Protein Modulator of Multidrug Efflux Gene Expression in Pseudomonas aeruginosa
| Autor: | Natalie C. J. Strynadka, Sebastien Fraud, Charles R. Dean, Angela Pacey, Mark S. Wilke, Rachael Klinoski, Denis M. Daigle, Lily Cao, Keith Poole |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
Models
Molecular Regulation of gene expression Operon Mutant Biological Transport Active Membrane Transport Proteins Repressor Gene Expression Regulation Bacterial Plasma protein binding Biology Microbiology Molecular biology Repressor Proteins Bacterial Proteins Transcription (biology) Drug Resistance Multiple Bacterial Two-Hybrid System Techniques Mutation Protein Interaction Mapping Pseudomonas aeruginosa Gene expression Gene Regulation Efflux Molecular Biology Protein Binding |
| Zdroj: | Journal of Bacteriology. 189:5441-5451 |
| ISSN: | 1098-5530 0021-9193 |
| DOI: | 10.1128/jb.00543-07 |
| Popis: | nalC multidrug-resistant mutants of Pseudomonas aeruginosa show enhanced expression of the mexAB-oprM multidrug efflux system as a direct result of the production of a ca. 6,100-Da protein, PA3719, in these mutants. Using a bacterial two-hybrid system, PA3719 was shown to interact in vivo with MexR, a repressor of mexAB-oprM expression. Isothermal titration calorimetry (ITC) studies confirmed a high-affinity interaction (equilibrium dissociation constant [ K D ], 158.0 ± 18.1 nM) of PA3719 with MexR in vitro. PA3719 binding to and formation of a complex with MexR obviated repressor binding to its operator, which overlaps the efflux operon promoter, suggesting that mexAB-oprM hyperexpression in nalC mutants results from PA3719 modulation of MexR repressor activity. Consistent with this, MexR repression of mexA transcription in an in vitro transcription assay was alleviated by PA3719. Mutations in MexR compromising its interaction with PA3719 in vivo were isolated and shown to be located internally and distributed throughout the protein, suggesting that they impacted PA3719 binding by altering MexR structure or conformation rather than by having residues interacting specifically with PA3719. Four of six mutant MexR proteins studied retained repressor activity even in a nalC strain producing PA3719. Again, this is consistent with a PA3719 interaction with MexR being necessary to obviate MexR repressor activity. The gene encoding PA3719 has thus been renamed armR ( a nti r epressor for M ex R ). A representative “noninteracting” mutant MexR protein, MexR I104F , was purified, and ITC confirmed that it bound PA3719 with reduced affinity (5.4-fold reduced; K D , 853.2 ± 151.1 nM). Consistent with this, MexR I104F repressor activity, as assessed using the in vitro transcription assay, was only weakly compromised by PA3719. Finally, two mutations (L36P and W45A) in ArmR compromising its interaction with MexR have been isolated and mapped to a putative C-terminal α-helix of the protein that alone is sufficient for interaction with MexR. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|