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The VieB auxiliary protein negatively regulates the VieSA signal transduction system in Vibrio cholerae.

Mitchell SL, Ismail AM, Kenrick SA, Camilli A - BMC Microbiol. (2015)

Bottom Line: Here we show that VieB binds to VieS and inhibits its autophosphorylation and phosphotransfer activity thus preventing phosphorylation of VieA.Additionally, we show that phosphorylation of the highly conserved Asp residue in the receiver domain of VieB regulates the inhibitory activity of VieB.Taken together, these data point to an inhibitory role of VieB on the VieSA phosphorelay, allowing for additional control over the signal output.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and the Department of Molecular Biology and Microbiology, Tufts University, School of Medicine, Boston, USA. stephanie.mitchell@tufts.edu.

ABSTRACT

Background: Vibrio cholerae is a facultative pathogen that lives in the aquatic environment and the human host. The ability of V. cholerae to monitor environmental changes as it transitions between these diverse environments is vital to its pathogenic lifestyle. One way V. cholerae senses changing external stimuli is through the three-component signal transduction system, VieSAB, which is encoded by the vieSAB operon. The VieSAB system plays a role in the inverse regulation of biofilm and virulence genes by controlling the concentration of the secondary messenger, cyclic-di-GMP. While the sensor kinase, VieS, and the response regulator, VieA, behave similar to typical two-component phosphorelay systems, the role of the auxiliary protein, VieB, is unclear.

Results: Here we show that VieB binds to VieS and inhibits its autophosphorylation and phosphotransfer activity thus preventing phosphorylation of VieA. Additionally, we show that phosphorylation of the highly conserved Asp residue in the receiver domain of VieB regulates the inhibitory activity of VieB.

Conclusion: Taken together, these data point to an inhibitory role of VieB on the VieSA phosphorelay, allowing for additional control over the signal output. Insight into the function and regulatory mechanism of the VieSAB system improves our understanding of how V. cholerae controls gene expression as it transitions between the aquatic environment and human host.

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VieB is a dose-dependent inhibitor of phosphotransfer. (A) Purified GST-VieS-C was incubated with 32P-ATP-γ either alone (lane 1) or in the presence of equimolar VieA-His6 (lane 2) and 5 μM VieB (lane 3) for 30 minutes at 30°C. (B) GST-VieS-C was incubated with 32P-ATP-γ in the presence of equimolar VieA-His6 and either 0, 0.25, 0.5, 1, 2, 4, or 8 μM VieB for 30 minutes at 30°C. Samples were stopped with the addition of 2X-denaturing sample buffer and separated using a 10% SDS-Page gel. Proteins labeled with 32P were observed by radioautography. Figure panels are from the same experiment and exposed for the same amount of time. The radioautographs shown are a representative of three replicates. The band just below GST-VieS-C is a VieS degradation band, which is present in all figures and should be ignored.
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Fig1: VieB is a dose-dependent inhibitor of phosphotransfer. (A) Purified GST-VieS-C was incubated with 32P-ATP-γ either alone (lane 1) or in the presence of equimolar VieA-His6 (lane 2) and 5 μM VieB (lane 3) for 30 minutes at 30°C. (B) GST-VieS-C was incubated with 32P-ATP-γ in the presence of equimolar VieA-His6 and either 0, 0.25, 0.5, 1, 2, 4, or 8 μM VieB for 30 minutes at 30°C. Samples were stopped with the addition of 2X-denaturing sample buffer and separated using a 10% SDS-Page gel. Proteins labeled with 32P were observed by radioautography. Figure panels are from the same experiment and exposed for the same amount of time. The radioautographs shown are a representative of three replicates. The band just below GST-VieS-C is a VieS degradation band, which is present in all figures and should be ignored.

Mentions: Since many other described auxiliary proteins of TCSs negatively modulate their cognate TCS, we hypothesized that VieB functions as an inhibitor of the VieSA TCS. To test this we examined the effect of VieB on phosphotransfer between GST-VieS-C (GST-tagged cytoplasmic portion of VieS) and VieA-His6. In vitro phosphotransfer between an MBP-VieS-C fusion protein and VieA-His6 was previously demonstrated [13]. Using purified GST-VieS-C, we found that autophosphorylation and phosphotransfer to VieA-His6 behaves as expected (Figure 1A). When a five-fold molar excess of VieB was added to the reaction, there was a complete loss of the production of phosphorylated VieA-His6 (Figure 1A). VieB does not become readily phosphorylated in this assay even though it harbors a Rec domain, suggesting VieB does not act as a phosphate sink or compete with VieA-His6 for phosphorylation. However, our data so far cannot rule out the possibility that VieB may harbor high phosphatase activity, which could result in no observable phosphorylated VieB if it is a phosphatase of itself. Together, these data show that VieB is an inhibitor of phosphotransfer between GST-VieS-C and VieA-His6.Figure 1


The VieB auxiliary protein negatively regulates the VieSA signal transduction system in Vibrio cholerae.

Mitchell SL, Ismail AM, Kenrick SA, Camilli A - BMC Microbiol. (2015)

VieB is a dose-dependent inhibitor of phosphotransfer. (A) Purified GST-VieS-C was incubated with 32P-ATP-γ either alone (lane 1) or in the presence of equimolar VieA-His6 (lane 2) and 5 μM VieB (lane 3) for 30 minutes at 30°C. (B) GST-VieS-C was incubated with 32P-ATP-γ in the presence of equimolar VieA-His6 and either 0, 0.25, 0.5, 1, 2, 4, or 8 μM VieB for 30 minutes at 30°C. Samples were stopped with the addition of 2X-denaturing sample buffer and separated using a 10% SDS-Page gel. Proteins labeled with 32P were observed by radioautography. Figure panels are from the same experiment and exposed for the same amount of time. The radioautographs shown are a representative of three replicates. The band just below GST-VieS-C is a VieS degradation band, which is present in all figures and should be ignored.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4352251&req=5

Fig1: VieB is a dose-dependent inhibitor of phosphotransfer. (A) Purified GST-VieS-C was incubated with 32P-ATP-γ either alone (lane 1) or in the presence of equimolar VieA-His6 (lane 2) and 5 μM VieB (lane 3) for 30 minutes at 30°C. (B) GST-VieS-C was incubated with 32P-ATP-γ in the presence of equimolar VieA-His6 and either 0, 0.25, 0.5, 1, 2, 4, or 8 μM VieB for 30 minutes at 30°C. Samples were stopped with the addition of 2X-denaturing sample buffer and separated using a 10% SDS-Page gel. Proteins labeled with 32P were observed by radioautography. Figure panels are from the same experiment and exposed for the same amount of time. The radioautographs shown are a representative of three replicates. The band just below GST-VieS-C is a VieS degradation band, which is present in all figures and should be ignored.
Mentions: Since many other described auxiliary proteins of TCSs negatively modulate their cognate TCS, we hypothesized that VieB functions as an inhibitor of the VieSA TCS. To test this we examined the effect of VieB on phosphotransfer between GST-VieS-C (GST-tagged cytoplasmic portion of VieS) and VieA-His6. In vitro phosphotransfer between an MBP-VieS-C fusion protein and VieA-His6 was previously demonstrated [13]. Using purified GST-VieS-C, we found that autophosphorylation and phosphotransfer to VieA-His6 behaves as expected (Figure 1A). When a five-fold molar excess of VieB was added to the reaction, there was a complete loss of the production of phosphorylated VieA-His6 (Figure 1A). VieB does not become readily phosphorylated in this assay even though it harbors a Rec domain, suggesting VieB does not act as a phosphate sink or compete with VieA-His6 for phosphorylation. However, our data so far cannot rule out the possibility that VieB may harbor high phosphatase activity, which could result in no observable phosphorylated VieB if it is a phosphatase of itself. Together, these data show that VieB is an inhibitor of phosphotransfer between GST-VieS-C and VieA-His6.Figure 1

Bottom Line: Here we show that VieB binds to VieS and inhibits its autophosphorylation and phosphotransfer activity thus preventing phosphorylation of VieA.Additionally, we show that phosphorylation of the highly conserved Asp residue in the receiver domain of VieB regulates the inhibitory activity of VieB.Taken together, these data point to an inhibitory role of VieB on the VieSA phosphorelay, allowing for additional control over the signal output.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and the Department of Molecular Biology and Microbiology, Tufts University, School of Medicine, Boston, USA. stephanie.mitchell@tufts.edu.

ABSTRACT

Background: Vibrio cholerae is a facultative pathogen that lives in the aquatic environment and the human host. The ability of V. cholerae to monitor environmental changes as it transitions between these diverse environments is vital to its pathogenic lifestyle. One way V. cholerae senses changing external stimuli is through the three-component signal transduction system, VieSAB, which is encoded by the vieSAB operon. The VieSAB system plays a role in the inverse regulation of biofilm and virulence genes by controlling the concentration of the secondary messenger, cyclic-di-GMP. While the sensor kinase, VieS, and the response regulator, VieA, behave similar to typical two-component phosphorelay systems, the role of the auxiliary protein, VieB, is unclear.

Results: Here we show that VieB binds to VieS and inhibits its autophosphorylation and phosphotransfer activity thus preventing phosphorylation of VieA. Additionally, we show that phosphorylation of the highly conserved Asp residue in the receiver domain of VieB regulates the inhibitory activity of VieB.

Conclusion: Taken together, these data point to an inhibitory role of VieB on the VieSA phosphorelay, allowing for additional control over the signal output. Insight into the function and regulatory mechanism of the VieSAB system improves our understanding of how V. cholerae controls gene expression as it transitions between the aquatic environment and human host.

Show MeSH
Related in: MedlinePlus