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ABSTRACTThe swine pathogen Actinobacillus pleuropneumoniaepossesses a 75-kDa outer membrane protein (OMP), FhuA, the receptor for ferrichrome, a hydroxamate-type siderophore. Polyclonal serum to FhuA reacted with OMP preparations from 12 serotypes of A. pleuropneumoniaeunder conditions of iron repletion and restriction. Reverse transcription-PCR confirmed that A. pleuropneumoniae fhuAexpression is not upregulated in response to low iron levels. An A. pleuropneumoniae fhuAdeletion mutant was generated and showed abolishment of ferrichrome uptake.

The major diffusion channel in the outer membrane of Haemophilus influenzaetype b (Hib) is porin (341 amino acids; Mr37 782). The Hibporin gene was cloned and overexpressed in Bacillus subtilis. Recombinant Hib porin (Bac porin), having aggregated into inclusion bodies, was purified under denaturing conditions and subsequently refolded. To compare Bac porin that is intrinsically devoid of lipooligosaccharides versus native Hib porin, the properties of Bac porin were assessed by the following four criteria: circular dichroism spectroscopy, channel formation in planar bilayers, resistance to trypsin digestion and formation of the conformational epitope recognized by an anti-Hib porin monoclonal antibody. We conclude that in the absence of lipooligosaccharides, Bac porin was refolded into a functional form which closely resembled the structure of Hib porin.

ABSTRACTThe mexCD-oprJand mexAB-oprMoperons encode components of two distinct multidrug efflux pumps inPseudomonas aeruginosa. To assess the contribution of individual components to antibiotic resistance and substrate specificity, these operons and their component genes were cloned and expressed in Escherichia coli. Western immunoblotting confirmed expression of the P. aeruginosaefflux pump components in E. colistrains expressing and deficient in the endogenous multidrug efflux system (AcrAB), although only the ΔacrABstrain, KZM120, demonstrated increased resistance to antibiotics in the presence of the P. aeruginosaefflux genes. E. coliKZM120 expressing MexAB-OprM showed increased resistance to quinolones, chloramphenicol, erythromycin, azithromycin, sodium dodecyl sulfate (SDS), crystal violet, novobiocin, and, significantly, several β-lactams, which is reminiscent of the operation of this pump in P. aeruginosa. This confirmed previous suggestions that MexAB-OprM provides a direct contribution to β-lactam resistance via the efflux of this group of antibiotics. An increase in antibiotic resistance, however, was not observed when MexAB or OprM alone was expressed in KZM120. Thus, despite the fact that β-lactams act within the periplasm, OprM alone is insufficient to provide resistance to these agents. E. coliKZM120 expressing MexCD-OprJ also showed increased resistance to quinolones, chloramphenicol, macrolides, SDS, and crystal violet, though not to most β-lactams or novobiocin, again somewhat reminiscent of the antibiotic resistance profile of MexCD-OprJ-expressing strains ofP. aeruginosa. Surprisingly, E. coliKZM120 expressing MexCD alone also showed an increase in resistance to these agents, while an OprJ-expressing KZM120 failed to demonstrate any increase in antibiotic resistance. MexCD-mediated resistance, however, was absent in a tolCmutant of KZM120, indicating that MexCD functions in KZM120 in conjunction with TolC, the previously identified outer membrane component of the AcrAB-TolC efflux system. These data confirm that a tripartite efflux pump is necessary for the efflux of all substrate antibiotics and that the P. aeruginosamultidrug efflux pumps are functional and retain their substrate specificity in E. coli.

ABSTRACTTonB couples the energized state of the cytoplasmic membrane to the operation of outer membrane receptors responsible for Fe(III) siderophore uptake across the outer membrane of gram-negative bacteria. A tonBmutant of Pseudomonas aeruginosadeficient in iron siderophore uptake was shown in the present study to be hypersusceptible to a wide variety of antibiotics, reminiscent of the phenotype of mutants defective in the mexAB-oprMantibiotic efflux operon. This was not related to influences of atonBmutation on the iron status of the cell, and indeed, intrinsic antibiotic susceptibility and mexAB-oprMexpression were unaffected by iron levels in the growth medium. The presence of tonBon a multicopy plasmid increased the level of resistance of a MexAB-OprM+strain but not that of a MexAB-OprM−strain to a variety of antimicrobial agents.mexAB-oprMexpression was not, however, altered in atonBdeletion mutant, indicating that any influence of TonB on MexAB-OprM-mediated multidrug resistance was at the level of pump activity. Consistent with this, drug accumulation assays revealed that the tonBdeletion mutant exhibited decreased levels of drug efflux. Still, the multidrug resistance of a nalBstrain was not wholly abrogated by a tonBmutation, indicating that it is likely not an essential component of the efflux apparatus. Similarly, elimination of tonBfrom an nfxBstrain only partially compromised MexCD-OprJ-mediated multidrug resistance. Intriguingly, the drug susceptibility of amexAB-oprMdeletion strain was increased following deletion of tonB, suggesting that TonB may also influence antibiotic resistance mediated by determinants other than MexAB-OprM (and MexCD-OprJ). Thus, TonB plays an important role in both intrinsic and acquired antibiotic resistance in P. aeruginosa.

ABSTRACTA Pseudomonas aeruginosastrain carrying an insertion of an ΩHg interposon in the mexBgene (mexB::ΩHg; strain K879) produced markedly reduced but still detectable levels of OprM, the product of the third gene of the mexAB-oprMmultidrug efflux operon. By using alacZtranscriptional fusion vector, promoter activity likely responsible for OprM expression in themexB::ΩHg mutant was identified upstream ofoprM. Introduction of the oprMgene, but not the mexABgenes, into a P. aeruginosamultidrug-susceptible ΔmexAB-oprMmutant increased resistance to quinolones, cephalosporins, erythromycin, and tetracycline. A ΔmexAB-oprMstrain carrying the oprMgene accumulated markedly less antibiotic than the deletion strain without oprM. Antibiotic accumulation by the MexAB−OprM+strain was markedly enhanced upon treatment of cells with the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), indicating that MexAB-independent OprM function likely involves an efflux process. Moreover, pretreatment of cells with CCCP prior to the accumulation assay abrogated any differences in accumulation levels between the MexAB−OprM+and MexAB−OprM−strains, indicating that reduced drug accumulation by the OprM+strain (in the absence of CCCP) cannot be due to OprM-mediated reduction in outer membrane permeability. It appears, therefore, that OprM can be expressed and function in a drug efflux capacity independent of MexAB.