Systematic Identification of Cyclic-di-GMP Binding Proteins in Vibrio cholerae Reveals a Novel Class of Cyclic-di-GMP-Binding ATPases Associated with Type II Secretion Systems

Abstract

Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial signaling molecule that regulates a variety of complex processes through a diverse set of c-di-GMP receptor proteins. We have utilized a systematic approach to identify c-di-GMP receptors from the pathogen Vibrio cholerae using the Differential Radial Capillary Action of Ligand Assay (DRaCALA). The DRaCALA screen identified a majority of known c-di-GMP binding proteins in V. cholerae and revealed a novel c-di-GMP binding protein, MshE (VC0405), an ATPase associated with the mannose sensitive hemagglutinin (MSHA) type IV pilus. The known c-di-GMP binding proteins identified by DRaCALA include diguanylate cyclases, phosphodiesterases, PilZ domain proteins and transcription factors VpsT and VpsR, indicating that the DRaCALA-based screen of open reading frame libraries is a feasible approach to uncover novel receptors of small molecule ligands. Since MshE lacks the canonical c-di-GMP-binding motifs, a truncation analysis was utilized to locate the c-di-GMP binding activity to the N-terminal T2SSE_N domain. Alignment of MshE homologs revealed candidate conserved residues responsible for c-di-GMP binding. Site-directed mutagenesis of these candidate residues revealed that the Arg9 residue is required for c-di-GMP binding. The ability of c-di-GMP binding to MshE to regulate MSHA dependent processes was evaluated. The R9A allele, in contrast to the wild type MshE, was unable to complement the ΔmshE mutant for the production of extracellular MshA to the cell surface, reduction in flagella swimming motility, attachment to surfaces and formation of biofilms. Testing homologs of MshE for binding to c-di-GMP identified the type II secretion ATPase of Pseudomonas aeruginosa (PA14_29490) as a c-di-GMP receptor, indicating that type II secretion and type IV pili are both regulated by c-di-GMP. Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial signaling molecule that regulates important bacterial functions, including virulence, antibiotic resistance, biofilm formation and cell division. The list of known c-di-GMP receptors is clearly incomplete. Here we utilized a systematic and unbiased biochemical approach to identify c-di-GMP receptors from the 3,812 genes of the Vibrio cholerae genome. Results from this analysis identified most known c-di-GMP receptors as well as MshE, a protein not known to interact with c-di-GMP. The c-di-GMP binding site was identified at the N-terminus of MshE and requires a conserved arginine residue in the 9^th position. MshE is the ATPase that powers the secretion of the MshA pili onto the surface of the bacteria. We show that c-di-GMP binding to MshE is required for MshA export and the function of the pili in attachment and biofilm formation. ATPases responsible for related processes such as type IV pili and type II secretion were also tested for c-di-GMP binding, which identified the P. aeruginosa ATPase PA14_29490 as another c-di-GMP binding protein. These findings reveal a new class of c-di-GMP receptor and raise the possibility that c-di-GMP regulate membrane complexes through direct interaction with related type II secretion and type IV pili ATPases.