Complete genome sequence of Nitrobacter hamburgensis X14 and comparative genomic analysis of species within the genus Nitrobacter

Abstract

The alphaproteobacterium Nitrobacter hamburgensis X14 is a gram-negative facultative chemolithoautotroph that conserves energy from the oxidation of nitrite to nitrate. Sequencing and analysis of the Nitrobacter hamburgensis X14 genome revealed four replicons comprised of one chromosome (4.4 Mbp) and three plasmids (294, 188, and 121 kbp). Over 20% of the genome is composed of pseudogenes and paralogs. Whole-genome comparisons were conducted between N. hamburgensis and the finished and draft genome sequences of Nitrobacter winogradskyi and Nitrobacter sp. strain Nb-311A, respectively. Most of the plasmid-borne genes were unique to N. hamburgensis and encode a variety of functions (central metabolism, energy conservation, conjugation, and heavy metal resistance), yet approximately 21 kb of a approximately 28-kb "autotrophic" island on the largest plasmid was conserved in the chromosomes of Nitrobacter winogradskyi Nb-255 and Nitrobacter sp. strain Nb-311A. The N. hamburgensis chromosome also harbors many unique genes, including those for heme-copper oxidases, cytochrome b(561), and putative pathways for the catabolism of aromatic, organic, and one-carbon compounds, which help verify and extend its mixotrophic potential. A Nitrobacter "subcore" genome was also constructed by removing homologs found in strains of the closest evolutionary relatives, Bradyrhizobium japonicum and Rhodopseudomonas palustris. Among the Nitrobacter subcore inventory (116 genes), copies of genes or gene clusters for nitrite oxidoreductase (NXR), cytochromes associated with a dissimilatory nitrite reductase (NirK), PII-like regulators, and polysaccharide formation were identified. Many of the subcore genes have diverged significantly from, or have origins outside, the alphaproteobacterial lineage and may indicate some of the unique genetic requirements for nitrite oxidation in Nitrobacter.

FIG. 1.

Genome of Nitrobacter hamburgensis X14 (ATCC 25391). The outer circle depicts the locations of genes conserved in all three sequenced Nitrobacter genomes. Genes that are conserved in Nitrobacter but not R. palustris or B. japonicum (the subcore) are indicated in the second circle. The third and fourth circles depict predicted protein-encoding and structural RNA genes in N. hamburgensis on the plus and minus strands, respectively (green, energy metabolism; red, DNA replication; magenta, transcription; yellow, translation; orange, amino acid metabolism; dark blue, carbohydrate metabolism; pale red, nucleotide metabolism; black, coenzyme metabolism; cyan, lipid metabolism; light blue, cellular processes; brown, general function; gray, hypothetical and conserved hypothetical genes; pale green, structural RNAs). Genes “unique” to N. hamburgensis (not present in N. winogradskyi or NB311A) are depicted in circle five (red). The sixth and seventh circles indicate the locations of all annotated pseudogenes (green) and paralogs (orange), respectively. The eighth circle indicates GC bias, and the ninth circle indicates GC skew. The highlighted region of pPB13 depicts the locations of key gene clusters: autotrophic island (peach), RuBisCO and pentose phosphate pathway genes (green), and the cytochrome oxidases (aa₃ and bd-ubiquinol types) (blue).

FIG. 2.

Global gene conservation in Nitrobacter. Each circle represents the total number of gene types in each genome. Overlapping regions depict the number of genes types shared between the respective genomes. The numbers outside the circles indicate the total number of genes identified in each genome, including paralogs/gene duplications.

FIG. 3.

Functional distribution of N. hamburgensis plasmid genes based on COG assignments. Genes in COG groups J, K, and L are included in “information storage and processing.” Genes in COG groups D, V, T, M, N, U, and O are included in “cellular processes.” Genes in COG groups, G, E, F, H, I, P, and Q are included in “metabolism.” Genes in COG groups R and S are included in “poorly characterized.” Of the 494 plasmid genes, 242 could be assigned to a COG group. The remaining plasmid genes were excluded from the analysis.