MexZ is a transcriptional regulator of the mexXY multidrug transporter operon, which confers aminoglycoside resistance on Pseudomonas aeruginosa. Highly purified MexZ showed direct binding with a specific site of the mexZ-mexX intergenic DNA when probed by a gel retardation assay. Both in vitro chemical cross-linking experiments and an in vivo two-hybrid expression system showed that the active form of MexZ, which is capable of binding the intergenic DNA, appeared to be a dimer. These results explain the mechanism by which MexZ represses transcription of the mexXY operon, but do not explain the substrate-induced hyperproduction of MexXY. The presence of inducer antibiotic in the gel-retardation assay mixture failed to detect altered MexZ-probe DNA interaction suggesting the possible involvement of an additional regulator.

To investigate the functional role of protein F of the outer membrane of Pseudomonas aeruginosa, we isolated mutants devoid of protein F, and the defective gene was transferred to a wild-type strain by plasmid FP5-mediated conjugation. Chemical analyses of the protein F-deficient outer membrane revealed that the amount of outer membrane protein was reduced to 72 to 74% of that of the protein F-sufficient strain and that lipopolysaccharides and phospholipids increased to 117 to 123% and 135 to 136%, respectively. The mutants and the transconjugant showed the following characteristics: (i) growth rates of protein F-deficient strains in low-osmolarity medium (e.g., L broth containing 0.1% NaCl) were less than 1/10 the rate of the protein F-sufficient strain; (ii) protein F-deficient cells were rounded, and the outer membrane formed large protruded blebs; and (iii) the outer membrane became physically fragile, since a significant amount of periplasmic proteins leaked out and the cells became highly sensitive to osmotic shock. The results suggested that protein F plays an important role in morphogenesis and in maintaining the integrity of the outer membrane.Determination of the diffusion rates of saccharides and IB-lactam antibiotics showed that the protein F-deficient outer membrane had no detectable transport defect compared with the protein F-sufficient outer membrane. The MICs of antibiotics for the protein F-deficient strains were nearly identical to those for the protein F-sufficient strain.The outer membrane proteins of gram-negative bacteria play roles in forming diffusion pores stabilizing the outer membrane; that is, they form porins (22) for small hydrophilic solutes. These outer membrane proteins, which include OmpF, OmpC, and OmpA (20), are involved in the receptor function for bacteriophages, bacteriocins, and sex pili (20). Pseudomonas aeruginosa, a pathogen in immunocompromised patients, is highly resistant to a wide range of antibiotics (44). This intrinsic drug resistance is known to be due mainly to the diffusion barrier at the outer membrane (1, 42). In permeability studies of the outer membrane of P. aeruginosa, protein F was reported to be the porin forming the large diffusion pore. This pore allows the passage of uncharged polysaccharides with Mrs of several thousands as determined by the equilibrium of the labeled polysaccharides in vitro (11) and in vivo (10) and rate measurement by the liposome-swelling method (43). Determination of ion conductivity in a black lipid bilayer supported these results, and the pore diameter was calculated to be about 2.2 nm (2). Nicas and Hancock had isolated a mutant devoid of protein F and showed that the diffusion rate of a chromogenic P-lactam, nitrocefin, across such an outer membrane was lowered to one-sixth that of the protein F-sufficient outer membrane, but the antibiotic susceptibility was essentially unchanged (8, 27). To explain this apparent discrepancy, they proposed that the majority of the protein F pores were nonfunctional, being in a closed sta...

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Determination of the rates of diffusion of P-lactam antibiotics through purified Pseudomonas aeruginosa porins C, D2, and E in liposomes yielded the following results. (i) The rates of carbapenem (imipenem and meropenem) diffusion through the protein D2 pore were roughly 2 to 70 times higher than those through other porin pores. It is not clear why the protein D2 pore allowed rapid diffusion of carbapenems. The rates of diffusion of glucosamine and triglycine through the protein D2 pore were about 14 and 4 times higher, respectively, than that of an uncharged test solute with a similar Mr, glucose.(ii) The rates of diffusion of antipseudomonal anionic j-lactams such as piperacillin, ceftazidime, cefsulodin, and aztreonam through the protein C pore were higher than those through other porin pores. This was probably due to the slightly larger pore size and the slight anion selectivity of protein C, since the apparent exclusion limit of the protein C pore for uncharged saccharides is higher than that of other porins and the rate of diffusion of gluconic acid through the protein C pore is about double that for glucose. (iii) The rates of diffusion of cefoperazone through all three species of porin were relatively high. These results indicate that the antipseudomonal (-lactams permeate the P. aeruginosa outer membrane via newly identified porins.

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