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

Abstract

ABSTRACTCRP (cyclic AMP receptor protein), encoded bycrp, is a global regulator that is activated by cAMP (cyclic AMP), a second messenger synthesized by a class I adenylate cyclase (AC-I) encoded bycyaAinEscherichia 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 thecrpandcyaAhomologs inVibrio parahaemolyticusand found that the Δcrpmutant did not grow in minimal media supplemented with non-preferred carbon sources, but the ΔcyaAmutant grew similar to wild type. Bioinformatics analysis of theV. parahaemolyticusgenome 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. BothV. parahaemolyticus cyaAandcyaBgenes functionally complemented anE. coliΔcyaAmutant. The Δcrpand ΔcyaB/ΔcyaAmutants showed defects in growth on non- preferred carbon sources, and in swimming and swarming motility, indicating cAMP- CRP is an activator. The ΔcyaAand ΔcyaBsingle mutants had no defects in these phenotypes indicating AC-IV complements AC-I. Capsule polysaccharide and biofilm production assays showed significant defects in Δcrp, ΔcyaB/ΔcyaA,and the ΔcyaBmutant, whereas ΔcyaAbehaved 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 inV. parahaemolyticus,showing cAMP-CRP was an activator of metabolism, motility, capsule and biofilm formation. These results are in contrast to cAMP-CRP inV. cholerae,which represses capsule and biofilm formation. Previously, only an AC-I CyaA had been identified inVibriospecies. Our data showed that an AC-IV CyaB homolog is present inV. parahaemolyticusand 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 thecyaBgene was widespread amongVibrionaceaespecies and several other Gamma-Proteobacteria, but in general, its phylogenetic distribution was limited. Our phylogenetic analysis also demonstrated that in some species thecyaBgene was acquired by horizontal gene transfer.