Growth of Escherichia coli is inhibited upon exposure to a large volume of a harmful solvent, and there is an inverse correlation between the degree of inhibition and the log P OW of the solvent, where P OW is the partition coefficient measured for the partition equilibrium established between the n-octanol and water phases. The AcrAB-TolC efflux pump system is involved in maintaining intrinsic solvent resistance. We inspected the solvent resistance of ⌬acrAB and/or ⌬tolC mutants in the presence of a large volume of solvent. Both mutants were hypersensitive to weakly harmful solvents, such as nonane (log P OW ‫؍‬ 5.5). The ⌬tolC mutant was more sensitive to nonane than the ⌬acrAB mutant. The solvent entered the E. coli cells rapidly. Entry of solvents with a log P OW higher than 4.4 was retarded in the parent cells, and the intracellular levels of these solvents were maintained at low levels. The ⌬tolC mutant accumulated n-nonane or decane (log P OW ‫؍‬ 6.0) more abundantly than the parent or the ⌬acrAB mutant. The AcrAB-TolC complex likely extrudes solvents with a log P OW in the range of 3.4 to 6.0 through a first-order reaction. The most favorable substrates for the efflux system were considered to be octane, heptane, and n-hexane.

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A constitutively active mutant of histidine kinase sensor EvgS was found to confer multi-drug resistance (MDR) to an acrA-deficient Escherichia coli, indicating the relationship between the two-component system EvgAS and the expression of the MDR system. The observed MDR also depended on an outer-membrane channel, TolC. Microarray and S1 mapping assays indicated that, in the presence of this constitutive mutant EvgS, the level of transcription increased for some MDR genes, including the drug efflux genes emrKY, yhiUV, acrAB, mdfA and tolC. Transcription in vitro of emrK increased by the addition of phosphorylated EvgA. Transcription activation of tolC by the activated EvgS was, however, dependent on both EvgAS and PhoPQ (Mg 2+ -responsive two-component system), in agreement with the presence of the binding site (PhoP box) for the regulator PhoP in the tolC promoter region. Transcription in vitro of yhiUV also appears to require an as-yet-unidentified additional transcriptional factor besides EvgA. Taken together we propose that the expression of the MDR system is under a complex regulatory network, including the phosphorylated EvgA serving as the master regulator.

Alkaliphilic Bacillus sp. strain 41M-1, isolated from soil, produced xylan-degrading enzymes extracellularly. Optimum pH for the crude xylanase preparation was about pH 9, confirming the production of novel alkaline xylanase(s) by the isolate. Xylanases were induced by xylan, but were not produced in the presence of xylose, arabinose or glucose. Xylanase productivity was influenced by culture pH, and production at pH 10.5 was higher than that at pH 8.0. Zymogram analysis of the culture supernatant showed the alkaline xylanase with a molecular mass of 36 kDa.

Escherichia coli K-12 OST3410 was isolated previously as a stable cyclohexane-tolerant mutant derived from cyclohexane-sensitive strain JA300. A plasmid which provides cyclohexane tolerance to strain JA300 was isolated from the OST3410 genomic library. Subcloning and sequence analysis showed that the plasmid contained the robA gene, whose gene product was reported to bind specifically to the right border of oriC. We observed that the robA gene on the multicopy plasmid generally increased the organic solvent tolerance of several E. coli strains. We also observed an increase in the organic solvent tolerance of JA300 carrying the lac-robA fusion gene on a low-copy plasmid by isopropyl-␤-D-thiogalactopyranoside induction. Strain JA300 carrying the multicopy robA plasmid also showed an increase in resistance to a number of unrelated antibiotics and heavy metal ions, and the spectrum of resistance was significantly similar to that of the soxS-overexpressing strain.

The AcrAB-TolC efflux pump plays an intrinsic role in resistance to hydrophobic solvents in Escherichia coli. E. coli OST5500 is hypersensitive to solvents due to inactivation of the acrB gene by insertion of IS30. Suppressor mutants showing high solvent resistance were isolated from OST5500. These mutants produced high levels of AcrE and AcrF proteins, which were not produced in OST5500, and in each mutant an insertion sequence (IS1 or IS2) was found integrated upstream of the acrEF operon, coding for the two proteins. The suppressor mutants lost solvent resistance on inactivation of the acrEF operon. The solvent hypersensitivity of OST5500 was suppressed by introduction of the acrEF operon with IS1 or IS2 integrated upstream but not by introduction of the operon lacking the integrated IS. It was concluded that IS integration activated acrEF, resulting in functional complementation of the acrB mutation. The acrB mutation was also complemented by a plasmid containing acrF or acrEF under the control of Plac. The wild-type tolC gene was found to be essential for complementation of the acrB mutation by acrEF. Thus, it is concluded that in these cells a combination of the proteins AcrA, AcrF, and TolC or the proteins AcrE, AcrF, and TolC is functional in solvent efflux instead of the AcrAB-TolC efflux pump.Hydrophobic organic solvents with log P OW values of 2 to 5 can inhibit the growth of microorganisms. When microorganisms are incubated in the presence of a large volume of solvent, the magnitude of growth inhibition is inversely correlated with the log P OW of the solvent (2, 9), which is the common logarithm of the partition coefficient (P OW ) of a solvent, measured in a two-liquid phase system composed of n-octanol and water. It is known that this value is correlated with solvent hydrophobicity (16).The solvent resistance of a microorganism is determined genetically. In Escherichia coli, the AcrAB-TolC efflux pump plays an intrinsic role in the solvent resistance mechanism (3,30,31). In E. coli exposed to a large volume of a particular solvent, the intracellular concentration of the solvent is maintained at a low level (30). This pump consists of three components, AcrA, AcrB, and TolC (8,18,19). The inner membrane protein AcrB belongs to the RND (resistance-nodulation-cell division) transporter family and is thought to be a proton antiporter (23,28,33). The periplasmic lipoprotein AcrA belongs to the membrane fusion protein family and is a highly asymmetric protein capable of spanning the periplasmic space (6, 32). The outer membrane protein TolC spans the membrane and the periplasm and functions as a channel tunnel (8,14). Deletion of acrAB or tolC decreases the solvent resistance of E. coli.The acrEF operon encodes the components of an efflux pump other than the AcrAB efflux pump. AcrE and AcrF are highly homologous to AcrA and AcrB, respectively (12, 18). On the chromosome of E. coli, the acrAB and acrEF loci are located at 10.5 and 73.5 min, respectively (5, 13, 26). The acrEF operon was cloned accidentall...

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In this study, we examined cell surface properties of mutants of Escherichia coli for which organic solvent tolerance levels were elevated. The cell surface of each mutant was less hydrophobic than that of the parent, probably due to an increase in lipopolysaccharide content. OmpF synthesis was repressed in the mutants. Organic solvent bound readily to viable E. coli cells in response to the polarity of the solvent. The mutants were bound less abundantly with the organic solvent than was the parent.