regulator, TsaQ, is involved with TsaR in regulation of transport during the degradation of p-toluenesulfonate in Comamonas testosteroni T-2. Arch Microbiol. 2003; 180:319-326 View ArticlePubMed
  40. Tralau T, Mampel J, Cook AM and Ruff J. Characterization of TsaR, an oxygen-sensitive LysR-type regulator for the degradation of p-toluenesulfonate in Comamonas testosteroni T-2. Appl Environ Microbiol. 2003; 69:2298-2305 View ArticlePubMed
  41. Mampel J, Maier E, Tralau T, Ruff J, Benz R and Cook AM. A novel outer-membrane anion channel (porin) as part of a putatively two-component transport system for 4-toluenesulphonate in Comamonas testosteroni T-2. Biochem J. 2004; 383:91-99 View ArticlePubMed
  42. Mampel J, Providenti MA and Cook AM. Protocatechuate 4,5-dioxygenase from Comamonas testosteroni T-2: biochemical and molecular properties of a new subgroup within class III of extradiol dioxygenases. Arch Microbiol. 2005; 183:130-139 View ArticlePubMed
  43. Providenti MA, O'Brien JM, Ruff J, Cook AM and Lambert IB. Metabolism of isovanillate, vanillate, and veratrate by Comamonas testosteroni strain BR6020. J Bacteriol. 2006; 188:3862-3869 View ArticlePubMed
  44. Sasoh M, Masai E, Ishibashi S, Hara H, Kamimura N, Miyauchi K and Fukuda M. Characterization of the terephthalate degradation genes of Comamonas sp. strain E6. Appl Environ Microbiol. 2006; 72:1825-1832 View ArticlePubMed
  45. Fukuhara Y, Inakazu K, Kodama N, Kamimura N, Kasai D, Katayama Y, Fukuda M and Masai E. Characterization of the isophthalate degradation genes of Comamonas sp. strain E6. Appl Environ Microbiol. 2010; 76:519-527 View ArticlePubMed
  46. Kasai D, Kitajima M, Fukuda M and Masai E. Transcriptional regulation of the terephthalate catabolism operon in Comamonas sp. strain E6. Appl Environ Microbiol. 2010; 76:6047-6055 View ArticlePubMed
  47. Kamimura N, Aoyama T, Yoshida R, Takahashi K, Kasai D, Abe T, Mase K, Katayama Y, Fukuda M and Masai E. Characterization of the protocatechuate 4,5-cleavage pathway operon in Comamonas sp. strain E6 and discovery of a novel pathway gene. Appl Environ Microbiol. 2010; 76:8093-8101 View ArticlePubMed
  48. Ni B, Zhang Y, Chen DW, Wang BJ and Liu SJ. Assimilation of aromatic compounds by Comamonas testosteroni: characterization and spreadability of protocatechuate 4,5-cleavage pathway in bacteria. Appl Microbiol Biotechnol. 2012 View ArticlePubMed
  49. Ma YF, Wu JF, Wang SY, Jiang CY, Zhang Y, Qi SW, Liu L, Zhao GP and Liu SJ. Nucleotide sequence of plasmid pCNB1 from Comamonas strain CNB-1 reveals novel genetic organization and evolution for 4-chloronitrobenzene degradation. Appl Environ Microbiol. 2007; 73:4477-4483 View ArticlePubMed
  50. Zhang Y, Ma YF, Qi SW, Meng B, Chaudhry MT, Liu SQ and Liu SJ. Responses to arsenate stress by Comamonas sp strain CNB-1 at genetic and proteomic levels. Microbiology-Sgm. 2007; 153:3713-3721 View ArticlePubMed
  51. Cai L, Liu G, Rensing C and Wang G. Genes involved in arsenic transformation and resistance associated with different levels of arsenic-contaminated soils. BMC Microbiol. 2009; 9:4 View ArticlePubMed
  52. Xiong J, Li D, Li H, He M, Miller SJ, Yu L, Rensing C and Wang G. Genome analysis and characterization of zinc efflux systems of a highly zinc-resistant bacterium, Comamonas testosteroni S44. Res Microbiol. 2011; 162:671-679 View ArticlePubMed
  53. Siunova TV, Siunov AV, Kochetkov VV and Boronin AM. The cnr-like operon in strain Comamonas sp. encoding resistance to cobalt and nickel. Genetika. 2009; 45:336-341PubMed
  54. Turek M, Vilimkova L, Kremlackova V, Paca JJ, Halecky M, Paca J and Stiborova M. Isolation and partial characterization of extracellular NADPH-dependent phenol hydroxylase oxidizing phenol to catechol in Comamonas testosteroni. Neuroendocrinol Lett. 2011; 32:137-145PubMed
  55. Godocíková J, Bohácová V, Zámocký M and Polek B. Production of catalases by Comamonas spp. and resistance to oxidative stress. Folia Microbiol (Praha). 2005; 50:113-118 View ArticlePubMed
  56. Oppermann UC, Belai I and Maser E. Antibiotic resistance and enhanced insecticide catabolism as consequences of steroid induction in the gram-negative bacterium Comamonas testosteroni. J Steroid Biochem Mol Biol. 1996; 58:217-223 View ArticlePubMed
  57. Markowitz VM and Chen IMA. Palaniappan, Chu K, Szeto E, Grechkin Y, Ratner A, Jacob B, Huang J, Williams P and others. IMG: the integrated microbial genomes database and comparative analysis system. Nucleic Acids Res. 2012; 40:D115-D122 View ArticlePubMed
  58. Thurnheer T, Kohler T, Cook AM and Leisinger T. Orthanilic acid and analogs as carbon-sources for bacteria - Growth physiology and enzymatic desulfonation. J Gen Microbiol. 1986; 132:1215-1220
  59. Field D, Garrity GM, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ and Angiuoli SV. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008; 26:541-547 View ArticlePubMed
  60. Woese CR, Kandler O and Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA. 1990; 87:4576-4579 View ArticlePubMed
  61. Garrity GM, Bell JA, Lilburn T. Phylum XIV. Proteobacteria phyl. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT (eds), Bergey's Manual of Systematic Bacteriology, Second Edition, Volume 2, Part B, Springer, New York, 2005, p. 1.
  62. . 107. List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol. 2006; 56:1-6 View ArticlePubMed
  63. Garrity GM, Bell JA, Lilburn T. Class II. Betaproteobacteria class. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT (eds), Bergey's Manual of Systematic Bacteriology, Second Edition, Volume 2, Part C, Springer, New York, 2005, p. 575.
  64. Garrity GM, Bell JA, Lilburn T. Order I. Burkholderiales ord. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT (eds), Bergey's Manual of Systematic Bacteriology, Second Edition, Volume 2, Part C, Springer, New York, 2005, p. 575.
  65. Willems A, De Ley J, Gillis M and Kersters K. Comamonadaceae, a new family encompassing the acidovorans rRNA complex, including Variovorax paradoxus gen. nov., comb. nov., for Alcaligenes paradoxus (Davis 1969). Int J Syst Bacteriol. 1991; 41:445-450 View Article
  66. De Vos P, Kersters K, Gillis M, Segers P and de Ley J. Comamonas Davis and Park 1962 gen. nov., nom. rev. emend., and Comamonas terrigena Hugh 1962 sp. nov., nom. rev. Int J Syst Bacteriol. 1985; 35:443-453 View Article
  67. Zhang J, Wang Y, Zhou S, Wu C, He J and Li F. Comamonas guangdongensis sp. nov., isolated from subterranean forest sediment, and emended description of the genus Comamonas. Int J Syst Evol Microbiol. 2013; 63:809-814 View ArticlePubMed
  68. Willems A, Pot B, Falsen E, Vandamme P, Gillis M, Kersters K and de Ley J. Polyphasic taxonomic study of the emended genus Comamonas: relationship to Aquaspirillum aquaticum, E. Falsen group 10, and other clinical isolates. Int J Syst Bacteriol. 1991; 41:427-444 View Article
  69. Davis GHG and Park RWA. A taxonomic study of certain bacteria currently classified as Vibrio species. J Gen Microbiol. 1962; 27:101-119 View ArticlePubMed
  70. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS and Eppig JT. Gene Ontology: tool for the unification of biology. Nat Genet. 2000; 25:25-29 View ArticlePubMed
  71. Buhmann M. Charakterisierung der Biofilmbildung durch eine Tensid-abbauende Bakteriengemeinschaft [Master's thesis]. Konstanz: University of Konstanz; 2008. 126 p.
  72. Li M, Peng L, Ji Z, Xu J and Li S. Establishment and characterization of dual-species biofilms formed from a 3,5-dinitrobenzoic-degrading strain and bacteria with high biofilm-forming capabilities. FEMS Microbiol Lett. 2008; 278:15-21 View ArticlePubMed
  73. Schleheck D, Barraud N, Klebensberger J, Webb JS, McDougald D, Rice SA and Kjelleberg S. Pseudomonas aeruginosa PAO1 preferentially grows as aggregates in liquid batch cultures and disperses upon starvation. PLoS ONE. 2009; 4:e5513 View ArticlePubMed
  74. Stelzer S, Egan S, Larsen MR, Bartlett DH and Kjelleberg S. Unravelling the role of the ToxR-like transcriptional regulator WmpR in the marine antifouling bacterium Pseudoalteromonas tunicata. Microbiology. 2006; 152:1385-1394 View ArticlePubMed
  75. Thomas T, Evans FF, Schleheck D, Mai-Prochnow A, Burke C, Penesyan A, Dalisay DS, Stelzer-Braid S, Saunders N and Johnson J. Analysis of the Pseudoalteromonas tunicata genome reveals properties of a surface-associated life style in the marine environment. PLoS ONE. 2008; 3:e3252 View ArticlePubMed
  76. Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam-Syed-Mohideen AS, McGarrell DM, Marsh T and Garrity GM. The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res. 2009; 37:D141-D145 View ArticlePubMed
  77. Tamura K, Dudley J, Nei M and Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol. 2007; 24:1596-1599 View ArticlePubMed
  78. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ and Angiuoli SV. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008; 26:541-547 View ArticlePubMed
  79. . Web Site
  80. Ewing B and Green P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 1998; 8:186-194PubMed
  81. Ewing B, Hillier L, Wendl MC and Green P. Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 1998; 8:175-185PubMed
  82. Gordon D, Abajian C and Green P. Consed: a graphical tool for sequence finishing. Genome Res. 1998; 8:195-202PubMed
  83. Han CS, Chain P. Finishing repeat regions automatically with Dupfinisher. In: Arabnia HR, Valafar H, editors. Proceeding of the 2006 international conference on bioinformatics & computational biology: CSREA Press; 2006. p 141-146.
  84. Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW and Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010; 11:119 View ArticlePubMed
  85. Pati A, Ivanova N, Mikhailova N, Ovchinikova G, Hooper SD, Lykidis A and Kyrpides NC. GenePRIMP: A Gene Prediction Improvement Pipeline for microbial genomes. Nat Methods. 2010; 7:6 View ArticlePubMed
  86. Lowe TM and Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997; 25:955-964PubMed
  87. Lagesen K, Hallin PF, Rødland E, Stærfeldt HH, Rognes T and Ussery DW. RNammer: consistent annotation of rRNA genes in genomic sequences. Nucleic Acids Res. 2007; 35:3100-3108 View ArticlePubMed
  88. Griffiths-Jones S, Bateman A, Marshall M, Khanna A and Eddy SR. Rfam: an RNA family database. Nucleic Acids Res. 2003; 31:439-441 View ArticlePubMed
  89. Krogh A, Larsson B, von Heijne G and Sonnhammer ELL. Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes. J Mol Biol. 2001; 305:567-580 View ArticlePubMed
  90. Dyrløv Bendtsen JD, Nielsen H, von Heijne G and Brunak S. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol. 2004; 340:783-795 View ArticlePubMed
  91. Integrated Microbial Genomes (IMG) platform. Web Site
  92. Markowitz VM, Szeto E, Palaniappan K, Grechkin Y, Chu K, Chen IMA, Dubchak I, Anderson I, Lykidis A and Mavromatis K. The Integrated Microbial Genomes (IMG) system in 2007: data content and analysis tool extensions. Nucleic Acids Res. 2008; 36:D528-D533 View ArticlePubMed
  93. Möbus E and Maser E. Molecular cloning, overexpression, and characterization of steroid-inducible 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni. A novel member of the short-chain dehydrogenase/reductase superfamily. J Biol Chem. 1998; 273:30888-30896 View ArticlePubMed
  94. Xiong G and Maser E. Regulation of the steroid-inducible 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase gene in Comamonas testosteroni. J Biol Chem. 2001; 276:9961-9970 View ArticlePubMed
  95. Pruneda-Paz JL, Linares M, Cabrera JE and Genti-Raimondi S. TeiR, a LuxR-type transcription factor required for testosterone degradation in Comamonas testosteroni. J Bacteriol. 2004; 186:1430-1437 View ArticlePubMed
  96. Birkenmaier A, Holert J, Erdbrink H, Moeller HM, Friemel A, Schoenenberger R, Suter MJ, Klebensberger J and Philipp B. Biochemical and genetic investigation of initial reactions in aerobic degradation of the bile acid cholate in Pseudomonas sp. strain Chol1. J Bacteriol. 2007; 189:7165-7173 View ArticlePubMed
  97. Birkenmaier A, Möller HM and Philipp B. Identification of a thiolase gene essential for beta-oxidation of the acyl side chain of the steroid compound cholate in Pseudomonas sp. strain Chol1. FEMS Microbiol Lett. 2011; 318:123-130 View ArticlePubMed
  98. Horinouchi M, Hayashi T, Koshino H, Malon M, Yamamoto T and Kudo T. Identification of genes involved in inversion of stereochemistry of a C-12 hydroxyl group in the catabolism of cholic acid by Comamonas testosteroni TA441. J Bacteriol. 2008; 190:5545-5554 View ArticlePubMed
  99. Pérez-Pantoja D, Donoso R, Agullo L, Cordova M, Seeger M, Pieper DH and Gonzalez B. Genomic analysis of the potential for aromatic compounds biodegradation in Burkholderiales. Environ Microbiol. 2012; 14:1091-1117 View ArticlePubMed
  100. Chang HK and Zylstra GJ. Examination and expansion of the substrate range of m-hydroxybenzoate hydroxylase. Biochem Biophys Res Commun. 2008; 371:149-153 View ArticlePubMed
  101. Arai H, Ohishi T, Chang MY and Kudo T. Arrangement and regulation of the genes for meta-pathway enzymes required for degradation of phenol in Comamonas testosteroni TA441. Microbiology. 2000; 146:1707-1715PubMed
  102. Arai H, Akahira S, Ohishi T, Maeda M and Kudo T. Adaptation of Comamonas testosteroni TA441 to utilize phenol: organization and regulation of the genes involved in phenol degradation. Microbiology. 1998; 144:2895-2903 View ArticlePubMed
  103. Arai H, Yamamoto T, Ohishi T, Shimizu T, Nakata T and Kudo T. Genetic organization and characteristics of the 3-(3-hydroxyphenyl)propionic acid degradation pathway of Comamonas testosteroni TA441. Microbiology. 1999; 145:2813-2820PubMed
  104. Wang YZ, Zhou Y and Zylstra GJ. Molecular analysis of isophthalate and terephthalate degradation by Comamonas testosteroni YZW-D. Environ Health Perspect. 1995; 103:9-12PubMed
  105. Chae JC and Zylstra GJ. 4-Chlorobenzoate uptake in Comamonas sp. strain DJ-12 is mediated by a tripartite ATP-independent periplasmic transporter. J Bacteriol. 2006; 188:8407-8412 View ArticlePubMed
  106. Lévy-Schil S, Soubrier F, Crutz-Le Coq AM, Faucher D, Crouzet J and Petre D. Aliphatic nitrilase from a soil-isolated Comamonas testosteroni sp.: gene cloning and overexpression, purification and primary structure. Gene. 1995; 161:15-20 View ArticlePubMed
  107. Yang Y, Chen T, Ma P, Shang G, Dai Y and Yuan S. Cloning, expression and functional analysis of nicotinate dehydrogenase gene cluster from Comamonas testosteroni JA1 that can hydroxylate 3-cyanopyridine. Biodegradation. 2010; 21:593-602 View ArticlePubMed
  108. Yee LN, Chuah JA, Chong ML, Phang LY, Raha AR, Sudesh K and Hassan MA. Molecular characterisation of phaCAB from Comamonas sp. EB172 for functional expression in Escherichia coli JM109. Microbiol Res. 2012; 167:550-557 View ArticlePubMed
  109. Jendrossek D, Backhaus M and Andermann M. Characterization of the extracellular poly(3-hydroxybutyrate) depolymerase of Comamonas sp. and of its structural gene. Can J Microbiol. 1995; 41(Suppl 1):160-169 View ArticlePubMed