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Structure, function and inhibition of RND efux pumps in Gram-negative bacteria: an update
Jessica MA Blair and Laura JV Piddock
Resistance nodulation division efux systems have a major role in both intrinsic and acquired multi-drug resistance in Gramnegative bacteria. The recent structure of an assembled tripartite system, AcrAB-TolC, revealed that AcrB is docked onto TolC, which remains in an open state once part of the assembled complex and three AcrA molecules complete the structure. This is in contrast to data for the MexAB-OprM system of P. aeruginosa that, depending on pH, has between two and six MexA molecules per OprM trimer. RND systems are also important for pathogenicity of several bacteria and for Salmonellae lacking components of AcrAB-TolC, expression of known virulence determinants were signicantly altered. The importance of these systems in both MDR and pathogenicity has made RND systems the target of new drugs aimed at inhibiting their function. The wealth of new structural and functional data will inform rational drug design.
Address Antimicrobial Agents Research Group, School of Immunity and Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK Corresponding author: Piddock, Laura JV (l.j.v.piddock@bham.ac.uk)

multiple classes of structurally distinct antimicrobials, including those that are clinically relevant. Lack of functional RND components renders the bacterium susceptible to these agents, whereas for example, overexpression of AcrB in laboratory mutants of Escherichia coli or Salmonella enterica confers multi-drug resistance (MDR) [14]. Overexpression of RND systems in clinical isolates is also associated with MDR [5]. In addition, MDR bacteria can over-produce more than one efux system simultaneously; for instance, of 450 clinical isolates of ticarcillin resistant Pseudomonas aeruginosa 28% of isolates simultaneously overexpressed MexAB-OprM and MexXY [6]. Similarly, expression of acrA, acrB, acrE and acrF and efux pump encoding genes of other classes was increased in both laboratory selected and clinical uoroquinolone resistant S. Typhimurium [7]. Enhanced efux is also implicated in rapidly emerging clinical resistance to new antimicrobials such as tigecycline, for which overexpression of AcrAB was detected in E. coli and Enterobacter cloacae [8,9]. Acriavine and ethidium bromide are known substrates of AcrAB-TolC. However, a recent model for the transport of these dyes showed that the single component transporters EmrE and MdfA are required for the intrinsic resistance of E. coli to both [10]. These plasma membrane proteins transport the dyes from the cytoplasm to the periplasm where they are captured by AcrAB-TolC and transported across the outer membrane [10].

Current Opinion in Microbiology 2009, 12:512519 This review comes from a themed issue on Antimicrobials Edited by Christopher Walsh and Gerry Wright Available online 5th August 2009 1369-5274/$ see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2009.07.003

Structure
RND pump structure

Introduction
The resistance nodulation division (RND) of efux pumps are found ubiquitously throughout the Bacteria, Archaea and Eukaryotes. In Gram-negative bacteria they are situated within the inner membrane and function in complex with two other proteins, an outer membrane channel and a periplasmic adaptor protein, to form a tripartite efux pump spanning both the inner and outer membrane (Figure 1). These multi-protein complexes transport a wide variety of substrates including antibiotics, dyes, detergents and host derived molecules from the periplasm to the extra-cellular space.

Impact of RND pumps on MDR

Active efux by RND systems plays an important role in the innate resistance of Gram-negative bacteria to
Current Opinion in Microbiology 2009, 12:512519

The crystal structures of the two of the best studied RND transporters, AcrB of E. coli and MexB of P. aeruginosa, have been solved [1113]. AcrB and MexB are closely related with 69% identity and 83% similarity and their structures share many common features [5,13]. Three identical monomers of AcrB and MexB form an integral membrane protein complex within the cytoplasmic membrane [11,13] (Figure 2a). Each monomer has a transmembrane domain composed of 12 membrane-spanning helices and a large periplasmic domain folded in a complex manner to give the pore or porter domain [1214]. The monomers exist in one of three conformations and conformational cycling is responsible for the pumping mechanism of AcrB [1216]. Biochemical evidence to support this hypothesis has emerged recently. Seeger et al. [16] introduced constraining di-sulde bonds between subdomains of AcrB; these inhibited movement and pump function and increased susceptibility to toxic
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Structure, function and inhibition of RND efflux pumps in Gram-negative bacteria: an update Blair and Piddock 513

Figure 1

studies in which sections of this domain from E. coli AcrB were replaced with the highly similar sections from AcrD showed that the substrate specicity of the chimeric protein resembled that of AcrD, not AcrB [25,26]. Furthermore, a single point mutation at Val-610 of E. coli YhiV, also within the periplasmic domain of this RND pump also signicantly altered substrate specicity [27]. The substrate binding pocket of AcrB is rich in aromatic residues, particularly phenylalanines, that interact with the structurally varied substrates [12,14]. AcrB has been crystallised in complex with bile acid; the binding site for this natural substrate is in a similar position to that for other published complexes including ethidium bromide, ciprooxacin and PAbN [21,28]. Site directed mutagenesis was used to show that mutation of different phenylalanine residues within the binding pocket had variable effects on susceptibility to substrates and highlighted the F610 residue as having a particularly signicant and broad impact on susceptibility [29]. Conversely, a substitution at the AcrB 616 residue was found to be particularly relevant for macrolide resistance [30] and a serine 715 substitution had a specic effect on bile resistance [28]. Redundancy of phenylalanines in the pocket may partially account for the varied consequences of substituting different residues. Some substrates are still accommodated in a mutated binding pocket while other larger substrates, such as the macrolides, are unable to adapt to changes caused by a single substitution [29]. The docking domain, which interacts with the outer membrane channel, is highly conserved between AcrB and MexB although the two corresponding outer membrane proteins (TolC and OprM, respectively) cannot complement the others function suggesting that it is the periplasmic component that is responsible for the specicity of the component interactions [13,26]. A protrusion from the docking region into the adjacent RND monomer holds the three monomers together and possibly stabilises the complex [13,31].
Periplasmic adaptor protein structure

Schematic diagram of a tripartite RND system, AcrAB-TolC of E. coli. In Gram-negative bacteria RND pumps (e.g. AcrB) are situated in the inner membrane and function in a complex with two other proteins, an outer membrane channel (e.g. TolC) and a periplasmic adaptor protein (e.g. AcrA). From [90].

compounds. This effect was reversed after exposure to the reducing agent dithiothreitol, removing the constraints [16]. Takatsuka and Nikaido [17] took the novel approach of creating a single giant gene containing three acrB sequences connected by short linker sequences enabling inactivation of a single protomer in the trimeric complex. Inactivation of one protomer abolished the activity of the whole complex conrming that the AcrB trimer is the functional unit and providing strong evidence for the functionally rotating hypothesis [17]. Efux by RND pumps is driven by the proton motive force. The proton translocation site of AcrB is a triplet of charged amino acid residues, Asp 407, Asp 408 and Lys 940 found on the fourth and tenth trans-membrane domains, in a spatially separate location from the substrate binding pocket [14,18,19]. The trans-membrane domains of AcrB and MexB are particularly well conserved with almost all residues being identical, including this amino acid triplet involved in proton translocation suggesting that the mechanism may be common to all RND systems [13]. The periplasmic domain is partially responsible for the substrate specicity of RND pumps [2024]. Chimeric
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The periplasmic adaptor proteins (PAP) (formerly described as membrane fusion proteins) AcrA from E. coli and MexA from P. aeruginosa share 62% sequence identity and 73% similarity but cannot complement each others function [26]. Partial crystal structures of these PAPs revealed three distinct domains: a b-barrel, a central lipoyl domain and an a-helical coiled coil hairpin [3235]. However, these structures lacked the 130 residues forming the N and C termini of the proteins. The structures of PAPs AcrA and MexA was completed in 2009 revealing that the N terminus (residues 1327) and C terminus (residues 262339) together form a membrane proximal (MP) domain that is a compact b-roll linked to the bbarrel domain by a b-ribbon linker (Figure 2b) [36]. This domain is highly conserved between the two
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514 Antimicrobials

Figure 2

The individual completed structures of MexB, MexA and OprM. The crystal structure of the individual components of MexAB-OprM have been solved. (a) MexB is a homotrimer in which each subunit adopts a different conformation. This structure is highly homologous to that of AcrB. From [13]. (b) The complete structure of MexA showing the four domains: the a-helical hairpin in blue, the lipoyl domain in green, the b-barrel in yellow and the newly modelled membrane proximal domain in orange. The blue dotted line represents the 12 residues of the N-terminal strand and the blue atom is the . From [46]. N-terminal cysteine with its lipid tail. (c) OprM is also a homotrimer forming a pore in the membrane with a diameter of 68 A

proteins and the similarity between this MP domain and the b-barrel domain suggests that the structure may have arisen owing to domain duplication [36]. Another study has conrmed the importance of the C-terminal region of AcrA [37]. Conformational exibility has been revealed in the structure of both AcrA [33,34] and MexA [38]. Conformational changes in AcrA were detected in vitro in response to acidic pH, similar to that present in the periplasm [34], and subsequently crystallisation revealed four different AcrA conformations [33]. This exibility comes from the hinges between the lipoyl domain and the a-helical hairpin and b-barrel domains. Further exibility was revealed upon completion of the crystal structure of MexA. The orientation of the newly modelled MP domain was found to differ by up to 858 in relation to the rest of the protein owing to conformational change in the b-linker [36]. The surface of AcrB, which associates with AcrA is relatively stable and does not undergo large conformational changes previously associated with the functionally rotating mechanism [14,31]. On the periplasmic surface the largest conformational change comes from the PC2 domain that is spatially separated from the AcrA attachment site suggesting that once docked, the conformational change required of the PAP would be limited [36].
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Until recently the stoichiometry of RND system components in vivo was unclear [39]. Following the completion of the AcrA crystal structure, evidence from modelling with the complete structures of the three components and validated by cross-linking at pH 7.5 has supported a stoichiometry of 3 AcrB:3 AcrA:3 TolC (Figure 3) [36]. This is consistent with predictions based upon known structures and predicting the most energetically favourable stoichiometric composition [40]. However, this is in contrast with recent data from Reffay et al. concerning the stoichiometry of MexA suggesting variation between two MexA molecules per OprM trimer at pH 7.5 to six MexA molecules per OprM trimer at pH 5.5 [41]. Two putative RND systems have been shown to contain two different PAPs. TriABC-OpmH of P. aeruginosa has TriA and TriB that are both required for efux pump function [42] while ZrpADBC of Serratia sp., includes ZrpA and ZrpD of which only the latter is essential for pump function [43].
Outer membrane channel

The structures of several outer membrane proteins that associate with RND efux systems have been solved [4446] (Figure 2c). TolC is trimeric and each protomer long b-barrel domain anchored to the outer has a 40 A long a-helical domain consisting membrane and a 100 A of 12 coiled coils that project into the periplasm. Once
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Structure, function and inhibition of RND efflux pumps in Gram-negative bacteria: an update Blair and Piddock 515

Figure 3

overall structure despite low sequence identity and lack of conservation of many functionally important residues between all members of this family [45]. Previous models of TolC opening involved the relaxation of the tightly packed helices that obstruct the pore entrance, allowing the helices to rearrange in an iris like movement [48]. More recently, molecular dynamics simulations have suggested that the iris opening analogy to describe TolC opening may be too simplistic and that an additional twisting motion in the upper portion of the periplasmic tunnel leads to a peristaltic action that may aid movement of the substrate along the tunnel [49]. Recent modelling based on extensive site-specic crosslinking, suggested that the interaction between TolC and AcrA was most favourable with TolC in its open state [36]. Symmons et al. [36] proposed that once AcrA is bound to AcrB, TolC is recruited to the complex causing TolC to open and remain in a constitutively open state, allowing the high-throughput transport displayed by this system. In this model, TolC closure is not required because substrates can pass straight from AcrB to TolC without leakage into the periplasm as the connection between the two components is successfully stabilised by AcrA [36] (Figure 3).
A fourth component?

Crystal structures of endogenous AcrB complexed with a novel transmembrane protein, termed YajC, have been reported predicting that AcrB formed a complex with YajC once the AcrAB-TolC efux complex had assembled [50]. This induces a functionally signicant rotation of the porter domain of AcrB relative to the transmembrane domain. Deletion of YajC resulted in only a modest increase in susceptibility to ampicillin and nafcillin. Tornroth-Horseeld [50] also suggested that the N-terminus of YajC could interact with AcrA, transmitting the rotation in AcrB to TolC, facilitating the opening of the channel.

Other functions of RND efux systems

The assembled tri-partite efflux pump AcrAB-TolC. From MF Symmons, V Koronakis, personal communication. The assembled structure of AcrAB-TolC with 3 AcrA:3 AcrB:3 TolC ratio. The AcrB subunits are shown in shades blue, TolC subunits in orange/yellow and AcrA is shown in green. Membrane exposed surfaces and the TolC equatorial domain are shown grey. This model, based on extensive site directed crosslinking data, shows TolC, in its open conformation, is directly docked onto AcrB [36].

assembled the monomers form an outer membrane pore and a periplasmic tunnel that projects into the periplasm in and narrows to a closed end approximately 2 A diameter [44,47]. TolC, OprM and VceC have a similar
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In addition to an established role in MDR, RND efux pumps also contribute to the basic biology of several pathogens. RND systems are required for the virulence of several species as mutants lacking functional RND efux pumps are attenuated [1,5160]. Partly, the contribution of these efux systems to pathogenicity is explained by their involvement in export of host derived substrates such as bile salts, fatty acids and steroid hormones thereby enabling bacteria to survive within the hostile environment of the host [28,6163]. AcrAB from E. coli has a far greater afnity for bile acids than for antibiotics suggesting that AcrB is better adapted for the efux of bile acids [64,65]. Furthermore, expression of acrAB in Salmonella Typhimurium and acrD, acrE, emrK, mdtA and mdtE of E. coli are induced by the presence of
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516 Antimicrobials

indole or bile, both of which are present in the human intestine, allowing these toxic compounds to be exported permitting bacterial survival within the host [66,67]. Another class of host derived molecules that are substrates of RND efux pumps are the antimicrobial peptides (AMPs) [6870]. The human AMP LL-37 and the porcine protegrin-1 are substrates of the MtrCDE complex of Neisseria gonorrhoeae leading to the hypothesis that efux may have evolved as a mechanism to circumvent the innate antimicrobial defences of the host [71]. Furthermore, mutation of mtrR, which represses mtrCDE transcription, led not only to increased resistance to antimicrobials including host derived peptides but also to an increase in tness in the in vivo mouse model of infection suggesting that resistance to AMPs could contribute to virulence [7173]. AMPs may not be substrates of all RND pumps or may have a species-specic role as resistance to several AMPs was not mediated by AcrAB of E. coli or MexAB of P. aeruginosa [74]. In support of the hypothesis that MDR efux pumps are involved in survival in the hostile host environment, during macrophage infection by Salmonella the major facilitator superfamily (MFS) genes emrAB and the RND pump genes mdsABC and mdtABC were increased in expression [75,76]. This suggests a role for these genes during infection. However, expression of acrAB, acrEF and tolC were unaffected [75]. Strains lacking functional RND pump components have an impaired ability to cause infection even when host derived molecules are absent. For Salmonellae the attenuation due to lack of TolC has been associated with reduced production of Salmonella Pathogenicity Island (SPI-1) genes [77,78]. Likewise, inactivation of acrB led to reduced expression of SPI-1 genes and its effectors and other pathogenicity related genes [78]. Webber et al. [78] also showed that lack of functional AcrA was associated with decreased expression of genes found in SPI-2.
Figure 4

Inactivation of acrA, acrB or tolC had different effects on the transcriptome suggesting that the attenuation of virulence in strains lacking AcrA, AcrB or TolC could be mediated by different mechanisms. P. aeruginosa lacking the MexAB-OprM efux system had a reduced ability to invade Madin-Darby Canine Kidney (MDCK) cells and invasiveness was restored not only by complementation of the disrupted genes but also by addition of culture supernatant from MDCK cells infected with wild-type P. aeruginosa. This suggests that the MexAB-OprM system exports a factor, present in the supernatant, that is able to ameliorate the virulence defect in the mutant [54]. It has also been suggested that AcrAB and its homologues export potentially toxic cellular metabolites, although evidence for this hypothesis is limited [7981]. A novel physiological role for AcrB in E. coli in contactdependent growth inhibition (CDI) has been proposed that is independent of both AcrA and TolC [82]. CDI is the regulation of growth via cell to cell contact and signicant numbers of mutants with resistance to CDI showed mutations in acrB or bamA, the latter of which is a member of the Omp85 family of essential outer membrane proteins. The specic role of AcrB in this process has not been elucidated but it may be a downstream target for the CDI signal [82].
Inhibition of efux

The role of RND systems in innate and acquired MDR and virulence makes these proteins attractive targets for development of combination therapy involving an antibiotic administered with an inhibitor of efux. In this way it is hoped that the clinical effectiveness of the remaining agents with activity against Gram-negative pathogens can ` s and Amaral be preserved. A recent review by Page discusses in detail the most recent advances in efux pump inhibitors (EPIs) so here we focus only on recent data for RND efux pumps [83].

The structures of PAbN and chlorpromazine. The chemical structure of (a) PAbN and (b) chlorpromazine. Current Opinion in Microbiology 2009, 12:512519 www.sciencedirect.com

Structure, function and inhibition of RND efflux pumps in Gram-negative bacteria: an update Blair and Piddock 517

PAbN was the rst molecule identied as an EPI against P. aeruginosa MexB but is now thought to active against many more RND systems (Figure 4a) [84,85]. This molecule is a competitive efux pump inhibitor that is preferentially pumped out by MexB. This means that less antibiotic is exported so intracellular accumulation is higher and toxicity is reached at lower concentrations leading to restoration of antibiotic sensitivity. PAbN is also thought to target C. jejuni CmeABC as addition increased susceptibility to bile and and oral administration to chickens 24 days post-inoculation signicantly reduced C. jejuni colonisation [86]. The anti-psychotic phenothiazine drugs have also emerged as possible EPIs with one member, chlorpromazine, showing EPI-like activity for Salmonella Typhimurium, Burkholderia pseudomallei and E. coli (Figure 4b) [8789]. Bailey et al. [88] showed that chlorpromazine reduced expression of acrB suggesting that the EPI-like activity was due to inhibition of AcrB production rather than interaction with the AcrB protein [88]. The wealth of new structural data including the publication of the rst complete structure of a tripartite RND pump complex has provided an unprecedented insight into the function of these systems. These data will inform future rational design of antimicrobials targeted against these efux systems.

6.

Hocquet D, Roussel-Delvallez M, Cavallo J-D, Plesiat P: MexABOprM- and MexXY-overproducing mutants are very prevalent among clinical strains of Pseudomonas aeruginosa with reduced susceptibility to ticarcillin. Antimicrob Agents Chemother 2007, 51:1582-1583. Chen S, Cui S, McDermott PF, Zhao S, White DG, Paulsen I, Meng J: Contribution of target gene mutations and efux to decreased susceptibility of Salmonella enterica serovar Typhimurium to uoroquinolones and other antimicrobials. Antimicrob Agents Chemother 2007, 51:535-542. Keeney D, Ruzin A, Bradford PA: RamA, a transcriptional regulator, and AcrAB, an RND-type efux pump, are associated with decreased susceptibility to tigecycline in Enterobacter cloacae. Microb Drug Resist 2007, 13:1-6. Keeney D, Ruzin A, McAleese F, Murphy E, Bradford PA: MarAmediated overexpression of the AcrAB efux pump results in decreased susceptibility to tigecycline in Escherichia coli. J Antimicrob Chemother 2008, 61:46-53.

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10. Tal N, Schuldiner S: A coordinated network of transporters with overlapping specicities provides a robust survival strategy. Proc Natl Acad Sci 2009, 106:9051-9056. 11. Murakami S, Nakashima R, Yamashita E, Yamaguchi A: Crystal structure of bacterial multidrug efux transporter AcrB. Nature 2002, 419:587-593. 12. Seeger MA, Schiefner A, Eicher T, Verrey F, Diederichs K, Pos KM: Structural asymmetry of AcrB trimer suggests a peristaltic pump mechanism. Science 2006, 313:1295-1298. tter MG: Crystal 13. Sennhauser G, Bukowska MA, Briand C, Gru structure of the multidrug exporter MexB from Pseudomonas aeruginosa. J Mol Biol 2009, 389:134-145. 14. Murakami S, Nakashima R, Yamashita E, Matsumoto T, Yamaguchi A: Crystal structures of a multidrug transporter reveal a functionally rotating mechanism. Nature 2006, 443:173-179. 15. Sennhauser G, Amstutz P, Briand C, Storchenegger O, tter MG: Drug export pathway of multidrug exporter AcrB Gro revealed by DARPin inhibitors. PLoS Biol 2007, 5:e7. 16. Seeger MA, von Ballmoos C, Eicher T, Brandstatter L, Verrey F, Diederichs K, Pos KM: Engineered disulde bonds support the functional rotation mechanism of multidrug efux pump AcrB. Nat Struct Mol Biol 2008, 15:199-205. 17. Takatsuka Y, Nikaido H: Covalently linked trimer of the AcrB  multidrug efux pump provides support for the functional rotating mechanism. J Bacteriol 2009, 191:1729-1737. These authors use a very novel methodology to provide support for the hypothesis of a functionally rotating mechanism of AcrB. 18. Takatsuka Y, Nikaido H: Threonine-978 in the transmembrane segment of the multidrug efux pump AcrB of Escherichia coli is crucial for drug transport as a probable component of the proton relay network. J Bacteriol 2006, 188:7284-7289. 19. Seeger MA, von Ballmoos C, Verrey F, Pos KM: Crucial role of Asp408 in the proton translocation pathway of multidrug transporter AcrB: evidence from site-directed mutagenesis and carbodiimide labeling. Biochemistry 2009, 48:5801-5812. 20. Yu EW, McDermott G, Zgurskaya HI, Nikaido H, Koshland DE Jr: Structural basis of multiple drug-binding capacity of the AcrB multidrug efux pump. Science 2003, 300:976-980. 21. Yu EW, Aires JR, McDermott G, Nikaido H: A periplasmic drugbinding site of the AcrB Multidrug efux pump: a crystallographic and site-directed mutagenesis study. J Bacteriol 2005, 187:6804-6815. 22. Hearn EM, Gray MR, Foght JM: Mutations in the central cavity and periplasmic domain affect efux activity of the resistancenodulation-division pump EmhB from Pseudomonas uorescens cLP6a. J Bacteriol 2006, 188:115-123. 23. Mao W, Warren MS, Black DS, Satou T, Murata T, Nishino T, Gotoh T, Lomovskaya O: On the mechanism of substrate specicity by resistance nodulation division (RND)-type Current Opinion in Microbiology 2009, 12:512519

Acknowledgements
We are very grateful to Martyn Symmons for helpful discussions and providing the diagram of the complete AcrAB-TolC system (Figure 3) and for the edited MexA structure (Figure 2b) that were presented in a different format to those in [36]. We also thank Vassillis Koronakis. tter and Atsushi Nakagawa for We are also grateful to Klaas Pos, Markus Gru permission to reproduce Figures 1 and 2a,c, respectively.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1. Nishino K, Lati T, Groisman EA: Virulence and drug resistance roles of multidrug efux systems of Salmonella enterica serovar Typhimurium. Mol Microbiol 2006, 59:126-141. Baucheron S, Tyler S, Boyd D, Mulvey MR, Chaslus-Dancla E, Cloeckaert A: AcrAB-TolC directs efux-mediated multidrug resistance in Salmonella enterica serovar Typhimurium DT104. Antimicrob Agents Chemother 2004, 48:3729-3735. Eaves DJ, Ricci V, Piddock LJV: Expression of acrB, acrF, acrD, marA, and soxS in Salmonella enterica serovar Typhimurium: role in multiple antibiotic resistance. Antimicrob Agents Chemother 2004, 48:1145-1150. Giraud E, Cloeckaert A, Kerboeuf D, Chaslus-Dancla E: Evidence for active efux as the primary mechanism of resistance to ciprooxacin in Salmonella enterica serovar Typhimurium. Antimicrob Agents Chemother 2000, 44:1223-1228. Piddock LJV: Clinically relevant chromosomally encoded multidrug resistance efux pumps in bacteria. Clin Microbiol Rev 2006, 19:382-402.

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multidrug resistance pumps: the large periplasmic loops of MexD from Pseudomonas aeruginosa are involved in substrate recognition. Mol Microbiol 2002, 46:889-901. 24. Middlemiss JK, Poole K: Differential impact of MexB mutations on substrate selectivity of the MexAB-OprM multidrug efux pump of Pseudomonas aeruginosa. J Bacteriol 2004, 186:1258-1269. 25. Elkins CA, Nikaido H: Substrate specicity of the RND-type multidrug efux pumps AcrB and AcrD of Escherichia coli is determined predominately by two large periplasmic loops. J Bacteriol 2002, 184:6490-6498. 26. Tikhonova EB, Wang Q, Zgurskaya HI: Chimeric analysis of the multicomponent multidrug efux transporters from gramnegative bacteria. J Bacteriol 2002, 184:6499-6507. 27. Bohnert JA, Schuster S, Fahnrich E, Trittler R, Kern WV: Altered spectrum of multidrug resistance associated with a single point mutation in the Escherichia coli RND-type MDR efux pump YhiV (MdtF). J Antimicrob Chemother 2007, 59:1216-1222. 28. Drew D, Klepsch MM, Newstead S, Flaig R, De Gier J-W, Iwata S, Beis K: The structure of the efux pump AcrB in complex with bile acid. Mol Membr Biol 2008, 25:677-682. 29. Bohnert JA, Schuster S, Seeger MA, Fahnrich E, Pos KM, Kern WV: Site-directed mutagenesis reveals putative substrate binding residues in the Escherichia coli RND efux pump AcrB. J Bacteriol 2008, 190:8225-8229. 30. Wehmeier C, Schuster S, Fahnrich E, Kern WV, Bohnert JA: Sitedirected mutagenesis reveals amino acid residues in the Escherichia coli RND efux pump AcrB that confer macrolide resistance. Antimicrob Agents Chemother 2009, 53:329-330. 31. Seeger MA, Diederichs K, Eicher T, Brandstatter L, Schiefner A, Verrey F, Pos KM: The AcrB efux pump: conformational cycling and peristalsis lead to multidrug resistance. Curr Drug Targets 2008, 9:729-749. 32. Akama H, Matsuura T, Kashiwagi S, Yoneyama H, Narita S-i, Tsukihara T, Nakagawa A, Nakae T: Crystal structure of the membrane fusion protein, MexA, of the multidrug transporter in Pseudomonas aeruginosa. J Biol Chem 2004, 279:25939-25942. 33. Mikolosko J, Bobyk K, Zgurskaya HI, Ghosh P: Conformational exibility in the multidrug efux system protein AcrA. Structure 2006, 14:577-587. 34. Ip H, Stratton K, Zgurskaya H, Liu J: pH-induced conformational changes of AcrA, the membrane fusion protein of Escherichia coli multidrug efux system. J Biol Chem 2003, 278:50474-50482. 35. Higgins MK, Bokma E, Koronakis E, Hughes C, Koronakis V: Structure of the periplasmic component of a bacterial drug efux pump. Proc Natl Acad Sci U S A 2004, 101:9994-9999. 36. Symmons MF, Bokma E, Koronakis E, Hughes C, Koronakis V: The  assembled structure of a complete tripartite bacterial multidrug efux pump. Proc Natl Acad Sci U S A 2009, 106:7173-7178. This excellent paper is the rst to solve the complete structure of the periplasmic adaptor protein MexA and also the complete structure of AcrAB-TolC. The stoichiometry of the components of the system is shown to be 3:3:3. 37. Ge Q, Yamada Y, Zgurskaya H: The C-terminal domain of AcrA is essential for the assembly and function of the multidrug efux pump AcrAB-TolC. J Bacteriol 2009, 191:4365-4371. 38. Vaccaro L, Koronakis V, Sansom MSP: Flexibility in a drug transport accessory protein: molecular dynamics simulations of MexA. Biophys J 2006, 91:558-564. 39. Zgurskaya HI, Yamada Y, Tikhonova EB, Ge Q, Krishnamoorthy G: Structural and functional diversity of bacterial membrane fusion proteins. Biochim Biophys Acta (BBA) Proteins Proteomics 2008, 1794:794-807. 40. Fernandez-Recio J, Walas F, Federici L, Venkatesh Pratap J, Bavro VN, Miguel RN, Mizuguchi K, Luisi B: A model of a Current Opinion in Microbiology 2009, 12:512519

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