Complete Genome Sequence of 3-Chlorobenzoate-Degrading Bacterium Cupriavidus necator NH9 and Reclassification of the Strains of the Genera Cupriavidus and Ralstonia Based on Phylogenetic and Whole-Genome Sequence Analyses

Abstract

Cupriavidus necator NH9, a 3-chlorobenzoate (3-CB)-degrading bacterium, was isolated from soil in Japan. In this study, the complete genome sequence of NH9 was obtained via PacBio long-read sequencing to better understand the genetic components contributing to the strain's ability to degrade aromatic compounds, including 3-CB. The genome of NH9 comprised two circular chromosomes (4.3 and 3.4 Mb) and two circular plasmids (427 and 77 kb) containing 7,290 coding sequences, 15 rRNA and 68 tRNA genes. Kyoto Encyclopedia of Genes and Genomes pathway analysis of the protein-coding sequences in NH9 revealed a capacity to completely degrade benzoate, 2-, 3-, or 4-hydroxybenzoate, 2,3-, 2,5-, or 3,4-dihydroxybenzoate, benzoylformate, and benzonitrile. To validate the identification of NH9, phylogenetic analyses (16S rRNA sequence-based tree and multilocus sequence analysis) and whole-genome sequence analyses (average nucleotide identity, percentage of conserved proteins, and tetra-nucleotide analyses) were performed, confirming that NH9 is a C. necator strain. Over the course of our investigation, we noticed inconsistencies in the classification of several strains that were supposed to belong to the two closely-related genera Cupriavidus and Ralstonia. As a result of whole-genome sequence analysis of 46 Cupriavidus strains and 104 Ralstonia strains, we propose that the taxonomic classification of 41 of the 150 strains should be changed. Our results provide a clear delineation of the two genera based on genome sequences, thus allowing taxonomic identification of strains belonging to these two genera.

Keywords: ANI (average nucleotide identity); Cupriavidus; Ralstonia; TNA (tetra-nucleotide analysis); aromatic degradation; reclassification.

Figure 1

Schematic representation of the four replicons of NH9. Circular maps of chromosome 1 (A), chromosome 2 (B), pENH91 (C), and pENH92 (D) are shown. The circles represent (from the inside): 1, GC skew; 2, GC content; 3, RNA genes (except for pENH91); 4, Clusters of Orthologous Groups (COG) assignments for coding sequences (CDSs) on the reverse strand; 5, COG assignments for CDSs on the forward strand; 6, scale in Mb or kb. Note that maps are not drawn to scale relative to the sizes of each replicon, very short RNA genes are enlarged to enhance visibility, and pENH91 does not contain any RNA genes.

Figure 2

Functional classification of proteins encoded on each replicon of the NH9 genome based on Clusters of Orthologous Groups (COG) categories. COG categories are shown on the horizontal axis, with the percentage of proteins belonging to each category for each replicon plotted on the vertical axis (percentages >25% are not shown). ^*p < 0.05 and ^**p < 0.01, respectively, as determined by Fisher's test with false discovery rate adjustments. COG functional annotations and specific values are summarized in Table S1.

Figure 3

Aromatic compound degradation pathways in NH9. Enzyme-encoding genes are shown by red text, including locus tag(s). 3-chlorobenzoate, benzoate, benzonitrile, and benzoylformate are shaded in yellow, magenta, green, and purple, respectively. Hydroxybenzoate and dihydroxybenzoate are shaded in blue and orange, respectively.

Figure 4

Maximum likelihood trees showing the phylogeny of the genera Cupriavidus and Ralstonia based on 16S rRNA gene sequences. Phylogenetic trees generated for both genera (A), the genus Cupriavidus (B), and the genus Ralstonia (C). Full-length 16S rRNA gene sequences were aligned and used in the phylogenetic analysis (see section Materials and Methods for detail). Proposed type-strains are designated with a “T.” Strain NH9 is marked with a red circle. Bootstrap values are represented at the branching points (only values >50% are shown).

Figure 5

Maximum likelihood phylogenetic trees constructed using concatenated atpD, leuS, rplB, and gyrB nucleotide sequences from strains belonging to the genera Cupriavidus and Ralstonia. Phylogenetic trees generated for both genera (A), the genus Cupriavidus (B), and the genus Ralstonia (C). The final dataset contained 6,333 positions (see section Materials and Methods for details). Strain NH9 is marked with a red circle. Bootstrap values are represented at the branching points (only values >50% are shown).

Figure 6

Heatmap and dendrogram of average nucleotide identity (ANI) values of the 48 putative Cupriavidus genomes. Strains with ANI values >90% are shown within blue squares (see Table 3 and Table S4 for a description of cluster designations). All squares were assigned numbers from 1 to 17.

Figure 7

Heatmap and dendrogram of average nucleotide identity (ANI) values of the 101 putative Ralstonia genomes. Strains with ANI values >90 and >95% are shown within blue and red squares, respectively (see Table 3 and Table S4 for a description of cluster designations). All squares were assigned a number from 18 to 25.

Figure 8

Comparison of average percentage of conserved proteins (POCP) values between Cupriavidus (A) and Ralstonia (B) named strains. Ralstonia group and Cupriavidus group strains are shown to the left and right of the vertical dotted bar, respectively. The two red dots indicate strains that are currently classified as Ralstonia but grouped into the genus Cupriavidus in this analysis. Blue dots indicate identical average POCP values when compared with Cupriavidus and Ralstonia strains. The red horizontal line indicates the genus boundary threshold (a POCP value of 60% was used in this study). Organism names are abbreviated as follows: 25fmcol4.1, Ralstonia sp. 25fmcol4.1; DTP0602, R. pickettii DTP0602; PBA, Ralstonia sp. PBA.

Figure 9

Three-dimensional plot of principal components analysis results for all 150 Cupriavidus and Ralstonia genomes. Differences in color between clusters indicates divergence using the first three principal components. Sixteen clusters (A to P) are shown (see Table 3 for a description of cluster assignments).