Journal of Bacteriology, March 2004, p . 1258-1269, Vol . 186, No . 5

Differential Impact of MexB Mutations on Substrate Selectivity of the MexAB-OprM Multidrug Efflux Pump of Pseudomonas aeruginosa

Jocelyn K . Middlemiss and Keith Poole^*

Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada K7L 3N6

Received 30 September 2003/ Accepted 13 November 2003

 
The integral inner membrane resistance-nodulation-division [RND] components of three-component RND-membrane fusion protein-outer membrane factor multidrug efflux systems define the substrate selectivity of these efflux systems . To gain a better understanding of what regions of these proteins are important for substrate recognition, a plasmid-borne mexB gene encoding the RND component of the MexAB-OprM multidrug efflux system of Pseudomonas aeruginosa was mutagenized in vitro by using hydroxylamine and mutations compromising the MexB contribution to antibiotic resistance identified in a mexB strain . Of 100 mutants that expressed wild-typelevels of MexB and showed increased susceptibility to one ormore of carbenicillin, chloramphenicol, nalidixic acid, andnovobiocin, the mexB genes of a representative 46 were sequenced,and 19 unique single mutations were identified . While the majorityof mutations occurred within the large periplasmic loops betweentransmembrane segment 1 [TMS-1] and TMS-2 and between TMS-7and TMS-8 of MexB, mutations were seen in the TMSs and in otherperiplasmic as well as cytoplasmic loops . By threading the MexBamino acid sequence through the crystal structure of the homologousRND transporter from Escherichia coli, AcrB, a three-dimensionalmodel of a MexB trimer was obtained and the mutations were mappedto it . Unexpectedly, most mutations mapped to regions of MexBpredicted to be involved in trimerization or interaction withMexA rather than to regions expected to contribute to substraterecognition . Intragenic second-site suppressor mutations thatrestored the activity of the G220S mutant version of MexB, which was compromised for resistance to all tested MexAB-OprM antimicrobial substrates, were recovered and mapped to the apparently distal portion of MexB that is implicated in OprM interaction . As theG220S mutation likely impacted trimerization, it appears thateither proper assembly of the MexB trimer is necessary for OprMinteraction or OprM association with an unstable MexB trimermight stabilize it, thereby restoring activity.

 
Bacterial antimicrobial resistance is attributable in part tothe activity of antimicrobial efflux transporters, which aregrouped into five families based primarily upon amino acid sequencehomology: the ATP-binding cassette [ABC] family, the major facilitator[MF] family, the small multidrug resistance family, the multidrugand toxic compound extrusion family, and the resistance-nodulation-division [RND] family [43, 44, 51] . Despite structural differences, multidrugefflux pumps have in common the ability to accommodate and expelfrom the bacterial cell a wide variety of structurally unrelatedantimicrobial compounds . RND family efflux systems, in particular,play an important role in the export of and resistance to clinicallysignificant antimicrobials [43].

In Pseudomonas aeruginosa, an opportunistic human pathogen whose innate resistance to antimicrobials has long complicated antipseudomonal chemotherapy, RND-type multidrug efflux systems contribute significantly to intrinsic, as well as acquired, resistance [42, 48] . To date,seven RND multidrug efflux systems have been characterized inP . aeruginosa, including MexAB-OprM [14, 27, 46, 47], MexCD-OprJ[45], MexEF-OprN [24], MexXY-OprM [2, 36, 63], MexJK-OprM [4], and, most recently, MexHI-OpmD [1, 56] and MexVW-OprM [31].The major system contributing to intrinsic multidrug resistanceis encoded by the mexAB-oprM operon, which is also hyperexpressedin so-called nalB-type multidrug-resistant mutants [18, 19, 23, 33, 34, 41, 49, 52, 68] . These mutants carry mutations inthe mexR gene [19, 49, 53, 60, 68], which encodes a repressor of mexAB-oprM [60] . MexAB-OprM accommodates a broad range ofstructurally diverse antimicrobials, including dyes and detergents[26, 59, 61], organic solvents [28, 30], disinfectants [55],and a number of clinically relevant antibiotics [5, 23, 29,35, 59] . This efflux system is also implicated in the exportof homoserine lactones involved in quorum sensing [11, 40] andmay export virulence factors [17].

RND transporters such as MexB, an inner membrane-spanning drug-H⁺ antiporter, function in association with accessory proteinsthat include a channel-forming outer membrane factor [OMF] [e.g.,OprM] and a periplasmic membrane fusion protein [MFP] [e.g.,MexA]; the latter is believed to facilitate association of theRND and OMF components during efflux [51, 66, 67] . Together,these proteins form a tripartite efflux system that is responsiblefor the transport of substrates from the periplasm and/or cytoplasmof P . aeruginosa to the external environment in a single step.MexB, like other RND family multidrug efflux transporters, consistsof 12 transmembrane segments [TMSs] with extensive periplasmicloops between TMS-1 and -2 and between TMS-7 and -8, with bothtermini residing on the cytoplasmic side of the inner membrane[15] . Three charged amino acids [D407 and D408 of TMS-4 andK939 of TMS-10] have been identified by site-directed mutagenesisas essential for MexB pump function, possibly participatingin proton translocation or energy coupling [16].

The RND components of tripartite RND-MFP-OMF multidrug efflux systems [e.g., MexB] are known to determine substrate specificity[39], but until recently, the regions involved in substraterecognition had not been identified . Construction and characterizationof AcrB-MexB [62] and AcrB-AcrD [10] chimeric RND transportersfrom Escherichia coli and P . aeruginosa recently confirmed that the extensive periplasmic loops in RND transporters determine substrate selectivity . In agreement with this, mutant studies confirmed the importance of specific amino acid residues withinthe large periplasmic loops [LPLs] of the P . aeruginosa RND transporter MexD in substrate selectivity [32] . Eda et al . [9] also demonstrated that both the N- and C-terminal halves of MexB are required for efflux of its antibiotic substrates, implying that both LPLs are essential for transporter activity.

Little is known, however, about the nature and number of substrate-binding sites in RND-type multidrug transporters or the identity of residues involved in substrate selectivity, although substrate recognition by other families of multidrug transporters hasbeen examined in some detail . Mutations differentially impactingthe substrate profile of Bmr, an MF family multidrug transporterfrom Bacillus subtilis, for example, were identified by a random mutagenesis approach, and the existence of multiple substrate-binding sites was postulated [22] . Additional studies support the occurrenceof multiple substrate-binding sites in MF family multidrug effluxtransporters in E . coli [MdfA] [25], Lactococcus lactis [LmrP][50], and Staphylococcus aureus [QacA] [37] and in human ABC multidrug transporter P-glycoprotein [6, 13] . Crystal structuresof multidrug-binding regulators of multidrug efflux systems[e.g., the QacR regulator of qacA gene expression [54a]] reveala large generalized binding "site" with different residues involvedin binding different substrates . Still, it is unclear whethermultidrug efflux systems themselves use a similar strategy.In this study, we used chemical mutagenesis to identify randommutations in MexB that do not alter protein production but compromiseresistance to one or more antimicrobial substrates of MexAB-OprM,in hopes of identifying residues important for individual substraterecognition.

 
Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in this study are describedin Table 1 . P . aeruginosa K1589 was constructed by introductionof in-frame mexR [60] and mexB [17] deletions into strain K870as described previously . Elimination of the chromosomal mexBgene was necessary to permit screening of mutated plasmid-bornemexB genes for changes impacting substrate selectivity [seebelow], while elimination of mexR served to ensure high-levelexpression of the chromosomally encoded MexA and OprM components.Together with the high-level expression of the plasmid-encodedMexB [under plac control [see below]], this served to ensuremaximal production of a MexAB-OprM efflux system and thus readyassessment of its activity [by using MIC assays] and any changesin activity resulting from mutations in mexB . E . coli and P.aeruginosa were routinely cultured in Luria-Bertani [LB] broth[15.5 g of Miller's Luria broth base [Difco] and 2 g of NaClper liter of H₂O] at 37°C with shaking [180 rpm], exceptduring growth of the conjugation recipient strains [42°Cwithout shaking] and during incubation of organic solvent susceptibilitytesting plates [30°C] . Strains carrying pRSP35, pJKM14,and their derivatives were cultured in the presence of tetracycline[10 µg/ml].

 
Plasmid protocols. Electrocompetent E . coli DH5 and S17-1 cells were prepared asdescribed by Dower et al . [7] . Electroporation of plasmids pRSP35,pJKM14, and their derivatives into 100 µl of electrocompetentE . coli was performed with the ECM399 electroporator [BTX, Holliston,Mass.] at 2,500 V for 5 ms . Plasmid DNA used for nucleotidesequencing was isolated from E . coli hosts by using plasmidmidikits [Qiagen Inc., Mississauga, Ontario, Canada] accordingto a protocol provided by the company for isolation of low-copy-numberplasmids.

Hydroxylamine hydrochloride mutagenesis. Chemical mutagenesis with hydroxylamine hydrochloride was adaptedfrom the method of Garinot-Schneider et al . [12] . Briefly, plasmid pRSP35 [10 µg] carrying the mexB gene was incubated at 70°C for 1.5 h in 500 µl of potassium phosphate buffer[pH 6.0; 44 mM] containing EDTA [5 mM] and hydroxylamine hydrochloride[0.46 M] . A 20-µl sample was removed at 1.5 h, and hydroxylaminehydrochloride was inactivated by addition of Tris-HCl [pH 8.0;90 mM final concentration] and EDTA [9 mM final concentration].Plasmid DNA was precipitated by using a standard protocol [54], washed in 70% [vol/vol] ethanol, and resuspended in 20 µlof H₂O. E . coli S17-1 was electroporated with 1 to 3 µlof hydroxylamine hydrochloride-treated pRSP35 and, followingaddition of 1 ml of LB broth, was permitted to recover at 37°Cwith shaking for 1 h in the absence of antibiotic selectiveagents . Electroporated cells [1 ml] were then added to 9 mlof LB broth containing tetracycline [10 µg/ml] and culturedovernight at 37°C with shaking.

Mutagenized pRSP35 was mobilized into P . aeruginosa from E. coli S17-1 via conjugation as described previously [47], withmodifications . Briefly, a 1.0-ml overnight culture of plasmid-containingE . coli S17-1 was mixed with 0.3 ml of an overnight LB cultureof P . aeruginosa K1589 and pelleted in a microcentrifuge tube.Following resuspension in 150 µl of LB broth and spottingonto the center of an LB agar plate, cells were incubated for4 h at 37°C . Bacterial growth was then resuspended in 1 ml of LB broth, and appropriate dilutions were plated on LB plates containing tetracycline [10 µg/ml] and streptomycin[250 µg/ml], the latter to counterselect the donor E.coli S17-1 . Colonies of P . aeruginosa K1589 with plasmid-bornemutated mexB were recovered from LB plates containing tetracyclineand streptomycin and screened for loss of MexB function by picking isolated colonies onto master plates of the same formulationand replica plating onto each of four LB plates containing chloramphenicol [64 µg/ml], carbenicillin [64 µg/ml], nalidixicacid [128 µg/ml], or novobiocin [128 µg/ml] . Antibioticswere chosen to be at the highest concentration at which P . aeruginosaK1589[pRSP35] containing wild-type mexB could grow . pRSP35-carryingP . aeruginosa K1589 organisms that were unable to grow on oneor more of the antibiotic plates were recovered and stored at-70°C.

Hydroxylamine mutagenesis of plasmid pJKM14 expressing MexB_G220S was performed as described above, with modifications . Plasmid pJKM14 DNA [7.5 µg] was incubated at 70°C for 1 hin 150 µl of potassium phosphate buffer [pH 6; 75 mM]containing EDTA [0.75 mM] and hydroxylamine hydrochloride [0.4M] . DNA was then precipitated without addition of Tris-EDTAand resuspended in 20 µl of H₂O as described above . Followingelectroporation into E . coli S17-1, plasmid pJKM14 and its derivativeswere then mobilized into P . aeruginosa K1589 as described above,although imipenem [0.5 µg/ml] served as counterselectionin this instance . P . aeruginosa K1589 containing pJKM14 withcompensatory mutations which partially or completely restoredwild-type MexB function was selected on LB broth with tetracycline[10 µg/ml] and carbenicillin [8 or 16 µg/ml] andstored at -70°C.

Antibiotic susceptibility testing. The susceptibility of P . aeruginosa K1589 carrying pRSP35, pJKM14,and their derivatives was assessed by using the twofold serialmicrotiter broth dilution method described previously [20],with an inoculum of 5 x 10⁵ cells per ml . MICs were recordedas the lowest concentration of antibiotic inhibiting visiblegrowth after an 18-h incubation at 37°C.

Organic solvent susceptibility testing. The ability of P . aeruginosa K1589 carrying mutated mexB plasmidsto grow in the presence of the organic solvent n-hexane wasassessed by the efficiency-of-plating approach outlined previously[28] . Briefly, 100 µl of a suspension of approximately10⁷ cells/ml was spread to cover the surface of a glass platecontaining LB agar and overlaid with 1 ml of 100% n-hexane.The plates were sealed and incubated for 20 h at 30°C, atwhich point the presence or absence of growth was recorded.

SDS-PAGE and Western immunoblotting. Whole-cell extracts of P . aeruginosa K1589 harboring pRSP35,pJKM14, and their derivatives were prepared by harvesting 1ml of overnight LB cultures [18 h, 37°C] and resuspendingthe cells in 300 µl of sodium dodecyl sulfate-polyacrylamidegel electrophoresis [SDS-PAGE] sample buffer [SDS [4% wt/vol],glycerol [20% vol/vol], Tris-HCl [pH 6.8; 0.25 M]] containing2-mercaptoethanol [10%, vol/vol] . Samples were heated at 95°Cfor 5 min, sonicated 20 s at 50% power with a Vibra Cell sonicator[Sonics and Materials, Inc., Danbury, Conn.], and centrifugedat 12,000 x g for 2 min to remove debris . Protein samples wereanalyzed by SDS-10% [vol/vol] PAGE and subsequently electroblottedand screened with MexB-specific antibodies as described previously[58], with the exception that proteins were transferred ontoImmobilon-P transfer membranes [Millipore, Bedford, Mass.] byusing a Trans-Blot SD semidry electrophoretic transfer cell[Bio-Rad Laboratories, Hercules, Calif.] at a 25-mA constantcurrent for 1 h at room temperature . Blots were developed byusing Western Lightning chemiluminescence reagents [Perkin-ElmerLife Sciences, Boston, Mass.], according to the manufacturer'sinstructions.

Nucleotide sequencing. The mexB genes of pRSP35, pJKM14, and their derivatives weresequenced by Cortec DNA Service Laboratories, Inc., Kingston,Ontario, Canada, by using custom primers mexB forward2 [5'-GAAGAATGTCGCGTCCGC-3'],mexB forward3 [5'-CAACGCGCAGTTCAACGG-3'], mexB forward4 [5'-CAGGCGCAGAACGTGCAG-3'], mexB reverse [5'-GGGGATCAGCGAGCAGC-3'], mexB reverse2 [5'-GCGTCACCGGAACTCAGG-3'],and mexB reverse3 [5'-GTACGCCCTGGTCCTCGTC-3'], which were synthesizedby the company . Mutations in mexB were identified followingsequence alignment with the wild-type gene by using DNAMAN version4.11 software.

Protein modeling. The three-dimensional structure of MexB was modeled [by M . Kuiperof the Department of Biochemistry, Queen's University, Kingston,Ontario, Canada] with SYBYL version 6.5 molecular modeling software[Tripos Associates, St . Louis, Mo.] by threading the MexB proteinsequence onto the crystal structure of the homologous RND transporter,AcrB, of E . coli [38] and refining the structure by minimizationand dynamics . Locations of mutations within MexB were pinpointedwithin the larger three-dimensional structure of MexB and hydrogenbonds were predicted, using both DeepView/Swiss-PdbViewer version3.7 and POV-RAY for Windows.

 
Isolation of MexB mutants exhibiting reduced antibiotic resistance. To identify mutations in mexB that compromised MexB activity, ca . 4,000 colonies of a mexB derivative of PAO1 [strain K1589]harboring hydroxylamine-mutated, plasmid-borne mexB were screenedfor increased susceptibility to one or more of four antibioticschosen as representative MexB antimicrobial substrates . Initially,249 colonies showed increased susceptibility to one or moreof chloramphenicol, carbenicillin, nalidixic acid, or novobiocinon plates . Following susceptibility testing in broth cultures,these were grouped into six families based upon common patternsof susceptibility or resistance to these four agents [Table2] . Western immunoblotting subsequently confirmed that in ca.100 instances [studied from this point forth], wild-type levelsof MexB were being produced [representative examples are shownin Fig . 1] despite the increased susceptibility of the mutantMexB-producing P . aeruginosa K1589 . Some defects in MexB impactedresistance to a single [family A, chloramphenicol; family C,carbenicillin; and family D, nalidixic acid]; or only two antibioticsused in the original screen [family B, chloramphenicol and carbenicillin],while others impacted three of four [family E, all but novobiocin]or all four [family F] antibiotics, with a subgroup of F beingtotally compromised for resistance [family F₂] [Table 2] . Aswe were interested in the possibility of separate binding sites for periplasmically acting ß-lactams from those for antimicrobials with cytoplasmic targets, the lone representativeof family C [encoded by pJKM9 [Table 2]] exhibiting increased susceptibility to carbenicillin alone was tested in broth culture for susceptibility to three other ß-lactams, i.e., piperacillin, meropenem, and aztreonam . Although it exhibited increased susceptibility to meropenem and aztreonam comparedto that provided by wild-type MexB, its resistance to the ß-lactam piperacillin was not impacted [Table 2] . Thus, MexB produced by pJKM9 was not generally compromised for ß-lactamresistance.

 
To assess the impact of MexB defects on resistance to nonantibiotic antimicrobials, we tested susceptibility to an organic solventby measuring the presence or absence of growth on plates overlaidwith n-hexane . In general, only hydroxylamine hydrochloride-induced mutations in mexB that impacted resistance to all or almost all antibiotics [Table 2, families E and F] also compromised solvent tolerance.

Identification of MexB mutations impacting antimicrobial resistance. To identify the mutations in the mexB gene that are responsible for the compromised resistance seen in families A to F [Table 2], plasmid-borne mexB was recovered from representatives ofeach family and sequenced . Of 46 sequenced mutants, 28 had asingle MexB mutation, of which 19 were unique . While the majorityof mutations occurred within the LPLs between TMS-1 and -2 [LPL-1]and between TMS-7 and -8 [LPL-2], mutations in the TMSs as wellas other periplasmic and cytoplasmic loops were also seen [Table2] . With the exception of family B, mutations occurring in TMSsand smaller loops were found mostly in families E and F, whichimpacted larger numbers of substrates . Of interest is that ourhydroxylamine mutagenesis approach identified two mutationswithin TMS-4, D407N and D408N [Table 2, family F₂], that werepreviously identified as essential for MexB function and predictedto participate in proton translocation or energy coupling [16].Both mutants exhibited susceptibility to all tested antimicrobialsat levels equivalent to that of the MexB-deficient strain.

Mapping of MexB mutations to a three-dimensional model of MexB. To gain a more complete understanding of the effects of these mutations on MexB function, we first modeled the MexB three-dimensional structure upon the X-ray crystal structure of AcrB, the homologous [70% amino acid identity] RND transporter from E . coli [38]. Like AcrB, MexB appears to be a trimer formed by the interlocking of a protruding thumb region of one monomer with a hole in the upper periplasmic region of the neighboring monomer [Fig . 2A]. The trimer is embedded in the inner membrane but forms a central cavity in the periplasmic domain which is fed by openings or vestibules that occur between adjacent monomers at the planeof the membrane and are contiguous with the periplasm [Fig.2B] . The vestibules are predicted to function as portals forsubstrates entering the AcrB-MexB central cavity from the periplasmand/or outer leaflet of the inner membrane . A pore at the distalend of the MexB trimer likely connects with the OMF componentof the efflux system [i.e., OprM] [Fig . 2B] to permit passageof substrates captured by MexB across the outer membrane andinto the extracellular milieu . With the exception of familyB, mutations impacting one or a few antimicrobials [Table 2, families A, C, and D] tended to map towards the top of the MexB structure [periplasmic domain], while mutations impacting the majority of antimicrobials [Table 2, families E and F] tended to map to the TMSs or loop regions near the inner membrane plane [Fig . 3] . Two exceptions were mutations G220S and T578I in MexBmutants of family F₁, which mapped to the periplasmic domain[Fig . 3F₁].

 
[i] Family A. Of the mutants with increased susceptibility to chloramphenicolalone, two [T60I and S183L] had mutations in the vicinity ofthe periplasmic hole [Fig . 3A] into which the thumb of a neighboringmonomer is predicted to insert [see Fig . 2A] . T60 bordered thehole itself and was oriented towards the center of the trimer,below the point of thumb insertion . While it is not entirelyclear how mutation of this residue might have impacted MexBfunction, T60 may have formed hydrogen bonds with T56 [2.8 Åaway] and V57 [3.2 Å away] in the wild-type protein, anda mutation to the hydrophobic isoleucine would have preventedthis . As a result, a disruption of the MexB tertiary structureimmediately below the thumb may have occurred, possibly interferingwith thumb insertion . Indeed, T60 of one monomer is predictedto be within close proximity of residues on the thumb of a neighboringmonomer [e.g., R239, 3.7Å away], and any changes in T60 may adversely affect thumb insertion . In contrast to T60, S183 occurred somewhat to the right of the hole region shown in Fig. 2A and towards the outer surface of MexB [Fig . 3A] . Perhapsmutation to the hydrophobic leucine indirectly [i.e., at a distance]impacted MexB structure in the vicinity of the hole.

Two mutations in members of family A [A618T and R716H] occurredon an outward-facing portion of the MexB trimer, within a groovethat extends from the hole where the thumb inserts to the innermembrane plane [Fig . 3A] and was previously predicted to be involved in MexA binding [38] . Thus, these mutations may have adversely affected MexA binding to MexB . The final member of family A carried a M395I mutation within TMS-4, potentiallyaltering the local conformation of MexB in the inner membrane[Fig. 3A] . It is not clear how this might have impacted function, although given the apparent importance of TMS-4 and TMS-10 in proton translocation [16], it may have adversely affected thisessential process in the operation of the MexB drug-H⁺ antiporter.

[ii] Family B. In contrast to members of families A, C, and D [descriptionsof families C and D are below], which showed susceptibilityto one antimicrobial and carried MexB mutations that tendedto map to the upper periplasmic region of the protein, MexB mutants belonging to family B [which also showed susceptibility to only a few agents] carried mutations that mapped to TMS-5[S450L] and the cytoplasmic loops between TMS-10 and -11 [E946Kand C966Y] [Fig . 3B] . How these mutations might have impacted MexB function, particularly with respect to efflux of or resistance to these two substrates only, is unclear.

[iii] Family C. Similar to the T60I mutation of family A, the G51D family Cmutation that resulted in increased susceptibility to carbenicillinwas located within the hole into which the thumb of the neighboringmonomer was predicted to insert [Fig . 3C] . G51 was, however,even closer [within 1.5 Å of S216] to the inserting thumbthan T60 . The change from a small and flexible residue [glycine]to one that is larger and negatively charged [aspartic acid]likely had a negative impact on thumb insertion and thus trimerformation.

[iv] Family D. The G754D mutation of family D that caused increased susceptibilityto nalidixic acid also occurred within the thumb insertion holeregion [Fig . 3D] . Again, too, the mutated residue is much closerto the inserting thumb of the neighboring monomer than is thewild type [e.g., G754 is within 3.3 Å of G217 of the thumb,while D754 is within less than 1 Å of G217] . This familyD mutation appears, therefore, also to negatively impact thumbinsertion and thus trimerization . In contrast, a second family D mutation, S977F, occurs in TMS-11 and may have had an impact on local packing of TMSs, since following the mutation, potential hydrogen bonds with two residues in the carboxy-terminal tail[M1009 and T1003] would be lost . How this would impact functionis at present unclear.

[v] Family E. Mutants susceptible to all antimicrobials used in the initialscreen except novobiocin [family E] possessed mutations withinTMS-10 [V928] or the vestibule region [S462F] [Fig. 4A] . Whilethe nature of the defect caused by the V928M change is uncertain,the substitution of a bulky, hydrophobic residue [F] for a small,polar one [S] at position 462 in the vestibule region may havedisrupted tertiary structure in such a way as to limit substraterecognition or entry, possibly through steric hindrance or lossof hydrogen bonds with the substrate itself . With all or mostsubstrates predicted to enter the MexB exporter via the vestibules,it might not be surprising to find that some changes here impacta large number of substrates.

 
[vi] Family F₁. The G220S mutation that compromised resistance to all testedantimicrobials occurred on the protruding thumb region of MexB[Fig . 3F₁], which is predicted to insert into the periplasmichole of the neighboring monomer during trimer formation . AsG220 is within approximately 3.6 to 4.2 Å of Q622, R779,and M780 of the periplasmic hole region of the neighboring monomer,changes in thumb secondary structure as a result of the G220Schange may, for steric reasons, have hindered thumb insertionand thus decreased efficiency of trimer formation . Similar toA618T and R716H of family A, the family F₁ T578I mutation occurredwithin the vicinity of the outward-facing periplasmic grooveof MexB, with which MexA could potentially interact [Fig . 3F₁].This mutation might thus adversely impact MexA binding to theMexB trimer . The remaining two members of family F₁ possessedmutations within TMS-12 [G1002D] or immediately above the membraneplane [E864K] [Fig. 3F₁] in the vestibule region that occurs between neighboring monomers [Fig . 4B] and serves as a predictedentry point for substrates from the periplasm to a central collectioncavity [38] . The introduction of a large, negatively chargedresidue [D1002] into the largely hydrophobic TMS-12 may haveinterfered with the packing of the neighboring helices, thushaving an indirect negative effect on proton translocation.Similarly, a mutation from negative [E864] to positive [K864]charge in the vestibule region between neighboring monomersmay have disrupted the local tertiary structure . Again, if thevestibules are the portals of entry for most if not all substratesaccommodated by MexB, changes here are expected to impact manyor most substrates.

[vii] Family F₂. Each F₂ mutant with completely compromised resistance to alltested antimicrobials contained a mutation in TMS-4 [D407N orD408N] [Fig . 3F₂], which was previously identified as beingessential for pump function [16] . These residues are proposedto interact with K939 on TMS-10 and to participate in protonpumping by this drug-H⁺ antiporter, by a mechanism that hasthus far not been characterized.

Identification of compensatory MexB mutations restoring antimicrobial resistance in a MexBG220S-expressing strain. To identify second-site suppressor mutations in mexB that restoreantimicrobial resistance to P . aeruginosa strain K1589 expressingthe G220S family F₁ mutant MexB protein [characterized by increased susceptibility to all tested antimicrobials], plasmid pJKM14, encoding MexB_G220S, was subjected to a second round of hydroxylamine mutagenesis . Plasmid pJKM14-carrying K1589 that expressed MexB with restored function was then selected on plates with carbenicillin at levels where the G220S mutant could not survive unless a compensatory mexB mutation had occurred . In order to confirma wild-type phenotype in the compensatory mutants, 24 compensatory mutants from the initial screen were subjected to susceptibility testing in broth culture with representative MexB antimicrobial substrates . Plasmid-borne mexB from 14 of these mutants that exhibited wild-type or near-wild-type levels of resistance toa number of the tested antimicrobials [Table 3] was recovered and subjected to nucleotide sequencing.

 
Nine of 10 mutants in which resistance to all tested antimicrobials was restored to wild-type levels contained the same compensatory mutation, E796K, which occurred within LPL-2 . As Table 3 indicates,the remaining mutant that exhibited wild-type levels of resistanceto all tested antimicrobials harbored two mutations, V203M andG581D, in LPL-1 and -2, respectively, although it was not clear if one or both were needed . Four additional compensatory mutants contained a single mutation in LPL-2, of which three were unique [A737V, L738F, and D793N] and showed only partially restored MexB function [i.e., resistance, although increased for most antimicrobials, was not restored to wild-type levels for all[Table 3].

Mapping of compensatory MexB mutations to a three-dimensional model of MexB. All single MexB mutations [Table 3] restoring antibiotic resistanceof a G220S MexB mutant to wild-type levels for some or all ofthe tested antimicrobial substrates mapped to the upper periplasmicportion of MexB in the region predicted to interact with theOMF OprM [Fig . 5] . The original G220S mutant may form alteredtrimers with which OprM docks with reduced efficiency, and compensatorymutations may enhance OprM docking with mutant trimers, restoringantimicrobial efflux . Alternatively, compensatory mutationsmay enhance OprM docking, facilitating more efficient MexB trimerformation in the face of the destabilizing G220S mutation, alsorestoring efflux.

 
Conservation in other RND transporters of residues whose mutation in MexB compromises function. In order to determine whether MexB residues whose mutation inthis study compromised antimicrobial resistance were conservedin other RND transporters from P . aeruginosa as well representativesfrom other bacteria [and thus were of broad functional significance],we performed alignments of the protein sequences of these transporters.As we predict that many of the mutations identified in thisstudy affect trimer formation and proton pumping, which shouldbe conserved functions within RND transporters, it was expectedthat amino acid residues involved in these functions would behighly conserved . With the exception of T60 and S183 [which were conserved in only 4 and 1 of the 11 RND transporters examined, respectively], residues predicted to be involved in MexB trimer formation [G51, G754, and G220] were highly conserved [in 8to 11 of the RND transporters to which they were compared] [Table 4] . Indeed, when the analysis was extended to seven additional RND pumps found in Pseudomonas putida, Burkholderia pseudomallei, Campylobacter jejuni, Porphyromonas gingivalis, and Stenotrophomonasmaltophilia, residues corresponding to G51, G220, and G754 inMexB were also very highly conserved [not shown] . Similarly,MexB residues predicted to impact efficiency of proton pumpingwere generally well conserved, occurring in 6 to 11 of the RNDtransporters [M395, V928, E946, S977, G1002, D407, and D408] [Table 4] . This was also true when the analysis was extendedto the additional RND transporters described above, with residuescorresponding to D407, D408, E946, G220, S977, and G1002 in particular being very well conserved . S450 and C966 were clearly exceptions, however, with serine and cysteine being very poorly represented at the corresponding positions of the RND transporters listed in Table 4 . Interestingly, all of the exceptions mentionedabove occurred at residues that, when mutated, decreased resistanceonly to the antimicrobials chloramphenicol and/or carbenicillin,which are predicted to be poorer substrates of MexB . Thus, althoughthese residues are predicted to participate in conserved functions[e.g., trimer formation and proton pumping], they may play comparativelyminor roles.

 
Mutations predicted to impact the interaction of MexB with theMFP MexA [T578, A618, and R716] occurred at residues that werepoorly conserved in the 11 RND transporters in Table 4, and this held true for T5781 and A618 when the analysis was extended to the above-mentioned 7 additional transporters [arginines corresponding to R716 were present in 5 of these transporters,making this residue somewhat better conserved in the RND transporters].This is not entirely unexpected, since subunit swapping studieshave indicated that MFP association with its cognate RND transporteris very specific and that MFPs cannot generally be exchangedand efflux function still retained [64] . Thus, it is probable that each RND transporter has unique amino acid residues inthose regions with which the MFP interacts . Similar to the situationwith mutations potentially impacting association of MexB withMexA, the two MexB mutations [S462F and E864K] within predicted substrate-binding sites in the vestibule regions occurred atamino acid residues that were less well conserved [E864; conservedin 2 of 11] and somewhat less well conserved [S462; conservedin 5 of 11] in the other RND transporters to which they werecompared [Table 4] . This observation was again supported by comparisons with the seven additional RND transporters and may reflect an expected variability in the vestibule regions ofdifferent RND transporters that allows different pumps to accommodatedifferent antimicrobial substrates . In any case, the two MexBmutations [listed above] affected susceptibilities to almostall tested antimicrobials [Table 2, families E and F₁], whichis evidence that although these residues are not highly conserved,they are important for substrate accommodation by MexB.

 
Although RND transporters have the ability to accommodate a surprisingly vast array of substrates, little is known aboutthe basis of their broad substrate specificity . The two substantial periplasmic loops in RND transporters MexB, MexD, AcrB, andAcrD have been implicated in substrate selectivity by others[8, 10, 32, 62] . Our hydroxylamine hydrochloride mutagenesisapproach also identified a range of mutations within the twoLPLs of MexB that negatively impacted the MexB contributionto resistance, consistent with their having a role in substrateselectivity . In contrast to the conclusions of the aforementionedstudies, however, we contend that most of our mutations in themajor periplasmic loop regions did not directly impact substratebinding . Instead, these mutations appeared to affect pump assemblyand/or activity, although how is not entirely clear . That suchmutations were, nonetheless, somewhat substrate specific mightbe explained by any reduction in pump assembly or activity havinga greater impact on those substrates that are not as efficientlyaccommodated by the MexB transporter . Thus, chloramphenicolis the only substrate affected by the family A mutations [T60Iand S183L] that are predicted to impact trimerization, possiblybecause it is a poorer substrate for MexAB-OprM and a reductionin efficiency of trimer formation affects its export to a greaterextent . Similarly, the family B mutations that apparently impactthe transmembrane region of MexB, the lone family C mutationthat possibly impacts trimer formation, and the family D mutationsthat impact trimerization or proton pumping are all fairly substrateselective, despite the apparent impact on MexB assembly and/orfunction and not substrate recognition per se . Again, the factthat mutations impacting assembly and/or activity of MexB do not affect all substrates suggests that that they must have only a modest impact on MexB assembly or function, with the compromised MexB able to accommodate some substrates betterthan others . Still, other mutations predicted to impact trimerization [e.g., G220S] do compromise resistance for all substrates, presumably because trimerization and thus pump assembly are impacted toa greater extent.

Beyond their contribution to substrate selectivity, the twolarge loops comprising the periplasmic domain of an RND transporterare presumably also involved in interaction with the cognateMFP . Using AcrB-MexB chimeric RND transporters, Tikhonova etal . demonstrated that the N-terminal half of AcrB is likelyinvolved in determining the specificity of interaction withthe MFP AcrA, with residues 590 to 612 apparently of particularimportance [62] . When these residues are mapped to our modelof MexB, they occur on the outward-facing surface of each monomer,to the right of a large groove that extends from the mid-periplasmicdomain to the inner membrane and was previously postulated tobe involved in binding [in the case of AcrB] to its cognateMFP, AcrA [38] . Interestingly, two of our mutations that arepredicted to impact interaction of MexA with MexB [i.e., T578Iand A618T] occur in a region of MexB that, in general, correspondsto the MFP-binding region of AcrB [62] . A third mutation inMexB that is also predicted to impact MexA association [R716H]occurs outside this . It is, however, in close proximity to T578and A618 in the three-dimensional structure of MexB and, assuch, may also play a role in determining MexA binding . Still,unlike T578 and A618, which are poorly conserved in other RNDtransporters [consistent with a role in determining the specificityof MexA binding by MexB], R716 is modestly conserved and maynot, in fact, determine the specificity of MexA interactionwith MexB . Nonetheless, it may be important for MexA binding.

Yu et al . [65] have very recently crystallized AcrB in the presenceof several antimicrobial substrates and demonstrated that differentsubstrates bind to different amino acid residues in AcrB, allof which, however, occur within the vestibule-central cavity region, at or immediately above the plane of the inner membrane. Only two of the mutations characterized here for MexB occurred in the vestibule region [and none occurred in the central cavity], although these did result in increased susceptibility to all[E864K] or most [S462F] tested substrates . In all cases, however, antimicrobial resistance was decreased from wild-type levelsbut not completely abolished . As mutation of a residue withina specific substrate-binding site of MexB would generally beexpected to completely abolish resistance to the substrate[s]it accommodates, it seems likely that MexB, like AcrB, has alarge and flexible binding pocket with which any given substratehas multiple points of interaction . A single mutation in thebinding region, then, is unlikely to eliminate binding or exportand thus will have only a modest effect on resistance.

As in our study, mutations in a second RND transporter of P. aeruginosa, MexD of MexCD-OprJ, have also been reported to impact transport of numerous substrates . Indeed, MexD mutations affecting susceptibility to a broad range of ß-lactams as wellas several cytoplasmically acting antimicrobials were identifiedand shown to occur within the two LPL regions of this RND transporter[32] . Upon mapping of these MexD mutations to our model of MexB [corresponding to Q34K, E89K, A292V, and P328L in MexB], itis evident that they occur at the beginning or end of LPL-1and line the vestibule or central cavity region . These residuesin MexD are thus likely involved in substrate recognition . Interestingly,these MexD mutations affected periplasmically and cytoplasmicallyacting antimicrobials . Similarly, we noted here that MexB mutationsnear the vestibules [S462F and E864K] also negatively impactresistance to both periplasmically [carbenicillin, meropenem,and aztreonam] and cytoplasmically [chloramphenicol and nalidixicacid] acting antimicrobials . Thus, it does not appear that thereare separate substrate-binding sites for antimicrobials withperiplasmic versus cytoplasmic targets.

Unexpectedly, we found that a possible decreased efficiencyof MexB trimer formation in the G220S mutant may also have hadan indirect negative effect on the ability of MexB to form afunctional association with the trimeric OprM component of theMexAB-OprM multidrug efflux system . Since the majority of mutationscompensating for the original G220S mutation on the thumb regionoccurred in the upper docking domain of MexB, it seems plausiblethat a reduction in efficiency of trimer formation [caused bythe G220S mutation] reduces the ratio of functional to nonfunctionalcouplings of MexB with OprM, its outer membrane counterpart.Potentially, trimer formation in wild-type MexB induces a conformationalchange in MexB that permits OprM recruitment and subsequentexport of antimicrobials across the outer membrane.

 
This work was supported by an operating grant from the Canadian Cystic Fibrosis Foundation [CCFF] . J.K.M . was supported by a studentship from the Natural Sciences and Engineering Research Council . K.P . is a CCFF Scholar.

 
* Corresponding author . Mailing address: Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada K7L 3N6 . Phone: [613] 533-6677 . Fax [613] 533-6796 . E-mail: poolek@post.queensu.ca.

1. Aendekerk, S., B . Ghysels, P . Cornelis, and C . Baysse. 2002 . Characterization of a new efflux pump, MexGHI-OpmD, from Pseudomonas aeruginosa that confers resistance to vanadium . Microbiology 148:2371-2381 .
2. 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 .
3. Ausubel, F . M., R . Brent, R . E . Kingston, D . D . Moore, J . G . Seidman, J . A . Smith, and K . Struhl. 1992 . Short protocols in molecular biology, 2nd ed . John Wiley & Sons, Inc., New York, N.Y.
4. Chuanchuen, R., C . T . Narasaki, and H . P . Schweizer. 2002 . The MexJK efflux pump of Pseudomonas aeruginosa requires OprM for antibiotic efflux but not for efflux of triclosan . J . Bacteriol . 184:5036-5044 .
5. Dean, C . R., M . A . Visalli, S . J . Projan, P.-E . Sum, and P . A . Bradford. 2003 . Efflux-mediated resistance to tigecycline [GAR-936] in Pseudomonas aeruginosa PAO1 . Antimicrob . Agents Chemother . 47:972-978 .
6. Dey, S., M . Ramachandra, I . Pastan, M . M . Gottesman, and S . V . Ambudkar. 1997 . Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein . Proc . Natl . Acad . Sci . USA 94:10594-10599 .
7. Dower, W . J., J . F . Miller, and C . W . Ragsdale. 1988 . High efficiency transformation of Escherichia coli by high voltage electroporation . Nucleic Acids Res . 16:6127-6145.
8. Eda, S., H . Maseda, and T . Nakae. 2003 . An elegant means of self-protection in Gram-negative bacteria by recognizing and extruding xenobiotics from the periplasmic space . J . Biol . Chem . 278:2085-2088 .
9. Eda, S., H . Yoneyama, and T . Nakae. 2003 . Function of the MexB efflux-transporter divided into two halves . Biochemistry 42:7238-7244.
10. Elkins, C . A., and H . Nikaido. 2002 . Substrate specificity of the RND-type multidrug efflux pumps AcrB and AcrD of Escherichia coli is determined predominately by two large periplasmic loops . J . Bacteriol . 184:6490-6498 .
11. Evans, K., L . Passador, R . Srikumar, E . Tsang, J . Nezezon, and K . Poole. 1998 . Influence of the MexAB-OprM multidrug efflux system on quorum-sensing in Pseudomonas aeruginosa . J . Bacteriol . 180:5443-5447 .
12. Garinot-Schneider, C., A . J . Pommer, G . R . Moore, C . Kleanthous, and R . James. 1996 . Identification of putative active-site residues in the DNase domain of colicin E9 by random mutagenesis . J . Mol . Biol . 260:731-742.
13. Garrigos, M., L . M . Mir, and S . Orlowski. 1997 . Competitive and non-competitive inhibition of the multidrug-resistance-associated P-glycoprotein ATPase—further experimental evidence for a multisite model . Eur . J . Biochem . 244:664-673.
14. Gotoh, N., H . Tsujimoto, K . Poole, J.-I . Yamagishi, and T . Nishino. 1995 . The outer membrane protein OprM of Pseudomonas aeruginosa is encoded by oprK of the mexA-mexB-oprK multidrug resistance operon . Antimicrob . Agents Chemother . 39:2567-2569.
15. Guan, L., M . Ehrmann, H . Yoneyama, and T . Nakae. 1999 . Membrane topology of the xenobiotic-exporting subunit, MexB, of the MexA, B-OprM extrusion pump in Pseudomonas aeruginosa . J . Biol . Chem . 274:10517-10522 .
16. Guan, L., and T . Nakae. 2001 . Identification of essential charged residues in transmembrane segments of the multidrug transporter MexB of Pseudomonas aeruginosa . J . Bacteriol . 183:1734-1739 .
17. Hirakata, Y., R . Srikumar, K . Poole, N . Gotoh, T . Suematsu, S . Kohno, S . Kamihira, R . E . Hancock, and D . P . Speert. 2002 . Multidrug efflux systems play an important role in the invasiveness of Pseudomonas aeruginosa . J Exp . Med . 196:109-118 .
18. Jalal, S., O . Ciofu, N . Hoiby, N . Gotoh, and B . Wretlind. 2000 . Molecular mechanisms of fluoroquinolone resistance in Pseudomonas aeruginosa isolates from cystic fibrosis . Antimicrob . Agents Chemother . 44:710-712 .
19. Jalal, S., and B . Wretlind. 1998 . Mechanisms of quinolone resistance in clinical strains of Pseudomonas aeruginosa . Microbiol . Drug Resist . 4:257-261.
20. Jo, J . T., F . S . Brinkman, and R . E . Hancock. 2003 . Aminoglycoside efflux in Pseudomonas aeruginosa: involvement of novel outer membrane proteins . Antimicrob . Agents Chemother . 47:1101-1111 .
21. Keen, N . T., S . Tamaki, D . Kobayashi, and D . Trollinger. 1988 . Improved broad-host-range plasmids for DNA cloning in Gram-negative bacteria . Gene 70:191-197.
22. Klyachko, K . A., S . Schuldiner, and A . A . Neyfakh. 1997 . Mutations affecting substrate specificity of the Bacillus subtilis multidrug transporter Bmr . J . Bacteriol . 179:2189-2193.
23. Kohler, T., M . Kok, M . Michea-Hamzehpour, P . Plesiat, N . Gotoh, T . Nishino, L . Kocjanici Curty, and J.-C . Pechere. 1996 . Multidrug efflux in intrinsic resistance to trimethoprim and sulfamethoxazole in Pseudomonas aeruginosa . Antimicrob . Agents Chemother . 40:2288-2290.
24. Kohler, T., M . Michea-Hamzehpour, U . Henze, N . Gotoh, L . K . Curty, and J.-C . Pechere. 1997 . Characterization of MexE-MexF-OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa . Mol . Microbiol . 23:345-354.
25. Lewinson, O., and E . Bibi. 2001 . Evidence for simultaneous binding of dissimilar substrates by the Escherichia coli multidrug transporter MdfA . Biochemistry 40:12612-12618.
26. Li, X.-Z., K . Poole, and H . Nikaido. 2003 . Contributions of MexAB-OprM and an EmrE homologue to intrinsic resistance of Pseudomonas aeruginosa to aminoglycosides and dyes . Antimicrob . Agents Chemother . 47:27-33 .
27. Li, X.-Z., H . Nikaido, and K . Poole. 1995 . Role of MexA-MexB-OprM in antibiotic efflux in Pseudomonas aeruginosa . Antimicrob . Agents Chemother . 39:1948-1953.
28. Li, X.-Z., L . Zhang, and K . Poole. 1998 . Role of the multidrug efflux systems of Pseudomonas aeruginosa in organic solvent tolerance . J . Bacteriol . 180:2987-2991 .
29. Li, X.-Z., L . Zhang, R . Srikumar, and K . Poole. 1998 . ß-Lactamase inhibitors are substrates of the multidrug efflux pumps of Pseudomonas aeruginosa . Antimicrob . Agents Chemother . 42:399-403 .
30. Li, X . Z., and K . Poole. 1999 . Organic solvent-tolerant mutants of Pseudomonas aeruginosa display multiple antibiotic resistance . Can . J . Microbiol . 45:18-22.
31. Li, Y., T . Mima, Y . Komori, Y . Morita, T . Kuroda, T . Mizushima, and T . Tsuchiya. 2003 . A new member of the tripartite multidrug efflux pumps, MexVW-OprM, in Pseudomonas aeruginosa . J Antimicrob . Chemother . 52:572-575 .
32. Mao, W., M . S . Warren, D . S . Black, T . Satou, T . Murata, T . Nishino, N . Gotoh, and O . Lomovskaya. 2002 . On the mechanism of substrate specificity by resistance nodulation division [RND]-type multidrug resistance pumps: the large periplasmic loops of MexD from Pseudomonas aeruginosa are involved in substrate recognition . Mol . Microbiol . 46:889-901.
33. Masuda, N., and S . Ohya. 1992 . Cross-resistance to meropenem, cephems, and quinolones in Pseudomonas aeruginosa . Antimicrob . Agents Chemother . 36:1847-1851.
34. Masuda, N., E . Sakagawa, and S . Ohya. 1995 . Outer membrane proteins responsible for multiple drug resistance in Pseudomonas aeruginosa . Antimicrob . Agents Chemother . 39:645-649.
35. Masuda, N., E . Sakagawa, S . Ohya, N . Gotoh, H . Tsujimoto, and T . Nishino. 2000 . Substrate specificities of MexAB-OprM, MexCD-OprJ, and MexXY-OprM efflux pumps in Pseudomonas aeruginosa . Antimicrob Agents Chemother . 44:3322-3327 .
36. Mine, T., Y . Morita, A . Kataoka, T . Mitzushima, and T . Tsuchiya. 1999 . Expression in Escherichia coli of a new multidrug efflux pump, MexXY, from Pseudomonas aeruginosa . Antimicrob . Agents Chemother . 43:415-417 .
37. Mitchell, B . A., I . T . Paulsen, M . H . Brown, and R . A . Skurray. 1999 . Bioenergetics of the staphylococcal multidrug export protein QacA . Identification of distinct binding sites for monovalent and divalent cations . J . Biol . Chem . 274:3541-3548 .
38. Murakami, S., R . Nakashima, E . Yamashita, and A . Yamaguchi. 2002 . Crystal structure of bacterial multidrug efflux transporter AcrB . Nature 419:587-593.
39. Murata, T., M . Kuwagaki, T . Shin, N . Gotoh, and T . Nishino. 2002 . The substrate specificity of tripartite efflux systems of Pseudomonas aeruginosa is determined by the RND component . Biochem . Biophys . Res . Commun . 299:247-251.
40. Pearson, J . P., C . Van Delden, and B . H . Iglewski. 1999 . Active efflux and diffusion are involved in transport of Pseudomonas aeruginosa cell-to-cell signals . J . Bacteriol . 181:1203-1210 .
41. Piddock, L . J . V., M . C . Hall, F . Bellido, M . Bains, and R . E . W . Hancock. 1992 . A pleiotropic, posttherapy, enoxacin-resistant mutant of Pseudomonas aeruginosa . Antimicrob . Agents Chemother . 36:1057-1061.
42. Poole, K. 2001 . Multidrug efflux pumps and antimicrobial resistance in Pseudomonas aeruginosa and related organisms . J . Mol . Microbiol . Biotechnol . 3:255-264.
43. Poole, K. 2001 . Multidrug resistance in Gram-negative bacteria . Curr . Opin . Microbiol . 4:500-508.
44. Poole, K. 2002 . Outer membranes and efflux: the path to multidrug resistance in gram-negative bacteria . Curr . Pharm . Biotechnol . 3:77-98.
45. Poole, K., N . Gotoh, H . Tsujimoto, Q . Zhao, A . Wada, T . Yamasaki, S . Neshat, J.-I . Yamagishi, X.-Z . Li, and T . Nishino. 1996 . Overexpression of the mexC-mexD-oprJ efflux operon in nfxB multidrug resistant strains of Pseudomonas aeruginosa . Mol . Microbiol. 21:713-724.
46. Poole, K., D . E . Heinrichs, and S . Neshat. 1993 . Cloning and sequence analysis of an EnvCD homologue in Pseudomonas aeruginosa: regulation by iron and possible involvement in the secretion of the siderophore pyoverdine . Mol . Microbiol . 10:529-544.
47. Poole, K., K . Krebes, C . McNally, and S . Neshat. 1993 . Multiple antibiotic resistance in Pseudomonas aeruginosa: evidence for involvement of an efflux operon . J . Bacteriol . 175:7363-7372.
48. Poole, K., and R . Srikumar. 2001 . Multidrug efflux in Pseudomonas aeruginosa: components, mechanisms and clinical significance . Curr . Top . Med . Chem . 1:59-71.
49. Poole, K., K . Tetro, Q . Zhao, S . Neshat, D . Heinrichs, and N . Bianco. 1996 . Expression of the multidrug resistance operon mexA-mexB-oprM in Pseudomonas aeruginosa: mexR encodes a regulator of operon expression . Antimicrob . Agents Chemother . 40:2021-2028.
50. Putman, M., L . A . Koole, H . W . van Veen, and W . N . Konings. 1999 . The secondary multidrug transporter LmrP contains multiple drug interaction sites . Biochemistry 38:13900-13905.
51. Putman, M., H . W . van Veen, and W . N . Konings. 2000 . Molecular properties of bacterial multidrug transporters . Microbiol . Mol . Biol . Rev . 64:672-693 .
52. Rella, M., and D . Haas. 1982 . Resistance of Pseudomonas aeruginosa PAO to nalidixic acid and low levels of ß-lactam antibiotics: mapping of chromosomal genes . Antimicrob . Agents Chemother . 22:242-249.
53. Saito, K., H . Yoneyama, and T . Nakae. 1999 . nalB-type mutations causing the overexpression of the MexAB-OprM efflux pump are located in the mexR gene of the Pseudomonas aeruginosa chromosome . FEMS Microbiol . Lett . 179:67-72.
54. Sambrook, J., and D . W . Russell. 2001 . Molecular cloning: a laboratory manual, 3rd ed . Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
54. Schumacher, M . A., M . C . Miller, S . Grkovic, M . H . Brown, R . A . Skurray, and R . G . Brennan. 2001 . Structural mechanisms of QacR induction and multidrug recognition . Science 294:2158-2163 .
55. Schweizer, H . P. 1998 . Intrinsic resistance to inhibitors of fatty acid biosynthesis in Pseudomonas aeruginosa is due to efflux: application of a novel technique for generation of unmarked chromosomal mutations for the study of efflux systems . Antimicrob . Agents Chemother . 42:394-398 .
56. Sekiya, H., T . Mima, Y . Morita, T . Kuroda, T . Mizushima, and T . Tsuchiya. 2003 . Functional cloning and characterization of a multidrug efflux pump, MexHI-OpmD, from a Pseudomonas aeruginosa mutant . Antimicrob . Agents Chemother . 47:2990-2992 .
57. Simon, R., U . Priefer, and A . Puehler. 1983 . A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria . Bio/Technology 1:784-791.
58. Srikumar, R., T . Kon, N . Gotoh, and K . Poole. 1998 . Expression of Pseudomonas aeruginosa multidrug efflux pumps MexA-MexB-OprM and MexC-MexD-OprJ in a multidrug-sensitive Escherichia coli strain . Antimicrob . Agents Chemother . 42:65-71 .
59. Srikumar, R., X.-Z . Li, and K . Poole. 1997 . Inner membrane efflux components are responsible for the ß-lactam specificity of multidrug efflux pumps in Pseudomonas aeruginosa . J . Bacteriol . 179:7875-7881.
60. Srikumar, R., C . J . Paul, and K . Poole. 2000 . Influence of mutations in the mexR repressor gene on expression of the MexA-MexB-OprM multidrug efflux system of Pseudomonas aeruginosa . J . Bacteriol . 182:1410-1414 .
61. Srikumar, R., and K . Poole. 1999 . Demonstration of ethidium bromide efflux by multiresistant pumps of Pseudomonas aeruginosa . Clin . Microbiol . Infect . 5:5S58-5S59.
62. Tikhonova, E . B., Q . Wang, and H . I . Zgurskaya. 2002 . Chimeric analysis of the multicomponent multidrug efflux transporters from Gram-negative bacteria . J . Bacteriol . 184:6499-6507 .
63. Westbrock-Wadman, S., D . R . Sherman, M . J . Hickey, S . N . Coulter, Y . Q . Zhu, P . Warrener, L . Y . Nguyen, R . M . Shawar, K . R . Folger, and C . K . Stover. 1999 . Characterization of a Pseudomonas aeruginosa efflux pump contributing to aminoglycoside impermeability . Antimicrob . Agents Chemother . 43:2975-2983 .
64. Yoneyama, H., A . Ocaktan, N . Gotoh, T . Nishino, and T . Nakae. 1998 . Subunit swapping in the Mex-extrusion pumps in Pseudomonas aeruginosa . Biochem . Biophys . Res . Commun . 244:898-902.
65. Yu, E . W., G . McDermott, H . I . Zgurskaya, H . Nikaido, and D . E . Koshland, Jr. 2003 . Structural basis of multiple drug-binding capacity of the AcrB multidrug efflux pump . Science 300:976-980 .
66. Zgurskaya, H . I. 2002 . Molecular analysis of efflux pump-based antibiotic resistance . Int . J . Med . Microbiol . 292:95-105.
67. Zgurskaya, H . I., and H . Nikaido. 2000 . Multidrug resistance mechanisms: drug efflux across two membranes . Mol . Microbiol . 37:219-225.
68. Ziha-Zarifi, I., C . Llanes, T . Koehler, J.-C . Pechere, and P . Plesiat. 1999 . In vivo emergence of multidrug-resistant mutants of Pseudomonas aeruginosa overexpressing the active efflux system MexA-MexB-OprM . Antimicrob . Agents Chemother . 43:287-291 .

Free Online Full-text Article

What Is Antibiotic?, What Is Dna?, What Is Molecular Microbiology?, What Is Pcr?, What Is Rhizobia?, i, Bacteria, o, Microorganism, o, Bacteriology, e, Microbes, s, Microbe, r, Microbial, i, Candida albicans, n, Escherichia coli, r, Antimicrobial, i, Yeasts, i, Campylobacter, o, Escherichia coli, s, S. cerevisiae, i, S. cerevisiae, c, Prokaryotes, s, Antibiotic treatment, r, Aeromonades, r, Microbial, o, Paenibacilli, i, Microorganism, n, Petri dish, s, Alcaligenes, o, Bacteria, i, Pseudomonas aeruginosa, r, Vibriosis, a, Escherichia coli




 

© 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com
 

Last modified: May 25, 2005