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

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 Ca²⁺ levels. Whole-genome expression profiling revealed that Ca²⁺ decreased the expression of genes required for biofilm matrix production. Luria-Bertani (LB) medium supplemented with Ca²⁺ (LBCa²⁺) caused V. cholerae to form biofilms with decreased thickness and increased roughness, as compared with biofilms formed in LB. Furthermore, addition of Ca²⁺ 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 LBCa²⁺. Analysis of null and overexpression alleles of carS and carR revealed that expression of vps ( V ibrio poly saccharide) 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.