Microarray and functional analysis of growth phase-dependent gene regulation in Bordetella bronchiseptica

Abstract

Growth phase-dependent gene regulation has recently been demonstrated to occur in Bordetella pertussis, with many transcripts, including known virulence factors, significantly decreasing during the transition from logarithmic to stationary-phase growth. Given that B. pertussis is thought to have derived from a Bordetella bronchiseptica-like ancestor, we hypothesized that growth phase-dependent gene regulation would also occur in B. bronchiseptica. Microarray analysis revealed and quantitative real-time PCR (qRT-PCR) confirmed that growth phase-dependent gene regulation occurs in B. bronchiseptica, resulting in prominent temporal shifts in global gene expression. Two virulence phenotypes associated with these gene expression changes were tested. We found that growth-dependent increases in expression of some type III secretion system (TTSS) genes led to a growth phase-dependent increase in a TTSS-dependent function, cytotoxicity. Although the transcription of genes encoding adhesins previously shown to mediate adherence was decreased in late-log and stationary phases, we found that the adherence of B. bronchiseptica did not decrease in these later phases of growth. Microarray analysis revealed and qRT-PCR confirmed that growth phase-dependent gene regulation occurred in both Bvg(+) and Bvg(-) phase-locked mutants, indicating that growth phase-dependent gene regulation in B. bronchiseptica can function independently from the BvgAS regulatory system.

FIG. 1.

Growth curves and changes in virulence gene expression of B. bronchiseptica strain RB50. (A) DNA concentrations (geq/ml) and CFU counts (CFU/ml) during growth of RB50 are plotted along the left y axis; optical densities (OD₆₀₀) are plotted along the right y axis; and time, given in hours, is plotted along the x axis. All data points represent averages obtained from triplicate cultures. (B) Change in gene expression (y axis) occurring at 6-h intervals (x axis), starting at early log phase (12 h) and continuing until late stationary phase (48 h), determined by using qRT-PCR. Data shown are averages obtained from triplicate cultures. The error bars represent standard deviations.

FIG. 2.

Hierarchical clustering of the transcriptional response of B. bronchiseptica strain RB50 during late-log-phase and stationary-phase growth. (A) Expression profiles representing global transcriptional changes. (B) Expression profiles representing transcriptional changes of genes positively regulated by BvgAS, along with 16S RNA and rpoH. Transcriptional changes identified by comparing cDNA from mid-log phase (15 h) to that from late log phase (24 h) are represented under “late-log phase,” and the transcriptional changes identified by comparing cDNA from mid-log phase (15 h) to that from late stationary phase (48 h) are represented under “stationary phase.” Data are mean centered for each array element and averaged from three biological replicates. All expression profiles of genes are in rows and are represented using the color scale at top, with gray indicating missing data.

FIG. 3.

Gene expression changes occurring in the bsc and btr loci from B. bronchiseptica strain RB50 during late-log-phase (A) and stationary-phase (B) growth. Data are mean centered for each array element and averaged from three biological replicates. All expression profiles are represented using the color scale at bottom. Genes encoding known effector proteins (bopN, bsp22, bopD, and bopB) are shown as bold arrows. The bscN gene, which encodes the known ATPase, is shown by an arrow with wavy lines. Regulatory elements btrS, btrU, btrW, and btrV are shown by arrows with slanted bold solid lines.

FIG. 4.

Growth phase-dependent changes in B. bronchiseptica TTSS-mediated cytotoxicity as measured by percent LDH release. J774 macrophages treated with medium alone (M) or infected with RB50 or RB50ΔbscN from cultures collected in mid-log phase (15 h), late log phase (24 h), and stationary phase (48 h) for 4 h at an MOI of 10. The error bars represent standard deviations. An asterisk indicates that the P value is <0.001.

FIG. 5.

Growth phase-dependent changes in B. bronchiseptica adherence to L2 epithelial cells. L2 cells were incubated with RB50, RB63 (ΔfimBCD), RBX9 (ΔfhaB), or SP5 (Δprn) from cultures collected in mid-log phase (15 h), late log phase (24 h), and stationary phase (48 h) for 40 min at an MOI of 100. Adherence is expressed as the proportion of adherent bacteria to that in the original inoculum. The error bars represent standard deviations. An asterisk indicates that the P value is <0.05.

FIG. 6.

Growth curves for B. bronchiseptica strain RB53, a Bvg⁺ phase-locked mutant. Optical densities (OD₆₀₀), DNA concentrations (geq/ml), and CFU counts (CFU/ml) during growth of RB53 are plotted along the y axis, and time, given in hours, is plotted along the x axis. All data points represent averages obtained from triplicate cultures, and error bars represent standard deviations.

FIG. 7.

Hierarchical clustering of the transcriptional response of B. bronchiseptica strain RB53, a Bvg⁺ phase-locked mutant, during late-log-phase and stationary-phase growth. (A) Expression profiles representing global transcriptional changes. (B) Expression profiles representing transcriptional changes of genes positively regulated by BvgAS, along with 16S RNA and rpoH. Transcriptional changes identified by comparing cDNA from mid-log phase (15 h) to that from late log phase (24 h) are represented under “late-log phase,” and the transcriptional changes identified by comparing cDNA from mid-log phase (15 h) to that from late stationary phase (48 h) are represented under “stationary phase.” Data are mean centered for each array element and averaged from three biological replicates. All expression profiles of genes are in rows and are represented using the color scale at top, with gray indicating missing data.

FIG. 8.

Growth curves and changes in gene expression of genes that are negatively regulated by BvgAS and maximally expressed when the BvgAS system is inactive for B. bronchiseptica strain RB54, a Bvg⁻ phase-locked mutant. (A) Optical densities (OD₆₀₀), DNA concentrations (geq/ml), and CFU counts (CFU/ml) during growth of RB50 are plotted along the y axis, and time, given in hours, is plotted along the x axis. All data points represent averages obtained from triplicate cultures. (B) Change in gene expression (y axis) occurring at 6-h intervals (x axis) starting at early logarithmic phase (12 h) and continuing until late stationary phase (48 h) determined by using qRT-PCR. Data shown are averages obtained from triplicate cultures. The error bars represent standard deviations.

FIG. 9.

Hierarchical clustering of the transcriptional response of B. bronchiseptica strain RB54, a Bvg⁻ phase-locked mutant during late-log-phase and stationary-phase growth. (A) Expression profiles representing global transcriptional changes. (B) Expression profiles representing transcriptional changes of genes negatively regulated by BvgAS, along with 16S RNA and rpoH. Transcriptional changes identified by comparing cDNA from mid-log phase (15 h) to that from late log phase (24 h) are represented under “late-log phase,” and the transcriptional changes identified by comparing cDNA from mid-log phase (15 h) to that from late stationary phase (48 h) are represented under “stationary phase.” Data are mean centered for each array element and averaged from three biological replicates. All expression profiles of genes are in rows and are represented using the color scale at top, with gray indicating missing data.