Natural Chromosome-Chromid Fusion across rRNA Operons in a Burkholderiaceae Bacterium

Abstract

Chromids (secondary chromosomes) in bacterial genomes that are present in addition to the main chromosome appear to be evolutionarily conserved in some specific bacterial groups. In rare cases among these groups, a small number of strains from Rhizobiales and Vibrionales were shown to possess a naturally fused single chromosome that was reported to have been generated through intragenomic homologous recombination between repeated sequences on the chromosome and chromid. Similar examples have never been reported in the family Burkholderiaceae, a well-documented group that conserves chromids. Here, an in-depth genomic characterization was performed on a Burkholderiaceae bacterium that was isolated from a soil bacterial consortium maintained on diesel fuel and mutagenic benzo[a]pyrene. This organism, Cupriavidus necator strain KK10, was revealed to carry a single chromosome with unexpectedly large size (>6.6 Mb), and results of comparative genomics with the genome of C. necator N-1^T indicated that the single chromosome of KK10 was generated through fusion of the prototypical chromosome and chromid at the rRNA operons. This fusion hypothetically occurred through homologous recombination with a crossover between repeated rRNA operons on the chromosome and chromid. Some metabolic functions that were likely expressed from genes on the prototypical chromid region were indicated to be retained. If this phenomenon-the bacterial chromosome-chromid fusion across the rRNA operons through homologous recombination-occurs universally in prokaryotes, the multiple rRNA operons in bacterial genomes may not only contribute to the robustness of ribosome function, but also provide more opportunities for genomic rearrangements through frequent recombination. IMPORTANCE A bacterial chromosome that was naturally fused with the secondary chromosome, or "chromid," and presented as an unexpectedly large single replicon was discovered in the genome of Cupriavidus necator strain KK10, a biotechnologically useful member of the family Burkholderiaceae. Although Burkholderiaceae is a well-documented group that conserves chromids in their genomes, this chromosomal fusion event has not been previously reported for this family. This fusion has hypothetically occurred through intragenomic homologous recombination between repeated rRNA operons and, if so, provides novel insight into the potential of multiple rRNA operons in bacterial genomes to lead to chromosome-chromid fusion. The harsh conditions under which strain KK10 was maintained-a genotoxic hydrocarbon-enriched milieu-may have provided this genotype with a niche in which to survive.

Keywords: Cupriavidus; chromid; chromosomal fusion; genomic rearrangement.

FIG 1

Circular maps of the Cupriavidus necator strain KK10 chromosome and plasmid with functional gene annotation. Rings from outside to the center are genes on forward strand and reverse strand (colored according to COG annotation categories, as listed in the bar graph with gene counts), rRNA operons (pink), GC content (gray), and GC skew (yellow and purple).

FIG 2

Heatmap of average nucleic acid identity (ANI) among C. necator strain KK10 and reference Cupriavidus genomes with cluster dendrograms. ANI values of each strain to strain KK10 are shown in parentheses in blue.

FIG 3

Sequencing comparisons between genomes of strain KK10 and N-1^T. Pink arrowheads indicate the locations of rRNA operons (rrn1 to -5). (A) Visualized sequencing similarities between KK10 and N-1 replicons. Local alignments are presented as ribbons with colors corresponding to the alignment bitscore in four quartiles (red, top 25%; orange, second 25%; green, third 25%; blue, worst 25%). (B) GC skew profiles of the chromosome of KK10 (above) and the chromosome and chromid of N-1 (below). GC skew peaks as potential replication termini are highlighted with red or blue lines.

FIG 4

Sequencing alignments between the GridION long-read sequencing raw reads from the KK10 chromosome and the chromosome and chromid of N-1. (A) Two representative raw reads of the GridION long-read sequencing of the KK10 genome covering the upstream and downstream regions of the rRNA operons (rrn1 or rrn3) provided evidence that chromosome-chromid fusion occurred across these rRNA operons. (B) Simplified gene map indicating the upstream and downstream regions of the rRNA operons; gltA, citrate synthase; nasA, assimilatory nitrate reductase catalytic subunit; nirB/nirD, assimilatory nitrite reductase large/small subunits; narK, nitrate/nitrite transporter; acd, acyl-CoA dehydrogenase; bcd, butyryl-CoA dehydrogenase; fadD, long-chain acyl-CoA synthetase; npd, nitronate monooxygenase; atoB, acetyl-CoA acetyltransferase; gst, glutathione S-transferase; dnaQ, DNA polymerase-3 subunit epsilon; osmC, organic hydroperoxide reductase. Functional genes are colored according to COG annotation categories.

FIG 5

Gene clusters responsible for biotransformation of aromatic hydrocarbons conserved in the KK10 chromosome. The pox-xyl gene cluster encoding proteins for benzene/phenol/toluene degradation was located in a region that originated from the prototypical chromid (blue), while the ben-cat gene cluster for benzoic acid degradation was located in a region on the prototypical main chromosome (red).

FIG 6

Hypothetical schematic model for the mechanism of chromosome-chromid fusion across rRNA operons in the KK10 genome. The process was initiated by a double-strand break in an rRNA operon (in either the prototypical chromosome or chromid). Through the typical repairing process of the double-strand break (generation of single-strand DNA [ssDNA] by 5′-end degradation, strand invasion, and DNA synthesis) and according to if the Holliday junctions were resolved via cleavage in the opposite orientations (black arrowheads), the regions upstream and downstream from the rRNA operon were swapped, resulting in a fused chromosome across the rRNA operons.