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Expression of the AcrAB Components of the AcrAB-TolC Multidrug Efflux Pump of Yersinia enterocolitica Is Subject to Dual Regulation by OmpR.

Raczkowska A, Trzos J, Lewandowska O, Nieckarz M, Brzostek K - PLoS ONE (2015)

Bottom Line: The identification of putative OmpR-binding sites and electrophoretic mobility shift assays confirmed that this regulator binds specifically to both promoter regions with different affinity.Examination of the activity of the acrR and acrAB promoters after the exposure of cells to different chemicals showed that bile salts can act as an OmpR-independent inducer.Taken together, our findings suggest that OmpR positively controls the expression of the AcrAB-TolC efflux pump involved in the adaptive response of Y. enterocolitica O:9 to different chemical stressors, thus conferring an advantage in particular ecological niches.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Microbiology, Institute of Microbiology, Faculty of Biology, University of Warsaw, Warsaw, Poland.

ABSTRACT
OmpR is a transcriptional regulator implicated in the control of various cellular processes and functions in Enterobacteriaceae. This study was undertaken to identify genes comprising the OmpR regulon in the human gastrointestinal pathogen Yersinia enterocolitica. Derivatives of an ompR-negative strain with random transposon insertions creating transcriptional fusions with the reporter gene lacZ were isolated. These were supplied with the wild-type ompR allele in trans and then screened for OmpR-dependent changes in β-galactosidase activity. Using this strategy, five insertions in genes/operons positively regulated by OmpR and two insertions in genes negatively regulated by this protein were identified. Genetic analysis of one of these fusion strains revealed that the gene acrR, encoding transcriptional repressor AcrR is negatively regulated by OmpR. Differential analysis of membrane proteins by SDS-PAGE followed by mass spectrometry identified the protein AcrB, a component of the AcrAB-TolC multidrug efflux pump, as being positively regulated by OmpR. Analysis of the activity of the acrR and acrAB promoters using gfp fusions confirmed their OmpR-dependent repression and activation, respectively. The identification of putative OmpR-binding sites and electrophoretic mobility shift assays confirmed that this regulator binds specifically to both promoter regions with different affinity. Examination of the activity of the acrR and acrAB promoters after the exposure of cells to different chemicals showed that bile salts can act as an OmpR-independent inducer. Taken together, our findings suggest that OmpR positively controls the expression of the AcrAB-TolC efflux pump involved in the adaptive response of Y. enterocolitica O:9 to different chemical stressors, thus conferring an advantage in particular ecological niches.

No MeSH data available.


Related in: MedlinePlus

Influence of OmpR activity on the Y. enterocolitica membrane protein profile.Proteins were isolated from strains Ye9 (wild type) and AR4 (ompR mutant) grown overnight in LB medium at 27°C or 37°C. In each case, 50 μg of protein were separated by SDS-PAGE and visualized by Coomassie blue staining. Putative OmpR-regulated proteins subsequently identified by LC-MS/MS are indicated by arrows. The bands were named according to their migration in the 12% polyacrylamide gel relative to the molecular weight standards.
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pone.0124248.g002: Influence of OmpR activity on the Y. enterocolitica membrane protein profile.Proteins were isolated from strains Ye9 (wild type) and AR4 (ompR mutant) grown overnight in LB medium at 27°C or 37°C. In each case, 50 μg of protein were separated by SDS-PAGE and visualized by Coomassie blue staining. Putative OmpR-regulated proteins subsequently identified by LC-MS/MS are indicated by arrows. The bands were named according to their migration in the 12% polyacrylamide gel relative to the molecular weight standards.

Mentions: In parallel with the mutant screen described above, we analyzed membrane proteins of the wild-type Y. enterocolitica Ye9 and strain AR4 (the ompR mutant), grown under different temperature conditions, using SDS-PAGE. Changes in the membrane protein profiles due to the growth temperature and the presence and activity of the OmpR regulator were observed (Fig 2). A differential analysis of band intensities was used to select proteins for identification by LC-MS/MS. Following this procedure, we identified six putative OmpR-controlled proteins that matched Y. enterocolitica proteins present in the NCBI protein databases (Table 2). The synthesis of four proteins was higher in the wild-type strain than in mutant AR4: p120 and p110 (cells grown at 27°C), and p100 and p85 (cells grown at 37°C). Two other proteins, p80 and p60, were present at lower levels in the wild-type strain compared to AR4. The proteins that appeared to be positively controlled by OmpR were AcrB (p120; an essential component of the AcrAB-TolC multidrug efflux pump), ferrichrome-iron receptor (p110), dehydratase (p100; a bifunctional aconitate hydratase 2/2-methylisocitrate dehydratase) and a putative ABC transporter ATP-binding protein (p85). The proteins presumed to be negatively regulated by OmpR were a 60-kDa heat shock protein (p80) and elongation factor Tu (p60).


Expression of the AcrAB Components of the AcrAB-TolC Multidrug Efflux Pump of Yersinia enterocolitica Is Subject to Dual Regulation by OmpR.

Raczkowska A, Trzos J, Lewandowska O, Nieckarz M, Brzostek K - PLoS ONE (2015)

Influence of OmpR activity on the Y. enterocolitica membrane protein profile.Proteins were isolated from strains Ye9 (wild type) and AR4 (ompR mutant) grown overnight in LB medium at 27°C or 37°C. In each case, 50 μg of protein were separated by SDS-PAGE and visualized by Coomassie blue staining. Putative OmpR-regulated proteins subsequently identified by LC-MS/MS are indicated by arrows. The bands were named according to their migration in the 12% polyacrylamide gel relative to the molecular weight standards.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403819&req=5

pone.0124248.g002: Influence of OmpR activity on the Y. enterocolitica membrane protein profile.Proteins were isolated from strains Ye9 (wild type) and AR4 (ompR mutant) grown overnight in LB medium at 27°C or 37°C. In each case, 50 μg of protein were separated by SDS-PAGE and visualized by Coomassie blue staining. Putative OmpR-regulated proteins subsequently identified by LC-MS/MS are indicated by arrows. The bands were named according to their migration in the 12% polyacrylamide gel relative to the molecular weight standards.
Mentions: In parallel with the mutant screen described above, we analyzed membrane proteins of the wild-type Y. enterocolitica Ye9 and strain AR4 (the ompR mutant), grown under different temperature conditions, using SDS-PAGE. Changes in the membrane protein profiles due to the growth temperature and the presence and activity of the OmpR regulator were observed (Fig 2). A differential analysis of band intensities was used to select proteins for identification by LC-MS/MS. Following this procedure, we identified six putative OmpR-controlled proteins that matched Y. enterocolitica proteins present in the NCBI protein databases (Table 2). The synthesis of four proteins was higher in the wild-type strain than in mutant AR4: p120 and p110 (cells grown at 27°C), and p100 and p85 (cells grown at 37°C). Two other proteins, p80 and p60, were present at lower levels in the wild-type strain compared to AR4. The proteins that appeared to be positively controlled by OmpR were AcrB (p120; an essential component of the AcrAB-TolC multidrug efflux pump), ferrichrome-iron receptor (p110), dehydratase (p100; a bifunctional aconitate hydratase 2/2-methylisocitrate dehydratase) and a putative ABC transporter ATP-binding protein (p85). The proteins presumed to be negatively regulated by OmpR were a 60-kDa heat shock protein (p80) and elongation factor Tu (p60).

Bottom Line: The identification of putative OmpR-binding sites and electrophoretic mobility shift assays confirmed that this regulator binds specifically to both promoter regions with different affinity.Examination of the activity of the acrR and acrAB promoters after the exposure of cells to different chemicals showed that bile salts can act as an OmpR-independent inducer.Taken together, our findings suggest that OmpR positively controls the expression of the AcrAB-TolC efflux pump involved in the adaptive response of Y. enterocolitica O:9 to different chemical stressors, thus conferring an advantage in particular ecological niches.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Microbiology, Institute of Microbiology, Faculty of Biology, University of Warsaw, Warsaw, Poland.

ABSTRACT
OmpR is a transcriptional regulator implicated in the control of various cellular processes and functions in Enterobacteriaceae. This study was undertaken to identify genes comprising the OmpR regulon in the human gastrointestinal pathogen Yersinia enterocolitica. Derivatives of an ompR-negative strain with random transposon insertions creating transcriptional fusions with the reporter gene lacZ were isolated. These were supplied with the wild-type ompR allele in trans and then screened for OmpR-dependent changes in β-galactosidase activity. Using this strategy, five insertions in genes/operons positively regulated by OmpR and two insertions in genes negatively regulated by this protein were identified. Genetic analysis of one of these fusion strains revealed that the gene acrR, encoding transcriptional repressor AcrR is negatively regulated by OmpR. Differential analysis of membrane proteins by SDS-PAGE followed by mass spectrometry identified the protein AcrB, a component of the AcrAB-TolC multidrug efflux pump, as being positively regulated by OmpR. Analysis of the activity of the acrR and acrAB promoters using gfp fusions confirmed their OmpR-dependent repression and activation, respectively. The identification of putative OmpR-binding sites and electrophoretic mobility shift assays confirmed that this regulator binds specifically to both promoter regions with different affinity. Examination of the activity of the acrR and acrAB promoters after the exposure of cells to different chemicals showed that bile salts can act as an OmpR-independent inducer. Taken together, our findings suggest that OmpR positively controls the expression of the AcrAB-TolC efflux pump involved in the adaptive response of Y. enterocolitica O:9 to different chemical stressors, thus conferring an advantage in particular ecological niches.

No MeSH data available.


Related in: MedlinePlus