Calcium-dependent site-switching regulates expression of the atypical iam pilus locus in Vibrio vulnificus

The opportunistic human pathogen Vibrio vulnificus inhabits warm coastal waters and asymptomatically colonizes seafood, most commonly oysters. We previously characterized an isolate that exhibited greater biofilm formation, aggregation and oyster colonization than its parent. This was due, in part, to the production of a Type IV Tad pilus (Iam). However, the locus lacked key processing and regulatory genes required for pilus production. Here, we identify a pilin peptidase iamP, and LysR-type regulator (LRTR) iamR, that fulfil these roles and show that environmental calcium, which oysters enrich for shell repair and growth, regulates iam expression. The architecture of the iam locus differs from the classical LRTR paradigm and requires an additional promoter to be integrated into the regulatory network. IamR specifically recognized the iamR promoter (P_iamR ) and the intergenic iamP-iamA region (P_iamP-A ). P_iamR exhibited classical negative auto-regulation but, strikingly, IamR inversely regulated the divergent iamP and iamA promoters in a calcium-dependent manner. Moreover, expression of the c-di-GMP and calcium-regulated, biofilm-promoting brp exopolysaccharide was IamA-dependent. These results support a scenario in which the calcium-enriched oyster environment triggers IamP-mediated processing of prepilin amassed in the periplasm for rapid pilin elaboration and subsequent BRP production to promote colonization.