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Structural and biochemical characterization of MepR, a multidrug binding transcription regulator of the Staphylococcus aureus multidrug efflux pump MepA.

Kumaraswami M, Schuman JT, Seo SM, Kaatz GW, Brennan RG - Nucleic Acids Res. (2009)

Bottom Line: DNA-binding data show that MepR uses a dual regulatory binding mode as the repressor binds the mepA operator as a dimer of dimers, but binds the mepR operator as a single dimer.Alignment of the six half sites reveals the consensus MepR binding site, 5'-GTTAGAT-3'. 'Drug' binding studies show that MepR binds to ethidium and DAPI with comparable affinities (K(d) = 2.6 and 4.5 microM, respectively), but with significantly lower affinity to the larger rhodamine 6G (K(d) = 62.6 microM).Mapping clinically relevant or in vitro selected MepR mutants onto the MepR structure suggests that their defective repressor phenotypes are due to structural and allosteric defects.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.

ABSTRACT
MepR is a multidrug binding transcription regulator that represses expression of the Staphylococcus aureus multidrug efflux pump gene, mepA, as well as its own gene. MepR is induced by multiple cationic toxins, which are also substrates of MepA. In order to understand the gene regulatory and drug-binding mechanisms of MepR, we carried out biochemical, in vivo and structural studies. The 2.40 A resolution structure of drug-free MepR reveals the most open MarR family protein conformation to date, which will require a huge conformational change to bind cognate DNA. DNA-binding data show that MepR uses a dual regulatory binding mode as the repressor binds the mepA operator as a dimer of dimers, but binds the mepR operator as a single dimer. Alignment of the six half sites reveals the consensus MepR binding site, 5'-GTTAGAT-3'. 'Drug' binding studies show that MepR binds to ethidium and DAPI with comparable affinities (K(d) = 2.6 and 4.5 microM, respectively), but with significantly lower affinity to the larger rhodamine 6G (K(d) = 62.6 microM). Mapping clinically relevant or in vitro selected MepR mutants onto the MepR structure suggests that their defective repressor phenotypes are due to structural and allosteric defects.

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Genetic organization of the mepR, mepA and mepB genes. The open rectangular boxes represent the region encoding the respective genes, which are labelled below, and the shaded rectangles indicate the relevant promoter sequences, which encompass MepR-binding sites. The inverted repeats within the MepR-binding sites are indicated by head-to-head inverted arrows. The thick bent arrows above the rectangles denote the transcription start sites and the thin curved arrows with the negative signs below, indicate the promoters negatively regulated by MepR. The mepB gene does have a MepR-binding site.
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Figure 1: Genetic organization of the mepR, mepA and mepB genes. The open rectangular boxes represent the region encoding the respective genes, which are labelled below, and the shaded rectangles indicate the relevant promoter sequences, which encompass MepR-binding sites. The inverted repeats within the MepR-binding sites are indicated by head-to-head inverted arrows. The thick bent arrows above the rectangles denote the transcription start sites and the thin curved arrows with the negative signs below, indicate the promoters negatively regulated by MepR. The mepB gene does have a MepR-binding site.

Mentions: Efflux pumps are categorized into five different families: the major facilitator super (MFS) family, ATP-binding cassette (ABC) transporters, small multidrug resistance (SMR) transporters, multidrug and toxic compound extrusion (MATE) family and resistance-nodulation-cell division (RND) family (6,7). Amongst the various mdr pumps identified in S. aureus, most belong to the MFS or SMR family (5) but recent genetic and microbiological studies on multidrug resistant S. aureus strains identified the first MATE family multidrug transporter, MepA (8,9). MepA confers resistance to a wide range of compounds, which include various dyes, biocides, fluoroquinolones and the glycylcycline, tigecycline (9,10). Genetic analysis of the open reading frame of mepA reveals that the gene is part of the three-gene cluster, mepRAB, which encodes, MepR, a MarR family transcription regulator, MepA, the MATE-family transporter with 12 transmembrane segments and MepB, a protein of unknown function that belongs to the uncharacterized but highly conserved COG4815 family (Figure 1) (9). Analogous to other described multidrug efflux systems MepR negatively regulates mepR and mepA (and possibly mepB) expression by binding to promoter sequences upstream of the mepR and mepA transcription start sites, respectively (Figure 1). Repression is relieved by MepR binding to one of a multitude of structurally and chemically different monovalent and bivalent cationic, lipophilic drugs (11). The significance of MepR-dependent mepA regulation and its role in multidrug resistance is underscored by the finding of multidrug-resistant clinical isolates of S. aureus, which contain mutations in the mepR gene that result in defective repressor phenotypes (8,12).Figure 1.


Structural and biochemical characterization of MepR, a multidrug binding transcription regulator of the Staphylococcus aureus multidrug efflux pump MepA.

Kumaraswami M, Schuman JT, Seo SM, Kaatz GW, Brennan RG - Nucleic Acids Res. (2009)

Genetic organization of the mepR, mepA and mepB genes. The open rectangular boxes represent the region encoding the respective genes, which are labelled below, and the shaded rectangles indicate the relevant promoter sequences, which encompass MepR-binding sites. The inverted repeats within the MepR-binding sites are indicated by head-to-head inverted arrows. The thick bent arrows above the rectangles denote the transcription start sites and the thin curved arrows with the negative signs below, indicate the promoters negatively regulated by MepR. The mepB gene does have a MepR-binding site.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651776&req=5

Figure 1: Genetic organization of the mepR, mepA and mepB genes. The open rectangular boxes represent the region encoding the respective genes, which are labelled below, and the shaded rectangles indicate the relevant promoter sequences, which encompass MepR-binding sites. The inverted repeats within the MepR-binding sites are indicated by head-to-head inverted arrows. The thick bent arrows above the rectangles denote the transcription start sites and the thin curved arrows with the negative signs below, indicate the promoters negatively regulated by MepR. The mepB gene does have a MepR-binding site.
Mentions: Efflux pumps are categorized into five different families: the major facilitator super (MFS) family, ATP-binding cassette (ABC) transporters, small multidrug resistance (SMR) transporters, multidrug and toxic compound extrusion (MATE) family and resistance-nodulation-cell division (RND) family (6,7). Amongst the various mdr pumps identified in S. aureus, most belong to the MFS or SMR family (5) but recent genetic and microbiological studies on multidrug resistant S. aureus strains identified the first MATE family multidrug transporter, MepA (8,9). MepA confers resistance to a wide range of compounds, which include various dyes, biocides, fluoroquinolones and the glycylcycline, tigecycline (9,10). Genetic analysis of the open reading frame of mepA reveals that the gene is part of the three-gene cluster, mepRAB, which encodes, MepR, a MarR family transcription regulator, MepA, the MATE-family transporter with 12 transmembrane segments and MepB, a protein of unknown function that belongs to the uncharacterized but highly conserved COG4815 family (Figure 1) (9). Analogous to other described multidrug efflux systems MepR negatively regulates mepR and mepA (and possibly mepB) expression by binding to promoter sequences upstream of the mepR and mepA transcription start sites, respectively (Figure 1). Repression is relieved by MepR binding to one of a multitude of structurally and chemically different monovalent and bivalent cationic, lipophilic drugs (11). The significance of MepR-dependent mepA regulation and its role in multidrug resistance is underscored by the finding of multidrug-resistant clinical isolates of S. aureus, which contain mutations in the mepR gene that result in defective repressor phenotypes (8,12).Figure 1.

Bottom Line: DNA-binding data show that MepR uses a dual regulatory binding mode as the repressor binds the mepA operator as a dimer of dimers, but binds the mepR operator as a single dimer.Alignment of the six half sites reveals the consensus MepR binding site, 5'-GTTAGAT-3'. 'Drug' binding studies show that MepR binds to ethidium and DAPI with comparable affinities (K(d) = 2.6 and 4.5 microM, respectively), but with significantly lower affinity to the larger rhodamine 6G (K(d) = 62.6 microM).Mapping clinically relevant or in vitro selected MepR mutants onto the MepR structure suggests that their defective repressor phenotypes are due to structural and allosteric defects.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.

ABSTRACT
MepR is a multidrug binding transcription regulator that represses expression of the Staphylococcus aureus multidrug efflux pump gene, mepA, as well as its own gene. MepR is induced by multiple cationic toxins, which are also substrates of MepA. In order to understand the gene regulatory and drug-binding mechanisms of MepR, we carried out biochemical, in vivo and structural studies. The 2.40 A resolution structure of drug-free MepR reveals the most open MarR family protein conformation to date, which will require a huge conformational change to bind cognate DNA. DNA-binding data show that MepR uses a dual regulatory binding mode as the repressor binds the mepA operator as a dimer of dimers, but binds the mepR operator as a single dimer. Alignment of the six half sites reveals the consensus MepR binding site, 5'-GTTAGAT-3'. 'Drug' binding studies show that MepR binds to ethidium and DAPI with comparable affinities (K(d) = 2.6 and 4.5 microM, respectively), but with significantly lower affinity to the larger rhodamine 6G (K(d) = 62.6 microM). Mapping clinically relevant or in vitro selected MepR mutants onto the MepR structure suggests that their defective repressor phenotypes are due to structural and allosteric defects.

Show MeSH
Related in: MedlinePlus