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

Hunter JL, Severin GB, Koestler BJ, Waters CM - BMC Microbiol. (2014)

Bottom Line: This activity was lost when the active site of VCA0965 was mutated to AGDAF, suggesting that VCA0965 synthesizes c-di-GMP.Furthermore, we found that purified VCA0965 was able to synthesize c-di-GMP in vitro.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA. watersc3@msu.edu.

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.

Show MeSH

Related in: MedlinePlus

VCA0965 requires an intact active site to repress motility. Quantification of motility as determined by quantifying colony area (cm2) in V. cholerae strains expressing the indicated proteins from a Ptac promoter on a plasmid. Proteins were induced with 0.1 mM IPTG (gray bars). The mean and standard deviation are indicated (* = p < 0.05, n = 3). In this and all subsequent figures the strain designations are pCMW75 (QrgB) (an active DGC from V. harveyi), pVCA0965 (WT VCA0965 expression plasmid), pVCA0965-I (I-site mutant), pVCA0965-A (A-site mutant), and pVCA0965-AI (I-site/A-site double mutant).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3974145&req=5

Figure 2: VCA0965 requires an intact active site to repress motility. Quantification of motility as determined by quantifying colony area (cm2) in V. cholerae strains expressing the indicated proteins from a Ptac promoter on a plasmid. Proteins were induced with 0.1 mM IPTG (gray bars). The mean and standard deviation are indicated (* = p < 0.05, n = 3). In this and all subsequent figures the strain designations are pCMW75 (QrgB) (an active DGC from V. harveyi), pVCA0965 (WT VCA0965 expression plasmid), pVCA0965-I (I-site mutant), pVCA0965-A (A-site mutant), and pVCA0965-AI (I-site/A-site double mutant).

Mentions: We hypothesized that VCA0965 was incapable of synthesizing c-di-GMP and functioned as a c-di-GMP receptor protein to repress motility. Consistent with previously studied DGC effectors, c-di-GMP could bind VCA0965 at its degenerate active site (referred to as the A-site) and/or its RXXD allosteric inhibition site (referred to as the I-site) [10,20,21]. To determine if the A-site and/or I-site were necessary for motility repression, we mutated the VCA0965 I-site from RXXD to AXXA (VCA0965-I) and the VCA0965 A-site from AGDEF to AGDAF (VCA0965-A). We also constructed an allele of VCA0965 which contained both the A-site and the I-site mutations (VCA0965-AI). These mutant VCA0965 alleles were ectopically expressed from a plasmid in V. cholerae and then tested for the ability to repress swimming motility (Figure 2). Confirming our previous findings, ectopic expression of WT VCA0965 repressed motility 3-fold in a motility assay, as did the positive control QrgB, a DGC from the related species Vibrio harveyi that has been shown to be active in V. cholerae[34] (Figure 2). The I-site mutant more robustly repressed motility than WT VCA0965. This result demonstrated that the I-site is not required for motility repression. However, the A-site was necessary for this regulation as expression of both the VCA0965-A and VCA0965-AI proteins failed to inhibit motility (Figure 2).


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

Hunter JL, Severin GB, Koestler BJ, Waters CM - BMC Microbiol. (2014)

VCA0965 requires an intact active site to repress motility. Quantification of motility as determined by quantifying colony area (cm2) in V. cholerae strains expressing the indicated proteins from a Ptac promoter on a plasmid. Proteins were induced with 0.1 mM IPTG (gray bars). The mean and standard deviation are indicated (* = p < 0.05, n = 3). In this and all subsequent figures the strain designations are pCMW75 (QrgB) (an active DGC from V. harveyi), pVCA0965 (WT VCA0965 expression plasmid), pVCA0965-I (I-site mutant), pVCA0965-A (A-site mutant), and pVCA0965-AI (I-site/A-site double mutant).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3974145&req=5

Figure 2: VCA0965 requires an intact active site to repress motility. Quantification of motility as determined by quantifying colony area (cm2) in V. cholerae strains expressing the indicated proteins from a Ptac promoter on a plasmid. Proteins were induced with 0.1 mM IPTG (gray bars). The mean and standard deviation are indicated (* = p < 0.05, n = 3). In this and all subsequent figures the strain designations are pCMW75 (QrgB) (an active DGC from V. harveyi), pVCA0965 (WT VCA0965 expression plasmid), pVCA0965-I (I-site mutant), pVCA0965-A (A-site mutant), and pVCA0965-AI (I-site/A-site double mutant).
Mentions: We hypothesized that VCA0965 was incapable of synthesizing c-di-GMP and functioned as a c-di-GMP receptor protein to repress motility. Consistent with previously studied DGC effectors, c-di-GMP could bind VCA0965 at its degenerate active site (referred to as the A-site) and/or its RXXD allosteric inhibition site (referred to as the I-site) [10,20,21]. To determine if the A-site and/or I-site were necessary for motility repression, we mutated the VCA0965 I-site from RXXD to AXXA (VCA0965-I) and the VCA0965 A-site from AGDEF to AGDAF (VCA0965-A). We also constructed an allele of VCA0965 which contained both the A-site and the I-site mutations (VCA0965-AI). These mutant VCA0965 alleles were ectopically expressed from a plasmid in V. cholerae and then tested for the ability to repress swimming motility (Figure 2). Confirming our previous findings, ectopic expression of WT VCA0965 repressed motility 3-fold in a motility assay, as did the positive control QrgB, a DGC from the related species Vibrio harveyi that has been shown to be active in V. cholerae[34] (Figure 2). The I-site mutant more robustly repressed motility than WT VCA0965. This result demonstrated that the I-site is not required for motility repression. However, the A-site was necessary for this regulation as expression of both the VCA0965-A and VCA0965-AI proteins failed to inhibit motility (Figure 2).

Bottom Line: This activity was lost when the active site of VCA0965 was mutated to AGDAF, suggesting that VCA0965 synthesizes c-di-GMP.Furthermore, we found that purified VCA0965 was able to synthesize c-di-GMP in vitro.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA. watersc3@msu.edu.

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.

Show MeSH
Related in: MedlinePlus