SummaryA major tripartite multidrug efflux pump of Escherichia coli, AcrAB-TolC, confers resistance to a wide variety of compounds. The drug molecule is captured by AcrB probably from the periplasm or the periplasm/inner membrane interface, and is passed through AcrB and then TolC to the medium. Currently, there exist numerous crystallographic and mutation data concerning the regions of AcrB and its homologues that may interact with substrates. Starting with these data, we devised fluorescence assays in whole cells to determine the entire substrate path through AcrB. We tested 48 residues in AcrB along the predicted substrate path and 25 gave positive results, based on the covalent labelling of cysteine residues by a lipophilic dye-maleimide and the blocking of Nile red efflux by covalent labelling with bulky maleimide reagents. These residues are all located in the periplasmic domain, in regions we designate as the lower part of the large external cleft, the cleft itself, the crystallographically defined binding pocket, and the gate between the pocket and the funnel. Our observations suggest that the substrate is captured in the lower cleft region of AcrB, then transported through the binding pocket, the gate and finally to the AcrB funnel that connects AcrB to TolC.

The TolC protein of Escherichia coli, through its interaction with AcrA and AcrB, is thought to form a continuous protein channel that expels inhibitors from the cell. Consequently, tolC null mutations display a hypersensitive phenotype. Here we report the isolation and characterization of tolC missense mutations that direct the synthesis of mutant TolC proteins partially disabled in their efflux role. All alterations, consisting of single amino acid substitutions, were localized within the periplasmic ␣-helical domain. In two mutants carrying an I106N or S350F substitution, the hypersensitivity phenotype may be in part due to aberrant TolC assembly. However, two other alterations, R367H and R390C, disrupted efflux function by affecting interactions among the helices surrounding TolC's periplasmic tunnel. Curiously, these two TolC mutants were sensitive to a large antibiotic, vancomycin, and exhibited a Dex ؉ phenotype. These novel phenotypes of TolC R367H and TolC R390C were likely the result of a general influx of molecules through a constitutively open tunnel aperture, which normally widens only when TolC interacts with other proteins during substrate translocation. An intragenic suppressor alteration (T140A) was isolated from antibiotic-resistant revertants of the hypersensitive TolC R367H mutant. T140A also reversed, either fully (R390C) or partially (I106N and S350F), the hypersensitivity phenotype of other TolC mutants. Our data suggest that this global suppressor phenotype of T140A is the result of impeded antibiotic influx caused by tapering of the tunnel passage rather than by correcting individual mutational defects. Two extragenic suppressors of TolC R367H , mapping in the regulatory region of acrAB, uncoupled the AcrR-mediated repression of the acrAB genes. The resulting overexpression of AcrAB reduced the hypersensitivity phenotype of all the TolC mutants. Similar results were obtained when the chromosomal acrR gene was deleted or the acrAB genes were expressed from a plasmid. Unlike the case for the intragenic suppressor T140A, the overexpression of AcrAB diminished hypersensitivity towards only erythromycin and novobiocin, which are substrates of the TolC-AcrAB efflux pump, but not towards vancomycin, which is not a substrate of this pump. This showed that the two types of suppressors produced their effects by fundamentally different means, as the intragenic suppressor decreased the general influx while extragenic suppressors increased the efflux of TolC-AcrAB pump-specific antibiotics.Escherichia coli cells lacking the outer membrane protein (OMP) TolC display hypersensitivity to a variety of inhibitors, including bile salts, detergents, and hydrophobic antibiotics (38). This was initially thought to be due to a defect in the outer membrane permeability barrier as a result of a defective lipopolysaccharide (LPS) (32). However, it was shown that tolC and rfa (LPS core) mutations produce an additive effect on hypersensitivity, thus suggesting that tolC mutations may confer hypersensitivity indepen...

The path of substrates in the multidrug efflux pump AcrB of Escherichia coli was examined by using labeling with a lipophilic substrate mimic, Bodipy FL maleimide. Four (out of eight) residues in the vestibule bound the dye, suggesting its role in substrate transport, whereas only one (out of nine) residue in the central cavity tested positive.

Selection for feoAB mutations as a result of metronidazole treatment will disable the pathogenic potential of B. fragilis and could contribute to the clinical efficacy of metronidazole. While mutations in feoAB are probably not a direct cause of clinical resistance, this study provides a key insight into intracellular metronidazole activity and the link with intracellular iron homeostasis.

This paper provides the biochemical evidence for physical interactions between the outer membrane component, TolC, and the membrane fusion protein component, AcrA, of the major antibiotic efflux pump of Escherichia coli. Cross-linking between TolC and AcrA was independent of the presence of any externally added substrate of the efflux pump or of the pump protein, AcrB. The biochemical demonstration of a TolC-AcrA interaction is consistent with genetic studies in which extragenic suppressors of a mutant TolC strain were found in the acrA gene.

Bacteroides fragilis, an important component of the human gastrointestinal microbiota, can cause lethal extra-intestinal infection upon escape from the gastrointestinal tract. We demonstrated transfer and recombination of large chromosomal segments from B. fragilis HMW615, a multidrug resistant clinical isolate, to B. fragilis 638R. In one example, the transfer of a segment of ~435 Kb/356 genes replaced ~413 Kb/326 genes of the B. fragilis 638R chromosome. In addition to transfer of antibiotic resistance genes, these transfers (1) replaced complete divergent polysaccharide biosynthesis loci; (2) replaced DNA inversion-controlled intergenic shufflons (that control expression of genes encoding starch utilization system outer membrane proteins) with more complex, divergent shufflons; and (3) introduced additional intergenic shufflons encoding divergent Type 1 restriction/modification systems. Conjugative transposon-like genes within a transferred segment and within a putative integrative conjugative element (ICE5) ~45 kb downstream from the transferred segment both encode proteins that may be involved in the observed transfer. These data indicate that chromosomal transfer is a driver of antigenic diversity and nutrient adaptation in Bacteroides that (1) contributes to the dissemination of the extensive B. fragilis pan-genome, (2) allows rapid adaptation to a changing environment and (3) can confer pathogenic characteristics to host symbionts.

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dTwo multidrug-resistant Bacteroides fragilis clinical isolates contain and express a novel nim gene, nimJ, that is not recognized by the "universal" nim primers and can confer increased resistance to metronidazole when introduced into a susceptible strain on a multicopy plasmid. HMW615, an appendiceal isolate, contains at least two copies of nimJ on its genome, while HMW616, an isolate from a patient with sepsis, contains one genomic copy of nimJ. B. fragilis NimJ is phylogenetically closer to Prevotella baroniae NimI and Clostridium botulinum NimA than to the other known Bacteroides Nim proteins. The predicted protein structure of NimJ, based on fold recognition analysis, is consistent with the crystal structures derived for known Nim proteins, and specific amino acid residues important for substrate binding in the active site are conserved. This study demonstrates that the "universal" nim primers will not detect all nim genes with the ability to confer metronidazole resistance, but nimJ alone cannot account for the very high metronidazole MICs of these resistant clinical isolates.