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

PLoS Pathogens

Mona W. Orr¹  Kevin G. Roelofs¹  Vincent T. Lee¹  Xiaoran Shang¹  Jonathan R. Goodson¹  Sarah R. Helman¹  Christopher J. Jones²  Fitnat H. Yildiz²  Michael Y. Galperin³ 

[1] Department of Cell Biology and Molecular Genetics, University of Maryland at College Park, Maryland, United States of America;Department of Microbiology and Environmental Toxicology, University of California, Santa Cruz, Santa Cruz, California, United States of America;National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, United States of America

关键词: Vibrio cholerae;    Adenosine triphosphatase;    Protein domains;    Library screening;    Pseudomonas aeruginosa;    Transcription factors;    Pili;    fimbriae;    Secretion systems;   

DOI  :  10.1371/journal.ppat.1005232

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

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.

【 授权许可】

CC BY