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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
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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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Table 1 General features of the genomes of C. testosteroni CNB-2 and KF-1. 569
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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
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* Data for the unfinished genome of strain KF-1 was retrieved from GenBank deposit. 572
NA, no analysis. 573
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Table 2. Mobile genetic elements on C. testosteroni CNB-2 genome 575
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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_1938,CtCNB1_1940,CtCNB1_2411,
CtCNB1_2336, CtCNB1_2006,tCNB1_2333,
CtCNB1_1888,CtCNB1_2002,CtCNB1_2003,
CtCNB1_2475,CtCNB1_2476,CtCNB1_3143,
CtCNB1_1900,CtCNB1_1901,CtCNB1_1888,
CtCNB1_2002,CtCNB1_2003,CtCNB1_2475,
CtCNB1_2476,CtCNB1_3143,CtCNB1_1900,
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_2461,CtCNB1_2571,CtCNB1_2765,
CtCNB1_3023,CtCNB1_2488,CtCNB1_2763
Tn3 CtCNB1_2516
Other-type CtCNB1_3808,CtCNB1_4037,CtCNB1_3731,
CtCNB1_2334,CtCNB1_2478,
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