Evidence for phenotypic bistability resulting from transcriptional interference of bvgAS in Bordetella bronchiseptica

Abstract

Summary Bordetella species cause respiratory infections in mammals. Their master regulatory system BvgAS controls expression of at least three distinct phenotypic phases in response to environmental cues. The Bvg+ phase is necessary and sufficient for respiratory infection while the Bvg− phase is required for survival ex vivo. We obtained large colony variants (LCVs) from the lungs of mice infected with B. bronchiseptica strain RBX9, which contains an in-frame deletion mutation in fhaB, encoding filamentous hemagglutinin. RBX9 also yielded LCVs when switched from Bvg− phase conditions to Bvg+ phase conditions in vitro. We determined that LCVs are composed of both Bvg+ and Bvg− phase bacteria and that they result from defective bvgAS positive autoregulation. The LCV phenotype was linked to the presence of a divergent promoter 5′ to bvgAS, suggesting a previously undescribed mechanism of transcriptional interference that, in this case, leads to feedback-based bistability (FBM). Our results also indicate that a small proportion of RBX9 bacteria modulates to the Bvg− phase in vivo. In addition to providing insight into transcriptional interference and FBM, our data provide an example of an in-frame deletion mutation exerting a ‘polar’ effect on nearby genes.

Bordetella PlrSR regulatory system controls BvgAS activity and virulence in the lower respiratory tract

“…S3). GFP levels in the ΔbvgS strain reflect activity from the bvgA P2 promoter, which is expressed at a low level under Bvg -phase conditions (providing the cell with a low amount of BvgAS so that it can respond, via positive autoregulation, when Bvg + phase conditions are encountered) (29). This low-level expression of P bvgA was not affected by the ΔplrS mutation.…”

“…In contrast to pGFLIP, which reports on the history of the bacteria, the pBAM plasmid reports on the status of the bacteria at the time of plating (29). The pBAM plasmid integrates within the bvgAS promoter region and causes the P2 promoter to be expressed at a lower-than-normal level such that, when bacteria are growing under Bvg -phase conditions, the amount of BvgAS in a small proportion of cells in the population (∼5%) is below the threshold required for positive autoregulation, "trapping" these cells in the Bvg -phase (29).…”

“…The pBAM plasmid integrates within the bvgAS promoter region and causes the P2 promoter to be expressed at a lower-than-normal level such that, when bacteria are growing under Bvg -phase conditions, the amount of BvgAS in a small proportion of cells in the population (∼5%) is below the threshold required for positive autoregulation, "trapping" these cells in the Bvg -phase (29). When WT B. bronchiseptica containing the pBAM plasmid are grown under Bvg -phase conditions and then plated onto BG-blood agar and incubated at 37°C (Bvg + phase conditions), the trapped bacteria yield colonies that are larger, flatter, and less hemolytic than colonies formed by Bvg + phase bacteria because these colonies contain ∼5% phenotypically Bvg -phase bacteria (as the colony grows, ∼95% of the daughter cells produce enough BvgAS to convert to the Bvg+ phase, whereas ∼5% remain phenotypically Bvg -phase).…”

“…When WT B. bronchiseptica containing the pBAM plasmid are grown under Bvg -phase conditions and then plated onto BG-blood agar and incubated at 37°C (Bvg + phase conditions), the trapped bacteria yield colonies that are larger, flatter, and less hemolytic than colonies formed by Bvg + phase bacteria because these colonies contain ∼5% phenotypically Bvg -phase bacteria (as the colony grows, ∼95% of the daughter cells produce enough BvgAS to convert to the Bvg+ phase, whereas ∼5% remain phenotypically Bvg -phase). We previously referred to these colonies, which are easily scored by simple visual inspection, as large colony variants (29). However, because these colonies result from phenotypic bistability and not a genetic change, it is more appropriate to refer to them as large colony phenotypes (LCPs), which we will do henceforth.…”

“…contain pBAM and are Bvg -phase at the time of plating form typical Bvg + phase-appearing (non-LCP) colonies (29). Hence, although the presence of LCPs indicates that bacteria have modulated to the Bvg -phase, it vastly underestimates the number of bacteria that are Bvg -phase at the time of plating.…”