on April 20, 2020 by guest · 9/4/2009 · Strain CNB-2 has a circular chromosome of 5,373,643 bp in length and G+C content of 61.4%. Totally, 4803 ORFs were identified, of which

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Complete Genome of Comamonas testosteroni Reveals Its Genetic Adaptations to 1

the Changing Environments 2

Ying-Fei Ma1,2

, Yun Zhang1, Jia-Yue Zhang

1, Dong-Wei Chen

1,Yongqian Zhu

3, 3

Huajun Zheng3, Sheng-Yue Wang

3, Cheng-Ying Jiang

1, Guo-Ping Zhao

3,4 and 4

Shuang-Jiang Liu1,2

5

6

1State Key Laboratory of Microbial Resources and

2Environmental Microbiology and 7

Biotechnology Research center at Institute of Microbiology, Beijing, 100101, P. R. 8

China; 3Shanghai-MOST key laboratory of Health and Disease Genomics, Chinese 9

National Human Genome Center at Shanghai; and 4Shanghai Institutes for Biological 10

Sciences, Shanghai, 201203, P. R. China 11

12

13

Running title: Complete genome of Comamonas testosteroni 14

15

16

For correspondence: 17

Dr. Shuang-Jiang Liu, Institute of Microbiology, Chinese Academy of Sciences, 18

Bei-Chen-Xi-Lu No. 1, Chao-Yang Disctrict, Beijing 100101, P. R. China. 19

20

Tel: +86-10-64807423, Fax: +86-10-64807421. 21

Email: [email protected] 22

Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.00933-09 AEM Accepts, published online ahead of print on 4 September 2009

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Abstract: Members of the Gram-negative, strictly aerobic genus Comamonas occur 23

in various environments. Here we report the complete genome of Comamonas 24

testosteroni strain CNB-2. Strain CNB-2 has a circular chromosome of 5,373,643 bp 25

in length and G+C content of 61.4%. Totally, 4803 ORFs were identified, of which 26

3514 ORFs were functionally assigned for energy production, cell growth, signal 27

transduction, or transportation; while 866 ORFs were conserved hypothetical proteins 28

and 423 ORFs were purely hypothetical proteins. The CNB-2 genome is abundant for 29

genes of transportation (22%) and signal transduction (6%), which found the basis for 30

cell to response and adapt to the changing environments. Strain CNB-2 does not 31

assimilate carbohydrates due to the lack of the corresponding genes involving in 32

glycolysis and pentose phosphate pathways, and it carries many genes involved in 33

degradation of aromatic compounds. We identified 66 Tct and 9 TRAP-T systems and 34

a complete tricarboxylic acid cycle, which may endow CNB-2 to uptake and 35

metabolize a range of carboxylic acids. This biased nutritional feature for carboxylic 36

acids and aromatic compounds enabled strain CNB-2 to occupy unique niches in 37

environments. Four different sets of terminal oxidases for respiratory system were 38

identified and they were putatively functioning at different oxygen concentrations. 39

Conclusively, this study revealed at genomic level that the genetic versatility of C. 40

testosteroni is vital to compete other bacteria in its special niches. 41

Keywords: Comomonas testosteroni, genome, adaptation, aromatic degradation, 42

testosterone degradation 43

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INTRODUCTION 44

The members of the genus Comamonas are Gram-negative, strict aerobes and 45

frequently occur in diverse habitats, including activated sludge, marshes, marine 46

habitats, and plant and animal tissues (4, 12). They grow on organic acids, amino 47

acids and peptone, but rarely attack carbohydrates. Some species such as Comamonas 48

testosteroni can also mineralize complex and xenobiotic compounds, such as 49

testosterone (17) and 4-chloronitrobenzene (CNB) (54). Their diversified niches make 50

Comamonas species environmentally important, and suggest also that the genus 51

Comamonas represents a group of bacteria that are very adaptive, both ecologically 52

and physiologically, to environments. 53

To understand better that how environmental microbes adapt to their 54

environments, many well-known environmental microbes such as Pseudomonas 55

putida (53) and Rhodococcus RAH1 (31) have been sequenced. The genome data of 56

these as well as other environmental microbes provide not only the understanding of 57

their physiological and environmental functions at genetic level, but also a start-point 58

for systems biology on those microbes. Up to date, none of the Comamonas species 59

has been sequenced, although they represent an important group of environmental 60

microbes. 61

C. testosteroni strain CNB-1 was isolated from CNB-contaminated activated 62

sludge and grows with CNB as sole source of carbon and nitrogen, and was exploited 63

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successfully in the rhizoremediation of CNB-polluted soil (25). Strain CNB-1 has a 64

circular chromosome and a large plasmid, and the genes involved in the degradation 65

of CNB were identified on plasmid pCNB1 (28). In the present study, the genome of 66

strain CNB-2 that derived from strain CNB-1 was sequenced, and the genome data 67

were analyzed in parallel with physiological experiments. This work was aimed to 68

provide genetic insights that how C. testosteroni adapts to changing and diverse 69

environments. 70

MATERIALS AND METHODS 71

Bacterial strain, physiological and biochemical tests. Strains CNB-2 is a 72

mutant of C. testosteroni CNB-1 that lost the degrading plasmid pCNB1 (28). The 73

physiological and biochemical properties of both the CNB-1 and CNB-2 were 74

determined by using API 20NE and API 50CH identification systems (bioMérieux, 75

Hazelwood, Mo.) at 30 oC. 76

Genome sequencing. The genome of CNB-2 was sequenced by using a 77

massively parallel pyrosequencing technology (454 GS 20) (30). All useful reads were 78

assembled into totally 545 contigs (> 500 bp), covered 25-folds of genome. The gaps 79

were closed by multiplex PCR technique (10, 48). Finally, the sequences were 80

assembled using the PHRED_PHRAP_CONSED software package on Unix system 81

(10, 48). The final consensus quality score of each base was more than 30. 82

Assessment of genome assembly was performed by long-rang PCR and GC-skew 83

analysis (supplementary Fig. S1). 84

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Sequence analysis. Coding sequences were predicted by combining results from 85

the software of Glimmer packages and GeneMark online (6, 27). Translational 86

products of the coding sequences were annotated by searching the GenBank database 87

using the basic local alignment search tool (BLAST) for proteins. Putative functions 88

of translation products were further confirmed by KEGG, TIGRFams, and Clusters of 89

Orthologous Groups (COGs) (14, 33, 46). The tRNA genes were identified by the 90

tRNAScan-SE tool (26). The ribosomal RNA (rRNA) genes were identified by 91

BLASTN using the 16S rRNA gene of strain CNB-1. Transporter genes were 92

predicted by analyzing all annotated genes (putative proteins) with the Transport 93

Classification Database (TCDB) (http://www.tcdb.org) (39). The metabolic pathway 94

of CNB-2 were constructed in silico by KEGG tools (33). The protein domains were 95

predicted in NCBI Conserved Domain Database (CDD) 96

(http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) (29). Transmembrane domains 97

and signal peptide regions were identified with TMHMM2.0 and SignalP3.0, 98

respectively (2, 23). 99

Genome comparison. The genome data of C. testosteroni KF-1 100

(NZ_AAUJ00000000), Escherichia coli ATCC 8739 (CP000946) and P. putida 101

KT2440 (AE015451) were retrieved at http://www.ncbi.nlm.nih.gov/genomes. 102

BLASTP was employed for protein sequence analysis. Significant similarity of 103

protein was defined to have e-value <1e-05 and identity >30%. 104

Nucleotide sequence accession number. The complete genome sequence and 105

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annotation of C. testosteroni CNB-2 were deposited at GenBank under accession 106

number CP001220 (Accessible to public upon acceptance of publication). 107

RESULTS AND DISCUSSION 108

General Genome Features. Strain CNB-2 has a circular chromosome of 109

5,373,643 bp in length with a G+C content of 61.4% (Fig. 1), encoding 4803 110

predicted open reading frames (ORFs), 3 rRNA operons, and 79 tRNA genes for all 111

20 amino acids (Table 1). Based on BLASTP searches (e-value<1e-5), 3514 ORFs 112

encode proteins with assigned functions, 866 of conserved proteins and 423 of 113

hypothetical proteins. With COG functional assignment, 4649 ORFs were predicted. 114

Totally, the gene coding sequences cover 86.4% of the genome, and the average 115

length of the CDSs is 963 bp. The likely origin of replication (oriC) was located 116

between ribosomal protein L34 and DnaA genes based on GC-skew analysis 117

(supplementary Fig. S1). Four putative DnaA boxes (DnaA binding sites) were found 118

between the L34 and the DnaA genes. 119

Mobile genetic elements (MGEs), horizontal gene transfer (HGT), and 120

adaptation to environments. The genome of CNB-2 contains large numbers of 121

mobile genetic elements (Table 2). Three putative prophages (I, II and III) were 122

identified. Remarkably, these prophages harbor accessory genes possibly acquired by 123

HGT from other bacterial species. For example, a gene cluster encoding putative 124

terephthalate-dioxygenase (CtCNB1_1493-1496) was identified on prophage I. 125

Totally 39 ORFs encode various transposases and integrases of the IS21, IS4, IS3, and 126

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IS110 families were discovered (Table 2). The occurrence of highly homologous or 127

even identical ORFs among the 39 ORFs suggests that recent gene transposition or 128

gene duplication events happened in the genome. MGEs on CNB-2 genome are 129

mainly located on the region near to DNA replication terminal site. 130

HGT might result in sharp changes of GC content on the genome and confer the 131

cells additional physiological capacity. In the region of 2.75-2.97 Mbp, the GC 132

content is 57.0%, which is lower than that of this whole genome (61.4%). Considering 133

the observation of many putative transposes in this region (Fig. 1), it was deduced that 134

strain CNB-2 acquired this region via HTG. Functionally, this region of the CNB-2 135

genome carries gene clusters putatively for xenobiotic degradation (e.g. 136

protocatechuate-degrading pathway) and for heavy metal resistance (copper and 137

arsenate resistance). 138

Comparative genome. A different strain of C. testosteroni, strain KF-1, is being 139

sequenced (unfinished, data available at http://www.ncbi.nlm.nih.gov). Based on 16S 140

rRNA gene analysis, strains CNB-2 and KF-1 are closely phylogenetic relatives (99% 141

identity). In addition, they occur in similar niches (activated sludge) and are able to 142

degrade xenobiotics. Comparison of the two genomes showed that they were highly 143

similar to each other, thus provides the genetic evidences for their ecological and 144

physiological alikeness: 4272 ORFs (totally 4803 ORFs) of strain CNB-2 are 145

significantly similar to their counterparts of strain KF-1 (totally 5492 ORFs). 146

However, the results also revealed different features between CNB-2 and KF-1 (Table 147

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1). The two genomes have ca. 0.7 Mbp difference in size. The three prophages of 148

CNB-2 were not found on the KF-1 genome. 531 ORFs of CNB-2 that frequently 149

associated with MGEs were not found for their corresponding ORFs in KF-1. The 150

genomic differences between CNB-2 and KF-1 are at least partially attributed to the 151

abundance of MGEs in CNB-2 genome. Further comparative analysis of genomes of 152

strains CNB-2 and KF-2, P. putida KT2440, and E. coli ATCC 8739 are provided in 153

supplementary Fig. S2. 154

General metabolism of strain CNB-2 and the core physiological features of 155

the genus Comamonas. The CNB-2 grows on mineral medium, indicating that it 156

owns the ability to synthesize all the cellular components such as fatty acids, purine 157

and pyrimidine nucleotides, and the 20 amino acids. The genomic analysis in silico 158

supported this hypothesis. More information relative to general metabolism is shown 159

in Fig. 2. 160

The genus Comamonas was defined as having poor ability to assimilate 161

carbohydrates. Of the 49 carbon sources tested in this study, CNB-2 was not able to 162

assimilate any sugars except for glycerol and gluconate. The glycolysis for glucose 163

catabolism was incomplete due to the lack of the hexokinase and glucokinase genes. 164

The genes encoding glucose-6-phosphate-1-dehydrogenase and 165

6-phosphogluconolactonase of the pentose phosphate pathway were not found in 166

CNB-2 genome. Thus, the oxidation of glucose via this pentose phosphate pathway 167

was not possible (Fig. 2). However, the non-oxidative part of the pentose phosphate 168

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pathway was complete, and 5-carbon sugars were generated for biosynthesis. An 169

almost complete Entner-Doudoroff (ED) pathway was constructed in silico for 170

gluconate assimilation (Fig. 2), and physiological tests showed that strain CNB-2 171

grew on gluconate. In contrast to this poor ability to metabolize carbohydrates by 172

Comamonas species, members of the Pseudomonas such as P. putida KT2440 are able 173

to metabolize many sugars (20). This genome project on the representative of 174

Comamonas species revealed that they do not have a complete sugar metabolizing 175

pathways. Based on these findings, it was proposed that the two groups of 176

environmental microbes, Comamonas and Pseudomonas species, might play different 177

roles in geobiochemical cycles of elements. 178

The C. testosteroni was named after its ability to metabolize testosterone. A 179

genetic cluster (CtCNB1_1351-1362) was found in the strain CNB-2 genome, which 180

was similar to the well-identified genes responsible for testosterone degradation in C. 181

testosteroni TA441 (1, 16-18) (Fig. 3). 182

As an important environmental microbe, strain CNB-2 has the ability to utilize 183

many other kinds of compounds such as aromatics and short-chain fatty acids for 184

carbon sources. On the CNB-2 genome, 37, 18 and 47 genes encoding putative 185

dioxygenases, hydroxylases and oxidoreductases, respectively, were annotated for 186

aromatic and cyclic hydrocarbon degradation. Strain CNB-2 was able to grow on 187

benzoate, gentisate, phenol, 3-hydroxybenzoate, 4-hydroxybenzoate, protocatechuate, 188

and vanillate, and the corresponding gene clusters were identified on the genome (Fig. 189

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3). The key genes involved in degradation of the following compounds on CNB-2 190

genome were found, including catechol (CtCNB1_3144-3155), protocatechuate 191

(CtCNB1_2740-2747) and gentisate (CtCNB1_2777-2780). A range of peripheral 192

enzymes that direct aromatic compounds into central pathways were also discovered, 193

such as benzoyl-CoA ligase (CtCNB1_0097), benzoyl-CoA oxygenase 194

(CtCNB1_0065, 0066, 0067), Phenol hydroxylase (CtCNB1_3158-3162), 195

3-hydroxybenzoate hydroxylase (CtCNB1_3410), 4-hydroxybenzoate hydroxylase 196

(CtCNB1_2156) and vanillate monoxygenase (CtCNB1_4192). Also, 66 Tct systems 197

and 9 TRAP-T (please see “Tables for reviewers”, Table S1) were identified for 198

transportation of carboxylic acids. These carboxylic acids were metabolized through 199

the TCA cycle to supply energy and key intermediates for the cell growth. 200

Strain CNB-2 uses nitrate and ammonium as nitrogen source, and 3 genes 201

(CtCNB1_0130, 0202, 0616) encoding ammonium channels and 2 gene clusters 202

(CtCNB1_0586-0588, CtCNB1_1234-1236) for ABC-type nitrate transporters were 203

found on the genome. Inorganic nitrogen is finally incorporated into glutamine by 204

glutamine synthetase (CtCNB1_3271). Additionally, genes encoding putative 205

transporters were identified on the CNB-2 genome for import of exogenous amino 206

acids, oligopeptides, and branched-chain amino acids. These compounds could be 207

metabolized through the urea cycle and TCA cycle, which could supply not only 208

adequate nitrogen source, but also carbon source for cell growth. 209

Transport systems. Strain CNB-2 encodes genes for a broad range of transport 210

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proteins (Fig. 2 and please see “Tables for reviewers”, Table S1). Totally 22% of all 211

genes were predicted to be involved in substrates transport. This value is higher than 212

that of E. coli, Haemophilus influenzae and Helicobacter pylori that live in relatively 213

closed environments, but is similar to that of microbes such as P. putida that live in 214

open environments. 10 genes were predicted as channel transporter (TC #1.A) 215

responsible for ammonia, magnesium/cobalt, and urea/amide. 378 genes are probably 216

carriers (TC #2) for sugars, polyols, drugs, neurotransmitters, Krebs cycle metabolites, 217

phosphorylated glycolytic intermediates, amino acids, peptides, osmolites, 218

siderophores, iron-siderophores, nucleosides, organic anions, and inorganic anions. 13 219

predicted APC family transporters (TC #2.A.3) may be involved in amino acid 220

transport for cell. 36 genes encode 12 protein complexes of RND family, which 221

catalyze drugs or heavy metals efflux via an H+ antiport mechanism. On the genome 222

of CNB-2, the group of transporters (TC #3.A.) driven by P~P-bond-hydrolysis is the 223

largest one, and such transporters export or import of the cell a large amount of 224

compounds, including amino acids, drugs, metal ion, inorganic anions, proteins, and 225

aromatic compounds. 226

Effective transportations are important for microbial evolution. Import of a 227

compound into cells is vital to evolve metabolic pathway, and export of a toxic 228

compound out of cells is also vital for bacterial survival in harsh environments. As 229

mentioned above, 22% genes of the CNB-2 genome were predicted to encoding 230

channels, electrochemical potential-driven transporters, ABC-type transporters, RND 231

efflux systems, protein secretion systems and other transportation-related proteins 232

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according to the result using BLASTP searching in TCDB (e<10-5). This number is 233

similar to those of bacteria living in soil or plant, for example, Streptomyces 234

coelicolor (35), P. putida KT2440 (53) and P. aeruginosa (24), but more than those of 235

symbiotic and pathogenic bacteria, such as Mycobacterium leprae (51). Further 236

analysis revealed that P. aeruginosa (24) and Agrobacterium tumefaciens (5) possess 237

high numbers of transporters for sugars, amino acids and peptides, and that C. 238

testosteroni CNB-2 and P. putida KT2440 (53) has much less transporters for sugars. 239

Interestingly, P. putida utilizes many sugars for growth, but Comamonas species 240

including strain CNB-2 assimilate few sugars. 241

Secretion systems. Protein exportation that controls secretion of toxins, 242

hydrolytic enzymes and adhesins plays important role for Comamonas species living 243

in various niches and colonizing different surfaces. Four types of protein secretion 244

were identified in Gram-negative bacteria (40), but only two types were found in 245

CNB-2 genome. The putative type I secretion system of strain CNB-2 includes five 246

gene clusters (CtCNB_2177-2179, CtCNB_2281-2285, CtCNB_2442-2446, 247

CtCNB1_3019- 3022, CtCNB1_3183- 3186), which consist of a pore-forming outer 248

membrane protein, a membrane fusion protein, and an inner membrane ATP-binding 249

cassette protein. Type I secretion system was also involved in exporting arabinose, 250

hemolysin and drug outside of the cell. The type II secretion system functions via Sec, 251

Tat and Pul export pathways (3, 52), and their genes are usually conserved in 252

Gram-negative bacteria (7). The Sec genes of CNB-2 were identified and similar to 253

that of E. coli, but were not clustered except SecD/SecF/YajC (CtCNB1_4737-4739) 254

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that were consecutively organized and encoded for a putative protein complex. The 255

Tat genes, tatABC (CtCNB1_0735-0737), were located in one putative operon, but the 256

tatE was not found. As suggested from previous studies (41), the function of TatE 257

might be replaced by TatA. A genetic cluster of 11 genes (CtCNB1_0617-0634) that 258

encoded proteins similar to that of Klebsiella oxytoca (38) was identified on the 259

CNB-2 genome. 260

Signal transduction, regulation and responses to environmental changes. In 261

order to respond environmental changes in a quick and efficient way, environmental 262

bacteria evolved various signal sensing and transduction systems (36, 44). According 263

to COG and CDD analyses (47), 300 ORFs of the CNB-2 genome were assigned to 264

functions for signal transduction and regulation. Furthermore, 58 ORFs were 265

predicted to be putative response regulators, 46 ORFs to be putative histidine kinases, 266

and 10 to be putative hybrid sensory kinases. These putative regulators and kinases 267

were further grouped into 43 complete two-component signal transduction systems 268

(please see “Tables for reviewers”, Table S2). Moreover, 64 ORFs that contained one 269

output domain (50) and were putatively functioning as one component signal 270

transduction were identified on the CNB-2 genome (please see “Tables for reviewers”, 271

Table S3). The high content of signal transduction and regulation genes on the 272

genome of CNB-2 is comparative to that of P. aeruginosa PA01 (45) and is higher 273

than that of E. coli (32). 274

The comprehensive and diversified signal transduction and regulation systems 275

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enable strain CNB-2 respond to various environmental changes. This is further 276

supported by analysis of functional domains of the signal transduction systems. 277

Various input (signal sensing) domains, such as HAMP, PAS, KdpD and CHASE of 278

one- and two-components system were discovered (please see “Tables for reviewers”, 279

Table S3 and Table S4) (8). Especially, the domain PAS (55) was frequently identified 280

(please see “Tables for reviewers”, Table S2 and Table S3) and even three PAS 281

domains were found in the putative protein CtCNB1_4372 (please see “Tables for 282

reviewers”, Table S3). The CtCNB1_4372, a putative one-component signal 283

transduction system neighboring a putative ammonium monooxygenase, might be 284

involved in response to changing oxygen concentration and in regulation of 285

ammonium assimilation by strain CNB-2. 286

Chemotaxises are important microbial responses to environmental signals and 287

chemotaxic responses to aromatic compounds were characterized in Pseudomonas 288

and Ralstonia species (11, 34). Our results showed that strain CNB-2 exhibited 289

chemotaxis towards succinate, while strain CNB-1 exhibited chemotaxis towards both 290

succinate and CNB. Although the putative genes linking the sensing of chemical 291

signals and cell movement were not able to be identified at this stage, some relative 292

genes were identified in the CNB-2 genome. A locus of CtCNB1_0471-0475 carrying 293

three putative regulators and one putative kinase localized between the genes of 294

flagellar biosynthesis and chemotaxis, and ORFs of putative proteins (CtCNB1_0475, 295

0476, 0479) that were homologous to CheA, CheY and CheB proteins were found in 296

CNB-2 genome. 297

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Drug and heavy metal resistances. Several loci of CNB-2 genome were 298

predicted responsible for drug and heavy metal export (please see “Tables for 299

reviewers”, Table S1). Three gene clusters (CtCNB1_2506-2509, 3138-3141, 300

3971-3974) encode the system for exporting arsenate. Analysis of the CNB-2 genome 301

revealed that this strain CNB-2 carried 6 CzcA-type heavy metal efflux pumps for 302

heavy metals and 11 RND-type efflux transporters for various drugs (Fig. 2, also 303

please see “Tables for reviewers”, Table S2). Those gene possibly conferred strain 304

CNB-2 additional resistances to heavy metals and drugs. Moreover, strain CNB-2 has 305

many chaperones helping cell to overcome the toxicity. The toxic drug and heavy 306

metals could induce heat shock proteins (42, 49), as those [HslJ (CtCNB1_3317), 307

IbpA (CtCNB1_3446), HslU(CtCNB1_4124), HslJ (CtCNB1_4571)] were identified 308

on the genome of strain CNB-2 (please see “Tables for reviewers”, Table S4.). 309

Interestingly, those drug and heavy metal resistance genes are associated with MGEs. 310

For examples, four gene clusters (CtCNB1_ORF2496-2499, 2507-2510, 2513-2520, 311

2521-2524) of P-type ATPase, the CzcA family efflux pump genes, and the 312

arsenate-resistance genes are flanked by the genes of Tn3-type transposases, 313

IS116/IS110/IS902-type transposases and IS4-type transposases, respectively. 314

Responses to the availability of electron acceptor and rhizospheric 315

association with plant. Strain CNB-2 is strictly aerobic, and needs oxygen molecules 316

as final electron acceptor. All the aerobic respiratory chain members were tentatively 317

identified on the CNB-2 genome. Four putative terminal oxidase complexes, 318

CtCNB1_1811-1814, CtCNB1_2157-2161, CtCNB1_3937-3941, and 319

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CtCNB1_3431-3432 that were highly similar to cbb3-type, bo3-type, aa3-type and 320

bd-type (19) were found. It was reported that cbb3-type and bd-type terminal oxidases 321

functioned at lower oxygen concentrations in endosymbiotic rhizobia (37) and at 322

microaerobic conditions in nitrogen fixing bacteria (15, 22) and in association with 323

animals (21, 43). Thus, the robust competitiveness of strain CNB-1 in soil rhizosphere 324

and wastewater treatment plants is possibly attributed to the occurrence of multiple 325

terminal oxidases (25). Genome analysis suggested a putative transhydrogenase 326

(CtCNB1_0257-0258) that might regulate energy metabolism in strain CNB-2 at 327

different oxygen concentrations. 328

Our physiological experiment showed that CNB-2 could respire with nitrate 329

aerobically to produce energy. Accordingly, a gene cluster covering five genes 330

(napABCDE), which is responsible for the first step of aerobically denitrification (9), 331

and encoding putative periplasmic nitrate reductases was identified. Thus, strain 332

CNB-2 is equipped with multiple terminal oxidases depending on oxygen 333

concentrations, and with the ability of using nitrate as electron acceptor. 334

CONCLUSION 335

C. testosteroni CNB-2 is the first member of genus Comamonas that has been 336

completely sequenced. Its genome size is close to the previously reported 337

Pseudomonas species, for examples, P. putida GB-1 (6.1 Mb) (CP000926), P. putida 338

KT2440 (6.2 Mb) (AE015451), and P. stutzeri A1501 (4.6 Mb) (CP000304). 339

Comamonas species were found ubiquitous in terrestrial and aquatic environments, 340

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and associated with plants and animals. They were also found in severely polluted 341

environments. The diverse niches occupied by Comamonas species reflect their 342

effective adaptation to various physiochemical dimensions. In this study, the high 343

genetic enrichment and capacity of C. testosteroni CNB-2 revealed its highly 344

evolutionary adaptations and its genetic foundations for living in diverse and complex 345

ecological niches. This first C. testosteroni genome will significantly facilitate further 346

basic and applied investigations on the bacterial group of Comamonas species. 347

ACKNOWLEDGMENTS 348

This work is supported by grants from the National Natural Science Foundation 349

of China (30725001, 30621005). 350

REFERENCE 351

352

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Figure legends: 524

Fig. 1. Circular chromosome representation of C. testosteroni CNB-2. Outer scale 525

indicates true size relative to the chromosome. The first two rings (outside) represent 526

the genes on the forward and reverse strands, respectively. Rings 3 and 4 represent the 527

genes of CNB-2 that have no significant similarity to the genes of C. testosteroni 528

KF-1 (green) and P. putida KT2440 (red). Rings 5 and 6 show the GC content and GC 529

skew (C-G/C+G), respectively. Ring 7 represents prophages (gray), transposases, 530

recombinases, and integrases (black). Ring 8 indicates tRNA genes (blue) and rrn 531

operons (tomato1). Base pair 1 of this chromosome was assigned to the first 532

nucleotide acid of DnaA gene. For Rings 1 and 2, the genes are colored according to 533

COG categories. 534

535

Fig. 2. Overview of metabolisms and transport in C. testosteroni CNB-2. 536

Transporters are grouped by substrate specificity: inorganic cations (green), inorganic 537

anions (pink), carbohydrates (yellow), amino acids/peptides/amines/purines/ 538

pyrimidines and other nitrogenous compounds (red), drug efflux and other (black). 539

Arrows indicate the direction of substrates transport. 540

541

Fig. 3. Gene clusters involved in aromatic compound metabolism in Comamonas 542

testosterone strain CNB-2. Abbreviations: AphA, catechol 2,3-dioxygenase; AphB, 543

protein involved in meta-pathway of phenol degradation; AphR, transcriptional 544

regulator; AphC, 2-hydroxymuconic semialdehyde dehydrogenase; AphD, 545

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2-hydroxymuconic semialdehyde hydrolase; AphE, 2-hydroxypent-2,4-dienoate 546

hydratase; AphF, acetaldehyde dehydrogenase (acetylating); AphG, 547

4-hydroxy-2-oxovalerate aldolase; AphH, 4-oxalocrotonate decarboxylase; AphI, 548

4-oxalocrotonatetautomerase; Bla, benzoate-CoA ligase family; BoxA, benzoyl-CoA 549

oxygenase/reductase, component A; BoxB, benzoyl-CoA oxygenase component B; 550

BoxC, enoyl-CoA hydratase/isomerase; CnbA, 4CNB nitroreductase; CnbCa, 551

2-amino-5-chlorophenol 1,6-dioxygenase alpha subunit; CnbCb, 2-aminophenol 552

1,6-dioxygenase beta subunit; CnbD, 2-amino-5 -chloromuconic semialdehyde 553

dehydrogenase; CnbZ, 2-amino-5-chloromuconic acid deaminase; CnbG, 554

2-hydroxy-5-chloromuconic acid tautomerase; CnbE, 2-keto-4-pentenoate hydratase; 555

CnbF, 4-oxalocrotonate decarboxylase; ModA, ABC-type molybdate transport system 556

periplasmic component; PmdA, protocatechuate 4,5-dioxygenase alpha subunit; 557

PmdB, protocatechuate 4,5-dioxygenase beta subunit; PmdC, 2-hydroxy, 558

4-carboxymuconic semialdehyde dehydrogenase; PmdD, 2-pyrone-4,6-dicarboxylic 559

acid hydrolase; PmdE, 4-oxalomesaconate hydratase; PmdF, 560

4-hydroxy-4-methyl-2-oxoglutarate aldolase; PmdK, transporter; PH, phenol 561

hydroxylase; MPBH, m-hydroxybenzoate hydroxylase; PHBH, p-hydroxybenzoate 562

hydroxylase; VanA, vanillate monooxygenase; GodA, gentisate 1,2-dioxygenase; 563

MaiA, maleylacetoacetate isomerase; Fah, fumarylacetoacetate (FAA) hydrolase. The 564

arrows indicate putative pathways of the compounds. The grey arrows show the genes 565

possibly functioning in degradation process, while the black ones show the genes with 566

unknown function. 567

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568

Table 1 General features of the genomes of C. testosteroni CNB-2 and KF-1. 569

570

CNB-2 KF-1*

Total size (bp) 5,373,643 6,026,527

GC content (%) 61 61

Coding density (%) 86 86

ORFs 4803 5492

With assigned function 3514 4266

Conserved protein 866 952

Hypothetical protein 423 NA

Average ORF length (bp) 963 952

rRNA operons 3 6

tRNA 79 9

571

* Data for the unfinished genome of strain KF-1 was retrieved from GenBank deposit. 572

NA, no analysis. 573

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574

Table 2. Mobile genetic elements on C. testosteroni CNB-2 genome 575

576

Prophages

I CtCNB1_1435-1533

II CtCNB1_2607-2676

III CtCNB1_3548-3599

Transposases

IS21 CtCNB1_1935/1936,CtCNB1_1978/1979,

CtCNB1_2459/2458,CtCNB1_2521/2522,

CtCNB1_2582/2583,CtCNB1_3734/3735,

CtCNB1_2520/2522,CtCNB1_1546/1547

Integrases CtCNB1_1933,CtCNB1_1982,CtCNB1_2289,

CtCNB1_2318,CtCNB1_3007,CtCNB1_4129,


CtCNB1_2336, CtCNB1_2006,tCNB1_2333,






CtCNB1_1901CtCNB1_2333,

IS116/IS110

/IS902

CtCNB1_2462,CtCNB1_2490,CtCNB1_2491

IS3/IS911 CtCNB1_1899,CtCNB1_2479,CtCNB1_4038,

CtCNB1_3809

IS4 CtCNB1_0110,CtCNB1_2435,CtCNB1_2460,


CtCNB1_3023,CtCNB1_2488,CtCNB1_2763

Tn3 CtCNB1_2516

Other-type CtCNB1_3808,CtCNB1_4037,CtCNB1_3731,

CtCNB1_2334,CtCNB1_2478,

577

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on April 20, 2020 by guest · 9/4/2009 · Strain CNB-2 has a circular chromosome of 5,373,643 bp in length and G+C content of 61.4%. Totally, 4803 ORFs were identified, of which