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

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.

Regulatory Small RNA Qrr2 Is Expressed Independently of Sigma Factor-54 and Can Function as the Sole Qrr Small RNA To Control Quorum Sensing in Vibrio parahaemolyticus.

Bacterial cells alter gene expression in response to changes in population density in a process called quorum sensing (QS). In Vibrio harveyi, LuxO, a low-cell-density activator of sigma factor-54 (RpoN), is required for transcription of five noncoding regulatory small RNAs (sRNAs), Qrr1 to Qrr5, which each repress translation of the master QS regulator, LuxR. Vibrio parahaemolyticus, the leading cause of bacterial seafoodborne gastroenteritis, also contains five Qrr sRNAs that control OpaR (the LuxR homolog), controlling capsule polysaccharide (CPS), motility, and metabolism. We show that in a ΔluxO deletion mutant, opaR was derepressed and CPS and biofilm were produced. However, in a ΔrpoN mutant, opaR was repressed, no CPS was produced, and less biofilm production was observed than in the wild type. To determine why opaR was repressed, expression analysis in ΔluxO showed that all five qrr genes were repressed, while in ΔrpoN the qrr2 gene was significantly derepressed. Reporter assays and mutant analysis showed that Qrr2 sRNA can act alone to control OpaR. Bioinformatics analysis identified a sigma-70 (RpoD) -35 -10 promoter overlapping the canonical sigma-54 (RpoN) -24 -12 promoter in the qrr2 regulatory region. The qrr2 sigma-70 promoter element was also present in additional Vibrio species, indicating that it is widespread. Mutagenesis of the sigma-70 -10 promoter site in the ΔrpoN mutant background resulted in repression of qrr2. Analysis of qrr quadruple deletion mutants, in which only a single qrr gene is present, showed that only Qrr2 sRNA can act independently to regulate opaR. Mutant and expression data also demonstrated that RpoN and the global regulator, Fis, act additively to repress qrr2. Our data have uncovered a new mechanism of qrr expression and show that Qrr2 sRNA is sufficient for OpaR regulation. IMPORTANCE The quorum sensing noncoding small RNAs (sRNAs) are present in all Vibrio species but vary in number and regulatory roles among species. In the Harveyi clade, all species contain five qrr genes, and in Vibrio harveyi these are transcribed by sigma-54 and are additive in function. In the Cholerae clade, four qrr genes are present, and in Vibrio cholerae the qrr genes are redundant in function. In Vibrio parahaemolyticus, qrr2 is controlled by two overlapping promoters. In an rpoN mutant, qrr2 is transcribed from a sigma-70 promoter that is present in all V. parahaemolyticus strains and in other species of the Harveyi clade, suggesting a conserved mechanism of regulation. Qrr2 sRNA can function as the sole Qrr sRNA to control OpaR.