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Review
. 2002 Dec;66(4):671-701, table of contents.
doi: 10.1128/MMBR.66.4.671-701.2002.

Regulation of bacterial drug export systems

Steve Grkovic  1 , Melissa H Brown, Ronald A Skurray
Affiliations
Review

Regulation of bacterial drug export systems

Steve Grkovic et al. Microbiol Mol Biol Rev. 2002 Dec.

Abstract

The active transport of toxic compounds by membrane-bound efflux proteins is becoming an increasingly frequent mechanism by which cells exhibit resistance to therapeutic drugs. This review examines the regulation of bacterial drug efflux systems, which occurs primarily at the level of transcription. Investigations into these regulatory networks have yielded a substantial volume of information that has either not been forthcoming from or complements that obtained by analysis of the transport proteins themselves. Several local regulatory proteins, including the activator BmrR from Bacillus subtilis and the repressors QacR from Staphylococcus aureus and TetR and EmrR from Escherichia coli, have been shown to mediate increases in the expression of drug efflux genes by directly sensing the presence of the toxic substrates exported by their cognate pump. This ability to bind transporter substrates has permitted detailed structural information to be gathered on protein-antimicrobial agent-ligand interactions. In addition, bacterial multidrug efflux determinants are frequently controlled at a global level and may belong to stress response regulons such as E. coli mar, expression of which is controlled by the MarA and MarR proteins. However, many regulatory systems are ill-adapted for detecting the presence of toxic pump substrates and instead are likely to respond to alternative signals related to unidentified physiological roles of the transporter. Hence, in a number of important pathogens, regulatory mutations that result in drug transporter overexpression and concomitant elevated antimicrobial resistance are often observed.

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Figures

FIG. 1.
FIG. 1.
Genetic organization of the mexR-mexAB-oprM, mexT-mexEF-oprN, nfxB-mexCD-oprJ, and mexZ-mexXY MDR loci from P. aeruginosa. Each operon contains genes (grey arrows) that encode a drug efflux complex and is regulated by the product of an upstream gene (black arrow) which either represses (−) or activates (+) operon expression, although this is yet to be confirmed for MexZ. The intergenic region separating the mexR and mexA genes is depicted in greater detail, with the positions of the binding sites for the two MexR dimers (black ovals) indicated relative to the −10 and −35 hexamers of the mexR promoter (PmexR), the mexA promoter (P2mexA), and a second potential mexA promoter (P1mexA). A schematic representation of the MexAB-OprM tripartite complex, which can efflux drugs simultaneously across both the cytoplasmic (CM) and outer (OM) membranes, is also shown. MexB, the RND component of the pump complex, transports drugs across the cytoplasmic membrane in exchange for protons (H+).
FIG. 2.
FIG. 2.
Schematic representation of the known regulatory controls governing the expression of the E. coli acrAB and tolC genes. The AcrAB-TolC transport complex extrudes drugs across both the cytoplasmic (CM) and outer (OM) membranes (pale yellow shaded boxes). Excessive production of AcrA and AcrB is prevented (−) by the local dimeric repressor protein AcrR (green), whereas a regulatory protein involved in cell division, SdiA (grey), can increase (+) acrAB expression. However, activation of acrAB and tolC transcription occurs primarily because of the global regulatory proteins MarA, SoxS, and Rob (purple), any one of which can bind to a marbox (cyan) upstream of these genes. The intracellular level of MarA is controlled by MarR (red), a dimeric protein which binds to marO (orange) and represses (−) the expression of its own gene and the two others that constitute the marRAB operon. Binding of inducing compounds (diamonds) such as salicylate by MarR, in addition to the possible phosphorylation of MarR via a putative signal transduction pathway involving the periplasmic binding protein MppA, is proposed to transform MarR into a non-DNA-binding conformation, thereby permitting marRAB transcription to proceed. Hence, MarA protein is produced which can then bind as a monomer to the marO marbox upstream of the marRAB promoter, where it activates (+) transcription of marRAB and enhances the production of MarA. The ensuing highly elevated intracellular levels of MarA can then bind to marboxes adjacent to the promoters of mar regulon genes, such as acrAB and tolC, and activate their transcription. The MarA homologs SoxS and Rob can also bind to the marO marbox and activate marRAB transcription. The positive regulation by all three proteins on marRAB is enhanced by FIS (yellow), an accessory activating protein. SoxS is only produced upon conversion of the SoxR effector protein (magenta) into its active form (SoxR*) by superoxide-generating agents (O2−). Rob, like SoxS, in addition to mediating increases in MarA synthesis, can also directly activate the expression of some genes that belong to the mar regulon, such as acrAB. The regulatory-protein-binding sites within marO are shown in finer detail in the bottom left corner. The positions of the −35 and −10 hexamers of the marRAB promoter, the ribosome-binding site (RBS) and transcription start points (tsps) for the marRAB operon are indicated. See text for other details. (Modified with permission from reference .)
FIG. 3.
FIG. 3.
Structures of Rob and MarA proteins bound to micF and marRAB marboxes, respectively. The highly homologous DNA-binding domains of Rob and MarA are colored orange, with their respective HTH recognition helices in brown and the additional C-terminal putative ligand-binding domain of Rob in blue. The N-terminal Rob HTH is inserted into the major groove, whereas the C-terminal HTH makes contacts only to the DNA backbone. In contrast, MarA induces a significant bend in the marRAB promoter to facilitate the placement of both HTH motifs in successive major grooves of marRAB marbox DNA. (Reprinted with permission from reference ; kindly provided by Tom Ellenberger.)
FIG. 4.
FIG. 4.
(A) Regulation of expression of B. subtilis MDR genes bmr and blt. A BmrR dimer concurrently bound to both bmr promoter DNA and drugs (D) can correctly orient the −10 and −35 hexamers of this promoter to facilitate the binding of RNA polymerase. White arrows indicate the locations of promoters and also the direction in which transcription occurs from these sequences. Because many of the substrates of the Bmr MDR transporter are also ligands of BmrR (red ovals), activation (+) of bmr expression can occur in response to the presence of these deleterious compounds, permitting drug efflux across the cytoplasmic membrane (pale yellow; CM) in exchange for protons (H+) to occur. The global regulatory protein Mta (purple ovals) and the local activator of the blt operon, BltR (green ovals), are likely to act in the same fashion as BmrR, although inducing ligands for these proteins have yet to be identified (?). Mta activates both the bmr and blt genes by binding to the same DNA sequence as the local regulators. The blt operon also encodes SpAT, a polyamine acetyltransferase, whereas Bmr expression can result from transcription initiated at either its own promoter or the promoter of bmrU, an upstream gene of unknown function. (B) The DNA sequence from the region indicated by an asterisk in A, which contains the bmr promoter (Pbmr). The −10 and −35 hexamers of Pbmr and the unusually large 19-bp spacing between these hexanucleotides are indicated, while the large arrows denote the imperfect inverted repeat (labeled −11 to +11) within Pbmr that constitutes the BmrR binding site (2). Bases protected from DNase I digestion by BmrR bound to Pbmr are highlighted in white. (C) DNA contacts made by BmrR to the half-site that encompasses positions −1 to −10 in B. Also shown is the thymine from position +1, which in the BmrR-DNA complex was observed to be no longer base-paired to its partner, whereas the adenine and thymine bases at position −1, although significantly displaced, still formed a distorted base pair. Thin dashed lines indicate hydrogen bonds, and thick broken lines indicate van der Waals interactions between BmrR amino acids and bases (grey boxes) or the phosphates (P) and deoxyribose rings (pentagons) that form the DNA backbone. Note that the sequence depicted in C differs at position −8 from that shown in B because the experimentally determined data best fit a GC base pair at this location rather than the AT that was actually present in the BmrR-DNA complex. (Panel A modified with permission from reference ; panel C reprinted with permission from reference .)
FIG. 5.
FIG. 5.
Structure of a BmrR dimer in the drug- and DNA-bound tripartite complex. One polypeptide chain is colored yellow for the N-terminal winged-helix DNA-binding domain, red for the α5 linker helix, and green for the C-terminal drug-binding domain. The locations of selected helices are indicated for this polypeptide, as are the positions of the α3′, α4′, and α5′ helices in the second (cyan) polypeptide. Also labeled for the first monomer is the HTH (H, α1; T, turn; H, α2 recognition helix) and the two wings, W1 (sheets β2 and β3) and W2 (helices α3 and α4). The DNA and drug (tetraphenylantimonium) molecules are represented as a ball and sticks (carbon, black; nitrogen, blue; oxygen, red; and phosphorus/antimony, green). (Reprinted with permission from reference ; kindly provided by Richard Brennan.)
FIG. 6.
FIG. 6.
Structure of a TPP molecule depicted relative to the location of BRC binding-pocket residues that interact with this ligand. The negatively charged Glu134 BRC residue (the equivalent BmrR amino acid number is shown in parentheses) provides the crucial electrostatic interaction with the delocalized positive charge carried by the TPP phenyl rings. Ile23, Val28, Ala53, Ile71, and Ile136 all form van der Waals contacts with TPP, whereas the aromatic side chains of Tyr51 and Tyr68 stack against TPP rings. Tyr68 also hydrogen bonds to Glu134 to stabilize its negative charge, as does Tyr110 and a water molecule (W1), which replaces the hydrogen bond of Tyr33 (BRC α2) that was broken because of the displacement of BRC α2 by the entry of TPP into the binding site. The stabilizing hydrogen bonds are indicated by broken lines, as is the crucial electrostatic contact, with distances given in angstroms. (Reprinted with permission from reference .)
FIG. 7.
FIG. 7.
Control of tetA transcription by TetR. (A) The DNA sequence of the Tn10 tet intergenic region containing the tet operators and promoters is shown, with the base pairs that form the O1 and O2 inverted repeats shaded light grey. The tetA promoter PtetA, the two tetR promoters PtetR1 and PtetR2, and their associated transcription start points (right angle arrows) are also indicated. The product of the tetR gene (black ovals) forms dimers and binds to O1 and O2, preventing the expression (−) of both genes. Binding by TetR of the intracellular form of tetracycline, a tetracycline-Mg2+ complex, results in a conformational change so that TetR can no longer bind the tet operators. The ensuing initiation of tetA transcription protects the cell from tetracycline because of subsequent production of the membrane-bound TetA protein, which exports tetracycline-Mg2+ complexes in exchange for protons (H+). See text for other details. (B) DNA contacts formed by operator-bound TetR. The nucleotides from a tet operator half-site, representing the 0 to +7 positions of O1 in A, are depicted as grey boxes attached to the phosphate-ribose backbone. The contacts made by the DNA-reading head of one monomer in a TetR dimer to the bases in that operator half-site are shown as thin dashed lines for hydrogen bonds and thick broken lines for van der Waals interactions. TetR amino acids from the HTH recognition helix (α3) are in boldface type, those from elsewhere in the HTH motif are in italics, and Thr26 and Lys48 are additional residues from outside the HTH which make tet operator DNA contacts. The proline (P39) in the TetR recognition helix transfers binding of the TetR reading head from nucleotides on one strand of the operator half-site to nucleotides on the other strand. A hydrogen bond formed between a water molecule (wat) and His44 in the crystal structure mimics an interaction that would otherwise take place between His44 and the DNA backbone phosphate at position +8 of O1. (Panel B reprinted with permission from reference .)
FIG. 8.
FIG. 8.
Structure of the TetR dimer-tet operator DNA complex. The α-helices in each polypeptide are colored blue for the DNA-binding domain (α1 to α3), yellow for the rigid α-helices (α5, α8, and α10), and green for those that undergo conformational changes upon induction (α4, α6, α7, and α9). The α3 and α3′ recognition helices fit into successive major grooves of tet operator DNA, which is represented as red for the phosphate-ribose backbone and grey for the bases. A grey line also delineates the curvature of operator DNA induced by TetR binding. (Reprinted with permission from reference ; kindly provided by Winfried Hinrichs.)
FIG. 9.
FIG. 9.
(A) Diagrammatic illustration of specific interactions between TetR(D) binding-tunnel residues and a tetracycline-Mg2+ complex. Tetracycline is colored orange, with its four rings labeled A to D and the methyl groups shown as Me. The three water molecules coordinated by the Mg2+ atom (red) are represented by W1 to W3 (blue). Residues from the TetR dimer that contribute to the binding of this tetracycline-Mg2+ complex are shown in italics for those from the first polypeptide and in bold type for those from the second polypeptide. The α-helices in which these amino acids are located are also indicated except for the Thr103, Arg104, and Pro105 residues, which form part of the interhelical loop connecting α6 to α7. Thin dotted lines indicate hydrogen bonds, and thick broken lines show hydrophobic interactions. Equivalent residues from the other binding tunnel in a TetR dimer contact a second tetracycline-Mg2+ complex; see text for other details. (B) TetR conformational changes that occur upon binding a tetracycline-Mg2+ complex. The α1 to α8 helices of one monomer and the α9′ helix from the second polypeptide in a dimer are represented in blue for DNA-bound TetR and yellow for the induced tetracycline-Mg2+-bound form. The DNA phosphate-ribose backbone is shown in red, bases in grey, tetracycline (Tc) in orange, the Mg2+ atom in red, and the chain of water molecules that constitute the water zipper in the induced conformation as blue spheres. The pendulum-like motion of α4 upon tetracycline-Mg2+ binding leads to significant displacement of the attached α1-α3 DNA-reading head, so that the α3 recognition helix can no longer contact the major groove of tet operator DNA at the same time as the α3′ recognition helix from the second polypeptide in a TetR dimer. The location of the entrance to the TetR binding tunnel is also indicated, whereas the sliding door motion of α9′ that closes this entrance in the induced form is also apparent. See text for other details. (Panel A reprinted with permission from reference ; ©1994, American Association for the Advancement of Science. Panel B reprinted with permission from reference ; kindly provided by Winfried Hinrichs.)
FIG. 10.
FIG. 10.
(A) Model for regulation of expression of S. aureus qacA MDR gene. QacR (black ovals) represses (−) transcription from the qacA promoter, PqacA, by binding as one dimer per IR1 half-site. Many of the lipophilic cationic drugs (D) exported from the cell by QacA in exchange for protons (H+) are also ligands of QacR. Conformational changes that occur in a QacR dimer upon drug binding result in the ligand-bound form of this protein being incapable of binding IR1, thereby mediating increases in qacA transcription in response to the presence of transporter substrates. The locations of the ribosome-binding site (RBS) and transcription start point (right-angle arrows) are indicated for both the qacA and qacR genes. Preliminary results indicate that an unknown regulatory protein (?) indirectly influences qacA expression by binding IR2, which overlaps the qacR promoter, PqacR. (B) Structurally diverse compounds that are both substrates of the QacA MDR pump and ligands of the QacR regulator. The chemical structures of representative compounds from four distinct chemical families are depicted. Dequalinium and benzalkonium (where R represents a mixture of alkyls, either C12H25, C14H29, or C16H33) are bivalent and monovalent quaternary ammonium compounds, respectively; rhodamine 6G, ethidium bromide, proflavine, and crystal violet are monovalent dyes; chlorhexidine is a bivalent guanidine; and berberine is a monovalent plant alkaloid.
FIG. 11.
FIG. 11.
Structure of the complex formed by a pair of operator-bound QacR dimers. The two QacR dimers bound to a symmetrical version of IR1 operator DNA are depicted as ribbons, whereas the DNA is shown with the phosphate, oxygen, carbon, and nitrogen atoms colored yellow, red, grey, and blue, respectively. The DNA-reading heads from the distal subunit (purple) of dimer 2 and the proximal subunit (green) of dimer 1 contact the first major groove, whereas the second major groove of IR1 DNA is contacted by the DNA-binding domains from the other two subunits, one from each dimer. The individual α-helices (α1 to α9) of the distal subunit from dimer 2 are labeled, as are the N and C termini of that polypeptide. Additionally, the α8′ and α9′ helices from the proximal subunit of dimer 2, which form the four-helix-bundle dimerization domain with α8 and α9, are also indicated. For the distal (purple) polypeptide of dimer 2, an arrow points to the yellow region at the N terminus of α5 that undergoes a coil-to-helix transition upon ligand binding. (Reprinted with permission from reference ; kindly provided by Maria Schumacher; ©2002, Oxford University Press.)
FIG. 12.
FIG. 12.
DNA contacts formed by QacR to an IR1 half-site. (A) Sequence of the qacA −10 promoter region and the downstream IR1 operator, with the bases that QacR protects from DNase I digestion highlighted (white) against a black background, the qacA transcription start point (tsp) circled, and the location of IR1 indicated by bold arrows that flank the central 6-bp IR1 spacer region (47). (B) The DNA contacts made by a pair of operator-bound QacR dimers to a single IR1 half-site. The bp −1 to −14, which constitute the depicted operator half-site, are shown as a mirror image of A. The QacR residues in bold that contact the DNA in this half-site are from the distal subunit of dimer 2, whereas those in italics are from the proximal subunit of dimer 1 (Fig. 11, purple and green polypeptides, respectively). The contacts made to the bases and DNA backbone in the operator half-site by these amino acids are shown as thin dashed lines for hydrogen bonds and thick broken lines for van der Waals interactions. Also indicated is a DNA contact made by the N terminus of α1. (Panel B reprinted with permission from reference .)
FIG. 13.
FIG. 13.
(A to D) Binding of structurally diverse compounds in the extended QacR ligand-binding pocket. The key QacR residues and relevant α-helices in the ligand-binding site that interact with the compounds rhodamine 6G (R6G, A), ethidium bromide (Eb, B), dequalinium (Dc, C), and crystal violet (Cv, D) are shown in cyan, whereas the QacR glutamate residue(s) involved in electrostatic interactions with the positive charge(s) of each bound drug is colored red. The carbon, nitrogen, and oxygen atoms of each ligand are colored grey, blue, and red, respectively. (E) The QacR DNA-bound conformation (yellow) has been superimposed on the conformation of the QacR subunit to which a ligand is bound (blue). This illustrates the coil-to-helix transition that extends the N terminus of α5 by a turn and the concomitant shoving effect (blue arrow) of α6 against the DNA-binding domain, which produces a dramatic alteration in the position of this three-helix bundle. The location of rhodamine 6G (red) in the drug-bound structure is also indicated, as are α4 to α9, the HTH recognition helix (Hr), and the N and C termini of the protein. (Reprinted with permission from reference ; kindly provided by Maria Schumacher.)
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References

    1. Adewoye, L., A. Sutherland, R. Srikumar, and K. Poole. 2002. The MexR repressor of the mexAB-oprM multidrug efflux operon in Pseudomonas aeruginosa: characterization of mutations compromising activity. J. Bacteriol. 184:4308-4312. - PMC - PubMed
    1. Ahmed, M., C. M. Borsch, S. S. Taylor, N. Vázquez-Laslop, and A. A. Neyfakh. 1994. A protein that activates expression of a multidrug efflux transporter upon binding the transporter substrates. J. Biol. Chem. 269:28506-28513. - PubMed
    1. Ahmed, M., L. Lyass, P. N. Markham, S. S. Taylor, N. Vázquez-Laslop, and A. A. Neyfakh. 1995. Two highly similar multidrug transporters of Bacillus subtilis whose expression is differentially regulated. J. Bacteriol. 177:3904-3910. - PMC - PubMed
    1. Aires, J. R., T. Köhler, H. Nikaido, and P. Plesiat. 1999. Involvement of an active efflux system in the natural resistance of Pseudomonas aeruginosa to aminoglycosides. Antimicrob. Agents Chemother. 43:2624-2628. - PMC - PubMed
    1. Alekshun, M. N., Y. S. Kim, and S. B. Levy. 2000. Mutational analysis of MarR, the negative regulator of marRAB expression in Escherichia coli, suggests the presence of two regions required for DNA binding. Mol. Microbiol. 35:1394-1404. - PubMed

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