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Research Article | Spotlight

Contact with the CsrA Core Is Required for Allosteric Inhibition by FliW in Bacillus subtilis

Reid T. Oshiro, Caroline M. Dunn, Daniel B. Kearns
Conrad W. Mullineaux, Editor
Reid T. Oshiro
aDepartment of Biology, Indiana University, Bloomington, Indiana, USA
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Caroline M. Dunn
aDepartment of Biology, Indiana University, Bloomington, Indiana, USA
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Daniel B. Kearns
aDepartment of Biology, Indiana University, Bloomington, Indiana, USA
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Conrad W. Mullineaux
Queen Mary University of London
Roles: Editor
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DOI: 10.1128/JB.00574-20
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ABSTRACT

The RNA-binding protein CsrA is a posttranscriptional regulator encoded by genomes throughout the bacterial phylogeny. In the gammaproteobacteria, the activity of CsrA is inhibited by small RNAs that competitively sequester CsrA binding. In contrast, the firmicute Bacillus subtilis encodes a protein inhibitor of CsrA called FliW, which noncompetitively inhibits CsrA activity but for which the precise mechanism of antagonism is unclear. Here, we take an unbiased genetic approach to identify residues of FliW important for CsrA inhibition and these residues fall into two distinct spatial and functional classes. Most loss-of-function alleles mutated FliW residues surrounding the critical regulatory CsrA residue N55 and abolished interaction between the two proteins. Two loss-of-function alleles, however, mutated FliW residues near the CsrA core dimerization domain and maintained interaction with CsrA. One of the FliW alleles reversed a residue charge to disrupt a salt bridge with the CsrA core, and a compensatory charge reversal in the CsrA partner residue restored both the salt bridge and antagonism. We propose a model in which the initial interaction between FliW and CsrA is necessary but not sufficient for antagonism, and for which salt bridge formation with, and deformation of, the CsrA core domain is likely required to allosterically abolish RNA-binding activity.

IMPORTANCE CsrA is a small dimeric protein that binds RNA and is one of the few known examples of transcript-specific protein regulators of translation in bacteria. A protein called FliW binds to and antagonizes CsrA to govern flagellin homeostasis and flagellar assembly. Despite having a high-resolution three-dimensional structure of the FliW-CsrA complex, the mechanism of noncompetitive inhibition remains unresolved. Here, we identify FliW residues required for antagonism and we find that the residues make a linear connection in the complex from initial binding interaction with CsrA to a critical salt bridge near the core of the CsrA dimer. We propose that the salt bridge represents an allosteric contact that distorts the CsrA core to prevent RNA binding.

FOOTNOTES

    • Received 15 October 2020.
    • Accepted 20 October 2020.
    • Accepted manuscript posted online 26 October 2020.
  • Supplemental material is available online only.

  • Copyright © 2020 American Society for Microbiology.

All Rights Reserved.

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Contact with the CsrA Core Is Required for Allosteric Inhibition by FliW in Bacillus subtilis
Reid T. Oshiro, Caroline M. Dunn, Daniel B. Kearns
Journal of Bacteriology Dec 2020, 203 (2) e00574-20; DOI: 10.1128/JB.00574-20

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Contact with the CsrA Core Is Required for Allosteric Inhibition by FliW in Bacillus subtilis
Reid T. Oshiro, Caroline M. Dunn, Daniel B. Kearns
Journal of Bacteriology Dec 2020, 203 (2) e00574-20; DOI: 10.1128/JB.00574-20
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KEYWORDS

FliW
CsrA
RsmA
motility
flagella

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