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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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RamA binds to the upstream region of acrA and tolC. A, β-galactosidase activity measured with acrAB-lac (NKS505) harboring a plasmid expressing ramA, truncated ramA, or the vector control (pMALc2X). The data correspond to mean values from three independent experiments. Error bars correspond to the standard deviation. Student's t test; *, p < 0.01 versus control. B and C, EMSA images for RamA binding to the upstream regions of acrA (B) and tolC (C). Upstream regions of acrA (pAcrA1, -795 to +16 region relative to the start codon of acrA; pAcrA2, -141 to +16) (A) and tolC (-250 to -1 region relative to the start codon of tolC)(C) were incubated with various concentrations of RamA or N-terminal truncated RamA. Protein concentrations are as follows: -, without protein; +, 1.0 μm; ++, 1.5 μm; +++, 2.0 μm.
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fig6: RamA binds to the upstream region of acrA and tolC. A, β-galactosidase activity measured with acrAB-lac (NKS505) harboring a plasmid expressing ramA, truncated ramA, or the vector control (pMALc2X). The data correspond to mean values from three independent experiments. Error bars correspond to the standard deviation. Student's t test; *, p < 0.01 versus control. B and C, EMSA images for RamA binding to the upstream regions of acrA (B) and tolC (C). Upstream regions of acrA (pAcrA1, -795 to +16 region relative to the start codon of acrA; pAcrA2, -141 to +16) (A) and tolC (-250 to -1 region relative to the start codon of tolC)(C) were incubated with various concentrations of RamA or N-terminal truncated RamA. Protein concentrations are as follows: -, without protein; +, 1.0 μm; ++, 1.5 μm; +++, 2.0 μm.

Mentions: RamA Binds to the Upstream Regions of acrA and tolC—The aforementioned results indicate that RamA plays a major role in inducing acrAB in response to environmental signals such as indole and bile. To understand the regulation of acrAB by RamA, electrophoretic mobility shift assays (EMSA) with the RamA protein were performed. Plasmids encoding the histidine-tagged RamA or the N terminus truncated RamA proteins were constructed (Table 2). Because it was reported that RamA overproduction was related to increased AcrAB expression in clinical Klebsiella pneumoniae isolates (49), we investigated the effect of histidine-tagged RamA on acrAB expression. Overproduction of histidine-tagged RamA significantly induced the expression of acrAB (Fig. 6A); however, the truncated RamA did not induce acrAB (Fig. 6A) and was used as a negative control in subsequent EMSA. Upstream regions of acrA were amplified by PCR, and the fragments were incubated with RamA or truncated RamA protein. RamA bound to pAcrA1, whereas truncated RamA did not (Fig. 6B). However, RamA did not bind to pAcrA2, indicating that the RamA-binding site is located between -795 and -142 upstream of acrA (Fig. 6B). RamA did bind to the upstream region of tolC, whereas the truncated RamA did not (Fig. 6C). These results indicate that RamA directly controls the expression of acrAB and tolC.


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)

RamA binds to the upstream region of acrA and tolC. A, β-galactosidase activity measured with acrAB-lac (NKS505) harboring a plasmid expressing ramA, truncated ramA, or the vector control (pMALc2X). The data correspond to mean values from three independent experiments. Error bars correspond to the standard deviation. Student's t test; *, p < 0.01 versus control. B and C, EMSA images for RamA binding to the upstream regions of acrA (B) and tolC (C). Upstream regions of acrA (pAcrA1, -795 to +16 region relative to the start codon of acrA; pAcrA2, -141 to +16) (A) and tolC (-250 to -1 region relative to the start codon of tolC)(C) were incubated with various concentrations of RamA or N-terminal truncated RamA. Protein concentrations are as follows: -, without protein; +, 1.0 μm; ++, 1.5 μm; +++, 2.0 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig6: RamA binds to the upstream region of acrA and tolC. A, β-galactosidase activity measured with acrAB-lac (NKS505) harboring a plasmid expressing ramA, truncated ramA, or the vector control (pMALc2X). The data correspond to mean values from three independent experiments. Error bars correspond to the standard deviation. Student's t test; *, p < 0.01 versus control. B and C, EMSA images for RamA binding to the upstream regions of acrA (B) and tolC (C). Upstream regions of acrA (pAcrA1, -795 to +16 region relative to the start codon of acrA; pAcrA2, -141 to +16) (A) and tolC (-250 to -1 region relative to the start codon of tolC)(C) were incubated with various concentrations of RamA or N-terminal truncated RamA. Protein concentrations are as follows: -, without protein; +, 1.0 μm; ++, 1.5 μm; +++, 2.0 μm.
Mentions: RamA Binds to the Upstream Regions of acrA and tolC—The aforementioned results indicate that RamA plays a major role in inducing acrAB in response to environmental signals such as indole and bile. To understand the regulation of acrAB by RamA, electrophoretic mobility shift assays (EMSA) with the RamA protein were performed. Plasmids encoding the histidine-tagged RamA or the N terminus truncated RamA proteins were constructed (Table 2). Because it was reported that RamA overproduction was related to increased AcrAB expression in clinical Klebsiella pneumoniae isolates (49), we investigated the effect of histidine-tagged RamA on acrAB expression. Overproduction of histidine-tagged RamA significantly induced the expression of acrAB (Fig. 6A); however, the truncated RamA did not induce acrAB (Fig. 6A) and was used as a negative control in subsequent EMSA. Upstream regions of acrA were amplified by PCR, and the fragments were incubated with RamA or truncated RamA protein. RamA bound to pAcrA1, whereas truncated RamA did not (Fig. 6B). However, RamA did not bind to pAcrA2, indicating that the RamA-binding site is located between -795 and -142 upstream of acrA (Fig. 6B). RamA did bind to the upstream region of tolC, whereas the truncated RamA did not (Fig. 6C). These results indicate that RamA directly controls the expression of acrAB and tolC.

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