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The Vibrio cholerae diguanylate cyclase VCA0965 has an AGDEF active site and synthesizes cyclic di-GMP.
Hunter, Jessica L; Severin, Geoffrey B; Koestler, Benjamin J; Waters, Christopher M.
Afiliación
  • Waters CM; Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA. watersc3@msu.edu.
BMC Microbiol ; 14: 22, 2014 Feb 04.
Article en En | MEDLINE | ID: mdl-24490592
ABSTRACT

BACKGROUND:

Diguanylate cyclases (DGCs) regulate biofilm formation and motility in bacteria by synthesizing the second messenger cyclic di-GMP (c-di-GMP) in response to environmental stimuli. DGC enzymatic activity is believed to be dependent on the presence of a GG(D/E)EF active site motif, however approximately 25% of known DGCs contain a degenerate active site. The Vibrio cholerae protein VCA0965 contains an AGDEF active site and is presumed to be an inactive DGC.

RESULTS:

Ectopic expression of VCA0965 in V. cholerae causes a 3-fold reduction in flagellar-based motility. Additionally, an RXXD allosteric inhibition mutant of VCA0965 strongly inhibited motility and stimulated biofilm formation. This activity was lost when the active site of VCA0965 was mutated to AGDAF, suggesting that VCA0965 synthesizes c-di-GMP. In support of this, ectopic expression of VCA0965 and VCA0965 containing a mutation in its RXXD motif significantly increased the intracellular c-di-GMP levels in V. cholerae and Escherichia coli. Furthermore, we found that purified VCA0965 was able to synthesize c-di-GMP in vitro. Systematic mutation of the first amino acid in the AGDEF motif of VCA0965 revealed that glycine, methionine, and histidine also produced an active DGC capable of inhibiting motility and increasing the intracellular concentration of c-di-GMP in V. cholerae.

CONCLUSIONS:

Based on these results, we conclude that VCA0965 is capable of c-di-GMP synthesis and that the first amino acid of the GG(D/E)EF motif is more tolerant of substitutions than currently appreciated.
Asunto(s)

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Vibrio cholerae / GMP Cíclico / Liasas de Fósforo-Oxígeno / Proteínas de Escherichia coli Idioma: En Revista: BMC Microbiol Asunto de la revista: MICROBIOLOGIA Año: 2014 Tipo del documento: Article

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Vibrio cholerae / GMP Cíclico / Liasas de Fósforo-Oxígeno / Proteínas de Escherichia coli Idioma: En Revista: BMC Microbiol Asunto de la revista: MICROBIOLOGIA Año: 2014 Tipo del documento: Article