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.
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.
Department of Cell Biology and Molecular Genetics, University of Maryland at College Park, Maryland, United States of America.
Kevin G
KG
Roelofs
Department of Microbiology and Environmental Toxicology, University of California, Santa Cruz, Santa Cruz, California, United States of America.
Christopher J
CJ
Jones
Department of Cell Biology and Molecular Genetics, University of Maryland at College Park, Maryland, United States of America.
Sarah R
SR
Helman
Department of Cell Biology and Molecular Genetics, University of Maryland at College Park, Maryland, United States of America.
Xiaoran
X
Shang
Department of Cell Biology and Molecular Genetics, University of Maryland at College Park, Maryland, United States of America.
Mona W
MW
Orr
Department of Cell Biology and Molecular Genetics, University of Maryland at College Park, Maryland, United States of America.
Jonathan R
JR
Goodson
National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland, United States of America.
Michael Y
MY
Galperin
Department of Microbiology and Environmental Toxicology, University of California, Santa Cruz, Santa Cruz, California, United States of America.
Fitnat H
FH
Yildiz
Department of Cell Biology and Molecular Genetics, University of Maryland at College Park, Maryland, United States of America.
Vincent T
VT
Lee
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism; D036781:Fimbriae Proteins / Q000378:metabolism*; D010861:Fimbriae, Bacterial / Q000502:physiology; D037601:Mannose-Binding Lectin / Q000378:metabolism; D016366:Open Reading Frames; D000069178:Type II Secretion Systems / Q000502:physiology*; D014734:Vibrio cholerae / Q000378:metabolism*
D037601:Mannose-Binding Lectin; C101032:MshA protein, Vibrio cholerae; D000069178:Type II Secretion Systems; D036781:Fimbriae Proteins; C062025:bis(3',5')-cyclic diguanylic acid; D000251:Adenosine Triphosphatases; D006152:Cyclic GMP