Global map of growth-regulated gene expression in Burkholderia pseudomallei, the causative agent of melioidosis

Abstract

Many microbial pathogens express specific virulence traits at distinct growth phases. To understand the molecular pathways linking bacterial growth to pathogenicity, we have characterized the growth transcriptome of Burkholderia pseudomallei, the causative agent of melioidosis. Using a fine-scale sampling approach, we found approximately 17% of all B. pseudomallei genes displaying regulated expression during growth in rich medium, occurring as broad waves of functionally coherent gene expression tightly associated with distinct growth phases and transition points. We observed regulation of virulence genes across all growth phases and identified serC as a potentially new virulence factor by virtue of its coexpression with other early-phase virulence genes. serC-disrupted B. pseudomallei strains were serine auxotrophs and in mouse infection assays exhibited a dramatic attenuation of virulence compared to wild-type B. pseudomallei. Immunization of mice with serC-disrupted B. pseudomallei also conferred protection against subsequent challenges with different wild-type B. pseudomallei strains. At a genomic level, early-phase genes were preferentially localized on chromosome 1, while stationary-phase genes were significantly biased towards chromosome 2. We detected a significant level of chromosomally clustered gene expression, allowing us to predict approximately 100 potential operons in the B. pseudomallei genome. We computationally and experimentally validated these operons by showing that genes in these regions are preferentially transcribed in the same 5'-->3' direction, possess significantly shorter intergenic lengths than the overall genome, and are expressed as a common mRNA transcript. The availability of this transcriptome map provides an important resource for understanding the transcriptional architecture of B. pseudomallei.

FIG. 1.

Profiling of the B. pseudomallei growth transcriptome. (A) Composite growth curve of B. pseudomallei as a function of optical density (y axis) and time (x axis). Different-colored symbols represent samples from distinct batch clusters; for example, pink squares represent a batch cluster of 16 flasks harvested for time points 9 h to 10.5 h, and lime green inverted triangles represent another batch cluster of 14 flasks that were harvested for time points 3.5 h, 5 h, 20 h, and 22.5 h. Graph points represent individual time points used for subsequent expression profiling. Early (Ea), log, and stationary (St) phases are depicted. (B) Heat map of the B. pseudomallei growth transcriptome. Rows represent individual array probes, columns represent individual time points (30 min to 48 h), and distinct k-means clusters are shown as separate map blocks (e.g., E1 to S4, where E, L, and S represent early, log, and stationary phases, respectively). Red and green bars represent clusters of high and low gene expression levels, respectively. (C) Experimental validation of microarray data by semiquantitative PCR. Each row represents an individual B. pseudomallei gene, and columns represent gene expression levels, normalized against 16S rRNA expression (bottom row).

FIG. 2.

Characterization of serC as a virulence gene. (A) Relationship of serC growth-regulated expression to expression of other virulence genes (see text for details). The serC gene is underlined. (B) Isolation of a serC mutant. The genome order of serC and neighboring genes, with insertion of an interrupting transposon, Tn5Km2, is shown. Also depicted is the confirmation of serC as an early-phase growth-regulated gene by semiquantitative PCR (see Fig. 1C). (C) Serine auxotrophy in serC mutants. Graphs represent growth curves of wild-type (WT) B. pseudomallei (blue) and serC mutants (red) in rich (left) and minimal (right) media. serC mutants exhibit slower growth in minimal medium, which can be rescued to wild-type levels by the addition of exogenous serine (pink). (D) Vaccination with serC mutants confers protection. Graphs represent rates of survival for animals either vaccinated with serC mutant B. pseudomallei (solid points) or not (open points) following subsequent challenge with different strains of B. pseudomallei (pink, strain 576; blue, strain K96243).

FIG. 3.

Inter- and intrachromosomal clustering of growth-regulated genes. (A) Chromosomal biases in gene expression at early, log, and stationary phases. The y axis represents the total percentage of growth-regulated genes as a proportion of either the entire genome, chromosome 1, or chromosome 2. P values represent the significance of differences between the chromosomes, as assessed by Fisher's exact test. (B) Chromosomal clustering of growth-regulated genes. The y axis represents the expression correlation between all pairs of adjacent array probes for chromosome 1 and 2 (Chr 1 and 2 actual) or a randomized genome (Chr 1 and 2 random). The correlation value of 0.6 represents a cutoff FDR of 5%.