Burkholderia xenovorans LB400 harbors a multi-replicon, 9.73-Mbp genome shaped for versatility

Abstract

Burkholderia xenovorans LB400 (LB400), a well studied, effective polychlorinated biphenyl-degrader, has one of the two largest known bacterial genomes and is the first nonpathogenic Burkholderia isolate sequenced. From an evolutionary perspective, we find significant differences in functional specialization between the three replicons of LB400, as well as a more relaxed selective pressure for genes located on the two smaller vs. the largest replicon. High genomic plasticity, diversity, and specialization within the Burkholderia genus are exemplified by the conservation of only 44% of the genes between LB400 and Burkholderia cepacia complex strain 383. Even among four B. xenovorans strains, genome size varies from 7.4 to 9.73 Mbp. The latter is largely explained by our findings that >20% of the LB400 sequence was recently acquired by means of lateral gene transfer. Although a range of genetic factors associated with in vivo survival and intercellular interactions are present, these genetic factors are likely related to niche breadth rather than determinants of pathogenicity. The presence of at least eleven "central aromatic" and twenty "peripheral aromatic" pathways in LB400, among the highest in any sequenced bacterial genome, supports this hypothesis. Finally, in addition to the experimentally observed redundancy in benzoate degradation and formaldehyde oxidation pathways, the fact that 17.6% of proteins have a better LB400 paralog than an ortholog in a different genome highlights the importance of gene duplication and repeated acquirement, which, coupled with their divergence, raises questions regarding the role of paralogs and potential functional redundancies in large-genome microbes.

Fig. 1.

Schematic representation of the large chromosome (chromosome 1) (A), small chromosome (chromosome 2) (B), and megaplasmid (C) of B. xenovorans LB400. Radii are scaled based on replicon size, except for the megaplasmid, an extra ring for which was added to indicate true size relative to the chromosomes. The outer two rings (1 and 2) represent the LB400 genes on the forward and reverse strands, respectively, colored by functional class. The next two sets of rings represent the CGH data of LB400 vs. LMG 16224 (rings 3 and 4) and LMG 21720 (rings 5 and 6), respectively (blue, gene absent; yellow, gene present). Ring 7 shows the locations of the genomic islands (identified based on bias in Karlin score, G+C% and G+C skew; Table 4). The next four sets of rings are based on reciprocal best BLAST hit analysis (cutoffs: 30% amino acid identity, alignment over at least 70% of the length) of a test genome vs. the LB400 genome, which was displayed by using GenomeViz (66) with bar height relative to the % amino acid identity: B. cenocepacia J2315 (red, rings 8 and 9) [the J2315 sequence data were produced by the Pathogen Sequencing Group at the Sanger Institute (http://www.sanger.ac.uk/Projects/B_cenocepacia/)]; B. pseudomallei (blue, rings 10 and 11); Bcc strain 383 (green, rings 12 and 13); and Ralstonia solanacearum (magenta, rings 14 and 15). Ring 16 (black) represents G+C content, and ring 17 represents the G+C skew.

Fig. 2.

Functional distribution over the three replicons based on the COG classifications. Presented is the percent of genes on each replicon belonging to each COG.

Fig. 3.

The ratio of asynonymous vs. synonymous nucleotide substitutions (K_a:K_s) between orthologous pairs in LB400 and Bcc strain 383, plotted conjointly for the three chromosomes. There is a significant trend toward more spread ratios (flattening of the distribution) going from the large chromosome to the smaller ones. Although no positive selection could be evidenced overall for any pair of genes compared (i.e., K_a > K_s), this trend can be interpreted as a progressive relaxation of the selection pressure for amino acid substitution (pressure Chr1>Chr2>MP) (cf. Fig. 5).

Fig. 4.

Schematic representation of all central aromatic pathways (gray background), based on their main substrate, present in LB400. Peripheral aromatic pathways are indicated in the outer sectors.