Shima Eda

Linkage of the efflux-pump expression level with substrate extrusion rate in the MexAB–OprM efflux pump of Pseudomonas aeruginosa

The amount of the subunit proteins of the MexAB-OprM efflux pump in Pseudomonas aeruginosa was quantified by the immunoblotting method. A single cell of the wild-type strain contained about 2500, 1000, and 1200 copies of MexA, MexB, and OprM, respectively, and their stoichiometry therefore was 2:1:1. The mexR mutant produced an eightfold higher level of these proteins than did wild-type cells. Assuming that MexB and OprM exist as a trimer in a pump assembly, the total number of MexAB-OprM per wild-type cell was calculated to be about 400 assemblies. The substrate efflux rate of MexAB-OprM was calculated from the fluorescent intensity of ethidium in intact cells that a single cell extruded ethidium at a maximum of about 3 x 10(-19) mol s(-1) and, therefore, the turnover rate of a single pump unit was predicted to be about 500 s(-1).

A novel assembly process of the multicomponent xenobiotic efflux pump in Pseudomonas aeruginosa

The nfxC‐type cells of Pseudomonas aeruginosa show resistance to a wide range of structurally and functionally diverse antibiotics, which is a phenomenon that is mainly attributable to the expression of the MexEF‐OprN xenobiotic transporter. The MexF, MexE and OprN subunits of this transporter are located on the inner membrane, the periplasm and the outer membrane, respectively, and are assumed to function as an energy‐dependent transporter, a bridge connecting the inner and outer membranes and outer membrane channel respectively. The nfxC‐type cells showed a single protein band of MexF and OprN, whereas MexE appeared as three distinct bands in an SDS‐polyacrylamide gel electrophoretogram. The mutant cells lacking MexF produced undetectable OprN and only a full‐size of MexE even though the cells had unimpaired oprN and mexE. Expression of the plasmid‐borne MexF in this mutant fully restored OprN and three MexE bands. Another class of mutants producing a full amount of MexF yielded undetectable OprN and two MexE bands lacking the smallest protein species suggesting that the presence of the smallest MexE subunit is required for stabilization of OprN. To identify which part of MexE was needed for stabilization and assembly of OprN, the carboxyl‐terminal‐truncated MexE tagged with polyhistidine was constructed and protein bands were visualized in the presence of MexF with an antibody raised against polyhistidine or MexE. The results revealed that the proteolytic processing of MexE would occur at carboxyl terminal amino acids between 11 and 16, thereby suggesting that the presence of the C‐terminal truncated MexE is essential for stabilization and the proper assembly of OprN. Nucleotide sequencing of mutant mexFs, which produce a wild‐type level of MexF but are unable to support the production of the smallest MexE, thereby destabilizing OprN, revealed that all the mutations were located within two large periplasmic domains of MexF between transmembrane segments 1–2 and 7–8. Taking these findings together, we concluded that two large periplasmic domains of MexF interact with MexE thereby promoting programmed processing of MexE, and this complex eventually assists the correct assembly and sorting of OprN.

Functional interaction sites of OprM with MexAB in the Pseudomonas aeruginosa multidrug efflux pump.

Subunit-swapping between Pseudomonas aeruginosa MexAB-OprM and MexEF-OprN efflux pumps has shown that OprM can interact with MexEF to produce a functional efflux pump, but that OprN cannot functionally interact with MexAB. Taking advantage of this subunit selectivity, we carried out experiments using chimeric proteins composed of OprM and OprN to determine which regions of OprM are necessary for functional interaction with MexAB. We constructed two types of chimeric proteins: one with the N-terminal half of OprM and the C-terminal half of OprN (OprMN), and the second with these halves reversed (OprNM). Introduction of either of the chimeric protein genes into a mutant expressing MexEF alone restored the functionality of the efflux pump. However, expression of OprMN or OprNM in the presence of MexAB did not restore the pump functionality, indicating that the both the N- and C-terminal halves of OprM are necessary for a functional interaction with MexAB.

Diversity in the oligomeric channel structure of the multidrug efflux pumps in Pseudomonas aeruginosa.

MexAB‐OprM, the multidrug efflux pump of Pseudomonas aeruginosa, contributes to the high resistance of this organism to a wide variety of antibiotics. To investigate the structure and function of OprM, the outer membrane channel of MexAB‐OprM, we examined the oligomeric states of OprM and its homologues OprJ and OprN. These proteins were treated with crosslinking reagent after their reconstitution into liposome membranes. The crosslinked products indicated that OprM and OprN formed trimers, while OprJ unexpectedly appeared to form a tetramer. In order to test whether differences in oligomeric structure might be intimately related to channel function, we examined the channel‐forming activity of these proteins by liposome swelling assay. However, no significant differences in channel characteristics were detected among OprM, OprJ, and OprN. We proposed the probable explanation for the diversity in the oligomeric structure of the channel proteins.

Organic Solvent-Selective Domain of the Resistance-Nodulation-Division-Type Xenobiotic-Antibiotic Transporters of Pseudomonas aeruginosa

The hydrophobic substrate‐selective domain of the resistance‐nodulation‐division‐type xenobiotic transporter of Pseudomonas aeruginosa was assigned based on the different organic‐solvent selectivities of MexB and MexY. The MexB‐MexY hybrid protein consisting of two large periplasmic domains of MexB and the transmembrane domains of MexY showed MexB‐type organic solvent selectivity. The results imply that the resistance‐nodulation‐division‐type xenobiotic transporters recognize hydrophobic substrates such as organic solvents by their periplasmic domains and expel them to the external milieu. This is an elegant way to protect the cytoplasmic membrane from membrane‐deteriorating agents.