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Author Notes:

Address correspondence to William M. Shafer, wshafer@emory.edu.

Editor Karen Bush, Indiana University Bloomington

We thank Elizabeth Ohneck and Lane Pucko for their reading and editing of the manuscript.

Subject:

Research Funding:

This work was supported by NIH grants AI021150-28; to W.M.S. and U19 AI031496 (P. F. Sparling, University of North Carolina-Chapel Hill) and a Merit Award from the Medical Research Service of the Department of Veterans Affairs (to W.M.S.).

NHMRC Biomedical Training Fellowship 569913 supported Y.M.Z.

W.M.S. is the recipient of a Senior Research Career Scholarship Award from the VA Medical Research Service.

Dueling Regulatory Properties of a Transcriptional Activator (MtrA) and Repressor (MtrR) That Control Efflux Pump Gene Expression in Neisseria gonorrhoeae

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Journal Title:

mBio

Volume:

Volume 3, Number 6

Publisher:

, Pages 1-7

Type of Work:

Article | Final Publisher PDF

Abstract:

MtrA is a member of the AraC family of transcriptional regulators and has been shown to play an important role in enhancing transcription of the mtrCDE operon, which encodes a tripartite multidrug efflux pump, when gonococci are exposed to a sublethal level of antimicrobials. Heretofore, the DNA-binding properties of MtrA were unknown. In order to understand how MtrA activates mtrCDE expression, we successfully purified MtrA and found that it could bind specifically to the mtrCDE promoter region. The affinity of MtrA for the mtrCDE promoter increased 2-fold in the presence of a known effector and substrate of the MtrCDE pump, the nonionic detergent Triton X-100 (TX-100). When placed in competition with MtrR, the transcriptional repressor of mtrCDE, MtrA was found to bind with apparent lower affinity than MtrR to the same region. However, preincubation of MtrA with TX-100 prior to addition of the promoter-containing DNA probe increased MtrA binding and greatly reduced its dissociation from the promoter upon addition of MtrR. Two independent approaches (DNase I footprinting and a screen for bases important in MtrA binding) defined the MtrA-binding site 20–30 bp upstream of the known MtrR-binding site. Collectively, these results suggest that the MtrA and MtrR-binding sites are sterically close and that addition of an effector increases the affinity of MtrA for the mtrCDE promoter such that MtrR binding is negatively impacted. Our results provide a mechanism for transcriptional activation of mtrCDE by MtrA and highlight the complexity of transcriptional control of drug efflux systems possessed by gonococci.

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© 2012 Zalucki et al.

This is an Open Access work distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License (http://creativecommons.org/licenses/by-nc-sa/3.0/).

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