Distinct sensory pathways in Vibrio cholerae El Tor and classical biotypes modulate cyclic dimeric GMP levels to control biofilm formation

Abstract

Quorum sensing (QS), or cell-cell communication in bacteria, is achieved through the production and subsequent response to the accumulation of extracellular signal molecules called autoinducers (AIs). To identify AI-regulated target genes in Vibrio cholerae El Tor (V. cholerae(El)), the strain responsible for the current cholera pandemic, luciferase expression was assayed in an AI(-) strain carrying a random lux transcriptional reporter library in the presence and absence of exogenously added AIs. Twenty-three genes were identified and shown to require the QS transcription factor, HapR, for their regulation. Several of the QS-dependent target genes, annotated as encoding hypothetical proteins, in fact encode HD-GYP proteins, phosphodiesterases that degrade the intracellular second messenger cyclic dimeric GMP (c-di-GMP), which is important for controlling biofilm formation. Indeed, overexpression of a representative QS-activated HD-GYP protein in V. cholerae(El) reduced the intracellular concentration of c-di-GMP, which in turn decreased exopolysaccharide production and biofilm formation. The V. cholerae classical biotype (V. cholerae(Cl)), which caused previous cholera pandemics and is HapR(-), controls c-di-GMP levels and biofilm formation by the VieA signaling pathway. We show that the VieA pathway is dispensable for biofilm formation in V. cholerae(El) but that restoring HapR in V. cholerae(Cl) reestablishes QS-dependent repression of exopolysaccharide production. Thus, different pandemic strains of V. cholerae modulate c-di-GMP levels and control biofilm formation in response to distinct sensory pathways.

FIG. 1.

Multiple regulatory pathways control biofilm formation in V. cholerae. (A) In V. cholerae_El, sensory inputs from two QS AIs, CAI-1 (squares) and AI-2 (circles), regulate HapR, which in turn modulates the levels of c-di-GMP, vps transcription, biofilm formation, and virulence. (B) In V. cholerae_Cl, the VieSAB sensory system, in response to an unknown signal (triangles), controls c-di-GMP levels to control vps transcription, biofilm formation, and virulence in a HapR-independent manner.

FIG. 2.

QS activates expression of four genes encoding HD-GYP proteins in V. cholerae. Expression of lux transcriptional fusions to the promoters of genes encoding predicted HD-GYP proteins was measured in the high-cell-density (WT and ΔluxO) and low-cell-density (luxO D47E) strains. Shown in each panel are the mean LUX/OD and the standard deviation from triplicate overnight cultures.

FIG. 3.

HD-GYP activity represses V. cholerae_El vpsL expression and biofilm formation. Expression of vpsL-lux in a V. cholerae_El low-cell-density mutant (luxO D47E) carrying a vector control (first pair of bars) or a vector carrying VCA0681 (second pair of bars), VCA0681-FLAG (third pair of bars), or VCA0681(D289A)-FLAG (fourth pair of bars) was measured in the presence (black bars) or absence (white bars) of IPTG. The mean LUX/OD and the standard deviation of triplicate cultures diluted 1/00 and subsequently grown for 8 h are shown. (B) Western blot of the strains shown in panel A visualized with anti-FLAG antibodies. (C) Biofilm formation of static cultures of the strains shown in panel A incubated for 48 h.

FIG. 4.

The VCA0681 protein controls c-di-GMP levels in V. cholerae_El. Relative c-di-GMP/1,000·OD₆₀₀ (c-di-GMP concentration) in V. cholerae_El WT, ΔluxO, and luxO D47E strains and in the luxO D47E strain carrying IPTG-inducible vectors harboring VCA0681 or VCA0681(D289A). Shown are the mean values for triplicate cultures, with error bars indicating the standard deviation of the mean.

FIG. 5.

Expression of vpsL and levels of c-di-GMP are controlled differently in V. cholerae_El and V. cholerae_Cl. (A and B) Expression of vpsL-lux (A) and relative c-di-GMP levels (B) were measured for V. cholerae_El and V. cholerae_Cl strains containing or lacking hapR, and containing or lacking vieA. (C) Expression of vpsL-lux in V. cholerae_Cl ΔvieA and ΔvieA HapR⁺ strains carrying an IPTG-inducible vector expressing VCA0681 without or with IPTG. LUX/OD and c-di-GMP/1,000·OD₆₀₀ (c-di-GMP concentration) are shown for triplicate cultures, with error bars indicating the standard deviation of the mean.