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AcrAB multidrug efflux pump regulation in Salmonella enterica serovar Typhimurium by RamA in response to environmental signals.

Nikaido E, Yamaguchi A, Nishino K - J. Biol. Chem. (2008)

Bottom Line: Among these pumps, AcrAB is effective in generating drug resistance and has wide substrate specificity.Other regulators of acrAB such as MarA, SoxS, Rob, SdiA, and AcrR did not contribute to acrAB induction by indole in Salmonella.Our results suggest that RamA controls the Salmonella AcrAB-TolC multidrug efflux system through dual regulatory modes in response to environmental signals.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan.

ABSTRACT
Salmonella enterica serovar Typhimurium has at least nine multidrug efflux pumps. Among these pumps, AcrAB is effective in generating drug resistance and has wide substrate specificity. Here we report that indole, bile, and an Escherichia coli conditioned medium induced the AcrAB pump in Salmonella through a specific regulator, RamA. The RamA-binding sites were located in the upstream regions of acrAB and tolC. RamA was required for indole induction of acrAB. Other regulators of acrAB such as MarA, SoxS, Rob, SdiA, and AcrR did not contribute to acrAB induction by indole in Salmonella. Indole activated ramA transcription, and overproduction of RamA caused increased acrAB expression. In contrast, induction of ramA was not required for induction of acrAB by bile. Cholic acid binds to RamA, and we suggest that bile acts by altering pre-existing RamA. This points to two different AcrAB regulatory modes through RamA. Our results suggest that RamA controls the Salmonella AcrAB-TolC multidrug efflux system through dual regulatory modes in response to environmental signals.

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Indole induction of multidrug efflux genes and drug tolerance of Salmonella enterica serovar Typhimurium. The data correspond to mean values from three independent experiments. Bars correspond to the standard deviation. Asterisks indicate statistically significant differences (*, p < 0.01) in the paired Student's t test. A, differences in β-galactosidase activity in tolC-lac (EG15109), acrAB-lac (NKS505), acrEF-lac (EG15114), acrD-lac (EG15120), mdtABC-lac (EG15124), mdsABC-lac (NKS517), emrAB-lac (NKS522), mdfA-lac (NKS524), mdtK-lac (EG15132), and macAB-lac (NKS530) strains grown in LB medium with (solid bars) or without (open bars) 2 mm indole. B, drug tolerance of S. enterica serovar Typhimurium induced by indole. WT strain (ATCC14028s) was incubated with or without 2 mm indole. Cells were then challenged to benzalkonium (500 mg/ml) for 10 min. The survival levels of the indole-treated cells were normalized to 100%, and untreated cells are displayed relative to those of the indole-treated cells. The actual survival of indole-treated cells was 0.025%.
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fig1: Indole induction of multidrug efflux genes and drug tolerance of Salmonella enterica serovar Typhimurium. The data correspond to mean values from three independent experiments. Bars correspond to the standard deviation. Asterisks indicate statistically significant differences (*, p < 0.01) in the paired Student's t test. A, differences in β-galactosidase activity in tolC-lac (EG15109), acrAB-lac (NKS505), acrEF-lac (EG15114), acrD-lac (EG15120), mdtABC-lac (EG15124), mdsABC-lac (NKS517), emrAB-lac (NKS522), mdfA-lac (NKS524), mdtK-lac (EG15132), and macAB-lac (NKS530) strains grown in LB medium with (solid bars) or without (open bars) 2 mm indole. B, drug tolerance of S. enterica serovar Typhimurium induced by indole. WT strain (ATCC14028s) was incubated with or without 2 mm indole. Cells were then challenged to benzalkonium (500 mg/ml) for 10 min. The survival levels of the indole-treated cells were normalized to 100%, and untreated cells are displayed relative to those of the indole-treated cells. The actual survival of indole-treated cells was 0.025%.

Mentions: Indole Induces Four Multidrug Efflux Pumps and Drug Tolerance of Salmonella—In E. coli, indole is produced from tryptophan by tryptophanase and is excreted from the cell (42). However, Salmonella does not produce indole because it lacks the tnaA gene encoding tryptophanase (43). Indole has also been reported to auto-regulate multidrug efflux genes in E. coli (35). We postulated that Salmonella multidrug efflux genes may respond to indole. To investigate the effect of indole on the expression of multidrug efflux pumps, Salmonella strains, in which the efflux genes were replaced with a reporter gene (lacZ), were inoculated into cultures, with or without indole. Expression levels of drug efflux pumps were measured by a β-galactosidase reporter assay. Indole significantly induced expression of the acrAB, emrAB, acrD, and mdtABC efflux genes in Salmonella (Fig. 1A). A survival assay using benzalkonium showed that indole enhanced drug tolerance of Salmonella (Fig. 1B).


AcrAB multidrug efflux pump regulation in Salmonella enterica serovar Typhimurium by RamA in response to environmental signals.

Nikaido E, Yamaguchi A, Nishino K - J. Biol. Chem. (2008)

Indole induction of multidrug efflux genes and drug tolerance of Salmonella enterica serovar Typhimurium. The data correspond to mean values from three independent experiments. Bars correspond to the standard deviation. Asterisks indicate statistically significant differences (*, p < 0.01) in the paired Student's t test. A, differences in β-galactosidase activity in tolC-lac (EG15109), acrAB-lac (NKS505), acrEF-lac (EG15114), acrD-lac (EG15120), mdtABC-lac (EG15124), mdsABC-lac (NKS517), emrAB-lac (NKS522), mdfA-lac (NKS524), mdtK-lac (EG15132), and macAB-lac (NKS530) strains grown in LB medium with (solid bars) or without (open bars) 2 mm indole. B, drug tolerance of S. enterica serovar Typhimurium induced by indole. WT strain (ATCC14028s) was incubated with or without 2 mm indole. Cells were then challenged to benzalkonium (500 mg/ml) for 10 min. The survival levels of the indole-treated cells were normalized to 100%, and untreated cells are displayed relative to those of the indole-treated cells. The actual survival of indole-treated cells was 0.025%.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2527123&req=5

fig1: Indole induction of multidrug efflux genes and drug tolerance of Salmonella enterica serovar Typhimurium. The data correspond to mean values from three independent experiments. Bars correspond to the standard deviation. Asterisks indicate statistically significant differences (*, p < 0.01) in the paired Student's t test. A, differences in β-galactosidase activity in tolC-lac (EG15109), acrAB-lac (NKS505), acrEF-lac (EG15114), acrD-lac (EG15120), mdtABC-lac (EG15124), mdsABC-lac (NKS517), emrAB-lac (NKS522), mdfA-lac (NKS524), mdtK-lac (EG15132), and macAB-lac (NKS530) strains grown in LB medium with (solid bars) or without (open bars) 2 mm indole. B, drug tolerance of S. enterica serovar Typhimurium induced by indole. WT strain (ATCC14028s) was incubated with or without 2 mm indole. Cells were then challenged to benzalkonium (500 mg/ml) for 10 min. The survival levels of the indole-treated cells were normalized to 100%, and untreated cells are displayed relative to those of the indole-treated cells. The actual survival of indole-treated cells was 0.025%.
Mentions: Indole Induces Four Multidrug Efflux Pumps and Drug Tolerance of Salmonella—In E. coli, indole is produced from tryptophan by tryptophanase and is excreted from the cell (42). However, Salmonella does not produce indole because it lacks the tnaA gene encoding tryptophanase (43). Indole has also been reported to auto-regulate multidrug efflux genes in E. coli (35). We postulated that Salmonella multidrug efflux genes may respond to indole. To investigate the effect of indole on the expression of multidrug efflux pumps, Salmonella strains, in which the efflux genes were replaced with a reporter gene (lacZ), were inoculated into cultures, with or without indole. Expression levels of drug efflux pumps were measured by a β-galactosidase reporter assay. Indole significantly induced expression of the acrAB, emrAB, acrD, and mdtABC efflux genes in Salmonella (Fig. 1A). A survival assay using benzalkonium showed that indole enhanced drug tolerance of Salmonella (Fig. 1B).

Bottom Line: Among these pumps, AcrAB is effective in generating drug resistance and has wide substrate specificity.Other regulators of acrAB such as MarA, SoxS, Rob, SdiA, and AcrR did not contribute to acrAB induction by indole in Salmonella.Our results suggest that RamA controls the Salmonella AcrAB-TolC multidrug efflux system through dual regulatory modes in response to environmental signals.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan.

ABSTRACT
Salmonella enterica serovar Typhimurium has at least nine multidrug efflux pumps. Among these pumps, AcrAB is effective in generating drug resistance and has wide substrate specificity. Here we report that indole, bile, and an Escherichia coli conditioned medium induced the AcrAB pump in Salmonella through a specific regulator, RamA. The RamA-binding sites were located in the upstream regions of acrAB and tolC. RamA was required for indole induction of acrAB. Other regulators of acrAB such as MarA, SoxS, Rob, SdiA, and AcrR did not contribute to acrAB induction by indole in Salmonella. Indole activated ramA transcription, and overproduction of RamA caused increased acrAB expression. In contrast, induction of ramA was not required for induction of acrAB by bile. Cholic acid binds to RamA, and we suggest that bile acts by altering pre-existing RamA. This points to two different AcrAB regulatory modes through RamA. Our results suggest that RamA controls the Salmonella AcrAB-TolC multidrug efflux system through dual regulatory modes in response to environmental signals.

Show MeSH
Related in: MedlinePlus