The PhoPQ two-component system of Salmonella enterica serovar Typhimurium produces a remodeling of the lipid A domain of the lipopolysaccharide, including the PagP-catalyzed addition of palmitoyl residue, the PmrAB-regulated addition of the cationic sugar 4-aminoarabinose and phosphoethanolamine, and the LpxOcatalyzed addition of a 2-OH group onto one of the fatty acids. By using the diffusion rates of the dyes ethidium, Nile red, and eosin Y across the outer membrane, as well as the susceptibility of cells to large, lipophilic agents, we evaluated the function of this membrane as a permeability barrier. We found that the remodeling process in PhoP-constitutive strains produces an outer membrane that serves as a very effective permeability barrier in an environment that is poor in divalent cations or that contains cationic peptides, whereas its absence in phoP null mutants produces an outer membrane severely compromised in its barrier function under these conditions. Removing combinations of the lipid A-remodeling functions from a PhoP-constitutive strain showed that the known modification reactions explain a major part of the PhoPQ-regulated changes in permeability. We believe that the increased barrier property of the remodeled bilayer is important in making the pathogen more resistant to the stresses that it encounters in the host, including attack by the cationic antimicrobial peptides. On the other hand, drug-induced killing assays suggest that the outer membrane containing unmodified lipid A may serve as a better barrier in the presence of high concentrations (e.g., 5 mM) of Mg 2؉ .Cells of gram-negative bacteria are surrounded by the outer membrane (OM), which functions primarily as a permeability barrier (35). Large, hydrophilic compounds are excluded by the narrow porin channels, and lipophilic compounds cross the unusual, asymmetric bilayer of this membrane only slowly. Our early analysis by using steroids as probes (37) showed that the diffusion across the OM bilayer is nearly 2 orders of magnitude slower than the diffusion across a typical phospholipid bilayer, such as the phospholipid bilayer that exists in the inner, cytoplasmic membrane. A recent study (11) suggested that this estimate likely needs to be adjusted somewhat downward. Nevertheless, the OM bilayer clearly functions as a formidable permeation barrier, because perturbing this bilayer results in a striking sensitization of Escherichia coli and Salmonella enterica serovar Typhimurium to various lipophilic inhibitors (44).One major factor that contributes to this barrier property is presumably the asymmetric structure of the OM bilayer, whose outer leaflet is composed nearly entirely of lipopolysaccharides (LPS) (25). (In fact, very low permeability was found in symmetric LPS bilayers assembled in the laboratory [40].) According to the lattice model for diffusion in liquids, a statistical average of rapidly fluctuating variable distances creates transient holes within the bilayer into which solutes can migrate (41). Thus, an effective memb...

SummaryTripartite efflux systems of Gram-negative bacteria that contain an inner membrane transporter belonging to the resistance nodulation division (RND) superfamily can extrude a large variety of structurally diverse compounds. To gain an insight into the molecular mechanisms of substrate recognition by these multidrug resistance (MDR) transporters, we isolated spontaneous mutations that altered the substrate specificity of the MexCD-OprJ pump from Pseudomonas aeruginosa. These mutations enabled the pump to extrude the normally non-transported β β β β -lactam antibiotic carbenicillin. All amino acid substitutions were mapped to the large periplasmic loops (LPLs) of the RND proper, MexD. Q34K, E89K, A292V and P328L were found in the first LPL, located between transmembrane domains (TMD) 1 and 2, whereas F608S and N673K were contained in the second LPL, located between TMD7 and TMD8. These mutations also had a substantial impact on the MexCD-OprJ-mediated transport of numerous other substrates. Subsequent replacement of amino acid residues identified above by cysteines rendered MexCD-OprJ susceptible to inhibition by a thiol-reactive agent, MIANS. Interestingly, MIANS inhibited the transport of some (pyronin, EtBr) but not other (ANS, Leu-Nap) substrates of the pump. Our results suggest that the precise structure of the periplasmic loops of MexD determines the rate of transport of individual substrates. These results are consistent with the hypothesis that, in the case of RND transporters, the LPLs are directly implicated in substrate recognition and contain multiple sites of interaction for various structurally diverse compounds.

Induction of the MexCD-OprJ multidrug efflux pump was investigated in wild-type Pseudomonas aeruginosa PAO1. MexCD-OprJ was induced by clinically important disinfectants such as benzalkonium chloride and chlorhexidine gluconate, and by some cytotoxic agents such as tetraphenylphosphonium chloride, ethidium bromide and rhodamine 6G. MexCD-OprJ was not induced by norfloxacin, tetracycline, chloramphenicol, streptomycin, erythromycin or carbenicillin, although they are substrates for the pump. Cells of PAO1 showed increased resistance to norfloxacin when grown in the presence of the inducers of the mexCD-oprJ operon mentioned above. These results indicate that MexCD-OprJ plays an important role in intrinsic multidrug resistance in wild-type P. aeruginosa in hospitals where disinfectants are used frequently.

MexJK requires OprM for erythromycin efflux but not for triclosan efflux. Deletion of 15 OprM family outer membrane proteins (OMPs) revealed that only the TolC homolog OpmH functions with MexJK for triclosan efflux. This is the first report of natural utilization of multiple OMPs by a given resistance nodulation cell division transporter/membrane fusion protein pair.

The enteropathogenicity of Providencia alcalifaciens, a member of the family Enterobacteriaceae, has not yet been well established. In November 1996, a large outbreak of foodborne infection occurred in Fukui, Japan. In this study, the etiology of the outbreak was investigated. No other recognized enteropathogens were detected in patient fecal samples, but P. alcalifaciens was detected in 7 of 18 samples. The isolates were found to be clonal by pulsed-field gel electrophoresis. The patients who presented with gastroenteritis had elevated levels of specific antibody against the isolated P. alcalifaciens. The isolates showed invasion of Caco-2 cells and fluid accumulation in rabbit ileal loops. This study strongly suggests that the outbreak was caused by P. alcalifaciens. This is the first report of a large outbreak of foodborne infection attributed to the organism and provides definitive evidence that P. alcalifaciens is a causative agent of gastroenteritis.