- Research article
- Open Access
High correlation between genotypes and phenotypes of environmental bacteria Comamonas testosteroni strains
© Liu et al.; licensee BioMed Central. 2015
Received: 30 September 2014
Accepted: 3 February 2015
Published: 21 February 2015
Members of Comamonas testosteroni are environmental microorganisms that are usually found in polluted environment samples. They utilize steroids and aromatic compounds but rarely sugars, and show resistance to multiple heavy metals and multiple drugs. However, comprehensive genomic analysis among the C. testosteroni strains is lacked.
To understand the genome bases of the features of C. testosteroni, we sequenced 10 strains of this species and analyzed them together with other related published genome sequences. The results revealed that: 1) the strains of C. testosteroni have genome sizes ranging from 5.1 to 6.0 Mb and G + C contents ranging from 61.1% to 61.8%. The pan-genome contained 10,165 gene families and the core genome contained 3,599 gene families. Heap’s law analysis indicated that the pan-genome of C. testosteroni may be open (α = 0.639); 2) by analyzing 31 phenotypes of 11 available C. testosteroni strains, 99.4% of the genotypes (putative genes) were found to be correlated to the phenotypes, indicating a high correlation between phenotypes and genotypes; 3) gene clusters for nitrate reduction, steroids degradation and metal and multi-drug resistance were found and were highly conserved among all the genomes of this species; 4) the genome similarity of C. testosteroni may be related to the geographical distances.
This work provided an overview on the genomes of C. testosteroni and new genome resources that would accelerate the further investigations of this species. Importantly, this work focused on the analysis of potential genetic determinants for the typical characters and found high correlation between the phenotypes and their corresponding genotypes.
Comamonas testosteroni is a bacterial species belonging to β-Proteobacteria which was originally named as Pseudomonas testosteroni using a soil bacterium ATCC 11996 as the type strain . In 1987, Tamaoka et al. reclassified this species into Comamonas testosteroni based on phylogenetic analysis . C. testosteroni strains are capable of utilizing testosterone, 4-hydroxybenzoate (4HBA), acetate and lactate as their sole carbon sources, but not glucose and most of the carbohydrates . They are strictly aerobic, highly motile and gram-negative rods and can perform nitrate reduction but cannot denitrify .
C. testosteroni strains have been paid a great attention duo to their characteristics of degradation for pollutants and resistance to heavy metals. For example, most strains of C. testosteroni were isolated from such environments as active sludge [4,5] and heavy metal-contaminated mining soil ; C. testosteroni T-2 could utilize 4-toluenesulphonic acid and 4-sulphobenzoic acid ; C. testosteroni SPB-2 (KF-1) could utilize 4-sulfophenylcarboxylates . C. testosteroni CNB-1 have successfully been applied in environment bioremediation to degrade 4-chloronitrobenzene (4CNB) in the soil .
The genetic determinants for the organic compound degradation and heavy metal resistance have been analyzed in several C. testosteroni strains. Horinouchi et al. discovered five gene clusters for steroid degradation in the genome of C. testosteroni TA441 [10-15]. Gene operons responsible for degrading isophthalate and terephthalate to protocatechuate were determined in C. testosteroni YZW-D by Wang et al. . The operon aph (KLMNOPQB) that encodes phenol hydroxylase and catechol 2,3-dioxygenase, and the operon aph (CEFGHJI) that encodes the enzymes for degrading 2-hydroxymuconic semialdehyde to TCA cycle intermediates were found in C. testosteroni TA441 [17,18]. A gene cluster for protocatechuate (PCA) 4,5-cleavage pathway, which is an important pathway for aromatic compounds, such as 4HBA degradation , were found in the genome of C. testosteroni CNB-2 . C. testosteroni CNB-1 showed resistance to arsenate and an ars (RPBC) operon was found on the pCNB1 plasmid [5,21]. Xiong et al.  have reported ZntA genes responsible for Zn resistance in C. testosteroni S44.
So far, the genomic sequences of four C. testosteroni strains have been published. Ma et al.  sequenced the whole genome of C. testosteroni CNB-2, analyzed its general genome features, and found genes for many functions such as aromatic compound metabolism, transportation system and drug and heavy metal resistance. Gong et al.  sequenced the genome of the type strain of C. testosteroni ATCC 11996. Weiss et al.  sequenced the genome of strain KF-1, and found genes for degradation of aromatic compounds such as a gene cluster for PCA 4,5-cleavage and a gene cluster for isophthalate degradation. In our previous study, a multiple heavy-metal resistant strain named C. testosteroni S44 was isolated and its genome was sequenced . A series of metal-resistant genes and gene clusters for aromatic compound degradation were found in its genome [6,24]. Regarding carbohydrates utilization, studies on both genomes of C. testosteroni strains CNB-2 and ATCC 11996 showed that the genes encoding hexokinase and glucokinase were missing [5,22]. However, a comprehensive genomic analysis among the C. testosteroni strains is lacked.
The objective of this study was to analyze the genetic basis responsible for important biological characters in multiple C. testosteroni strains. Recently, we have identified ten C. testosteroni strains from different mining soils [24,25] according to the 16S rRNA gene and physiological and biochemical analysis. In order to gain a comprehensive genotypic and phonotypic understanding, we sequenced the ten C. testosteroni strains and analyzed them together with the four published C. testosteroni genomes [5,6,22,23]. This study is a first comparative genomic analysis for C. testosteroni strains. The results have revealed information to better understand this bacterial species at genomic level, and importantly, have pointed out potential genetic determinants for the typical characters and high correlation between the phenotypes and their corresponding genotypes (genes).
Genome analysis of C. testosteroni and related strains
General features of the genomes of the Comamonas testosteroni strains used in this study
No. of ORFs
Avg. ORF length (bp)
Accession number (reference)
C. testosteroni JC8
Coal mine soil
AWOS00000000 (this study)
C. testosteroni JC9
Coal mine soil
AWOT00000000 (this study)
C. testosteroni JC12
Coal mine soil
AWOU00000000 (this study)
C. testosteroni JC13
Coal mine soil
AWOV00000000 (this study)
C. testosteroni JL14
Sb mine soil
AWTN00000000 (this study)
C. testosteroni JL40
Sb mine soil
AWOR00000000 (this study)
C. testosteroni D4
As polluted soil
AWTQ00000000 (this study)
C. testosteroni DS1
Cu-Fe mine soil
AWTM00000000 (this study)
C. testosteroni DF1
Fe mine soil
AWTO00000000 (this study)
C. testosteroni DF2
Fe mine soil
AWTP00000000 (this study)
C. testosteroni S44
Sb mine soil
C. testosteroni CNB-2
C. testosteroni ATCC 11996
C. testosteroni KF-1
Ortholog clustering analysis
The OrthoMCL package  was used to determine the core-genome and unique genes of each genome. All predicted protein sequences were grouped together and compared with each other using blastp of NCBI-blast 2.27+ program . The homologous protein pairs with E-value cutoff 1e-5 and percent of match ≥ 50% were parsed, and the normalized homologous scores were calculated. We screened the homologous protein pairs by a custom Perl script with percent of identity greater than 50%. Then the proteins were grouped into orthologous families by cluster tool MCL (MCL; Van Dongen 2000; http://micans.org/mcl/), with the inflation value of 1.5. The unique genes of each genome were identified based on the resultant orthologous families by a custom Perl script. The core-genome sizes were estimated by summing the orthologous families that contained genes from all the selected genomes. The pan-genome sizes were estimated by summing all the orthologous families and the single genes together. Heap’s law  was used to calculate the constant α in power law regression analysis n = κN ‐ a which could determine whether pan-genome is open (α ≤ 1) or closed (α > 1) (where n is the number of new genes, N is the number of genomes, κ is another constant).
Consulting the method of Li et al.  and Collins et al. , the single copy orthologous gene families (contain one gene from one strain), which were extracted from the genomes of 14 C. testosteroni strains and from the genomes of 22 strains of family Comamonadaceae, were used to build phylogenomic trees to present the phylogenetic relationship. The predicted amino acid sequences of each gene family were aligned using Clustal W . The independent alignments were concatenated to form a pseudo amino acid sequence alignment. The result was submitted to MEGA6  to build Neighbor-joining (NJ) trees with p-distance. The bootstrap method of 1,000 bootstrap repetitions was used to assess tree reliability. Average Nucleotide Identity (ANI) analysis was performed among the 14 genomes from different sampling areas using Jspecies1.2.1 .
Analysis of important physiological and biochemical characteristics of 11 C. testosteroni strains
Most of physiological and biochemical characteristics were determined using the API 20NE system (bioMe’rieux, Marcyl’Etoile, France), except for the utilization of citrate that was detected using Simmons’ citrate assimilation test . For utilization of acetate, testosterone and 4HBA, the strains were cultured in basal medium containing standard minerals based on Stanier et al.  and with 0.3% (wt/vol) sodium acetate trihydrate, 0.2% (wt/vol) testosterone and 0.2% (wt/vol) sodium 4HBA, respectively, and incubated at 28°C for 10 days. The Minimum Inhibitory Concentrations (MICs) of Hg(II), Cu(II) and Zn(II) were detected in CDM solid medium  containing increasing concentrations of HgCl2, CuSO4•5H2O, and ZnCl2, respectively . Antibiotic susceptibility tests for penicillins (penicillin, prostaphlin and ampicillin), kanamycin, and tetracycline were detected using disk diffusion test on Mueller-Hinton agar with 10 μg penicillin, 1 μg prostaphlin, 10 μg ampicillin, 30 μg kanamycin and 30 μg tetracycline disks, respectively (HangZhou Microbial Reagent Co. LTD, China).
Construction of putative metabolic pathways based on the genomic sequences
The predicted proteins of each genome were submitted to KEGG Automatic Annotation Server (KAAS)  to perform KEGG Orthology (KO) functional annotation. The results were then submitted to Mapper of Kyoto Encyclopedia of Genes and Genomes (KEGG)  to reconstruct metabolic pathways.
Identification of heavy metal- and drug-resistant genes and virulence factors
Genes involved in heavy metal- and drug-resistance were identified base on the SEED gene functional classification of the “resistance to antibiotics and toxic compounds” subsystem on RAST server [35-37]. The identified genes were grouped into gene clusters. Orthologous genes from each genome were aligned by Clustal W , concatenated, and then submitted to MEGA6  to build NJ trees with p-distance and bootstrap method of 1000 bootstrap repetitions.
The virulence factors were identified by aligning the predicted protein sequences from the genomes of 14 C. testosteroni strains against the Virulence Factors Data Bases (VFDB)  using blastp of NCBI-blast 2.27+ with the standard of E-value ≤1e-5, identity ≥ 50%, match ≥ 70%.
Identification of putative genes responsible for the physiological and biochemical characteristics
Genes for nitrate/nitrite reduction were identified base on the SEED gene functional classification of the “Nitrate and nitrite ammonification” subsystem on RAST server [35-37]. Five gene clusters for steroid degradation that were found in C. testosteroni strain TA441 (GenBank: AB489116, AB040808, AB063482, AB076368, AB186487) [10-15] were searched against each genome sequence using blastn of NCBI-blast2.27+ program . Protein sequences from these 5 gene clusters were also searched against all the predicted proteins in the 22 genomes using blastp. Gene cluster of PCA 4,5-cleavage  that is potentially involved in 4HBA degradation was searched against the predicted proteins of genomes of C. testosteroni using blastp. The orthologous genes for steroid degradation, PCA 4,5-cleavage and nitrate/nitrite reduction were used to build evolutionary trees. Amino acid sequences of orthologous genes were aligned by Clustal W , concatenated, and submitted to MEGA6  to build NJ trees with p-distance and bootstrap method of 1000 bootstrap repetitions. The correlation value between genotypes and phenotypes is calculated based on the phenotypic results compared with the presence or absence of the putative corresponding genes.
Genome sequencing and features of 14 C. testosteroni strains
Ten genomes of C. testosteroni (strains JC8, JC9, JC12, JC13, JL14, JL40, D4, DS1, DF1 and DF2) were sequenced in this study using Illumina HiSeq2000. After trimming the raw data, we obtained 7,130,708 to 16,794,780 paired reads and 147,354 to 383,694 single reads with the average read lengths from 94 bp to 96 bp for each genome. The sequencing depths were from 116.2x to 283.2x. The reads of each genome were assembled into 75 to 238 contigs, and the total length of each draft genome was from 5,061,365 bp to 5,956,604 bp. The contig N50 was from 98,152 bp to 242,732 bp, and the contig N90 was from 18,558 bp to 56,606 bp. The details about the sequencing and assembly of each genome are showed in Additional file 3: Table S3.
Features of the ten newly sequenced and four published genomes of C. testosteroni were showed in Table 1. The sizes of C. testosteroni genomes are from 5.06 Mb to 6.03 Mb, and the G + C contents are from 61.1% to 61.8%. The predicted numbers of open reading frames (ORFs) are from 4674 to 5645 with the average lengths from 897 bp to 938 bp. These coding sequences cover 85% to 86% of their genome sequences.
The core- and pan-genomes of C. testosteroni
Phylogenetic relationships between C. testosteroni and other species in family Comamonadacea and among C. testosteroni strains
To understand the phylogenetic relationship between C. testosteroni and other species in family Comamonadacea, we performed phylogenetic analysis among the 14 C. testosteroni strains together with eight more sequenced strains from different species of family Comamonadacea (Additional file 2: Table S2). A NJ tree based on a total of 1,003 single copy orthologous gene families of 22 strains was built (Figure 2B). All the 14 C. testosteroni strains are grouped together. Strain Delftia acidovorans SPH-1, which was classified to genus Comamonas before 1999 , showed a closer relationship with strains of C. testosteroni than with the other seven species of family Comamonadacea.
The relationship between the genome similarity and the geographic distance
High correlation between phenotypes and genotypes in C. testosteroni
In all, we found that 99.4% of a total of 31 × 11 biophysical and biochemical characters were concordant between phenotypes and genotypes. The only two exclusions were that arginine double hydrolysis test showed positive on strain JC9 and kanamycin resistance test showed positive on strain DS1, but the genes for these metabolisms were not found in their genomes.
Important genotypes of C. testosteroni
The conservative gene clusters of nitrate/nitrite reduction founded in all the C. testosteroni genomes and other genomes of Comamonadacea
The key genes for hexose phosphorylation missed in the C. testosteroni genomes
Genomic bases of utilization of steroids and aromatic compounds in C. testosteroni
The genes for antibiotics and heavy metal resistance
Our previous study showed that the strain S44 of C. testosteroni has multiple heavy metal resistances . In this study, we found that all strains of C. testosteroni can resist Cu(II), Zn(II), penicillin, prostaphlin and ampicillin. Six of them can resist Hg(II). Based on RAST annotation result, gene clusters and scattered genes relating to heavy metal and medico resistances were found in the genomes of C. testosteroni. Of these gene clusters, 11 were shared in all of the 14 genomes (Additional file 8: Table S8). They included homologous genes for RND transporter families such as Czc family of cobalt-zinc-cadmium RND efflux transporter, Cme family of multidrug resistance RND efflux system and multidrug efflux RND membrane fusion protein MexC, and homologous genes for copper homeostasis such as copper homeostasis protein CutE, copper chaperone and copper-translocation P-type ATPase. Besides, there were many types of gene clusters that were not shared in all of these genomes (Additional file 8: Table S8). Genes in these clusters included those resistant to mercury, arsenic and chromium. RND efflux systems of cobalt-zinc-cadmium (Czc family) and multidrug were also found. In addition, many scattered genes such as those for penicillins and fosfomycin resistances were found.
Antibiotics and metal resistances are usually co-selected. The main mechanisms of this phenomenon are co-resistance and cross-resistance . Co-resistance means that the genes for antibiotics and metal resistances are located in the same genetic element. Cross-resistance means that the different resistant phenotypes are controlled by the same gene or the same group of genes. In the genomes of C. testosteroni, we found that the genes for different resistance functions were usually clustered in accord with co-resistance. For example, a gene for copper binding protein and a gene for Cu(I) responsive transcriptional regulator are located near some RND efflux Czc family genes (eg. strain D4 Locus Tag: P609_08845 ~ P609_08895 GenBank: AWTQ00000000). Also, the genes for the cross-resistance can be found in these genomes, such as the cobalt-zinc-cadmium efflux system Czc family  and multidrug resistance system . Notably, most of these efflux systems belong to the Resistance-Nodulation-cell Division (RND) family.
The phylogenetic relationships of the genotypes consistent among the strains of C. testosteroni
Virulence factors in C. testosteroni
In order to identify potential virulence factors (VFs) in the genomes of C. testosteroni, we aligned proteins from the 14 genomes against Virulence Factors Data Base (VFDB)  using blastp with the standard E-value ≤1e-5, identity ≥ 50%, and match ≥ 70%. As result, we identified 24 types of VFs that were involved in 13 functions such as adherence, anti-phagocytosis, invasion, and secretion system. Of them, 11 VFs are owned by all of the strains. These VFs include 2 types of secretion system (Bsa T3SS, xcp secretion system), urease, flagella of invasion, alginate used for anti-phagocytosis, type IV pili for adherence. Comparing the shared VFs between every two strains, we found that the average identity of the orthologous VFs from different combinations is 99%. The gene numbers of all the found VFs are showed in Additional file 9: Table S9.
The concept of pan-genome was first introduced in 2005 . It is the collection of the total genes that could be found in a species. The pan-genome could reflect the gene pool size of a bacterium . In this study, Heap’s law  analysis showed that the pan-genome of C. testosteroni is open. However, there are limitations in this analysis. First, the strains were not randomly sampled. Some genomes are very similar (e.g. JC8, JC9, JC12, JC13), and some genomes are significantly different from the others (e.g. KF-1). Therefore, we could notice a “break” in Figure 1A. Second, because most of the genomic sequences are draft, some genes may be lost in the sequences (e.g. in Figure 8, the gene for PCA 4,5-dioxygenase beta chain is lost in some strains due to interruption in the coding region of this gene). This may result in an underestimated core-genome and an overestimated pan-genome.
According to the natural selection theory, environment could affect organisms’ genetic characters. In our study, we found: a) the strains isolated from the same areas (Jixi and Daye) are always clustered together (this indicates the highest similarity), and b) ANIs among strains from different continents are lower than those from the same continents. These results revealed that the genome similarity of most C. testosteroni strains followed a certain degree of distance-similarity decay relationship.
In previous studies, strains of C. testosteroni showed abilities for nitrate reduction, and utilization of testosterone, 4HBA, acetate and lactate, but they cannot utilize glucose and most of the carbohydrates . In this study, we found that most of the above phenotypes of C. testosteroni are consistent with previous studies and consistent among all the tested strains of C. testosteroni, indicating that these important phenotypes are very stable and common in C. testosteroni, and could be used in the polyphasic taxonomy in identification of this species.
The key step of aromatic compounds degradation is the benzene ring cleavage and the main pathway for this reaction in strains of genus Comamonas is PCA 4,5-cleavage . In previous study, genes or potential genes for PCA 4,5-cleavage were found in many strains of C. testosteroni such as C. testosteroni BR6020  and C. testosteroni CNB-2 . In this study, potential genes for this function could be found in all of the genomes of C. testosteroni (Additional file 7: Table S7) and the gene cluster for PCA 4,5-cleavage is very conserved among the C. testosteroni strains (Figure 9C). This indicated that the strains of C. testosteroni would have potential abilities to degrade various aromatic compounds.
Ma et al.  built a main metabolic pathway map of C. testosteroni CNB-2. They found that the glycolysis is incomplete due to lack of the hexokinase and glucokinase genes so that the strain cannot utilize most types of sugars. Studies on the genomes of C. testosteroni strains ATCC 11996  and KF-1  showed the same results. We built metabolic pathway maps of 14 strains of C. testosteroni (including above strains) and showed that all genes for hexose phosphorylation were missed in all genomes of this species (Figure 6). This result effectively supports the previous studies and indicates that lack of these genes should be a common genome feature of C. testosteroni.
Comparison between the phylogenomic tree of C. testosteroni (Figure 2A) and the phylogenetic trees of the genes from the gene clusters for nitrate/nitrite reduction, steroids degradation, antibiotics and heavy metal resistance (Figure 9A, B, D, E and F) revealed that the gene clusters for these functions are conserved and their evolutions are consistent with the evolution of the core-genome. This indicated that the shared gene clusters for these functions are inherent in the genomes of C. testosteroni, and they may have suffered little natural selection pressure during the evolutionary process of C. testosteroni.
As an environmental bacterium, C. testosteroni is often considered to be nonpathogenic. However, according to the survey by Farshad et al. , up to 2012, there were 35 reported cases of human infection by this bacterium. C. testosteroni infected central nervous system , blood [63,64], mitral valve , subcutaneous tissue , eye , abdominal cavity  and urinary tract , that caused purulent meningitis, bacteremia, cellulitis, infectious endocarditis, post-operative endophthalmitis and other diseases. Nevertheless, few molecular biological investigations were taken on the pathogenicity of C. testosteroni. In this study, 24 types of VFs were found in the genomes of C. testosteroni with high protein-to-protein identify (≥50%) and match (match ≥70%). Further, we found that the shared VFs among the C. testosteroni are highly conserved. This result proposed the molecular biological basis of the potential pathogenicity of this bacterium.
Genotype determines phenotype. Jung et al.  found many genotypes relating to antibiotic resistance, oxidative stress, motility, and pectin metabolism in Alishewanella species, and confirmed the phenotypes of them. Ma et al.  examined the ability of the utilization of many sugars of C. testosteroni CNB-2 and confirmed the genome features that support these phenotypes. However, such a study that focuses on all the main recognition properties and their genome bases on an environmental bacterium is rare. In this study, we found a high correlation between phenotypes and genotypes of this species. The related genes may be useful to study the molecular mechanisms of C. testosteroni.
In conclusion, we generated the sequences of 10 genomes of C. testosteroni and performed comparative genomics analysis among the available genomes of this bacterium. We found that the pan-genome of C. testosteroni may be open. Physiological and biochemical investigation showed that C. testosteroni has the main properties of nitrate reduction, ability of utilizing testosterone, 4HBA, acetate and lactate, and disability of utilizing glucose and most carbohydrates. Genomic analysis revealed the molecular biological bases of these phenotypes. The phenotypes and genotypes of these features could be used in polyphasic taxonomy and molecular identification of this species. This study is the first comprehensive genomic analysis for C. testosteroni and provides information for better understanding this bacterial species at genomic level. Importantly, this study focused on the analysis of potential genetic determinants for the typical characters, and found the high correlation between the phenotypes and their corresponding genotypes (e.g. genes).
This work was supported by the National High Technology Research and Development Program of China (2012AA101402) and the National Natural Science Foundation of China (31170106).
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