Species- and strain-specific control of a complex, flexible regulon by Bordetella BvgAS

Abstract

The Bordetella master virulence regulatory system, BvgAS, controls a spectrum of gene expression states, including the virulent Bvg(+) phase, the avirulent Bvg(-) phase, and at least one Bvg-intermediate (Bvg(i)) phase. We set out to define the species- and strain-specific features of this regulon based on global gene expression profiling. Rather than functioning as a switch, Bvg controls a remarkable continuum of gene expression states, with hundreds of genes maximally expressed in intermediate phases between the Bvg(+) and Bvg(-) poles. Comparative analysis of Bvg regulation in B. pertussis and B. bronchiseptica revealed a relatively conserved Bvg(+) phase transcriptional program and identified previously uncharacterized candidate virulence factors. In contrast, control of Bvg(-)- and Bvg(i)-phase genes diverged substantially between species; regulation of metabolic, transporter, and motility loci indicated an increased capacity in B. bronchiseptica, compared to B. pertussis, for ex vivo adaptation. Strain comparisons also demonstrated variation in gene expression patterns within species. Among the genes with the greatest variability in patterns of expression, predicted promoter sequences were nearly identical. Our data suggest that the complement of transcriptional regulators is largely responsible for transcriptional diversity. In support of this hypothesis, many putative transcriptional regulators that were Bvg regulated in B. bronchiseptica were deleted, inactivated, or unregulated by BvgAS in B. pertussis. We propose the concept of a "flexible regulon." This flexible regulon may prove to be important for pathogen evolution and the diversification of host range specificity.

FIG. 1.

Distribution of differentially expressed genes. The distributions of Bvg-activated and Bvg-repressed genes among B. bronchiseptica RB50 and B. pertussis GMT-1 (A), three B. bronchiseptica strains (B), and two B. pertussis strains (C) are shown. Areas within Venn diagrams are drawn approximately to scale, and the number of ORFs in each is indicated. In parentheses are numbers of genes absent from the other genome(s) as determined by genome sequence comparison (RB50 and Tohama I) or comparative genome hybridization (GMT-1).

FIG. 2.

Validation of B. bronchiseptica microarray data by real-time PCR. Ratios of transcript abundance in RB53 (Bvg⁺) versus RB54 (Bvg⁻), obtained by microarray analysis (x axis) or real-time PCR (y axis). Red, Bvg activated; green, Bvg repressed. Selected genes are shown for illustrative purposes.

FIG. 3.

Patterns of Bvg-regulated gene expression in B. bronchiseptica RB50. Within each experiment, data are centered and shown from Bvg⁺ (left) to Bvg⁻ (right). Rows correspond to array probes, and columns correspond to experiments. +, phase-locked Bvg⁺; i, phase-locked Bvgⁱ; −, phase-locked Bvg⁻. Red, increased transcript abundance; green, decreased transcript abundance; black, no significant change in transcript abundance; gray, no data. NA concentrations (millimolar) are indicated along the bottom. The five major gene expression classes, with some representative loci, and the Bvg-independent NA-induced class are indicated on the right. Purple arrowhead, bipA. The set of genes represented by the gray bar on the right passed filtering criteria but showed no discernible pattern.

FIG. 4.

Patterns of Bvg-regulated gene expression in B. pertussis GMT-1. Data are centered within each experiment. Results obtained with NA modulation and phase-locked strain analysis are shown from Bvg⁺ (left) to Bvg⁻ (right), whereas the phase shift experiment is depicted from Bvg⁻ (left) to Bvg⁺ (right). Rows correspond to array probes, and columns correspond to experiments. +, phase-locked Bvg⁺; i, phase-locked Bvgⁱ; −, phase-locked Bvg⁻. Red, increased transcript abundance; green, decreased transcript abundance; black, no significant change in transcript abundance; gray, no data. NA concentrations (millimolar) and time elapsed after shift (minutes) are indicated along the bottom. The four major gene expression classes, with some representative loci, and the Bvg-independent NA-induced class are indicated on the right. Purple arrowhead, bipA. The set of genes represented by the gray bar on the right passed filtering criteria but showed no discernible pattern.

FIG. 5.

Functional categorization of ORFs in core and flexible BvgAS regulons. Bvg-regulated genes (red, Bvg activated; green, Bvg repressed) were grouped by functional categories (43, 50). Data are expressed as the percentage that is regulated by BvgAS among all annotated ORFs in each class. (A) B. bronchiseptica. ORFs in the core regulon were Bvg regulated by strains RB50, Bbr77, and Bbr81. Those in the flexible regulon were Bvg regulated in either one or two of the three strains. (B) B. pertussis. ORFs in the core regulon were Bvg regulated by strains GMT-1 and Tohama I. Those in the flexible regulon were Bvg regulated in only one of the two strains.

FIG. 6.

Putative cis-regulatory motifs. Multiple alignments are represented as sequence logos. x axis, nucleotide position within motif; y axis, bit score. Arrows indicate putative half-sites. (A) BvgA-binding motif from regions upstream of Bvg-activated genes. Dashed line, variable-length (0- to 2-nt) spacer between half-sites. (B) Fim box. Solid lines, box 1 and box 2; dashed line, beginning of poly(C) tract. (C and D) FNR box (C) and palindromic motif (D) from regions upstream of Bvgⁱ-b genes.