Research Article: 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

Date Published: October 27, 2015

Publisher: Public Library of Science

Author(s): Kevin G. Roelofs, Christopher J. Jones, Sarah R. Helman, Xiaoran Shang, Mona W. Orr, Jonathan R. Goodson, Michael Y. Galperin, Fitnat H. Yildiz, Vincent T. Lee, Samuel I Miller.


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.

Partial Text

Cyclic diguanosine monophosphate (c-di-GMP) is a ubiquitous bacterial nucleotide secondary signaling molecule that regulates cellular processes in response to environmental and cellular stimuli. The elements of this canonical pathway of signal production, signal transduction, altered activity and signal removal were elegantly described by the Benziman lab over twenty-five years ago [1, 2]. C-di-GMP is synthesized by diguanylate cyclases (DGCs) via a catalytic GGDEF domain [1, 3, 4]. Once made in the cell, c-di-GMP binds to macromolecule receptors to allosterically alter their activities. C-di-GMP signaling is terminated through hydrolysis by phosphodiesterases (PDEs) that contain catalytic EAL or HD-GYP domains [5–8]. In the characterization of bacterial cellulose synthase in Komagataeibacter xylinus, the Benziman lab demonstrated the importance of c-di-GMP in the allosteric activation of the cellulose synthase complex [1, 9, 10]. Recent structure elucidation of the BcsA-BcsB-c-di-GMP complex validated these early finding and provided a molecular mechanism for c-di-GMP activation of cellulose biosynthesis [11]. Since this initial description of c-di-GMP regulation of cellulose biosynthesis, genome sequencing has revealed genes for DGCs in diverse bacteria indicating that c-di-GMP is a ubiquitous and important signaling molecule in prokaryotes that regulates a variety of phenotypes [12].




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