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The Staphylococcus aureus QacA protein is a multidrug transporter that confers resistance to a broad range of antimicrobial agents via proton motive force-dependent efflux of the compounds. Primer extension analysis was performed to map the transcription start points of theqacA and divergently transcribed qacRmRNAs. Each gene utilized a single promoter element, the locations of which were confirmed by site-directed mutagenesis. Fusions of theqacA and qacR promoters to a chloramphenicol acetyl transferase reporter gene were used to demonstrate that QacR is a trans-acting repressor of qacA transcription that does not autoregulate its own expression. An inverted repeat overlapping the qacA transcription start site was shown to be the operator sequence for control of qacA gene expression. Removal of one half of the operator prevented QacR-mediated repression of the qacA promoter. Purified QacR protein bound specifically to this operator sequence in DNase I-footprinting experiments. Importantly, addition of diverse QacA substrates was shown to induce qacA expression in vivo, as well as inhibit binding of QacR to operator DNA in vitro, by using gel-mobility shift assays. QacR therefore appears to interact directly with structurally dissimilar inducing compounds that are substrates of the QacA multidrug efflux pump. The Staphylococcus aureus QacA protein is a multidrug transporter that confers resistance to a broad range of antimicrobial agents via proton motive force-dependent efflux of the compounds. Primer extension analysis was performed to map the transcription start points of theqacA and divergently transcribed qacRmRNAs. Each gene utilized a single promoter element, the locations of which were confirmed by site-directed mutagenesis. Fusions of theqacA and qacR promoters to a chloramphenicol acetyl transferase reporter gene were used to demonstrate that QacR is a trans-acting repressor of qacA transcription that does not autoregulate its own expression. An inverted repeat overlapping the qacA transcription start site was shown to be the operator sequence for control of qacA gene expression. Removal of one half of the operator prevented QacR-mediated repression of the qacA promoter. Purified QacR protein bound specifically to this operator sequence in DNase I-footprinting experiments. Importantly, addition of diverse QacA substrates was shown to induce qacA expression in vivo, as well as inhibit binding of QacR to operator DNA in vitro, by using gel-mobility shift assays. QacR therefore appears to interact directly with structurally dissimilar inducing compounds that are substrates of the QacA multidrug efflux pump. Closely following the discovery of mammalian P-glycoprotein (1Kartner N. Riordan J.R. Ling V. Science. 1983; 221: 1285-1288Crossref PubMed Scopus (884) Google Scholar,2Gros P. Croop J. Housman D. Cell. 1986; 47: 371-380Abstract Full Text PDF PubMed Scopus (856) Google Scholar), the phenomenon of multidrug resistance was also described for a bacterial system, the Staphylococcus aureus QacA pump (3Tennent J.M. Lyon B.R. Gillespie M.T. May J.W. Skurray R.A. Antimicrob. Agents Chemother. 1985; 27: 79-83Crossref PubMed Scopus (97) Google Scholar), and has since been found to be widespread among both Gram-negative and Gram-positive bacteria (4Lewis K. Trends Biochem. Sci. 1994; 19: 119-123Abstract Full Text PDF PubMed Scopus (252) Google Scholar, 5Nikaido H. Science. 1994; 264: 382-388Crossref PubMed Scopus (1262) Google Scholar, 6Paulsen I.T. Brown M.H. Skurray R.A. Microbiol. Rev. 1996; 60: 575-608Crossref PubMed Google Scholar). Resistance involves the active transport of a structurally diverse range of toxic compounds, typically hydrophobic cations, from the cell by a single efflux system. In the case of P-glycoprotein, the ability to export many anticancer agents (7Gottesman M.M. Hrycyna C.A. Schoenlein P.V. Germann U.A. Pastan I. Annu. Rev. Genet. 1995; 29: 607-649Crossref PubMed Scopus (463) Google Scholar) has prompted investigations into its mode of action. Biochemical studies and the generation of mutants with altered drug binding capabilities have suggested P-glycoprotein interacts directly with various substrates (7Gottesman M.M. Hrycyna C.A. Schoenlein P.V. Germann U.A. Pastan I. Annu. Rev. Genet. 1995; 29: 607-649Crossref PubMed Scopus (463) Google Scholar, 8Gottesman M.M. Pastan I. Annu. Rev. Biochem. 1993; 62: 385-427Crossref PubMed Scopus (3564) Google Scholar). Additionally, recent progress has been made toward determining the structure of P-glycoprotein (9Rosenberg M.F. Callaghan R. Ford R.C. Higgins C.F. J. Biol. Chem. 1997; 272: 10685-10694Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). Despite these advances, the basis of multidrug recognition by P-glycoprotein is still not understood. The functional similarities of bacterial multidrug efflux systems with P-glycoprotein, together with their presence in significant human pathogens, such as S. aureus (3Tennent J.M. Lyon B.R. Gillespie M.T. May J.W. Skurray R.A. Antimicrob. Agents Chemother. 1985; 27: 79-83Crossref PubMed Scopus (97) Google Scholar),Pseudomonas aeruginosa (10Li X.-Z. Nikaido H. Poole K. Antimicrob. Agents Chemother. 1995; 39: 1948-1953Crossref PubMed Scopus (508) Google Scholar), Neisseria gonorrhoeae (11Hagman K.E. Pan W. Spratt B.G. Balthazar J.T. Judd R.C. Shafer W.M. Microbiology. 1995; 141: 611-622Crossref PubMed Scopus (291) Google Scholar), and Mycobacterium tuberculosis (12Doran J.L. Pang Y.J. Mdluli K.E. Moran A.J. Victor T.C. Stokes R.W. Mahenthiralingam E. Kreiswirth B.N. Butt J.L. Baron G.S. Treit J.D. Kerr V.J. Vanhelden P.D. Roberts M.C. Nano F.E. Clin. Diagn. Lab. Immunol. 1997; 4: 23-32Crossref PubMed Google Scholar), makes elucidation of their molecular mechanisms an important research goal. Some progress has been made toward delineating the significance that various motifs and individual amino acids hold for determining the specificity of transport and overall mechanism of action for the QacA (13Paulsen I.T. Brown M.H. Littlejohn T.G. Mitchell B.A. Skurray R.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3630-3635Crossref PubMed Scopus (238) Google Scholar) and Bacillus subtilis Bmr (14Ahmed M. Borsch C.M. Neyfakh A.A. Schuldiner S. J. Biol. Chem. 1993; 268: 11086-11089Abstract Full Text PDF PubMed Google Scholar) multidrug transporter proteins (reviewed in Ref. 6Paulsen I.T. Brown M.H. Skurray R.A. Microbiol. Rev. 1996; 60: 575-608Crossref PubMed Google Scholar), but the strong association of efflux pumps with the membrane makes isolation and in depth analysis of these proteins difficult. Considerable effort has also been directed toward identifying the factors involved in regulation of multidrug transporter gene expression. For mammalian P-glycoprotein (15Kato S. Nishimura J. Yufu Y. Ideguchi H. Umemura T. Nawata H. FEBS Lett. 1992; 308: 175-178Crossref PubMed Scopus (23) Google Scholar, 16Chaudhary P.M. Roninson I.B. J. Natl. Cancer Inst. 1993; 85: 632-639Crossref PubMed Scopus (366) Google Scholar), as well as the Bmr (17Ahmed M. Borsch C.M. Taylor S.S. Vázquez-Laslop N. Neyfakh A.A. J. Biol. Chem. 1994; 269: 28506-28513Abstract Full Text PDF PubMed Google Scholar), and Escherichia coli EmrB (18Lomovskaya O. Lewis K. Matin A. J. Bacteriol. 1995; 177: 2328-2334Crossref PubMed Scopus (205) Google Scholar) bacterial multidrug efflux systems, increased gene expression followed the addition of some of the structurally diverse compounds exported by these pumps. Indeed, a certain degree of regulatory control over the genes for membrane transport proteins is to be expected, given their toxic nature toward the cell if overexpressed, as has been observed for the Gram-negative bacterial tetracycline resistance gene, tetA (19Hillen W. Berens C. Annu. Rev. Microbiol. 1994; 48: 345-369Crossref PubMed Scopus (426) Google Scholar). Control of tetA expression by the specific repressor protein, TetR, is the best understood example of the regulation of a gene encoding a drug transporter (19Hillen W. Berens C. Annu. Rev. Microbiol. 1994; 48: 345-369Crossref PubMed Scopus (426) Google Scholar). Induction of tetA expression occurs when TetR binds a tetracycline/divalent metal cation complex, inducing a conformational change in the protein such that TetR no longer binds the tetA operator, thereby liberating the promoter site (20Hinrichs W. Kisker C. Duvel M. Muller A. Tovar K. Hillen W. Saenger W. Science. 1994; 264: 418-420Crossref PubMed Scopus (335) Google Scholar). A similar style of regulation also be for the of expression observed for some bacterial multidrug efflux for this to be both the transporter and the regulatory protein to the structurally diverse compounds that these systems efflux from the Despite regulation of some bacterial multidrug efflux genes involves regulatory such as the subtilis (17Ahmed M. Borsch C.M. Taylor S.S. Vázquez-Laslop N. Neyfakh A.A. J. Biol. Chem. 1994; 269: 28506-28513Abstract Full Text PDF PubMed Google Scholar), E. coli (18Lomovskaya O. Lewis K. Matin A. J. Bacteriol. 1995; 177: 2328-2334Crossref PubMed Scopus (205) Google Scholar), and N. gonorrhoeae Balthazar J.T. K.E. Shafer W.M. J. Bacteriol. 1997; PubMed Google Scholar) Importantly, the subtilis protein binds directly to some of the compounds that both induce and are also substrates for the Bmr transporter (17Ahmed M. Borsch C.M. Taylor S.S. Vázquez-Laslop N. Neyfakh A.A. J. Biol. Chem. 1994; 269: 28506-28513Abstract Full Text PDF PubMed Google Scholar, M. Neyfakh A.A. J. Bacteriol. 1996; PubMed Scopus Google Neyfakh A.A. Sci. 1997; PubMed Scopus Google Scholar). of the from the S. aureus qacA gene a regulatory element, D. Littlejohn T.G. Skurray R.A. Microbiol. 4: PubMed Scopus Google Scholar). together with TetR, to a of regulatory proteins which with a their and have to be involved in the binding of inducing compounds D. Littlejohn T.G. Skurray R.A. Microbiol. 4: PubMed Scopus Google Scholar, H. N. M. 1995; PubMed Scopus Google Scholar). In this the toward QacR the expression by is a that both binds to an operator site to the qacA promoter and also appears to interact directly with a diverse range of inducing compounds. The bacterial and used in this are described in For performed was the E. was used for used chloramphenicol inverted transcription start QacR and in were in of of of of The DNA from D. Littlejohn T.G. Skurray R.A. Microbiol. 4: PubMed Scopus Google Scholar), was the for The used to promoter to the chloramphenicol reporter gene, was by the in J. 27: PubMed Scopus Google Scholar) with the from to of DNA from and and B.R. May J.W. Skurray R.A. Genet. PubMed Scopus Google Scholar) J. T. A and multidrug D. Littlejohn T.G. Skurray R.A. Microbiol. 4: PubMed Scopus Google Scholar) coli C. J. J. 1985; PubMed Scopus Google Scholar) coli with with PubMed Scopus Google Scholar) coli expression with PubMed Scopus Google Scholar) coli promoter a J. 27: PubMed Scopus Google Scholar) coli expression with for of with from for of from into J.M. Lyon B.R. Gillespie M.T. May J.W. Skurray R.A. Antimicrob. Agents Chemother. 1985; 27: 79-83Crossref PubMed Scopus (97) Google Scholar) from into qacR in in with the and in with the and in with the and in with the and directed in the from to with the and of in for directed in the from to qacR in in with the and qacR in with one half of from of to the in that to a in the sequence which is the as used in Some their for the of recognition an binding a encoding The of to the in that to a in the sequence D. Littlejohn T.G. Skurray R.A. Microbiol. 4: PubMed Scopus Google Scholar), which is the as used in Some their for the of recognition an binding a encoding in a the and which their encoding were performed to a encoding was into with the promoter for of the gene binding was with gene expression the control of the promoter for were in the of the qacR and promoters by site-directed and The used to was to qacA and that for was to qacA An with qacR in to was by the of a with the and into one half of the qacA operator sequence a from was using the site the of and site in the to the of qacR in was into the site of and a transcription the control of The which was to the expression in a by a strong binding site promoter of PubMed Scopus Google Scholar), a with the the and of the and were used to a that of the qacR gene by a strong binding site and also a sequence the was into the E. coli expression PubMed Scopus Google Scholar) to expression the control of the strong promoter. from the of were to that no been was from S. aureus and the by the as described by in Scholar), for with S. aureus by an in for to and DNA DNA and site-directed were by J. T. A Scholar). For of the was was performed using to the Primer extension analysis was performed as described by the and and for were with with of by for and for by the addition of and The extension were a and with the of E. a promoter together with qacR in were in of and with the and in some the addition of a of a inducing were by in of and by for The was by the of was using to the of PubMed Scopus Google Scholar). were performed An of E. coli with was in of and to an of which of QacR was by the addition of the were by and in of The were and addition of to and for were by of followed by the addition of A to and DNase to and for The was by for with of metal for The was into a and a of with of and with of Purified QacR was in The were by using the of PubMed Scopus Google Scholar) and proteins by with R. were from and protein was by the of M.M. Biochem. PubMed Scopus Google Scholar), using as a was performed a with as the molecular were from was performed as described in Ref. D. of analysis using a to proteins was performed to the Purified QacR in a of was in the of metal for The was by the addition of of were for for and by followed by to the by using with were with the DNA utilized a to the with the and in which substrates of QacA were to some of the binding used a which the qacA promoter and of the with the and A from the of gene that as a specificity control was using the and The QacR from the were into by a with this and in DNA were with the of QacR in of and of in a of Some also substrates of QacA metal the of the were by and as described by of the utilized the DNA used in gel-mobility shift using with and For of the qacA a was using the with and of DNA was in and for in a of also of of DNase in of DNase was and the to for with the addition of of The were with and the DNA The was with and in of for of the DNA were a were in case with the as was for the of the sequence of qacA a divergently transcribed to by binding to a of that the sequence for the qacA promoter D. Littlejohn T.G. Skurray R.A. Microbiol. 4: PubMed Scopus Google Scholar). Additionally, a promoter for qacR in was which and qacR expression to D. Littlejohn T.G. Skurray R.A. Microbiol. 4: PubMed Scopus Google Scholar). analysis of for in that to the start of the gene and has its own of that expression of be from the used by gene, the for and qacR were in the S. aureus B.R. May J.W. Skurray R.A. Genet. PubMed Scopus Google Scholar) D. Littlejohn T.G. Skurray R.A. Microbiol. 4: PubMed Scopus Google Scholar) using extension For one extension was The in S. aureus to a and that for qacR to be a for gene, transcription is from a single promoter element, by operator for proteins and the was used when the was in E. extension using the as described in were performed from E. coli The that the promoters were utilized in both for both genes the in E. coli was found to be an one from the for S. aureus not a since A is the start for the of transcription by E. R. J.L. W. M. M. Escherichia coli and and for Scholar). the observed change be an of from The expression of some regulatory (19Hillen W. Berens C. Annu. Rev. Microbiol. 1994; 48: 345-369Crossref PubMed Scopus (426) Google Scholar), from suggested the qacR promoter D. Littlejohn T.G. Skurray R.A. Microbiol. 4: PubMed Scopus Google Scholar) also be functional as an promoter. extension analysis using was also performed from aureus that been with a that as an of QacR-mediated qacA gene expression A was not observed the that the qacR gene is transcribed from a single promoter. was confirmed by to extension not for which was a to the which transcription be for the qacR promoter. in the promoter and DNA were utilized to the of the promoter The and were such that and be to control transcription of the reporter the described was used to change and of the and from and to and that were to have to as of the by that the qacR promoter was The in the expression of the gene and for the qacR promoter when with the promoter thereby the of the of promoter and of one half of the of various and promoter of the of the and of for are as for the for the qacR in qacR in in in one half of was as described of E. coli the For of the of the and The for are as for the for the in a The of was as described of E. coli the the regulation of qacA by gene were E. coli was that in the case of the presence of gene in to qacA in a significant in transcription from when with and the which qacR in were in the QacR expression from the qacA in no expected, the presence of the no transcription from not these that the observed of repression by was a of qacR the control of the strong to of The presence of was found to have no significant the of transcription from promoter in that the expression of qacR is not the of the of overlapping the qacA promoter to as an operator were performed with which one half of the the as described from the qacA promoter in to the to the observed for which qacR the of as a operator to which QacR binds are of QacR the qacR gene was from a strong binding site and the promoter in the E. coli in of QacR of a the of QacR to by The QacR protein in with an observed molecular of the for a QacR D. Littlejohn T.G. Skurray R.A. Microbiol. 4: PubMed Scopus Google Scholar) with a amino of the of the protein confirmed its The molecular of QacR was by to be in with the molecular for a of the in the of molecular the to the for a QacR QacR be from these molecular by with followed by but with the addition of a not suggested that of the in QacR and D. Littlejohn T.G. Skurray R.A. Microbiol. 4: PubMed Scopus Google Scholar) was for the A the and of proteins is the of from the of by metal and H. Biol. 1995; Google Scholar). this the were into their from the followed by in to QacR as a following for in in the of in the of of the and in with the in that QacR is from the cell as a and the observed are the which following using a to proteins were performed cell by QacR was not that the which of the protein are to be of to a of of QacR from the was to the of not the of metal in QacR the of metal cations, QacR was with various of and which in an increased of a for and for In with of in the of a significant to the observed which suggested that of from the the be to the the of the in the presence of was their A QacR an was also to the addition of to a of the in of and no the of not shift were used to QacR bound specifically to a DNA to and not to a control from the of gene and not control was to QacR from gel-mobility shift binding the specificity of QacR for the DNA Additionally, of the the site to in the that QacR is specific for the qacA promoter and not to a DNA the qacR promoter and to the that expression is not In the protein from E. coli the QacR but not the control was to shift DNA not that QacR is for the observed of The addition of no gel-mobility shift binding not that which increased the of these metal not to have the binding of QacR to DNase I-footprinting confirmed that QacR bound specifically to in the inverted repeat overlapping the qacA shown in QacR from DNase the in the with the of a both the qacA and The QacA protein confers resistance to a range of structurally dissimilar compounds from of the and if these substrates the expression of in of inducing compounds were with E. coli which the qacR gene in to theqacA promoter. A significant in was observed for many of the QacA substrates for which a degree of of no in was observed when the qacA promoter was a range of inducing compounds not for their ability to qacA gene expression were and both of which no not of the compounds were to the strong repression of the qacA promoter in by qacR in from the not of expression from a with qacR in the addition of various inducing of for of in of was by the of with the addition of inducing compounds by the in the of inducing of was for E. coli which the qacA promoter to with qacR in in the of inducing compounds, were performed as described The of was by the of with the addition of inducing compounds by the in the of inducing compounds. in a The of was for E. coli which the qacA promoter to with qacR in in the of inducing compounds, were performed as described for the in of many QacA substrates were also to an in binding of QacR to theqacA operator a strong of QacR from the DNA when of the were in gel-mobility shift experiments. QacR from the operator a the for S. aureus of the was of the as well as a not The and both binding of QacR to the operator, a and the for S. was shown to inhibit binding of QacR to operator DNA a that was the of this for S. aureus The addition of QacA substrates the used in the to DNA not its the to demonstrate that the expression of the S. aureus multidrug efflux is by a repressor protein, the of a divergently transcribed both in gel-mobility shift and DNase and in analysis by of one half of the which in expression of the operator site for QacR binding was shown to have been from the with studies systems such as that of the E. coli Cell. Full Text PDF PubMed Scopus Google Scholar, M. H. Proc. Natl. Acad. Sci. U. S. A. 85: PubMed Scopus Google Scholar), the binding of QacR to its operator not inhibit the binding of but the of the into a repression of qacA The of QacR to autoregulate expression of its own gene was by qacR in to transcription from the no its own promoter to a reporter gene was confirmed by that QacR not to a DNA the qacR promoter of the of regulatory proteins with QacR that are divergently transcribed to the expression of their own such from a J.L. Genet. PubMed Scopus Google Scholar), a repressor of in H. Y. H. N. T. J. Bacteriol. 1995; 177: PubMed Google Scholar), the repressor of the resistance gene J. Bacteriol. 1992; PubMed Google Scholar), and TetR (19Hillen W. Berens C. Annu. Rev. Microbiol. 1994; 48: 345-369Crossref PubMed Scopus (426) Google Scholar). QacR appears to be for this of proteins in that expression of its own gene was not to was not bound by an for this operator sequence in the control of and qacA gene The repression of qacA transcription by QacR was to be by the addition of a range of structurally dissimilar QacA in of qacA expression shift suggested that for many QacA of qacA expression involved QacR directly with the recognition of structurally dissimilar compounds, the of a has also been shown for (17Ahmed M. Borsch C.M. Taylor S.S. Vázquez-Laslop N. Neyfakh A.A. J. Biol. Chem. 1994; 269: 28506-28513Abstract Full Text PDF PubMed Google Scholar, M. Neyfakh A.A. J. Bacteriol. 1996; PubMed Scopus Google Scholar, Neyfakh A.A. Sci. 1997; PubMed Scopus Google Scholar). Some in of in binding of QacR operator DNA their for S. the that QacA substrates to be hydrophobic such in these compounds with the which were to the (17Ahmed M. Borsch C.M. Taylor S.S. Vázquez-Laslop N. Neyfakh A.A. J. Biol. Chem. 1994; 269: 28506-28513Abstract Full Text PDF PubMed Google Scholar). QacA substrates which no in of qacA gene and were to inhibit binding of QacR to operator but in of their for S. For and and no the expression of the binding of QacR to operator DNA were that the of transcription from the qacA promoter when qacR is in efflux of substrates such as the compounds not by the repressor protein the basis of the and that QacR is as a The to in of cell is a strong that the which of QacR not in the cell and are therefore to have The ability of and to induce together with the for a to the the of metal in the of and to the isolation of of the of proteins has been shown to be for the of human T. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar), R. PubMed Scopus Google Scholar), and J. Biol. Chem. Full Text PDF PubMed Google Scholar). The observed in the of both QacR and is the of of and which also occurs for human T. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). of following the of the bacterial resistance has also been a PubMed Scopus Google Scholar). for H. PubMed Scopus Google Scholar) and also the repressor of the E. coli and W. J. R.A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), are for the binding of the metal that as of these systems, in conformational in the regulatory thereby inducing expression. The in QacR a in the ability of this protein to the and compounds that as of qacA gene expression. The ability of QacR to interact with and diverse compounds makes an for of their in the of the multidrug efflux regulatory proteins are the membrane bound transporter for studies directed structurally diverse compounds are by a single the of a QacR and inducing compounds, analysis of individual and of the toward studies the structure of QacR bound to its operator DNA inducing compounds is in
Grkovic et al. (Wed,) studied this question.
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