Identification of a calcium-controlled negative regulatory system affecting Vibrio cholerae biofilm formation

Abstract

Vibrio cholerae's capacity to cause outbreaks of cholera is linked to its survival and adaptability to changes in aquatic environments. One of the environmental conditions that can vary in V. cholerae's natural aquatic habitats is calcium (Ca(+2)). In this study, we investigated the response of V. cholerae to changes in extracellular Ca(2+) levels. Whole-genome expression profiling revealed that Ca(2+) decreased the expression of genes required for biofilm matrix production. Luria-Bertani (LB) medium supplemented with Ca(2+) (LBCa(2+)) caused V. cholerae to form biofilms with decreased thickness and increased roughness, as compared with biofilms formed in LB. Furthermore, addition of Ca(2+) led to dissolution in biofilms. Transcription of two genes encoding a two-component regulatory system pair, now termed calcium-regulated sensor (carS) and regulator (carR), was decreased in cells grown in LBCa(2+). Analysis of null and overexpression alleles of carS and carR revealed that expression of vps (Vibriopolysaccharide) genes and biofilm formation are negatively regulated by the CarRS two-component regulatory system. Through epistasis analysis we determined that CarR acts in parallel with HapR, the negative regulator of vps gene expression.

Fig. 1

Effect of Ca²⁺ on vps gene expression and biofilm formation. (A) Comparison of transcription of vpsA–lacZ, vpsL–lacZ, vpsR–lacZ and vpsT–lacZ genes as determined by β-galactosidase assay in wild-type cells that were grown to mid-exponential phase in LB and LBCa²⁺ at 30°C. The result shown is the representative of three independent experiments and error bars represent standard deviations. (B, D) Changes in the architecture of the biofilms in response to increase in [Ca²⁺] in the gfp-tagged wild-type cells (B) and Δvps mutant (D). Biofilms of the wild-type cells and Δvps mutant were grown in flow cells for 48 h in LB and LBCa²⁺. Images were acquired at 24 and 48 h time points using a CSLM, and processed using Imaris software. 0.5% SDS was administered into each chamber at a 48 h time point to test biofilm stability. White bars represent 30 μm. The result shown is representative of three independent experiments. Quantification of changes in the structural properties of biofilms that were formed by the wild-type cells (C) and by Δvps mutant (E) was performed using COMSTAT. Results shown were calculated from three image stacks that were representative of three independent experiments.

Fig. 2

Effects of VpsR, VpsT and HapR on Ca²⁺-mediated repression of vps gene expression. Comparison of vpsL–lacZ transcription as determined by β-galactosidase assay in wild-type, ΔvpsR, ΔvpsT and ΔhapR strains that were grown to mid-exponential phase in LB and LBCa²⁺ at 30°C. The result shown is the representative of three independent experiments. Error bars represent standard deviation.

Fig. 3

CarS and CarR negatively regulate vps gene expression. A. Comparison of transcription of carR–lacZ gene, as determined by β-galactosidase assay in wild-type cells that were grown to mid-exponential phase in LB and LBCa²⁺ at 30°C. The result shown is the representative of three independent experiments and error bars represent standard deviations. B and C. Comparison of vpsA–lacZ, vpsL–lacZ, vpsR–lacZ and vpsT–lacZ gene expression as determined by β-galactosidase assay in the wild-type, ΔcarS or ΔcarR strains that were grown to mid-exponential phase in LB. The result shown is the representative of three independent experiments and error bars represent standard deviations. D. Comparison of vpsA–lacZ and vpsL–lacZ gene expressions in the wild-type, ΔcarR and ΔcarS strains that were grown in LB and LBCa²⁺ at 30°C. The result shown is the representative of three independent experiments and error bars represent standard deviation.

Fig. 4

Comparison of biofilms of the wild-type, ΔcarS and ΔcarR mutant strains. A. Biofilm structures of the gfp-tagged wild-type, ΔcarS and ΔcarR strains that were grown under non-flow static conditions. Biofilms were grown in chambered cover-slides for 8 h at 30°C, images were acquired using a CSLM and processed using Imaris software. White bars represent 30 μm. The result shown is representative of three independent experiments. B. Biofilm structures of the gfp-tagged wild-type, ΔcarS and ΔcarR strains in a flow-cell system. Biofilms were grown in flow cells for 48 h in LB. Biofilm images were acquired 24 and 48 h after the inoculation using a CSLM and processed using Imaris software. The result shown is representative of three independent experiments. White bars represent 30 μm. C. Biofilm structures of the gfp-tagged wild-type strains harbouring the empty vector pBAD/Myc-His C (wild-type), and overexpression plasmids for carS (CarS) and carR (CarR) in the flow-cell system. Biofilms were grown in flow cells for 48 h in LB supplemented with 0.2% arabinose. Images were taken 8, 24 and 48 h after the inoculation. White bars represent 30 μm. Images were acquired using a CSLM and processed using Imaris software. The result shown is representative of three independent experiments.

Fig. 5

Epistasis analysis of carR, vpsR, vpsT and hapR. A. Comparison of transcription of vpsL–lacZ as determined by β-galactosidase assay in the wild-type, ΔcarR, ΔvpsR, ΔvpsRΔcarR, ΔvpsT, ΔvpsTΔcarR, ΔhapR and ΔhapRΔcarR strains that were grown to mid-exponential phase in LB at 30°C. The results shown are representatives of three independent experiments and error bars represent standard deviations. B. A model of CarR regulation of biofilm formation in V. cholerae. Expression of vps structural genes, vpsR and vpsT is negatively regulated by CarR. CarR acts in parallel pathway with HapR.