A Class IV Adenylate Cyclase, CyaB, Is Required for Capsule Polysaccharide Production and Biofilm Formation in Vibrio parahaemolyticus.

ABSTRACT

Cyclic AMP (cAMP) receptor protein (CRP), encoded by crp, is a global regulator that is activated by cAMP, 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 nonpreferred carbon sources and is a global regulator. We constructed in-frame nonpolar deletions of the crp and cyaA homologs in Vibrio parahaemolyticus and found that the Δcrp mutant did not grow in minimal media supplemented with nonpreferred carbon sources, but the ΔcyaA mutant grew similarly to the 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 that CyaB was present in Gammaproteobacteria and Alphaproteobacteria as well as Planctomycetes and Archaea. Only the bacterial CyaB proteins contained an N-terminal motif, HFxxxxExExK, indicative of adenylyl cyclase activity. Both V. parahaemolyticus cyaA and cyaB genes functionally complemented an E. coli ΔcyaA mutant. The Δcrp and ΔcyaB ΔcyaA mutants showed defects in growth on nonpreferred carbon sources and in swimming and swarming motility, indicating that cAMP-CRP is an activator. The ΔcyaA and ΔcyaB single mutants had no defects in these phenotypes, indicating that AC-IV complements AC-I. Capsule polysaccharide and biofilm production assays showed significant defects in the Δcrp, ΔcyaBΔcyaA, and ΔcyaB mutants, whereas the ΔcyaA strain behaved similarly to the 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. IMPORTANCE Here, we characterized the roles of CRP and CyaA in V. parahaemolyticus, showing that cAMP-CRP is an activator of metabolism, motility, capsule production, 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 is required for optimal growth. The data demonstrated that CyaB is 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 Gammaproteobacteria, 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.