Dueling Regulatory Properties of a Transcriptional Activator (MtrA) and Repressor (MtrR) That Control Efflux Pump Gene Expression in <named-content content-type="genus-species">Neisseria gonorrhoeae</named-content>

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, th...

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Autores principales:	Yaramah M. Zalucki, Vijaya Dhulipala, William M. Shafer
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2012
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/0343a906424e463596d443af3c521280
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spelling	oai:doaj.org-article:0343a906424e463596d443af3c5212802021-11-15T15:39:11ZDueling Regulatory Properties of a Transcriptional Activator (MtrA) and Repressor (MtrR) That Control Efflux Pump Gene Expression in <named-content content-type="genus-species">Neisseria gonorrhoeae</named-content>10.1128/mBio.00446-122150-7511https://doaj.org/article/0343a906424e463596d443af3c5212802012-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00446-12https://doaj.org/toc/2150-7511ABSTRACT 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. IMPORTANCE Antibiotic resistance in Neisseria gonorrhoeae has been increasing in recent years, such that in 2007 the Centers for Disease Control and Prevention listed N. gonorrhoeae as a “superbug.” One of the major contributors to antibiotic resistance in N. gonorrhoeae is the MtrCDE efflux pump. Until now, most work on the regulation of the genes encoding this efflux pump has been done on the transcriptional repressor, MtrR. This study is the first one to purify and define the DNA-binding ability of the transcriptional activator, MtrA. Understanding how levels of the MtrCDE efflux pump are regulated increases our knowledge of gonococcal biology and how the gonococcus can respond to various stresses, including antimicrobials.Yaramah M. ZaluckiVijaya DhulipalaWilliam M. ShaferAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 3, Iss 6 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Yaramah M. Zalucki Vijaya Dhulipala William M. Shafer Dueling Regulatory Properties of a Transcriptional Activator (MtrA) and Repressor (MtrR) That Control Efflux Pump Gene Expression in <named-content content-type="genus-species">Neisseria gonorrhoeae</named-content>
description	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. IMPORTANCE Antibiotic resistance in Neisseria gonorrhoeae has been increasing in recent years, such that in 2007 the Centers for Disease Control and Prevention listed N. gonorrhoeae as a “superbug.” One of the major contributors to antibiotic resistance in N. gonorrhoeae is the MtrCDE efflux pump. Until now, most work on the regulation of the genes encoding this efflux pump has been done on the transcriptional repressor, MtrR. This study is the first one to purify and define the DNA-binding ability of the transcriptional activator, MtrA. Understanding how levels of the MtrCDE efflux pump are regulated increases our knowledge of gonococcal biology and how the gonococcus can respond to various stresses, including antimicrobials.
format	article
author	Yaramah M. Zalucki Vijaya Dhulipala William M. Shafer
author_facet	Yaramah M. Zalucki Vijaya Dhulipala William M. Shafer
author_sort	Yaramah M. Zalucki
title	Dueling Regulatory Properties of a Transcriptional Activator (MtrA) and Repressor (MtrR) That Control Efflux Pump Gene Expression in <named-content content-type="genus-species">Neisseria gonorrhoeae</named-content>
title_short	Dueling Regulatory Properties of a Transcriptional Activator (MtrA) and Repressor (MtrR) That Control Efflux Pump Gene Expression in <named-content content-type="genus-species">Neisseria gonorrhoeae</named-content>
title_full	Dueling Regulatory Properties of a Transcriptional Activator (MtrA) and Repressor (MtrR) That Control Efflux Pump Gene Expression in <named-content content-type="genus-species">Neisseria gonorrhoeae</named-content>
title_fullStr	Dueling Regulatory Properties of a Transcriptional Activator (MtrA) and Repressor (MtrR) That Control Efflux Pump Gene Expression in <named-content content-type="genus-species">Neisseria gonorrhoeae</named-content>
title_full_unstemmed	Dueling Regulatory Properties of a Transcriptional Activator (MtrA) and Repressor (MtrR) That Control Efflux Pump Gene Expression in <named-content content-type="genus-species">Neisseria gonorrhoeae</named-content>
title_sort	dueling regulatory properties of a transcriptional activator (mtra) and repressor (mtrr) that control efflux pump gene expression in <named-content content-type="genus-species">neisseria gonorrhoeae</named-content>
publisher	American Society for Microbiology
publishDate	2012
url	https://doaj.org/article/0343a906424e463596d443af3c521280
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Dueling Regulatory Properties of a Transcriptional Activator (MtrA) and Repressor (MtrR) That Control Efflux Pump Gene Expression in <named-content content-type="genus-species">Neisseria gonorrhoeae</named-content>

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