Diguanylate Cyclase GdpX6 with c-di-GMP Binding Activity Involved in the Regulation of Virulence Expression in pv. .

Cyclic diguanylate monophosphate (c-di-GMP) is a secondary messenger present in bacteria. The GGDEF-domain proteins can participate in the synthesis of c-di-GMP as diguanylate cyclase (DGC) or bind with c-di-GMP to function as a c-di-GMP receptor. In the genome of pv. (), the causal agent of bacterial blight of rice, there are 11 genes that encode single GGDEF domain proteins. The GGDEF domain protein, PXO_02019 (here GdpX6 [GGDEF-domain protein of 6]) was characterized in the present study. Firstly, the DGC and c-di-GMP binding activity of GdpX6 was confirmed in vitro. Mutation of the crucial residues D residue of the I site in GGDEF motif and E residue of A site in GGDEF motif of GdpX6 abolished c-di-GMP binding activity and DGC activity of GdpX6, respectively. Additionally, deletion of significantly increased the virulence, swimming motility, and decreased sliding motility and biofilm formation. In contrast, overexpression of GdpX6 in wild-type PXO99 strain decreased the virulence and swimming motility, and increased sliding motility and biofilm formation. Mutation of the E residue but not D residue of the GGDEF domain in GdpX6 abolished its biological functions, indicating the DGC activity to be imperative for its biological functions. Furthermore, GdpX6 exhibited multiple subcellular localization in bacterial cells, and D or E did not contribute to the localization of GdpX6. Thus, we concluded that GdpX6 exhibits DGC activity to control the virulence, swimming and sliding motility, and biofilm formation in