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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.

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Overexpression of the VCA0965-I mutant induces biofilm formation in an A-site dependent manner. (A) Mean biofilm formation of V. cholerae overexpressing wild type VCA0965 and its mutant derivatives were measured using a MBEC assay. (B) Biofilm formation as measured by flow cytometry was determined by multiplying the mean forward scatter value of the aggregate population by the percentage of aggregates relative to the total events measured. The mean and standard deviation are indicated (* = p < 0.05, n = 3).
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Figure 3: Overexpression of the VCA0965-I mutant induces biofilm formation in an A-site dependent manner. (A) Mean biofilm formation of V. cholerae overexpressing wild type VCA0965 and its mutant derivatives were measured using a MBEC assay. (B) Biofilm formation as measured by flow cytometry was determined by multiplying the mean forward scatter value of the aggregate population by the percentage of aggregates relative to the total events measured. The mean and standard deviation are indicated (* = p < 0.05, n = 3).

Mentions: Like many bacterial species, biofilm formation in V. cholerae is induced by increased intracellular c-di-GMP [9]. As ectopic expression of VCA0965 was able to repress motility, we determined whether ectopic expression of VCA0965 would induce biofilm formation. Biofilm formation was quantified using two approaches. First we measured biofilms with the minimum biofilm eradication concentration (MBEC) biofilm assay. Expression of the positive control DGC, QrgB, induced biofilm formation four fold above the vector control (Figure 3A). Although overexpression of VCA0965 significantly inhibited motility, overexpression of VCA0965 did not affect biofilm formation. This result could be due to high-specificity signaling or differential activity of the DGC in these two experiments (see Discussion). In contrast, overexpression of VCA0965-I stimulated a 3-fold increase in biofilm formation (Figure 3A). Analogous to our results measuring motility, overexpression of VCA0965-A and VCA0965-AI did not induce biofilms. This result indicated that the ability of the I-site mutant to induce biofilm formation was dependent on the presence of an intact active site motif.


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)

Overexpression of the VCA0965-I mutant induces biofilm formation in an A-site dependent manner. (A) Mean biofilm formation of V. cholerae overexpressing wild type VCA0965 and its mutant derivatives were measured using a MBEC assay. (B) Biofilm formation as measured by flow cytometry was determined by multiplying the mean forward scatter value of the aggregate population by the percentage of aggregates relative to the total events measured. The mean and standard deviation are indicated (* = p < 0.05, n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Overexpression of the VCA0965-I mutant induces biofilm formation in an A-site dependent manner. (A) Mean biofilm formation of V. cholerae overexpressing wild type VCA0965 and its mutant derivatives were measured using a MBEC assay. (B) Biofilm formation as measured by flow cytometry was determined by multiplying the mean forward scatter value of the aggregate population by the percentage of aggregates relative to the total events measured. The mean and standard deviation are indicated (* = p < 0.05, n = 3).
Mentions: Like many bacterial species, biofilm formation in V. cholerae is induced by increased intracellular c-di-GMP [9]. As ectopic expression of VCA0965 was able to repress motility, we determined whether ectopic expression of VCA0965 would induce biofilm formation. Biofilm formation was quantified using two approaches. First we measured biofilms with the minimum biofilm eradication concentration (MBEC) biofilm assay. Expression of the positive control DGC, QrgB, induced biofilm formation four fold above the vector control (Figure 3A). Although overexpression of VCA0965 significantly inhibited motility, overexpression of VCA0965 did not affect biofilm formation. This result could be due to high-specificity signaling or differential activity of the DGC in these two experiments (see Discussion). In contrast, overexpression of VCA0965-I stimulated a 3-fold increase in biofilm formation (Figure 3A). Analogous to our results measuring motility, overexpression of VCA0965-A and VCA0965-AI did not induce biofilms. This result indicated that the ability of the I-site mutant to induce biofilm formation was dependent on the presence of an intact active site motif.

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