Interaction of the MexA and MexB Components of the MexAB-OprM Multidrug Efflux System of
            <i>Pseudomonas aeruginosa</i>
            : Identification of MexA Extragenic Suppressors of a T578I Mutation in MexB

Nehme, Dominic; Poole, Keith

doi:10.1128/aac.49.10.4375-4378.2005

Cited by 20 publications

(24 citation statements)

References 23 publications

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“…Plasmid pDN40, encoding OprM, was constructed by amplifying the oprM gene from the chromosome of P. aeruginosa K870 via PCR and cloning it (as a SacI-HindIII fragment) into pMMB206. Amplification was achieved using the primers Fragment B-forward (5Ј-GAGCTCGAGCTCTCCA ACGACGTGTTCTTCCAGGT-3Ј; tandem SacI sites are underlined) and OprMR-HindIII [5Ј-AAGCTTAAGCTTAGGCCGA-GCGGGTCCGTGACG C-3Ј; tandem HindIII sites are underlined) and reaction conditions and parameters described previously (extension time increased to 2 min) for the amplification of the EcoRI-tagged mexA gene (30). Plasmid pDN42, encoding MexA(V129M), was constructed by amplifying the mexA(V129M) gene from plasmid pDN7 using the primers pMMB-MexA-For (5Ј-CCCGGGCCCGGGT GAATGTAAGTATTTTGCCTGC-3Ј; tandem SmaI sites are underlined) and pMMB-MexA-Rev (5Ј-GGATCCGGATCCGATCACCCACGCGAAAATGG-3Ј; tandem BamHI sites are underlined) and cloning it (as a SmaI-BamHI fragment) into pMMB206.…”

Section: Methodsmentioning

confidence: 99%

“…Mutagenic PCR of the mexA gene was conducted by PCR amplification of the mexA gene from plasmid pDN3, exactly as described previously (30), using the primers JT-28 (5Ј-AAGCTTAAGCTTTGAATGTAAGTATTTTGCCTGC-3Ј; tandem HindIII sites are underlined) and JT-27 (5Ј-GAGCTCGAGCTCGATC ACCCACGCGAAAATGG-3Ј; tandem SacI sites are underlined). The resultant mutagenized mexA-containing PCR products were digested with HindIII and SacI and cloned en masse into pRK415, with the recombinant plasmids electroporated into E. coli S17-1 and mobilized into P. aeruginosa K2275 (⌬mexR ⌬mexA ⌬mexB) harboring plasmid pDN43 [pMMB206::mexB(G220S)] via conjugation as described previously (29).…”

Section: Methodsmentioning

confidence: 99%

“…Following construction of pDN57, genes for wild-type and suppressor mutant (V66M and V259F) MexA proteins were amplified from plasmids pDN3, pDN47, and pDN49, respectively, and individually cloned upstream of mexB (G220S)-His exactly as described previously (30) to yield plasmids pDN44, pDN45, and pDN46. These plasmids, on which the mexA and mexB(G220S)-His genes are coexpressed from the same IPTG-inducible vector-borne promoter, were then introduced into P. aeruginosa K2275 (⌬mexR ⌬mexA ⌬mexB).…”

Section: Mexa-mexb Interaction Assaymentioning

confidence: 99%

“…To evaluate an interaction between MexB(G220S) and wild-type and suppressor mutant MexA proteins, a C-terminal polyhistidine tag was first engineered onto MexB(G220S) encoded by pDN43 (to yield pDN57) using an approach described elsewhere (30), and corecovery of MexA with MexB(G220S)-His on Ni-nitrilotriacetic acid (NTA) agarose (QIAGEN) was assessed as described previously without cross-linker (29). Following construction of pDN57, genes for wild-type and suppressor mutant (V66M and V259F) MexA proteins were amplified from plasmids pDN3, pDN47, and pDN49, respectively, and individually cloned upstream of mexB (G220S)-His exactly as described previously (30) to yield plasmids pDN44, pDN45, and pDN46.…”

Section: Mexa-mexb Interaction Assaymentioning

confidence: 99%

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Assembly of the MexAB-OprM Multidrug Pump of Pseudomonas aeruginosa : Component Interactions Defined by the Study of Pump Mutant Suppressors

Nehme

Poole

2007

J Bacteriol

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In an effort to identify key domains of the Pseudomonas aeruginosa MexAB-OprM drug efflux system involved in component interactions, extragenic suppressors of various inactivating mutations in individual pump constituents were isolated and studied. The multidrug hypersusceptibility of P. aeruginosa expressing MexB with a mutation in a region of the protein implicated in oligomerization (G220S) was suppressed by mutations in the ␣/␤ domain of MexA. MexB(G220S) showed a reduced ability to bind MexA in vivo while representative MexA suppressors (V66M and V259F) restored the MexA-MexB interaction. Interestingly, these suppressors also restored resistance in P. aeruginosa expressing OprM proteins with mutations at the proximal (periplasmic) tip of OprM that is predicted to interact with MexB, suggesting that these suppressors generally overcame defects in MexA-MexB and MexB-OprM interaction. The multidrug hypersusceptibility arising from a mutation in the helical hairpin of MexA implicated in OprM interaction (V129M) was suppressed by mutations (T198I and F439I) in the periplasmic ␣-helical barrel of OprM. Again, the MexA mutation compromised an in vivo interaction with OprM that was restored by the T198I and F439I substitutions in OprM, consistent with the hairpin domain mediating MexA binding to this region of OprM. Interestingly, these OprM suppressor mutations restored multidrug resistance in P. aeruginosa expressing MexB(G220S). Finally, the oprM(T198I) suppressor mutation enhanced the yields of all three constituents of a MexA-MexB-OprM(T198I) pump as detected in whole-cell extracts. These data highlight the importance of MexA and interactions with this adapter in promoting MexAB-OprM pump assembly and in stabilizing the pump complex.Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an innate resistance to multiple antimicrobials (10), resistance increasingly attributable to the operation of broadly specific, multidrug efflux systems of the resistance-nodulation-division (RND) family (33, 34). Several RND family multidrug efflux systems have been described in P. aeruginosa (34, 35) although the major system contributing to intrinsic multidrug resistance is encoded by the mexAB-oprM operon (19). This system also contributes to acquired multidrug resistance as a result of its hyperexpression in nalB (i.e., mexR) (45, 53), nalC (6, 21), and nalD (42) mutants. The MexAB-OprM efflux system, like other tripartite RND family pumps, consists of an inner membrane drug-proton antiporter (the RND component; MexB), an outer membrane (OM) channel-forming component (also called outer membrane factor [OMF]; OprM) and a periplasmic membrane fusion protein ([MFP]; MexA) (32, 52).Crystal structures have now been reported for MexA (2, 11) and OprM (1), with the OprM structure reminiscent of that of TolC, the homologous OM component of the Escherichia coli AcrAB-TolC multidrug efflux system (18). The OprM channel is trimeric and is comprised of an OM-spanning ␤-barrel and a periplasmic ␣-helical barrel, wi...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Mexa-mexb Interaction Assaymentioning

confidence: 99%

Section: Mexa-mexb Interaction Assaymentioning

confidence: 99%

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Assembly of the MexAB-OprM Multidrug Pump of Pseudomonas aeruginosa : Component Interactions Defined by the Study of Pump Mutant Suppressors

Nehme

Poole

2007

J Bacteriol

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“…In contrast to earlier predictions that suggested that MFPs might span the periplasm with their N and C termini bound to the inner and outer membranes, respectively (548), the structures suggest that both the N and C termini are likely to lie in close proximity to the inner membrane. Mutagenesis and suppressor analyses suggest that the C termini of the MFPs interact with the inner membrane transporters (at least in the case of the RND transporters Mex and Acr) (139,329,330), while the N terminus may be involved in oligomerization of the MFPs (329).…”

Section: Protein Trafficking Between and Across Membranesmentioning

confidence: 99%

Structure, Function, and Evolution of Bacterial ATP-Binding Cassette Systems

Davidson

Dassa

Orelle

et al. 2008

Microbiol Mol Biol Rev

1,168

1,137

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SUMMARY ATP-binding cassette (ABC) systems are universally distributed among living organisms and function in many different aspects of bacterial physiology. ABC transporters are best known for their role in the import of essential nutrients and the export of toxic molecules, but they can also mediate the transport of many other physiological substrates. In a classical transport reaction, two highly conserved ATP-binding domains or subunits couple the binding/hydrolysis of ATP to the translocation of particular substrates across the membrane, through interactions with membrane-spanning domains of the transporter. Variations on this basic theme involve soluble ABC ATP-binding proteins that couple ATP hydrolysis to nontransport processes, such as DNA repair and gene expression regulation. Insights into the structure, function, and mechanism of action of bacterial ABC proteins are reported, based on phylogenetic comparisons as well as classic biochemical and genetic approaches. The availability of an increasing number of high-resolution structures has provided a valuable framework for interpretation of recent studies, and realistic models have been proposed to explain how these fascinating molecular machines use complex dynamic processes to fulfill their numerous biological functions. These advances are also important for elucidating the mechanism of action of eukaryotic ABC proteins, because functional defects in many of them are responsible for severe human inherited diseases.

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