Entry into and Release of Solvents by Escherichia coli in an Organic-Aqueous Two-Liquid-Phase System and Substrate Specificity of the AcrAB-TolC Solvent-Extruding Pump

Abstract

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.

Ultrasonic Disintegration of Microalgal Biomass and Consequent Improvement of Bioaccessibility/Bioavailability in Microbial Fermentation

“…To achieve this aim, the E. coli K-12 JA300, which has been used in several studies in solvent resistance [8,[18][19][20], was manipulated to generate the acrB-inactivated derivative JA300A. The acrB gene was cloned into the low copy number plasmid pMW119 to create pAcrB.…”

Highly Solvent Tolerance in Serratia marcescens IBBPo15

“…5A-5D, only HAE1 and acrAB genes were detected in the genome of S. marcescens IBB Po15 control cells. Nevertheless the used primers (i.e., ompF-f, ompF-r; A24f2, A577r2; acrA-f, acrB-r; recA-f, recA-r) are specific for ompF 6 , HAE1 23 , acrAB 7 and recA 24 genes formerly described, they were not specially designed for the bacteria investigated in this study causing unspecific amplification of other fragments.…”

“…For PCR amplification, 1 μl of DNA extract was added to a final volume of 25 μl reaction mixture, containing: 5×GoTaq flexi buffer, MgCl 2 , dNTP mix, specific primers for ompF, HAE1, acrAB transporter genes (ompF-f and ompF-r, 6 ; A24f2 and A577r2, 23 ; acrA-f and acrB-r, 7 ), recA gene (recA-f and recA-r, 24 ), and GoTaq G2 hot start polymerase (Promega). The PCR program consisted in initial denaturation for 10 min at 94°C, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 50°C or 55°C for 30 sec, extension at 72°C for 2 min, and a final extension at 72°C for 10 min.…”

“…Porins OmpF (with molecular weight of 41 kDa) and OmpC (40 kDa) which exist in outer membrane of S. marcescens cells are two non-specific protein channels that serve to take in nutrients and toxic compounds, and export waste products 5,6 . Furthermore, the AcrAB-TolC efflux system is a tripartite complex and is a member of the resistance-nodulation-division (RND) family which is considered the major efflux system in members of Enterobacteriaceae family 7 responsible for tolerance to toxic compounds, including organic solvents 8 . The drug:proton antiporter AcrB (110 kDa), which is located in the inner membrane, captures its substrates and transports them into the external medium via the outer membrane channel TolC (53 kDa) 8,9 .…”

Mechanism of recognition of compounds of diverse structures by the multidrug efflux pump AcrB ofEscherichia coli

“…Based on the comparison of susceptibility of acrAB + and ΔacrAB strains [measured by minimal inhibitory concentration (MIC)], AcrAB-TolC can pump out not only basic dyes (such as acriflavine and ethidium) but also most antibiotics (such as macrolides, fluoroquinolones, β-lactams, tetracyclines, chloramphenicol, rifampin, novobiocin, fusidic acid, but not aminoglycosides), detergents (such as bile salts, Triton X-100, SDS) (13). Even simple solvents such as hexane, heptane, octane, and nonane (14) or cyclohexane (15) are pumped out by AcrAB-TolC (Fig. 1).…”