Organic solvents can be toxic to microorganisms, depending on the inherent toxicity of the solvent and the intrinsic tolerance of the bacterial species and strains. The toxicity of a given solvent correlates with the logarithm of its partition coefficient in n-octanol and water (log Pow). Organic solvents with a log Pow between 1.5 and 4.0 are extremely toxic for microorganisms and other living cells because they partition preferentially in the cytoplasmic membrane, disorganizing its structure and impairing vital functions. Several possible mechanisms leading to solvent-tolerance in gram-negative bacteria have been proposed: (a) adaptive alterations of the membrane fatty acids and phospholipid headgroup composition, (b) formation of vesicles loaded with toxic compounds, and (c) energy-dependent active efflux pumps belonging to the resistance-nodulation-cell division (RND) family, which export toxic organic solvents to the external medium. In these mechanisms, changes in the phospholipid profile and extrusion of the solvents seem to be shared by different strains. The most significant changes in phospholipids are an increase in the melting temperature of the membranes by rapid cis-to-trans isomerization of unsaturated fatty acids and modifications in the phospholipid headgroups. Toluene efflux pumps are involved in solvent tolerance in several gram-negative strains, e.g., Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa. The AcrAB-TolC and AcrEF-TolC efflux pumps are important for n-hexane tolerance in E. coli. A number of P. putida strains have been isolated that tolerate toxic hydrocarbons such as toluene, styrene, and p-xylene. At least three efflux pumps (TtgABC, TtgDEF, and TtgGHI) are present in the most extensively characterized solvent-tolerant strain, P. putida DOT-T1E, and the number of efflux pumps has been found to correlate with the degree of solvent tolerance in different P. putida strains. The operation of these efflux pumps seems to be coupled to the proton motive force via the TonB system, although the intimate mechanism of energy transfer remains elusive. Specific and global regulators control the expression of the efflux pump operons of E. coli and P. putida at the transcriptional level.

In Pseudomonas putida DOT-T1E multidrug efflux pumps of the resistance-nodulation-division family make a major contribution to solvent resistance. Two pumps have been identified: TtgABC, expressed constitutively, and TtgDEF, induced by aromatic hydrocarbons. A double mutant lacking both efflux pumps was able to survive a sudden toluene shock if and only if preinduced with small amounts of toluene supplied via the gas phase. In this article we report the identification and characterization in this strain of a third efflux pump, named TtgGHI. The ttgGHI genes form an operon that is expressed constitutively at high levels from a single promoter. In the presence of toluene the operon is expressed at an even higher level from two promoters, the constitutive one and a previously unreported one that is inducible and that partially overlaps the constitutive promoter. By site-directed mutagenesis we constructed a single ttgH mutant which was shown to be unable to survive sudden 0.3% (vol/vol) toluene shocks regardless of the preculture conditions. The mutation was transferred to single and double mutants to construct mutant strains in which two or all three pumps are knocked out. Survival analysis of induced and noninduced cells revealed that the TtgABC and TtgGHI pumps extruded toluene, styrene, m-xylene, ethylbenzene, and propylbenzene, whereas the TtgDEF pump removed only toluene and styrene. The triple mutant was hypersensitive to toluene, as shown by its inability to grow with toluene supplied via the vapor phase.Organic solvents with a partition coefficient in an octanolwater mixture (log P ow ) between 1.5 and 3 are extremely toxic to microorganisms (34). Organic solvents cause irreversible lesions to cell membranes (loss of ions, metabolites, lipids, and proteins, dissipation of the pH gradient and electrical potential, etc.) followed by cell lysis and death (5,36,37).Several Pseudomonas putida strains have been isolated as able to grow in the presence of high concentrations of toxic organic solvents, such as toluene, styrene, and p-xylene (4,10,11,31,40). Tolerance to organic solvents in these P. putida strains is mainly achieved by a series of efflux pumps that actively remove the organic solvent from the cell membranes (10-14, 19, 25, 32, 33, 36). These efflux pumps, which belong to the resistance-nodulation-division (RND) family, are formed by a translocase made of an inner membrane transporter and a periplasmic fusion protein anchored in the inner membrane plus an outer membrane protein that spans the periplasm and forms a tunnel. It has been proposed that this tunnel is contacted by the cytoplasmic membrane pump, presumably assisted by the inner membrane-periplasmic fusion protein to facilitate the direct removal of drugs across the two membranes and the intervening periplasmic space (17). Evidence for this mode of operation has been obtained with antibiotic efflux pumps in Escherichia coli (17,46) and Pseudomonas aeruginosa (43,44).We previously identified two efflux pumps that expel toluene in the solvent...

Pseudomonas putida is well known for its metabolic capabilities, but recently, it has been shown to exhibit resistance to a wide range of antibiotics. In P. putida DOT-T1E, the TtgABC efflux pump, which has a broad substrate specificity, extrudes antibiotics such as ampicillin, carbenicillin, tetracycline, nalidixic acid, and chloramphenicol. We have analyzed the expression of the ttgABC efflux pump operon and its regulatory gene, ttgR, in response to several structurally unrelated antibiotics at the transcriptional level and investigated the role of the TtgR protein in this process. ttgABC and ttgR are expressed in vivo at a moderate basal level, which increases in the presence of hydrophobic antibiotics like chloramphenicol and tetracycline. In vitro experiments show that, in the absence of inducers, TtgR binds to a palindromic operator site which overlaps both ttgABC and ttgR promoters and dissociates from it in the presence of chloramphenicol and tetracycline. These results suggest that the TtgR repressor is able to bind to structurally different antibiotics, which allows induction of TtgABC multidrug efflux pump expression in response to these antimicrobial agents. This is the first case in which the expression of a drug transporter of the resistance-nodulation-division family has been shown to be regulated directly by antibiotics.