pH-induced Conformational Changes of AcrA, the Membrane Fusion Protein of Escherichia coli Multidrug Efflux System

The multidrug efflux system AcrA-AcrB-TolC of Escherichia coli expels a wide range of drugs directly into the external medium from the bacterial cell. The mechanism of the efflux process is not fully understood. Of an elongated shape, AcrA is thought to span the periplasmic space coordinating the concerted operation of the inner and outer membrane proteins AcrB and TolC. In this study, we used site-directed spin labeling (SDSL) EPR (electron paramagnetic resonance) spectroscopy to investigate the molecular conformations of AcrA in solution. Ten AcrA mutants, each with an alanine to cysteine substitution, were engineered, purified, and labeled with a nitroxide spin label. EPR analysis of spin-labeled AcrA variants indicates that the side chain mobilities are consistent with the predicted secondary structure of AcrA. We further demonstrated that acidic pH induces oligomerization and conformational change of AcrA, and that the structural changes are reversible. These results suggest that the mechanism of action of AcrA in drug efflux is similar to the viral membrane fusion proteins, and that AcrA actively mediates the efflux of substrates. The multidrug efflux system AcrA-AcrB-TolC of Escherichia coli expels a wide range of drugs directly into the external medium from the bacterial cell. The mechanism of the efflux process is not fully understood. Of an elongated shape, AcrA is thought to span the periplasmic space coordinating the concerted operation of the inner and outer membrane proteins AcrB and TolC. In this study, we used site-directed spin labeling (SDSL) EPR (electron paramagnetic resonance) spectroscopy to investigate the molecular conformations of AcrA in solution. Ten AcrA mutants, each with an alanine to cysteine substitution, were engineered, purified, and labeled with a nitroxide spin label. EPR analysis of spin-labeled AcrA variants indicates that the side chain mobilities are consistent with the predicted secondary structure of AcrA. We further demonstrated that acidic pH induces oligomerization and conformational change of AcrA, and that the structural changes are reversible. These results suggest that the mechanism of action of AcrA in drug efflux is similar to the viral membrane fusion proteins, and that AcrA actively mediates the efflux of substrates. Emergence of multidrug-resistant bacterial strains not only has hampered the current treatment of bacterial infections but also hindered the development of new therapeutic agents. Resistance mediated by multidrug efflux pumps as a major mechanism has been increasingly recognized. Available clinical data showed that 40–90% of some bacterial pathogens (Streptococcus pneumoniae, Streptococcus pyogenes, and Pseudomonas aeruginosa) bear efflux mechanisms for the major classes of available antibiotics (1.Brenwald N.P. Gill M.J. Wise R. Antimicrob. Agents Chemother. 1998; 42: 2032-2035Crossref PubMed Google Scholar, 2.Limia A. Jimenez M.L. Delgado T. Sanchez I. Lopez S. Lopez B. Rev. Esp. Quimioter. 1998; 11: 216-220PubMed Google Scholar, 3.Nikaido H. Clin. Infect. Dis. 1998; 27 (Suppl. 1, –S41): S32Crossref PubMed Scopus (288) Google Scholar, 4.Orden B. Perez T. Montes M. Martinez R. Pediatr. Infect. Dis. J. 1998; 17: 470-473Crossref PubMed Scopus (44) Google Scholar, 5.Shortridge V.D. Doern G.V. Brueggemann A.B. Beyer J.M. Flamm R.K. Clin. Infect. Dis. 1999; 29: 1186-1188Crossref PubMed Scopus (157) Google Scholar). Many drug efflux pumps have broad substrate specificity and expel a wide range of completely unrelated chemotherapeutic drugs. The AcrA-AcrB-TolC efflux system of Escherichia coli is such an example and is largely responsible for the intrinsic resistance of E. coli to most lipophilic antibiotics, detergents, and dyes (6.Nikaido H. J. Bacteriol. 1996; 178: 5853-5859Crossref PubMed Scopus (873) Google Scholar, 7.Nikaido H. Zgurskaya H.I. J. Mol. Microbiol. Biotechnol. 2001; 3: 215-218PubMed Google Scholar). This system consists of a resistance-nodulation-cell division (RND) type efflux pump, AcrB, a periplasmic, membrane fusion protein (MFP), 1The abbreviations used are: MFPmembrane fusion proteinDTTdithiothreitolMES4-morpholineethanesulfonic acidHAhemagglutininMTSLmethanethiosulfonate spin labelSDSLsite-directed spin labelingDSCdifferential scanning calorimetry. AcrA, and a multifunctional outer membrane channel, TolC. Such organization allows the bacterium expel a wide variety of noxious compounds from the cell directly into the medium, bypassing the periplasm (8.Zgurskaya H.I. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7190-7195Crossref PubMed Scopus (304) Google Scholar). Similar multicomponent efflux systems have been found in other Gram-negative bacteria including P. aeruginosa, Enterobacter aerogenes, and Neisseria gonorrhoeae (3.Nikaido H. Clin. Infect. Dis. 1998; 27 (Suppl. 1, –S41): S32Crossref PubMed Scopus (288) Google Scholar, 6.Nikaido H. J. Bacteriol. 1996; 178: 5853-5859Crossref PubMed Scopus (873) Google Scholar). membrane fusion protein dithiothreitol 4-morpholineethanesulfonic acid hemagglutinin methanethiosulfonate spin label site-directed spin labeling differential scanning calorimetry. Major progresses have been made for understanding the efflux mechanism in Gram-negative bacteria, which are highlighted by recent publications of the crystal structures of TolC and AcrB (9.Koronakis V. Sharff A. Koronakis E. Luisi B. Hughes C. Nature. 2000; 405: 914-919Crossref PubMed Scopus (870) Google Scholar, 10.Murakami S. Nakashima R. Yamashita E. Yamaguchi A. Nature. 2002; 419: 587-593Crossref PubMed Scopus (766) Google Scholar, 11.Yu E.W. McDermott G. Zgurskaya H.I. Nikaido H. Koshland Jr., D. Science. 2003; 300: 976-980Crossref PubMed Scopus (338) Google Scholar). The TolC trimer comprises of two barrel-like structures joined together, the outer membrane β-barrel and the periplasmic α-barrel. The long α-barrel (∼100 Å) is thought to traverse the periplasm and interact with AcrB or inner membrane (9.Koronakis V. Sharff A. Koronakis E. Luisi B. Hughes C. Nature. 2000; 405: 914-919Crossref PubMed Scopus (870) Google Scholar). Consistently, the AcrB protein, also as a trimer, contains two structural domains, the transmembrane domain (50 Å in thickness) and a headpiece that protrudes about 70 Å in depth into the periplasm (10.Murakami S. Nakashima R. Yamashita E. Yamaguchi A. Nature. 2002; 419: 587-593Crossref PubMed Scopus (766) Google Scholar). It is thought that AcrB and TolC may be directly docked with each other, forming a continuous pathway across the periplasm and the outer membrane. Substrates may gain access to the AcrB central cavity either from the cell interior through the transmembrane region, or from the periplasm through the vestibules of AcrB protein, which are then actively transported through the AcrB pore into the TolC tunnel (10.Murakami S. Nakashima R. Yamashita E. Yamaguchi A. Nature. 2002; 419: 587-593Crossref PubMed Scopus (766) Google Scholar). Indeed, AcrB structures with four structurally diverse substrates demonstrated that they bind to the large central cavity of AcrB (11.Yu E.W. McDermott G. Zgurskaya H.I. Nikaido H. Koshland Jr., D. Science. 2003; 300: 976-980Crossref PubMed Scopus (338) Google Scholar). An important question remains to be addressed is the role of AcrA in the efflux process. AcrA is essential for drug efflux, but how AcrA participates in this process is not fully understood. In its mature form, AcrA carries a diacylglycerol group and a palmitic acid chain linked to the N-terminal cysteine residue, which is believed to anchor the protein to the inner membrane. However, the lipid-deficient variant of AcrA carrying a His tag at the C-terminal is functional and has been used for biochemical studies (12.Zgurskaya H.I. Nikaido H. J. Mol. Biol. 1999; 285: 409-420Crossref PubMed Scopus (173) Google Scholar). The secondary structure predictions of AcrA suggest that AcrA and its MFP homologs contain two regions of high coiled-coil probability of approximately equal length, flanked by two lipoyl/biotin-binding motifs that are likely β-strands (13.Johnson J.M. Church G.M. J. Mol. Biol. 1999; 287: 695-715Crossref PubMed Scopus (143) Google Scholar). Although the high resolution structure is not available (14.Avila S. Misaghi S. Wilson K. Downing K.H. Zgurskaya H. Nikaido H. Nogales E. J. Struct. Biol. 2001; 136: 81-88Crossref PubMed Scopus (39) Google Scholar), AcrA was found to be a highly asymmetric molecule with an elongated shape of about 200 Å in length (12.Zgurskaya H.I. Nikaido H. J. Mol. Biol. 1999; 285: 409-420Crossref PubMed Scopus (173) Google Scholar). Cross-linking experiments showed that AcrA forms a complex with AcrB (15.Zgurskaya H.I. Nikaido H. J. Bacteriol. 2000; 182: 4264-4267Crossref PubMed Scopus (141) Google Scholar), and may interact with TolC. Therefore, AcrA could provide a seamless link between AcrB and TolC. Alternatively, it may simply bring the outer and inner membranes closer for substrate transfer. A better knowledge of the structure and function of AcrA is essential for understanding the efflux process. In this study, we used a sensitive biophysical method, the site-directed spin labeling (SDSL), for studying the structure and dynamics of AcrA. SDSL utilizes site-directed mutagenesis to replace the residue of interest in a protein with a cysteine, which is then modified with a sulfhydryl-specific nitroxide to introduce the paramagnetic side chain (Fig. 1). Electron paramagnetic resonance (EPR) spectroscopic analysis of the spin label yields spectral characteristics that are dependent on the local environment, which in turn provide information on the structure and dynamics of the protein (16.Hubbell W.L. Gross A. Langen R. Lietzow M.A. Curr. Opin. Struct. Biol. 1998; 8: 649-656Crossref PubMed Scopus (500) Google Scholar, 17.Hubbell W.L. Cafiso D.S. C. Struct. Biol. 2000; PubMed Scopus Google Scholar). this method, we that AcrA is a protein that conformational by changes of Such conformational changes may be important for the action of AcrA the drug efflux process. and E. coli strains were at in were to the and were by and in the of E. a of was into a and AcrB protein the The (12.Zgurskaya H.I. Nikaido H. J. Mol. Biol. 1999; 285: 409-420Crossref PubMed Scopus (173) Google was used to for the and of cysteine fusion protein, the of protein was to the mature of AcrA the AcrA and the N-terminal cysteine The tag was at the C-terminal of AcrA. cysteine were into the were by of and of functional of variants was by of this variants were into carrying H. D. Nikaido H. J. Bacteriol. 1996; 178: PubMed Scopus Google Scholar). at of were at a of into medium in the of of the drug was at The of of were by analysis to E. coli were by and by in and to In the of the was from the and E. coli or strains with cysteine were The was into of medium with antibiotics and at of protein was by the of at were by with pH and by AcrA protein was from the cell to the with a the of in the was protein was pH protein is for at at was and protein was at The protein was as by and were at the or the protein and EPR was to the protein in the and at for were then the label was at with to the AcrA and for at spin were by experiments at pH pH was used for were by and to labeled AcrA in was with AcrA at protein for at The was then and to EPR EPR were a The were and a of and was G. EPR were the by the experiments were on a The protein is at and the is analysis was by the and in AcrA protein has a cysteine residue, which is the for D. M. Nikaido H. J. Bacteriol. PubMed Google Scholar). However, analysis that residue are essential for AcrA function (12.Zgurskaya H.I. Nikaido H. J. Mol. Biol. 1999; 285: 409-420Crossref PubMed Scopus (173) Google Scholar). We have used the variant with a tag on the as a to cysteine AcrA were by a and by analysis predicted that AcrA has a highly central domain with a high to a coiled-coil and a C-terminal domain (13.Johnson J.M. Church G.M. J. Mol. Biol. 1999; 287: 695-715Crossref PubMed Scopus (143) Google Scholar, T. J. Bacteriol. PubMed Google Scholar). In the central of AcrA is by to lipoyl/biotin-binding domain (13.Johnson J.M. Church G.M. J. Mol. Biol. 1999; 287: 695-715Crossref PubMed Scopus (143) Google Scholar). We cysteine in of AcrA (Fig. could be in E. coli to the similar of AcrA not We not which could protein by most cysteine were by the AcrA-AcrB-TolC proteins multidrug-resistant of the of In two carrying and only resistance to and with the AcrA. However, fully resistance to other including and and Although data not of drug efflux are they suggest that the proteins are not of for E. coli AcrA coli AcrA in a new and of gain into the molecular and dynamics of AcrA in variants were and by the and to spin labeling by a nitroxide with the by and EPR that they are not the structure and are to which is in to a most cysteine not the function of AcrA We also the of spin label AcrA by its with spin-labeled a at a of as by the in The cysteine and the AcrA protein were also The data were to a and the are in It is that changes of and in the of spin label the range of found for at by cysteine with the of which is by the of These results with from studies that a of the or function of a protein Lietzow M.A. K. W.L. 1996; PubMed Scopus Google of AcrA and in a new of the EPR of spin-labeled AcrA at pH The are in of a of the of in the the central and the spectral that is by the of between the two have been to the of spin which the and of W.L. Cafiso D.S. C. Struct. Biol. 2000; PubMed Scopus Google Scholar). Such is by the of a residue that its protein between side chain and the of protein structure and dynamics have been in proteins including Lietzow M.A. K. W.L. 1996; PubMed Scopus Google Scholar), W.L. Sci. 1999; 8: PubMed Scopus Google Scholar), R. J.M. W.L. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar), and the domain of H. C. K. W.L. Science. 1996; PubMed Scopus Google Scholar). In are highly at or have complex multicomponent at and have high at or in The side chain of AcrA, as was in The of and are by and a high of This that the of and of AcrA are largely or with the of the This is consistent with the that AcrA is in an in and the and not interact with each other but are on two of the protein molecule (12.Zgurskaya H.I. Nikaido H. J. Mol. Biol. 1999; 285: 409-420Crossref PubMed Scopus (173) Google Scholar). In other which are characteristics of side structures or in The and that are of The of is an about an to the nitroxide was for residue of which is on the of a and with side Lietzow M.A. K. W.L. 1996; PubMed Scopus Google Scholar). The for the of in has been and was to the W.L. Sci. 2002; PubMed Scopus Google Scholar). with the EPR and are at the predicted two regions with high probability of forming coiled-coil (Fig. chain of spin-labeled AcrA in a new and have two spectral two of mobilities and in other spin label to a cysteine in protein of at two the structural The crystal structures of spin-labeled proteins that the which are by of the side chain with side or R. D. W.L. 2000; PubMed Scopus Google Scholar). and be in and may also have some of (Fig. and are to the predicted and which are β-strands (Fig. (13.Johnson J.M. Church G.M. J. Mol. Biol. 1999; 287: 695-715Crossref PubMed Scopus (143) Google Scholar). and are to the C-terminal domain T. J. Bacteriol. PubMed Google Scholar). Although secondary structures were predicted for regions of and the EPR that are In the EPR are with the predicted structure of AcrA on of AcrA by of most bacterial efflux utilizes the as the for drug (6.Nikaido H. J. Bacteriol. 1996; 178: 5853-5859Crossref PubMed Scopus (873) Google Scholar). In E. coli about of the across the membrane from a with the pH the external pH by about pH H. Antimicrob. Agents Chemother. PubMed Scopus Google Scholar). The in studies showed that AcrA the of AcrB by between two (8.Zgurskaya H.I. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7190-7195Crossref PubMed Scopus (304) Google Scholar). In AcrA could of of membrane the is a pH across the of interior is and external (8.Zgurskaya H.I. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7190-7195Crossref PubMed Scopus (304) Google Scholar). of membrane fusion by pH has been in the viral membrane fusion protein, hemagglutinin is thought be in a at pH and into its by to an acidic Rev. 2000; PubMed Scopus Google Scholar). Therefore, we AcrA conformational the is the pH from to changes of EPR of and but or change of the and and of not further spectral changes not The most spectral change by acidic pH was at and pH the of and a in that the local protein structure in the of a conformational change and residue was in a and a similar conformational they in at pH the EPR of at pH and pH the at pH was as the of spectral and the of the which are characteristics of that spin are at AcrA variants are labeled proteins, this indicates that acidic pH induces oligomerization of AcrA, and that residue is in further protein was with AcrA protein in and the Indeed, at pH of spin-labeled AcrA to spectral (Fig. that residue is a AcrA of protein at pH with AcrA also the at pH AcrA may an of other spin-labeled proteins including and with protein not change either at pH or (Fig. consistent with the that the spectral changes of conformational The conformational of AcrA by acidic pH is the pH was from to the EPR of (Fig. and were to at pH we found that the pH from to not change the of (Fig. that the conformational changes of AcrA is not by the but may a a pH of viral membrane fusion protein This with a that the of of pH of the is only at external pH of but not at pH (8.Zgurskaya H.I. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7190-7195Crossref PubMed Scopus (304) Google Scholar), the conformational changes of AcrA is for membrane of AcrA in in studies that in the periplasm AcrA as an most a trimer (15.Zgurskaya H.I. Nikaido H. J. Bacteriol. 2000; 182: 4264-4267Crossref PubMed Scopus (141) Google Scholar). investigate of cysteine of AcrA through the of we also cell from E. coli by by with We found that variants in the of the The analysis has that with the of and high molecular which (Fig. We the of by the of in the of AcrA was the only protein in high molecular (Fig. are most by in and These are to the and of AcrA. The molecular of by is about which is to the molecular of AcrA from the acid The and by protein with molecular of about These high molecular could to AcrA (Fig. the other the in the could as a of in of variants by Indeed, the molecular of by in other is of high molecular could to either AcrA or These results the that in AcrA as an (15.Zgurskaya H.I. Nikaido H. J. Bacteriol. 2000; 182: 4264-4267Crossref PubMed Scopus (141) Google Scholar). protein oligomerization is sensitive to protein the of that in the conformations of and variants are AcrA are in a the are between cysteine in the in the of AcrA. The of Gram-negative bacteria as an noxious compounds in the However, the outer which not have access to and the periplasmic which is about Å in depth J. B. J. Bacteriol. PubMed Google Scholar, R. J. Bacteriol. PubMed Google Scholar), have to be this a multicomponent that the inner the and the outer membrane is (6.Nikaido H. J. Bacteriol. 1996; 178: 5853-5859Crossref PubMed Scopus (873) Google Scholar). Such such as the AcrA-AcrB-TolC of E. allows bacterial to expel substrates directly into the AcrA is thought to the concerted operation of AcrB and TolC such that the substrates are through the complex into the AcrA this is not fully understood. In this study, we the that AcrA is a protein that oligomerization and conformational and suggest that AcrA an role in the drug efflux process. Although we not at the conformations of AcrA the in its conformational in it is that AcrA could conformational with AcrB, and changes of the local the which the of AcrB and TolC. of AcrA action were K. Curr. Biol. 2000; PubMed Scopus Google Scholar, H.I. Nikaido H. Mol. Microbiol. 2000; PubMed Scopus Google Scholar). that AcrA as an to TolC and AcrB, which forms a with TolC and a for the substrates to through the periplasm K. Curr. Biol. 2000; PubMed Scopus Google Scholar). The other that AcrA simply the outer and inner membranes into to substrate H.I. Nikaido H. Mol. Microbiol. 2000; PubMed Scopus Google Scholar). This was on the AcrA to a of membrane fusion protein found in Gram-negative bacteria T. J. Bacteriol. PubMed Google Scholar), which with a membrane fusion protein, In structural of such as the of coiled-coil and two regions the and which are of viral membrane fusion proteins (13.Johnson J.M. Church G.M. J. Mol. Biol. 1999; 287: 695-715Crossref PubMed Scopus (143) Google Scholar). This is further by including that AcrA is a highly asymmetric molecule with a length of about 200 to span the periplasm (12.Zgurskaya H.I. Nikaido H. J. Mol. Biol. 1999; 285: 409-420Crossref PubMed Scopus (173) Google Scholar), and that the AcrA protein is to the of two and a (8.Zgurskaya H.I. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7190-7195Crossref PubMed Scopus (304) Google Scholar). In the crystal structure of AcrB that which protrudes Å into the AcrB contains an that into the periplasm by 70 Å (9.Koronakis V. Sharff A. Koronakis E. Luisi B. Hughes C. Nature. 2000; 405: 914-919Crossref PubMed Scopus (870) Google Scholar, 10.Murakami S. Nakashima R. Yamashita E. Yamaguchi A. Nature. 2002; 419: 587-593Crossref PubMed Scopus (766) Google Scholar). The of the periplasmic length of AcrB and TolC is about which could be to the periplasmic in this provide new into the mechanism of AcrA action in the efflux process. data suggest that AcrA could in a cysteine in in of AcrA that in AcrA is an (15.Zgurskaya H.I. Nikaido H. J. Bacteriol. 2000; 182: 4264-4267Crossref PubMed Scopus (141) Google Scholar). in the AcrA are in the are between at the in the of AcrA. In AcrA in as a (12.Zgurskaya H.I. Nikaido H. J. Mol. Biol. 1999; 285: 409-420Crossref PubMed Scopus (173) Google Scholar). We found that acidic pH induces oligomerization of AcrA in solution. pH that only spin to was at residue and was by protein, that AcrA forms this and the residue is in The was the of protein was but was not at not that AcrA as a at The for the was to be by EPR of at and The of in AcrA remains resolution structure of AcrA is consistent with the of AcrA (14.Avila S. Misaghi S. Wilson K. Downing K.H. Zgurskaya H. Nikaido H. Nogales E. J. Struct. Biol. 2001; 136: 81-88Crossref PubMed Scopus (39) Google Scholar). However, in studies showed that with the Å AcrA (15.Zgurskaya H.I. Nikaido H. J. Bacteriol. 2000; 182: 4264-4267Crossref PubMed Scopus (141) Google Scholar). is also by analysis of coiled-coil of (13.Johnson J.M. Church G.M. J. Mol. Biol. 1999; 287: 695-715Crossref PubMed Scopus (143) Google Scholar). pH not only the oligomerization but also conformational changes of AcrA, which were in local structures of and This is most in the The EPR of at pH was and a that a of side chain to a environment, with side or residue the spectral of at pH was not by with protein, further the that the local structure of residue conformational of in Similar results were for and and Church (13.Johnson J.M. Church G.M. J. Mol. Biol. 1999; 287: 695-715Crossref PubMed Scopus (143) Google mechanism by which a MFP could bring the outer and inner membranes It was that is a of the coiled-coil and regions and that a MFP could simply on at the between the forming an (13.Johnson J.M. Church G.M. J. Mol. Biol. 1999; 287: 695-715Crossref PubMed Scopus (143) Google Scholar). the of AcrA that conformational and are in the predicted coiled-coil or such conformational change of AcrA could current data are not to directly the of AcrA are in or conformational EPR of to the such as and as as to the including and showed or change the pH was from to The four that spectral and are in the predicted coiled-coil and the that the coiled-coil and the motifs are of in the AcrA or conformational at the and such as and EPR that are of regions and are not by of consistent with the that AcrA an elongated shape in solution. the other and as by (Fig. the structural of the and C-terminal of AcrA to the between and a the be Å in a with this is of the structurally and In results from this provide that AcrA pH conformational changes and conformational changes are reversible. These are the across the inner membrane for The of pH in the periplasm the drug efflux could as a to the action of AcrA, which conformational Such action of AcrA could bring the two membranes closer to substrate or further conformational changes of AcrB or TolC such that the periplasmic of TolC to the TolC α-barrel be by a of its substrates that are of AcrB central cavity to gain to the tunnel of TolC (9.Koronakis V. Sharff A. Koronakis E. Luisi B. Hughes C. Nature. 2000; 405: 914-919Crossref PubMed Scopus (870) Google Scholar, 11.Yu E.W. McDermott G. Zgurskaya H.I. Nikaido H. Koshland Jr., D. Science. 2003; 300: 976-980Crossref PubMed Scopus (338) Google Scholar). This how of the efflux system are from the membrane to the outer that the membrane be to of a periplasmic a structural between AcrB and AcrA could actively the of substrates through the periplasm and across the outer membrane. the available knowledge of AcrB and TolC studies to the dynamics of the AcrA-AcrB-TolC system the mechanism of efflux process in Gram-negative

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