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.

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...

Escherichia coli mutants with improved organic solvent tolerance levels showed high levels of outer membrane protein TolC and inner membrane protein AcrA. The TolC level was regulated positively by MarA, Rob, or SoxS. A possible mar-rob-sox box sequence was found upstream of the tolC gene. These findings suggest that tolC is a member of the mar-sox regulon responsive to stress conditions. When a defective tolC gene was transferred to n-hexane- or cyclohexane-tolerant strains by P1 transduction, the organic solvent tolerance level was lowered dramatically to the decane-tolerant and nonane-sensitive level. The tolerance level was restored by transformation of the transductants with a wild-type tolC gene. Therefore, it is evident that TolC is essential for E. coli to maintain organic solvent tolerance.

Gene expression profiles were collected from Escherichia coli strains (OST3410, TK33, and TK31) before and after exposure to organic solvents, and the six genes that showed higher gene expression were selected. Among these genes, glpC encoding the anaerobic glycerol-3-phosphate dehydrogenase subunit C remarkably increased the organic solvent tolerance.

Strain KBC1, an anaerobic bacterium, that dechlorinates tetrachloroethene (PCE) to trichloroethene was isolated. This strain also dechlorinated high concentrations of PCE at a temperature range of 10 to 40 degrees C and showed high oxygen tolerance. Based on the 16S rRNA gene sequence analysis, this microorganism was identified as a species of the genus Desulfitobacterium. Several species of this genus have been reported to be potent ortho-chlorophenol and PCE dechlorinators; however, the gene coding PCE-specific dehalogenase had not been cloned thus far. In this report, we identified a novel PCE reductive dehalogenase (PrdA) gene from the Desulfitobacterium sp. strain KBC1. These prd genes, including putative membrane anchor protein, were classified as novel type of PCE reductive dehalogenase (approximately 40% homology with the general PCE dehalogenase). It was revealed that the two open reading frames had been transcribed as identical mRNA and were induced strictly in the presence of PCE. This transcriptional regulation appeared to be controlled by the transcriptional activator located downstream of prdAB operon. According to the substrate utility of the strain KBC1 and phylogenetic analysis of PrdA, this microorganism may be expected to play the role of a primary dechlorinator of PCE in the environment.