The Vibrio cholerae diguanylate cyclase VCA0965 has an AGDEF active site and synthesizes cyclic di-GMP.
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.
Jessica L
JL
Hunter
Geoffrey B
GB
Severin
Benjamin J
BJ
Koestler
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA. watersc3@msu.edu.
Christopher M
CM
Waters
D020134:Catalytic Domain; D003001:Cloning, Molecular; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D016297:Mutagenesis, Site-Directed; D019761:Phosphorus-Oxygen Lyases / Q000235:genetics* / Q000378:metabolism*; D014734:Vibrio cholerae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; C062025:bis(3',5')-cyclic diguanylic acid; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase; D006152:Cyclic GMP