Comparative genomics of Burkholderia multivorans, a ubiquitous pathogen with a highly conserved genomic structure

Abstract

The natural environment serves as a reservoir of opportunistic pathogens. A well-established method for studying the epidemiology of such opportunists is multilocus sequence typing, which in many cases has defined strains predisposed to causing infection. Burkholderia multivorans is an important pathogen in people with cystic fibrosis (CF) and its epidemiology suggests that strains are acquired from non-human sources such as the natural environment. This raises the central question of whether the isolation source (CF or environment) or the multilocus sequence type (ST) of B. multivorans better predicts their genomic content and functionality. We identified four pairs of B. multivorans isolates, representing distinct STs and consisting of one CF and one environmental isolate each. All genomes were sequenced using the PacBio SMRT sequencing technology, which resulted in eight high-quality B. multivorans genome assemblies. The present study demonstrated that the genomic structure of the examined B. multivorans STs is highly conserved and that the B. multivorans genomic lineages are defined by their ST. Orthologous protein families were not uniformly distributed among chromosomes, with core orthologs being enriched on the primary chromosome and ST-specific orthologs being enriched on the second and third chromosome. The ST-specific orthologs were enriched in genes involved in defense mechanisms and secondary metabolism, corroborating the strain-specificity of these virulence characteristics. Finally, the same B. multivorans genomic lineages occur in both CF and environmental samples and on different continents, demonstrating their ubiquity and evolutionary persistence.

Fig 1. Phylogenomic analysis showing the relatedness of the genomes in terms of sequence divergence of the panorthologs.

The maximum likelihood tree was inferred using the GTRGAMMA substitution model and is based on a concatenated nucleotide alignment of 4,503 CDS (4,457,847 positions). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap analyses (1,000 replicates) are shown next to the branches. Scale bar represents number of substitutions per site. The tree was rooted on the branch with the largest branch length.

Fig 2. The frequency of orthologous versus non-orthologous CDS varies among chromosomes and COG categories.

Bar plots show the number of orthologous and non-orthologous CDS per chromosome (X²(2) = 213.4, p<0.001) (a) and COG category (X²(22) = 5101.2, p<0.001) (c). Mosaic plots show the standardized residuals of the Pearson chi-square analysis for the number of orthologous and non-orthologous CDS per chromosome (b). Solid and dashed boundaries represent positive and negative residuals, respectively. Rectangles are colored only if the standardized residual is significant at p<0.05 (outside ±1.96). COG categories: J, translation, ribosomal structure and biogenesis; K, transcription; L, replication, recombination and repair; B, chromatin structure and dynamics; D, cell cycle control, cell division, chromosome partitioning; V, defense mechanisms; T, signal transduction mechanisms; M, cell wall/membrane/envelope biogenesis; N, cell motility; W, extracellular structures; U, intracellular trafficking, secretion, and vesicular transport; O, posttranslational modification, protein turnover, chaperones; X, mobilome: prophages, transposons; C, energy production and conversion; G, carbohydrate transport and metabolism; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism; R, general function prediction only; S, function unknown.

Fig 3. Venn diagram showing the number of core and ST-specific ortholog families.

ST, multilocus sequence type.

Fig 4. Ortholog specificity varies among chromosomes and COG categories.

Bar plots show the number of orthologs per specificity group for different chromosomes (X²(8) = 469.8, p<0.001) (a) and COG categories (X²(88) = 649.8, p<0.001) (c). Mosaic plots show the standardized residuals of the Pearson chi-square analysis on the number of orthologs per specificity group per chromosome (b). Solid and dashed boundaries represent positive and negative residuals, respectively. Rectangles are colored only if the standardized residual is significant at p<0.05 (outside ±1.96). COG categories: J, translation, ribosomal structure and biogenesis; K, transcription; L, replication, recombination and repair; B, chromatin structure and dynamics; D, cell cycle control, cell division, chromosome partitioning; V, defense mechanisms; T, signal transduction mechanisms; M, cell wall/membrane/envelope biogenesis; N, cell motility; W, extracellular structures; U, intracellular trafficking, secretion, and vesicular transport; O, posttranslational modification, protein turnover, chaperones; X, mobilome: prophages, transposons; C, energy production and conversion; G, carbohydrate transport and metabolism; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism; R, general function prediction only; S, function unknown.