The cyclic AMP receptor protein modulates colonial morphology in Vibrio cholerae

Abstract

Inactivation of the quorum-sensing regulator HapR causes Vibrio cholerae El Tor biotype strain C7258 to adopt a rugose colonial morphology that correlates with enhanced biofilm formation. V. cholerae mutants lacking the cyclic AMP (cAMP) receptor protein (CRP) produce very little HapR, which results in elevated expression of Vibrio exopolysaccharide (vps) genes and biofilm compared to the wild type. However, Deltacrp mutants still exhibited smooth colonial morphology and expressed reduced levels of vps genes compared to isogenic hapR mutants. In this study we demonstrate that deletion of crp and cya (adenylate cyclase) converts a rugose DeltahapR mutant to a smooth one. The smooth DeltahapR Deltacrp and DeltahapR Deltacya double mutants could be converted back to rugose by complementation with crp and cya, respectively. CRP was found to enhance the expression of VpsR, a strong activator of vps expression, but to diminish transcription of VpsT. Ectopic expression of VpsR in smooth DeltahapR Deltacrp and DeltahapR Deltacya double mutants restored rugose colonial morphology. Lowering intracellular cAMP levels in a DeltahapR mutant by the addition of glucose diminished VpsR expression and colonial rugosity. On the basis of our results, we propose a model for the regulatory input of CRP on exopolysaccharide biosynthesis.

FIG. 1.

Scanning electron microscopy of Δcrp, Δcya, and ΔhapR colonies. (A) Strain WL7258 (Δcrp); (B) strain AJB51 (ΔhapR); (C) strain WL51 (ΔhapR Δcrp); (D) WL51 transformed with pBADCRP7; (E) WL52 (ΔhapR Δcya); (F) WL52 (ΔhapR Δcya) transformed with pTT-Cya. Strains were streaked onto LB agar, and colonies were allowed to develop at 30°C for 24 h. Expression of crp was induced by the addition of 0.02% l-arabinose. Scanning electron microscopy was performed with a magnification of ×50.

FIG. 2.

Expression of regulators of rugose colonial morphology in V. cholerae ΔhapR and Δcrp mutants. Strains C7258 (wild type [WT]), AJB51 (ΔhapR), WL7258 (Δcrp), and WL51 (ΔhapR Δcrp) were grown in LB medium to an OD₆₀₀ of 1.5. Abundance of mRNAs encoding cytR (A), vpsT (B), and vpsR (C) was determined by qRT-PCR. Results were normalized to recA mRNA expression. Error bars indicate the standard deviations of at least three independent cultures.

FIG. 3.

Spot colonial morphology of Δcrp and Δcya mutants expressing VpsR. Overnight cultures of strain WL51 (ΔhapR Δcrp) and WL52 (ΔhapR Δcya) were diluted 1:200, and 2 μl of each dilution was spotted onto LB agar containing 100 μg/ml ampicillin as required. (A) Strain WL51 with empty vector pTTQ18; (B) WL51 with pTTVpsR; (C) WL51 with pTTVpsR induced with 10 mM IPTG; (D) strain WL52 with empty vector pTTQ18; (E) WL52 with pTTVpsR; (F) WL52 with pTTVpsR induced with 10 mM IPTG. Plates were incubated for 24 h at 30°C, and colonies were photographed with a Canon EOS camera with a 60-mm macro lens.

FIG. 4.

Repression of VpsR by glucose. Strain WL51 (ΔhapR) was grown in LB (control) and LB supplemented with 5% glucose. Expression of vpsR, vpsA, and vpsL was determined by qRT-PCR. Error bars indicate the standard deviations of at least three independent cultures.

FIG. 5.

Model for the regulatory input of cya and crp in exopolysaccharide biosynthesis and rugose colonial morphology.