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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 inhibits VieS intra-molecular phosphotransfer. Purified GST-VieS-C was incubated with 32P-ATP-γ in the absence of VieA-His6 and VieB for 30 minutes at 30°C. Excess 32P-ATP-γ was removed from phosphorylated VieS-C constructs by gel filtration. P-VieS-C constructs were then incubated with additional MnCl2 and either buffer, 1 μM VieA-His6 or pre-mixed 1 μM VieA-His6 and 5 μM VieB for 60 minutes. Samples were stopped at indicated time points with the addition of 2X-denaturing sample buffer and separated using a 10% SDS-Page gel. VieS-C constructs labeled with 32P were observed by radioautography. The radioautograph shown is a representative of three replicates.
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Fig3: VieB inhibits VieS intra-molecular phosphotransfer. Purified GST-VieS-C was incubated with 32P-ATP-γ in the absence of VieA-His6 and VieB for 30 minutes at 30°C. Excess 32P-ATP-γ was removed from phosphorylated VieS-C constructs by gel filtration. P-VieS-C constructs were then incubated with additional MnCl2 and either buffer, 1 μM VieA-His6 or pre-mixed 1 μM VieA-His6 and 5 μM VieB for 60 minutes. Samples were stopped at indicated time points with the addition of 2X-denaturing sample buffer and separated using a 10% SDS-Page gel. VieS-C constructs labeled with 32P were observed by radioautography. The radioautograph shown is a representative of three replicates.

Mentions: Since VieB is only able to partially inhibit GST-VieS-C autophosphorylation, yet can completely block phosphotransfer between VieS and VieA, we hypothesize that VieB must also be affecting either the VieS intra-molecular phosphorelay or phosphotransfer from the VieS Hpt domain to VieA. To test this hypothesis, the inhibitory effect of VieB on phosphotransfer between GST-VieS-C and VieA-His6 was observed in the absence of ATP. In order to specifically address phosphotransfer in the absence of autophosphorylation, ATP was removed from the reaction after GST-VieS-C was robustly phosphorylated to prevent further autophosphorylation. As seen in Figure 3, phosphorylated GST-VieS-C was able to transfer phosphate to VieA-His6 in the absence of ATP. However, when VieB was present, the amount of P-VieA-His6 was greatly reduced (Figure 3). This suggests that VieB is able to block phosphotransfer between VieS and VieA-His6. While we hypothesize that VieB may be inhibiting the intra-molecular phosphorelay between the VieS HK and REC domains, the data presented here cannot distinguish if VieB is acting intra- or inter-molecularly.Figure 3


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 inhibits VieS intra-molecular phosphotransfer. Purified GST-VieS-C was incubated with 32P-ATP-γ in the absence of VieA-His6 and VieB for 30 minutes at 30°C. Excess 32P-ATP-γ was removed from phosphorylated VieS-C constructs by gel filtration. P-VieS-C constructs were then incubated with additional MnCl2 and either buffer, 1 μM VieA-His6 or pre-mixed 1 μM VieA-His6 and 5 μM VieB for 60 minutes. Samples were stopped at indicated time points with the addition of 2X-denaturing sample buffer and separated using a 10% SDS-Page gel. VieS-C constructs labeled with 32P were observed by radioautography. The radioautograph shown is a representative of three replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: VieB inhibits VieS intra-molecular phosphotransfer. Purified GST-VieS-C was incubated with 32P-ATP-γ in the absence of VieA-His6 and VieB for 30 minutes at 30°C. Excess 32P-ATP-γ was removed from phosphorylated VieS-C constructs by gel filtration. P-VieS-C constructs were then incubated with additional MnCl2 and either buffer, 1 μM VieA-His6 or pre-mixed 1 μM VieA-His6 and 5 μM VieB for 60 minutes. Samples were stopped at indicated time points with the addition of 2X-denaturing sample buffer and separated using a 10% SDS-Page gel. VieS-C constructs labeled with 32P were observed by radioautography. The radioautograph shown is a representative of three replicates.
Mentions: Since VieB is only able to partially inhibit GST-VieS-C autophosphorylation, yet can completely block phosphotransfer between VieS and VieA, we hypothesize that VieB must also be affecting either the VieS intra-molecular phosphorelay or phosphotransfer from the VieS Hpt domain to VieA. To test this hypothesis, the inhibitory effect of VieB on phosphotransfer between GST-VieS-C and VieA-His6 was observed in the absence of ATP. In order to specifically address phosphotransfer in the absence of autophosphorylation, ATP was removed from the reaction after GST-VieS-C was robustly phosphorylated to prevent further autophosphorylation. As seen in Figure 3, phosphorylated GST-VieS-C was able to transfer phosphate to VieA-His6 in the absence of ATP. However, when VieB was present, the amount of P-VieA-His6 was greatly reduced (Figure 3). This suggests that VieB is able to block phosphotransfer between VieS and VieA-His6. While we hypothesize that VieB may be inhibiting the intra-molecular phosphorelay between the VieS HK and REC domains, the data presented here cannot distinguish if VieB is acting intra- or inter-molecularly.Figure 3

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