The Vibrio cholerae diguanylate cyclase VCA0965 has an AGDEF active site and synthesizes cyclic di-GMP

Abstract

BackgroundDiguanylate 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.ResultsEctopic 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.ConclusionsBased 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.

The Inhibitory Site of a Diguanylate Cyclase Is a Necessary Element for Interaction and Signaling with an Effector Protein

“…2C), indicating that protein instability is not responsible for the loss of biofilm formation observed. Previous studies suggested that disruption of a DGC's I-site leads to an inability to form the inactive dimer conformation (22) and that a DGC will still readily adopt its active dimer conformation, leading to c-di-GMP overproduction (8). However, we could not immediately rule out the possibility that the mutant variants of GcbC experienced a loss of the ability to produce c-di-GMP; thus, their catalytic ability is assessed below.…”

“…Our initial results showing that mutation of the I-site of GcbC reduced biofilm formation was somewhat surprising, given that other studies have found that I-site disruption leads to the overproduction of c-di-GMP and enhanced biofilm formation relative to proteins with functional I-sites (7,8). To analyze the impact of these I-site mutations on c-di-GMP levels, we employed two different assays.…”

“…Cultures of P. fluorescens grown overnight were subcultured at a 1:75 dilution in 45 ml of K10 minimal medium supplemented with 0.1% arabinose. Subcultures were shaken for 6 h and pelleted by centrifugation, followed by resuspension in lysis buffer (20 mM Tris [pH 8.0] and 25 mM MgCl 2 ) and sonication. Sample values were normalized to the protein concentration via a bicinchoninic acid (BCA) assay, and 6 g of the protein sample was loaded onto a 10% polyacrylamide gel.…”

“…Many, but not all, DGCs have a characteristic I-site that provides this kind of negative-feedback regulation, which has been described as characteristic of systems that require fast up-kinetics in their enzymatic reactions (7). Studies focused on disrupting the I-site have previously found that deregulation of a DGC leads to increased production of c-di-GMP and a concomitant increase in c-di-GMPrelated phenotypes, including increases in biofilm formation and decreases in motility (7,8). Additionally, effectors have been thought to provide one mechanism of signaling specificity by differentially activating at various levels of c-di-GMP, as demonstrated in Salmonella enterica serovar Typhimurium (9), with a complementary study identifying various response curves for cdi-GMP-dependent processes in Caulobacter crescentus (10).…”

Progress in Understanding the Molecular Basis Underlying Functional Diversification of Cyclic Dinucleotide Turnover Proteins

“…Recent experimentally characterized proteins with a G¡A or G¡S substitution still exhibit significant functionality, demonstrating unexpected flexibility in the GGDEF containing active-site hairpin ( Fig. 2) (17)(18)(19).…”