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Mutations within the mepA operator affect binding of the MepR regulatory protein and its induction by MepA substrates in Staphylococcus aureus.

Publication ,  Journal Article
Schindler, BD; Seo, SM; Birukou, I; Brennan, RG; Kaatz, GW
Published in: J Bacteriol
March 2015

The expression of mepA, encoding the Staphylococcus aureus MepA multidrug efflux protein, is repressed by the MarR homologue MepR. Repression occurs through binding of two MepR dimers to an operator with two homologous and closely approximated pseudopalindromic binding sites (site 1 [S1] and site 2 [S2]). MepR binding is impeded in the presence of pentamidine, a MepA substrate. The effects of various mepA operator mutations on MepR binding were determined using electrophoretic mobility shift assays and isothermal titration calorimetry, and an in vivo confirmation of the effects observed was established for a fully palindromic operator mutant. Altering the S1-S2 spacing by 1 to 4 bp severely impaired S2 binding, likely due to a physical collision between adjacent MepR dimers. Extension of the spacing to 9 bp eliminated the S1 binding-mediated DNA allostery required for efficient S2 binding, consistent with positive cooperative binding of MepR dimers. Binding of a single dimer to S1 was maintained when S2 was disrupted, whereas disruption of S1 eliminated any significant binding to S2, also consistent with positive cooperativity. Palindromization of binding sites, especially S2, enhanced MepR affinity for the mepA operator and reduced MepA substrate-mediated MepR induction. As a result, the on-off equilibrium between MepR and its binding sites was shifted toward the on state, resulting in less free MepR being available for interaction with inducing ligand. The selective pressure(s) under which mepA expression is advantageous likely contributed to the accumulation of mutations in the mepA operator, resulting in the current sequence from which MepR is readily induced by MepA substrates.

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Published In

J Bacteriol

DOI

EISSN

1098-5530

Publication Date

March 2015

Volume

197

Issue

6

Start / End Page

1104 / 1114

Location

United States

Related Subject Headings

  • Staphylococcus aureus
  • Protein Conformation
  • Protein Binding
  • Nucleic Acid Conformation
  • Mutation
  • Models, Molecular
  • Microbiology
  • Inverted Repeat Sequences
  • Gene Expression Regulation, Enzymologic
  • Gene Expression Regulation, Bacterial
 

Citation

APA
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Schindler, B. D., Seo, S. M., Birukou, I., Brennan, R. G., & Kaatz, G. W. (2015). Mutations within the mepA operator affect binding of the MepR regulatory protein and its induction by MepA substrates in Staphylococcus aureus. J Bacteriol, 197(6), 1104–1114. https://doi.org/10.1128/JB.02558-14
Schindler, Bryan D., Susan M. Seo, Ivan Birukou, Richard G. Brennan, and Glenn W. Kaatz. “Mutations within the mepA operator affect binding of the MepR regulatory protein and its induction by MepA substrates in Staphylococcus aureus.J Bacteriol 197, no. 6 (March 2015): 1104–14. https://doi.org/10.1128/JB.02558-14.
Schindler, Bryan D., et al. “Mutations within the mepA operator affect binding of the MepR regulatory protein and its induction by MepA substrates in Staphylococcus aureus.J Bacteriol, vol. 197, no. 6, Mar. 2015, pp. 1104–14. Pubmed, doi:10.1128/JB.02558-14.
Schindler BD, Seo SM, Birukou I, Brennan RG, Kaatz GW. Mutations within the mepA operator affect binding of the MepR regulatory protein and its induction by MepA substrates in Staphylococcus aureus. J Bacteriol. 2015 Mar;197(6):1104–1114.

Published In

J Bacteriol

DOI

EISSN

1098-5530

Publication Date

March 2015

Volume

197

Issue

6

Start / End Page

1104 / 1114

Location

United States

Related Subject Headings

  • Staphylococcus aureus
  • Protein Conformation
  • Protein Binding
  • Nucleic Acid Conformation
  • Mutation
  • Models, Molecular
  • Microbiology
  • Inverted Repeat Sequences
  • Gene Expression Regulation, Enzymologic
  • Gene Expression Regulation, Bacterial