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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

The NvarAL suppressor strains show abnormal responses to the NRES mix. Strains are labeled according to the csrA suppressor mutation present, but all mutant strains additionally carry the ΔvarA::cam mutation. Strains N16961 (wild type [WT]), NvarAL-00 (SD*), NvarAL-1 (R6H), NvarAL-2 (R6L), NvarAL-3 (T11P), NvarAL-4 (I14T), NvarAL-6 (A36S), and NvarAL-7 (P37L) 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 mid-log phase, and whole-cell preparations were resolved by SDS-PAGE (10%) and stained by Coomassie blue (A) or immunoblotted using polyclonal anti-ToxR antisera (B).
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fig5: The NvarAL suppressor strains show abnormal responses to the NRES mix. Strains are labeled according to the csrA suppressor mutation present, but all mutant strains additionally carry the ΔvarA::cam mutation. Strains N16961 (wild type [WT]), NvarAL-00 (SD*), NvarAL-1 (R6H), NvarAL-2 (R6L), NvarAL-3 (T11P), NvarAL-4 (I14T), NvarAL-6 (A36S), and NvarAL-7 (P37L) 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 mid-log phase, and whole-cell preparations were resolved by SDS-PAGE (10%) and stained by Coomassie blue (A) or immunoblotted using polyclonal anti-ToxR antisera (B).

Mentions: Since loss of VarA is predicted to result in overaccumulation of active CsrA due to low levels of the CsrA-sequestering sRNAs (Fig. 1), we reasoned that suppressor mutations could arise in the csrA gene itself. The csrA gene from the NvarAL strain was sequenced and was found to contain a point mutation that replaces the arginine residue at amino acid position 6 with a histidine (R6H). Several independently derived NvarAL isolates were analyzed to determine whether they also carried suppressor mutations in csrA and whether there was variation in the type of csrA point mutation that could arise in the absence of functional VarA. All of the NvarAL isolates tested carried point mutations affecting csrA. In contrast, none of the small colony phenotype NvarAS strains had mutations in csrA. Most of the NvarAL strain mutations were in the csrA coding region, but several isolates carried mutations in the predicted Shine-Dalgarno (SD) sequence upstream of the csrA translational start (Fig. 4). All coding region mutations resulted in a single amino acid substitution in CsrA (Fig. 4). The OMP profiles of several NvarAL suppressor mutants were analyzed. Many of these mutants did not produce OmpU in response to the NRES mix (Fig. 5A). Rather, they produced OmpT at high levels in minimal medium both with and without NRES supplementation. Thus, these mutants were not stimulated to switch OMPs in the presence of NRES. The amino acid substitutions in this group of mutants generally clustered in the N-terminal half of CsrA (R6H, R6L, T11P, I14T, and T19P) (Fig. 4). Another class of point mutants produced both OmpT and OmpU in T medium alone, and this pattern did not change with the addition of NRES, suggesting defects in OMP regulation in response to environmental cues (Fig. 5A). Several mutants of this class had amino acid substitutions localizing to a region within the C-terminal half of the CsrA protein (A36S and P37L) (Fig. 4). Some of the suppressor strains had mutations in the putative SD sequence (Fig. 4). These mutants were phenotypically similar to the class of mutants exhibiting no OmpU production in response to NRES (Fig. 5A; also data not shown).


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

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

The NvarAL suppressor strains show abnormal responses to the NRES mix. Strains are labeled according to the csrA suppressor mutation present, but all mutant strains additionally carry the ΔvarA::cam mutation. Strains N16961 (wild type [WT]), NvarAL-00 (SD*), NvarAL-1 (R6H), NvarAL-2 (R6L), NvarAL-3 (T11P), NvarAL-4 (I14T), NvarAL-6 (A36S), and NvarAL-7 (P37L) 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 mid-log phase, and whole-cell preparations were resolved by SDS-PAGE (10%) and stained by Coomassie blue (A) or immunoblotted using polyclonal anti-ToxR antisera (B).
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Related In: Results  -  Collection

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fig5: The NvarAL suppressor strains show abnormal responses to the NRES mix. Strains are labeled according to the csrA suppressor mutation present, but all mutant strains additionally carry the ΔvarA::cam mutation. Strains N16961 (wild type [WT]), NvarAL-00 (SD*), NvarAL-1 (R6H), NvarAL-2 (R6L), NvarAL-3 (T11P), NvarAL-4 (I14T), NvarAL-6 (A36S), and NvarAL-7 (P37L) 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 mid-log phase, and whole-cell preparations were resolved by SDS-PAGE (10%) and stained by Coomassie blue (A) or immunoblotted using polyclonal anti-ToxR antisera (B).
Mentions: Since loss of VarA is predicted to result in overaccumulation of active CsrA due to low levels of the CsrA-sequestering sRNAs (Fig. 1), we reasoned that suppressor mutations could arise in the csrA gene itself. The csrA gene from the NvarAL strain was sequenced and was found to contain a point mutation that replaces the arginine residue at amino acid position 6 with a histidine (R6H). Several independently derived NvarAL isolates were analyzed to determine whether they also carried suppressor mutations in csrA and whether there was variation in the type of csrA point mutation that could arise in the absence of functional VarA. All of the NvarAL isolates tested carried point mutations affecting csrA. In contrast, none of the small colony phenotype NvarAS strains had mutations in csrA. Most of the NvarAL strain mutations were in the csrA coding region, but several isolates carried mutations in the predicted Shine-Dalgarno (SD) sequence upstream of the csrA translational start (Fig. 4). All coding region mutations resulted in a single amino acid substitution in CsrA (Fig. 4). The OMP profiles of several NvarAL suppressor mutants were analyzed. Many of these mutants did not produce OmpU in response to the NRES mix (Fig. 5A). Rather, they produced OmpT at high levels in minimal medium both with and without NRES supplementation. Thus, these mutants were not stimulated to switch OMPs in the presence of NRES. The amino acid substitutions in this group of mutants generally clustered in the N-terminal half of CsrA (R6H, R6L, T11P, I14T, and T19P) (Fig. 4). Another class of point mutants produced both OmpT and OmpU in T medium alone, and this pattern did not change with the addition of NRES, suggesting defects in OMP regulation in response to environmental cues (Fig. 5A). Several mutants of this class had amino acid substitutions localizing to a region within the C-terminal half of the CsrA protein (A36S and P37L) (Fig. 4). Some of the suppressor strains had mutations in the putative SD sequence (Fig. 4). These mutants were phenotypically similar to the class of mutants exhibiting no OmpU production in response to NRES (Fig. 5A; also data not shown).

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