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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

An NvarAL suppressor mutant cannot be complemented by supplying wild-type csrA on a plasmid. Expression of wild-type CsrA in the absence of functional VarA is toxic to the cells, and leads to production of OmpU in the absence of NRES. (A) Strains N16961 (WT), NvarAL-3 (ΔvarA::cam csrA.T11P), NvarAL-3/pWKS30, and NvarAL-3/pWCsrA were grown overnight in LB broth and then subcultured 1:100 into fresh LB broth and grown to the stationary phase. (B) Strains N16961 (wild type [WT]), NvarAL-3/pWKS30, and NvarAL-3/pWCsrA 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.
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fig6: An NvarAL suppressor mutant cannot be complemented by supplying wild-type csrA on a plasmid. Expression of wild-type CsrA in the absence of functional VarA is toxic to the cells, and leads to production of OmpU in the absence of NRES. (A) Strains N16961 (WT), NvarAL-3 (ΔvarA::cam csrA.T11P), NvarAL-3/pWKS30, and NvarAL-3/pWCsrA were grown overnight in LB broth and then subcultured 1:100 into fresh LB broth and grown to the stationary phase. (B) Strains N16961 (wild type [WT]), NvarAL-3/pWKS30, and NvarAL-3/pWCsrA 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.

Mentions: Complementation experiments were carried out in order to rescue the csrA defect of the NvarAL mutant strains by supplying the csrA gene on a low-copy-number vector; however, the mutant strains did not tolerate the csrA plasmid well, and most of the strains failed to maintain the plasmid. This is not surprising, since the varA mutation impairs the ability of the strain to control the level of free CsrA, which is likely detrimental for growth, as shown above. Only one of the NvarAL strains, NvarAL-3 (ΔvarA::cam csrA.T11P), maintained the pWCsrA plasmid well enough for analysis. This strain exhibited very poor growth when carrying the pWCsrA plasmid but normal growth when carrying the vector alone (Fig. 6A). The presence of the pWCsrA plasmid did not restore the OMP profile to normal. Rather, OmpU was inappropriately expressed when the mutant strain carrying this plasmid was grown in T medium alone (Fig. 6B), suggesting overproduction of ToxR, even in the absence of an environmental cue. This is likely due to constitutively high levels of free CsrA in the absence of a functional VarA system and suggests that CsrA acts as a positive regulator of ToxR synthesis. The pWCsrA plasmid had no effect on the OMP profile of the wild-type strain grown with or without the NRES supplement (see Fig. S2 in the supplemental material). This is consistent with tight regulation of CsrA levels by the VarS/A-CsrB/C/D system in the wild-type strain to prevent toxic levels of free CsrA.


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

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

An NvarAL suppressor mutant cannot be complemented by supplying wild-type csrA on a plasmid. Expression of wild-type CsrA in the absence of functional VarA is toxic to the cells, and leads to production of OmpU in the absence of NRES. (A) Strains N16961 (WT), NvarAL-3 (ΔvarA::cam csrA.T11P), NvarAL-3/pWKS30, and NvarAL-3/pWCsrA were grown overnight in LB broth and then subcultured 1:100 into fresh LB broth and grown to the stationary phase. (B) Strains N16961 (wild type [WT]), NvarAL-3/pWKS30, and NvarAL-3/pWCsrA 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526715&req=5

fig6: An NvarAL suppressor mutant cannot be complemented by supplying wild-type csrA on a plasmid. Expression of wild-type CsrA in the absence of functional VarA is toxic to the cells, and leads to production of OmpU in the absence of NRES. (A) Strains N16961 (WT), NvarAL-3 (ΔvarA::cam csrA.T11P), NvarAL-3/pWKS30, and NvarAL-3/pWCsrA were grown overnight in LB broth and then subcultured 1:100 into fresh LB broth and grown to the stationary phase. (B) Strains N16961 (wild type [WT]), NvarAL-3/pWKS30, and NvarAL-3/pWCsrA 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.
Mentions: Complementation experiments were carried out in order to rescue the csrA defect of the NvarAL mutant strains by supplying the csrA gene on a low-copy-number vector; however, the mutant strains did not tolerate the csrA plasmid well, and most of the strains failed to maintain the plasmid. This is not surprising, since the varA mutation impairs the ability of the strain to control the level of free CsrA, which is likely detrimental for growth, as shown above. Only one of the NvarAL strains, NvarAL-3 (ΔvarA::cam csrA.T11P), maintained the pWCsrA plasmid well enough for analysis. This strain exhibited very poor growth when carrying the pWCsrA plasmid but normal growth when carrying the vector alone (Fig. 6A). The presence of the pWCsrA plasmid did not restore the OMP profile to normal. Rather, OmpU was inappropriately expressed when the mutant strain carrying this plasmid was grown in T medium alone (Fig. 6B), suggesting overproduction of ToxR, even in the absence of an environmental cue. This is likely due to constitutively high levels of free CsrA in the absence of a functional VarA system and suggests that CsrA acts as a positive regulator of ToxR synthesis. The pWCsrA plasmid had no effect on the OMP profile of the wild-type strain grown with or without the NRES supplement (see Fig. S2 in the supplemental material). This is consistent with tight regulation of CsrA levels by the VarS/A-CsrB/C/D system in the wild-type strain to prevent toxic levels of free CsrA.

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