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Vibrio cholerae CsrA Regulates ToxR Levels in Response to Amino Acids and Is Essential for Virulence.

Mey AR, Butz HA, Payne SM - MBio (2015)

Bottom Line: Conversely, specific amino acid substitutions in CsrA were associated with defects in ToxR production in response to NRES.Unlike previously described effects of CsrA on virulence gene regulation, the effects of CsrA on ToxR were not mediated through quorum sensing and HapR.By linking environmental sensing to the ToxR regulon, CsrA effectively acts as a switch that controls pathogenesis in response to specific signals.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, USA armey@austin.utexas.edu.

No MeSH data available.


Related in: MedlinePlus

(A) The NvarAL mutant did not increase OmpU or ToxR levels in response to NRES. This phenotype could not be complemented with pVarA, suggesting a suppressor mutation. Strains N16961 (WT), NvarAL, NvarAL/pWKS30 (vector control), and NvarAL/pVarA (complemented strain) were grown overnight in LB broth and then subcultured 1:100 into T medium with or without 12.5 mM NRES mix. Cells were harvested in the mid-log phase, and whole-cell preparations were resolved by SDS-PAGE (10%) and stained by Coomassie blue (top panel) or immunoblotted using polyclonal anti-ToxR antisera (bottom panel). (B) The NvarAL strain did not have an inherent defect in OmpU production, since OmpU was made exclusively in response to the bile acid cholate. Strains N16961 (WT) and NvarAL were grown overnight in LB broth and then subcultured 1:100 into T medium with or without 0.1% cholate. Cells were harvested in the mid-log phase, and whole-cell preparations were resolved by SDS-PAGE (10%) and stained with Coomassie blue to visualize the OMPs.
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fig3: (A) The NvarAL mutant did not increase OmpU or ToxR levels in response to NRES. This phenotype could not be complemented with pVarA, suggesting a suppressor mutation. Strains N16961 (WT), NvarAL, NvarAL/pWKS30 (vector control), and NvarAL/pVarA (complemented strain) were grown overnight in LB broth and then subcultured 1:100 into T medium with or without 12.5 mM NRES mix. Cells were harvested in the mid-log phase, and whole-cell preparations were resolved by SDS-PAGE (10%) and stained by Coomassie blue (top panel) or immunoblotted using polyclonal anti-ToxR antisera (bottom panel). (B) The NvarAL strain did not have an inherent defect in OmpU production, since OmpU was made exclusively in response to the bile acid cholate. Strains N16961 (WT) and NvarAL were grown overnight in LB broth and then subcultured 1:100 into T medium with or without 0.1% cholate. Cells were harvested in the mid-log phase, and whole-cell preparations were resolved by SDS-PAGE (10%) and stained with Coomassie blue to visualize the OMPs.

Mentions: In contrast to NvarAS, the large-colony NvarA strain (NvarAL) had a normal growth phenotype compared with its wild-type parental strain (see Fig. S1 in the supplemental material) but did not produce any OmpU in response to NRES when grown in a minimal medium (Fig. 3A). This was not due to an inherent inability to synthesize OmpU, since NvarAL made OmpU exclusively when grown in medium containing the bile salt cholate (Fig. 3B), a condition known to promote the production of OmpU and repress the synthesis of OmpT (8). These data suggest that NvarAL specifically cannot respond to NRES. The switch from ompT to ompU expression in response to NRES requires an increase in the level of ToxR, unlike the response to bile acids, in which the ToxR level is unchanged (8). To determine whether the inability to produce OmpU in the NvarAL strain is due to failure to upregulate the production of ToxR in response to the NRES mix, ToxR levels were assessed. The results show that ToxR levels did not increase in NvarAL grown with NRES (Fig. 3A), suggesting that the level of ToxR is insufficient to promote a switch in omp gene expression from ompT to ompU under these conditions.


Vibrio cholerae CsrA Regulates ToxR Levels in Response to Amino Acids and Is Essential for Virulence.

Mey AR, Butz HA, Payne SM - MBio (2015)

(A) The NvarAL mutant did not increase OmpU or ToxR levels in response to NRES. This phenotype could not be complemented with pVarA, suggesting a suppressor mutation. Strains N16961 (WT), NvarAL, NvarAL/pWKS30 (vector control), and NvarAL/pVarA (complemented strain) were grown overnight in LB broth and then subcultured 1:100 into T medium with or without 12.5 mM NRES mix. Cells were harvested in the mid-log phase, and whole-cell preparations were resolved by SDS-PAGE (10%) and stained by Coomassie blue (top panel) or immunoblotted using polyclonal anti-ToxR antisera (bottom panel). (B) The NvarAL strain did not have an inherent defect in OmpU production, since OmpU was made exclusively in response to the bile acid cholate. Strains N16961 (WT) and NvarAL were grown overnight in LB broth and then subcultured 1:100 into T medium with or without 0.1% cholate. Cells were harvested in the mid-log phase, and whole-cell preparations were resolved by SDS-PAGE (10%) and stained with Coomassie blue to visualize the OMPs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4526715&req=5

fig3: (A) The NvarAL mutant did not increase OmpU or ToxR levels in response to NRES. This phenotype could not be complemented with pVarA, suggesting a suppressor mutation. Strains N16961 (WT), NvarAL, NvarAL/pWKS30 (vector control), and NvarAL/pVarA (complemented strain) were grown overnight in LB broth and then subcultured 1:100 into T medium with or without 12.5 mM NRES mix. Cells were harvested in the mid-log phase, and whole-cell preparations were resolved by SDS-PAGE (10%) and stained by Coomassie blue (top panel) or immunoblotted using polyclonal anti-ToxR antisera (bottom panel). (B) The NvarAL strain did not have an inherent defect in OmpU production, since OmpU was made exclusively in response to the bile acid cholate. Strains N16961 (WT) and NvarAL were grown overnight in LB broth and then subcultured 1:100 into T medium with or without 0.1% cholate. Cells were harvested in the mid-log phase, and whole-cell preparations were resolved by SDS-PAGE (10%) and stained with Coomassie blue to visualize the OMPs.
Mentions: In contrast to NvarAS, the large-colony NvarA strain (NvarAL) had a normal growth phenotype compared with its wild-type parental strain (see Fig. S1 in the supplemental material) but did not produce any OmpU in response to NRES when grown in a minimal medium (Fig. 3A). This was not due to an inherent inability to synthesize OmpU, since NvarAL made OmpU exclusively when grown in medium containing the bile salt cholate (Fig. 3B), a condition known to promote the production of OmpU and repress the synthesis of OmpT (8). These data suggest that NvarAL specifically cannot respond to NRES. The switch from ompT to ompU expression in response to NRES requires an increase in the level of ToxR, unlike the response to bile acids, in which the ToxR level is unchanged (8). To determine whether the inability to produce OmpU in the NvarAL strain is due to failure to upregulate the production of ToxR in response to the NRES mix, ToxR levels were assessed. The results show that ToxR levels did not increase in NvarAL grown with NRES (Fig. 3A), suggesting that the level of ToxR is insufficient to promote a switch in omp gene expression from ompT to ompU under these conditions.

Bottom Line: Conversely, specific amino acid substitutions in CsrA were associated with defects in ToxR production in response to NRES.Unlike previously described effects of CsrA on virulence gene regulation, the effects of CsrA on ToxR were not mediated through quorum sensing and HapR.By linking environmental sensing to the ToxR regulon, CsrA effectively acts as a switch that controls pathogenesis in response to specific signals.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, USA armey@austin.utexas.edu.

No MeSH data available.


Related in: MedlinePlus