Standards in Genomic Sciences (2013) 8:239-254 DOI:10.4056/sigs.3847890 Permanent draft genome sequence of Comamonas testosteroni KF-1 Michael Weiss1,2, Anna I. Kesberg1, Kurt M. LaButti3, Sam Pitluck3, David Bruce4, Loren Hauser5, Alex Copeland3, Tanja Woyke3, Stephen Lowry3, Susan Lucas3, Miriam Land5, Lynne Goodwin3,4, Staffan Kjelleberg6, Alasdair M. Cook1,2, Matthias Buhmann1, Torsten Thomas6, and David Schleheck1,2* 1 Department of Biological Sciences, University of Konstanz, Germany 2 Konstanz Research School Chemical Biology, University of Konstanz, Germany 3 DOE Joint Genome Institute, Walnut Creek, California, USA 4 Los Alamos National Laboratory, Bioscience Division, Los Alamos, New Mexico, USA 5 Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA 6 Centre for Marine Bio-Innovation and School of Biotechnology and Biomolecular Science, University of New South Wales, Sydney, Australia *Corresponding author: David Schleheck ([email protected]) Keywords: Comamonas testosteroni KF-1, aerobic, Gram-negative, Comamonadaceae, xeno- biotic surfactant biodegradation Comamonas testosteroni KF-1 is a model organism for the elucidation of the novel biochemical degra- dation pathways for xenobiotic 4-sulfophenylcarboxylates (SPC) formed during biodegradation of syn- thetic 4-sulfophenylalkane surfactants (linear alkylbenzenesulfonates, LAS) by bacterial communities. Here we describe the features of this organism, together with the complete genome sequence and an- notation. 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. Introduction Comamonas testosteroni strain KF-1 (DSM14576) testosteroni KF-1. In particular, strain KF-1 was iso- was isolated for its ability to degrade xenobiotic lated from a laboratory trickling filter that had been sulfophenylcarboxylates (SPC), which are degrada- used to enrich a bacterial community from sewage tion intermediates of the synthetic laundry surfac- sludge that completely degraded commercial LAS tants linear alkylbenzenesulfonates (LAS) [1]. LAS and SPCs [1,6]. Strain KF-1 is able to utilize four is in use worldwide (appr. 3 × 106 tons per year individual SPCs (both enantiomers), namely R/S-3- [2]) and consists of a complex mixture of linear al- (4-sulfopenyl)butyrate (3-C4-SPC), enoyl-3-C4-SPC, kanes (C10-C13) sub-terminally substituted by 4- R/S-3-(4-sulfopenyl)pentanoate (3-C5-SPC), and sulfophenyl rings (i.e., 38 different compounds) [2]. enoyl-3-C5-SPC (see therefore also below), as novel Commercial LAS is completely biodegradable, as carbon an energy sources for its heterotrophic aer- known for more than 50 years [3], e.g., in sewage obic growth [1,9,10]. treatment plants, and its degradation is catalyzed The first Comamonas testosteroni (formerly Pseu- by heterotrophic aerobic bacterial communities in domonas testosteroni [11]) strain, type-strain ATCC two steps. First, an initial degradation step is cata- 11996, was enriched from soil and isolated in 1952 lyzed by bacteria such as Parvibaculum for its ability to degrade testosterone [12,13]. Since lavamentivorans DS-1T [4] through activation and then, the physiology, biochemistry, genetics, and shortening of the alkyl-chains of LAS, and many regulation of steroid degradation in this and in oth- short-chain degradation intermediates are excreted er C. testosteroni strains have been elucidated in by these organisms, i.e., approximately 50 different great detail [e.g., 14-21]. Most recently, the genome SPCs and related compounds [1,5-8]. Secondly, the of C. testosteroni ATCC 11996T has been sequenced ultimate degradation step, i.e., mineralization of all in order to further improve the understanding of SPCs, is catalyzed by other bacteria in the commu- the molecular basis for the degradation of steroids nity, and one representative of these is Comamonas [22]. The Genomic Standards Consortium Comamonas testosteroni KF-1 In the environment, members of the genus genome sequence and its annotation have been es- Comamonas may also be important degraders of tablished as part of the Microbial Genomics Pro- aromatic compounds other than steroids, especially gram 2006 of the DOE Joint Genome Institute, and of xenobiotic pollutants, since they have frequently are accessible via the IMG platform [57]. been enriched and isolated for their ability to uti- lize (xenobiotic) aromatic compounds. For exam- Classifications and features ple, Comamonas sp. strain JS46 is able to grow with Morphology and growth conditions 3-nitrobenzoate [23], Comamonas sp. strain CNB-1 C. testosteroni KF-1 is a rod-shaped (size, appr. 0.5 with 4-chloronitrobenzene [24], C. testosteroni T-2 x 2 µm, Figure 1) Gram-negative bacterium that with 4-toluenesulfonate and 4-sulfobenzoate [25], can be motile and grows strictly aerobically with C. testosteroni WDL7 with chloroaniline [26], complex medium (e.g., in LB- or peptone medium) Comamonas sp. strain JS765 with nitrobenzene or in a prototrophic manner when cultivated in [27], Comamonas sp. strain B-9 with lignin-polymer mineral-salts medium [58] with a single carbon fragments [28], C. testosteroni B-356 with biphenyl source (e.g., acetate). Strain KF-1 grows overnight and 4-chlorobiphenyl [29], Comamonas sp. strain on LB-agar plates and forms whitish-beige colo- KD-7 with dibenzofuran [30], Comamonas sp. strain nies [Table 1]. The strain grew with all amino ac- 4BC with naphthalene-2-sulfonate [31], or C. ids tested (D-alanine, L-alanine, L-aspartate, L- testosteroni SPB-2 (as well as strain KF-1) with phenylalanine, L-valine, glycine, L-histidine, L- 4-sulfophenylcarboxylates [1]. In several C. methionine), but not with any of the sugars tested testosteroni strains, the physiology, biochemistry, (D-glucose, D-fructose, D-galactose, D-arabinose, genetics, and/or regulation of the utilization of ar- and D-maltose). Strain KF-1 utilized the following omatic compounds have been elucidated [e.g., alcohols and carboxylic acids when tested (in this 10,23,25,27,29,32-48]. Furthermore, the genome study): ethanol, acetate, glycerol, glycolate, sequence of (plasmid-cured) C. testosteroni CNB-2 glyoxylate, butanol, butyrate, isobutyrate, succin- has been published [24], and the sequence of its ate, meso-tartaric acid, D- and L-malate, plasmid pCNB1 (of C. testosteroni CNB-1) [49], in mesaconate, and nicotinate. Furthermore, strain order to further improve the understanding of the KF-1 was positive for growth with poly-beta- molecular basis for the ability of C. testosteroni to hydroxybutyrate (this study). Strain KF-1 is able degrade such a large array of aromatic compounds. to utilize the steroids testosterone and progester- Members of the genus Comamonas are able to cope one (confirmed in this study), as well as with harsh environmental conditions such as high taurocholate and cholate (and taurine and N- concentrations of arsenate [50,51], zinc [52], cobalt methyl taurine) [19], and taurodeoxycholate; and nickel [53], or phenol [54], and can exhibit in- strain KF-1 was tested negative for growth with creased resistance to oxidative stress [55] or anti- cholesterol, er -estradiol and biotics [56]. Another C. testosteroni genome se- ethinylestradiol (this study), correlating with the quence, of strain S44, has recently been established findings for C. testosteronigosterol, strain17β TA441 [20]. in order to improve the understanding of the mo- In respect to other aromatic compounds, strain lecular basis for its resistance to increased concen- KF-1 is known to utilize benzoate, 3- and 4- trations of zinc [52]. Notably, an increased antibi- hydroxybenzoate, protocatechuate (3,4- otic resistance (and enhanced insecticide catabo- dihydroxybenzoate), gentisate (2,5- lism) as a consequence of induction of the steroid dihydroxybenzoate), phthalate, terephthalate, degradation pathway has been shown for C. vanillate, isovanillate, veratrate, 2- and 3- T testosteroni ATCC 11996 [56]. hydroxyphenylacetate (tested in this study, and Here, we present a summary classification and a set ref. 1). Xenobiotic aromatic substrates for strain of features for another C. testosteroni strain, strain KF-1 known are the 4-sulfophenylcarboxylates KF-1, which has been genome-sequenced in order R/S-3-(4-sulfophenyl)butyrate (R/S-3-C4-SPC), 3- to improve the understanding of the molecular ba- (4-sulfophenyl)- 2-enoylbutyrate (enoyl-3-C4- sis for its ability to degrade xenobiotic compounds, SPC), R/S-3-(4-sulfophenyl)pentanoate (R/S-3-C5- particularly xenobiotic, chiral 3-C4-SPC, and how SPC), 3-(4-∆sulfophenyl)- -enoylpentanoate this novel degradation pathway has been assem- (enoyl-3-C5-SPC), as well as the three xenobiotic bled in this organism, together with the description metabolites in the 3-C4∆2-SPC-pathway, 4- of its draft genome sequence and annotation. The sulfoacetophenone (4-acetylbenzenesulfonate), 4- 240 Standards in Genomic Sciences Weiss et al. sulfophenol acetate, and 4-sulfophenol [1,9]. Fi- Pseudomonas aeruginosa PAO1 [73], or C. nally, strain KF-1 did not utilize the following, oth- testosteroni SPB-2 [1]. er carbon sources tested (this study and refs. 1,9): No significant production of siderophores could n-alkanes (C6-C12), cycloalkanes (C8-C12), second- be observed for C. testosteroni KF-1 when grown ary-4-sulfophenylalkanes (LAS surfactants), sec- in presence of non-inhibitory levels of iron ondary alkanesulfonates (SAS surfactants), chelator 2,2'-dipyridyl [see 74], in comparison to dodecylsulfate (SDS