Antibiotic-Sensitive TolC Mutants and Their Suppressors

Abstract

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...

Reversal of the Drug Binding Pocket Defects of the AcrB Multidrug Efflux Pump Protein of Escherichia coli

“…In our genetic setup, the chromosomally deleted acrAB genes are complemented by acrAB expressed under the control of the native promoter from the low-copy-number plasmid pACYC184 (35,41). The plasmidcomplemented strain confers a drug phenotype indistinguishable from that of a strain expressing chromosomally encoded acrAB (data not shown).…”

“…The acrAB genes were expressed from the low-copynumber plasmid pACYC184 (34) under the control of their natural promoter (35). Plasmids were purified using a QIAprep Spin miniprep kit from Qiagen.…”

A periplasmic coiled-coil interface underlying TolC recruitment and the assembly of bacterial drug efflux pumps

“…3). This arrangement could be supported by the interaction of a cluster of specific hydrophobic residues within the TolC equatorial domain, which mutational studies suggest to be functionally important (33)(34)(35). Owing to the limited contacts between TolC and the IM proton antiporter AcrB (6,16,17), a substantial interaction between TolC and the adaptor protein is likely to be required to stabilize the tripartite complex.…”

Small RNA Regulation of TolC, the Outer Membrane Component of Bacterial Multidrug Transporters

“…SdsR repression of TolC leads to changes in antibiotic sensitivity. One major role of TolC is in the export of antibiotics from the cell; tolC deletion mutants are significantly more sensitive to a broad set of antibiotics than are strains without the deletion (47). We tested the effects of both multicopy SdsR and the deletion of sdsR on sensitivity to three different antibiotics and one cationic dye.…”