A class IV adenylate cyclase CyaB is required for capsule polysaccharide production and biofilm formation in Vibrio parahaemolyticus

Abstract

CRP (cyclic AMP receptor protein), encoded by crp, is a global regulator that is activated by cAMP (cyclic AMP), a second messenger synthesized by a class I adenylate cyclase (AC-I) encoded by cyaA in Escherichia coli. cAMP-CRP is required for growth on non-preferred carbon sources and is a global regulator. We constructed in-frame non- polar deletions of the crp and cyaA homologs in Vibrio parahaemolyticus and found that the {Delta}crp mutant did not grow in minimal media supplemented with non-preferred carbon sources, but the {Delta}cyaA mutant grew similar to wild type. Bioinformatics analysis of the V. parahaemolyticus genome identified a 181 amino acid protein annotated as a class IV adenylate cyclase (AC-IV) named CyaB, a member of the CYTH protein superfamily. AC-IV phylogeny showed CyaB was present in Gamma- and Alpha- Proteobacteria as well as Planctomycete and Archaea. Only the bacterial CyaB proteins contained an N-terminal motif HFxxxExExK indicative of adenylyl cyclase activity. Both V. parahaemolyticus cyaA and cyaB genes functionally complemented an E. coli {Delta}cyaA mutant. The {Delta}crp and {Delta}cyaB/{Delta}cyaA mutants showed defects in growth on non- preferred carbon sources, and in swimming and swarming motility, indicating cAMP- CRP is an activator. The {Delta}cyaA and {Delta}cyaB single mutants had no defects in these phenotypes indicating AC-IV complements AC-I. Capsule polysaccharide and biofilm production assays showed significant defects in {Delta}crp, {Delta}cyaB/{Delta}cyaA, and the {Delta}cyaB mutant, whereas {Delta}cyaA behaved similar to wild type. This is consistent with a role of cAMP-CRP as an activator of these phenotypes and establishes a cellular role for AC-IV in capsule and biofilm formation, which to date has been unestablished. IMPORTANCEHere, we characterized the roles of CRP and CyaA in V. parahaemolyticus, showing cAMP-CRP was an activator of metabolism, motility, capsule and biofilm formation. These results are in contrast to cAMP-CRP in V. cholerae, which represses capsule and biofilm formation. Previously, only an AC-I CyaA had been identified in Vibrio species. Our data showed that an AC-IV CyaB homolog is present in V. parahaemolyticus and was required for optimal growth. The data demonstrated that CyaB was essential for capsule production and biofilm formation uncovering a physiological role of AC-IV in bacteria. The data showed that the cyaB gene was widespread among Vibrionaceae species and several other Gamma-Proteobacteria, but in general, its phylogenetic distribution was limited. Our phylogenetic analysis also demonstrated that in some species the cyaB gene was acquired by horizontal gene transfer.