Three Efflux Pumps Are Required To Provide Efficient Tolerance to Toluene in Pseudomonas putida DOT-T1E

Abstract

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...

Effector-Repressor Interactions, Binding of a Single Effector Molecule to the Operator-bound TtgR Homodimer Mediates Derepression

“…This hence suggests the existence of other pump(s) that are impaired by MC 207110 and expel the same compounds, accounting for the high level of resistance of P. putida DOT-T1E to these natural antimicrobials. Furthermore, the low MIC values obtained for coumestrol (0.78 g/ml) or quercetin (4.2 g/ml) are comparable with those found for clinically relevant antibiotics (35,37), which points to the antibacterial potential of these natural compounds against Pseudomonas sp. strains when used in combination with MDR inhibitors.…”

“…In Vivo Susceptibility of P. putida DOT-T1E to Plant AntimicrobialsAs mentioned above, the implication of the TtgABC/TtgR system in the organic solvent tolerance and multidrug resistance of DOT-T1E was previously reported (21,35,37). However, the particularly broad substrate specificity of this efflux system and the multidrug binding capacity of its cognate repressor TtgR raised the question concerning their physiological role(s).…”

“…putida is a non-pathogenic bacterium present in water and soil and able to colonize plant roots and seeds (31)(32)(33). The DOT-T1E strain was isolated for its particularly high resistance to toxic organic solvents (34) and three RND efflux pumps, TtgABC, TtgDEF, and TtgGHI, were found to be essential for this resistance (35). This study will focus on the TtgABC efflux pump and its cognate regulator TtgR.…”

“…TtgABC has been shown to play an important role in the intrinsic tolerance of P. putida DOT-T1E to organic solvents (35,36). In addition, TtgABC is able to extrude several structurally different antimicrobial compounds, such as antibiotics (chloramphenicol, tetracycline, nalidixic acid, norfloxacin, streptomycin, ampicillin, and cefotaxime), biocides (triclosan), and dyes (ethidium bromide) (35,37,38), and hence confers an MDR phenotype to P. putida DOT-T1E.…”

Viability states of bacteria—Specific mechanisms of selected probes

“…Unspecific transporters were responsible for immediate export of NBDT from E. coli cells (55). Proton motive force dependent transporters of the RND (resistance/nodulation/division) superfamily were supposed to pump NBDT out, because they are known to be involved in toluene resistance (56) or export of fluorescence probes (57). Deactivation of the efflux pumps using the proton motive force inhibitor and protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was necessary to stabilize cellular NBDT fluorescence (55).…”