Efflux Pumps Involved in Toluene Tolerance in Pseudomonas putida DOT-T1E

The basic mechanisms underlying solvent tolerance in Pseudomonas putida DOT-T1E are efflux pumps that remove the solvent from bacterial cell membranes. The solvent-tolerant P. putida DOT-T1E grows in the presence of high concentrations (e.g., 1% [vol/vol]) of toluene and octanol. Growth of P. putida DOT-T1E cells in LB in the presence of toluene supplied via the gas phase has a clear effect on cell survival: the sudden addition of 0.3% (vol/vol) toluene to P. putida DOT-T1E pregrown with toluene in the gas phase resulted in survival of almost 100% of the initial cell number, whereas only 0.01% of cells pregrown in the absence of toluene tolerated exposure to this aromatic hydrocarbon. One class of toluene-sensitive octanol-tolerant mutant was isolated after Tn5-′phoA mutagenesis of wild-type P. putida DOT-T1E cells. The mutant, called P. putida DOT-T1E-18, was extremely sensitive to 0.3% (vol/vol) toluene added when cells were pregrown in the absence of toluene, whereas pregrowth on toluene supplied via the gas phase resulted in survival of about 0.0001% of the initial number. Solvent exclusion was tested with 1,2,4-[¹⁴C]trichlorobenzene. The levels of radiochemical accumulated in wild-type cells grown in the absence and in the presence of toluene were not significantly different. In contrast, the mutant was unable to remove 1,2,4-[¹⁴C]trichlorobenzene from the cell membranes when grown on Luria-Bertani (LB) medium but was able to remove the aromatic compound when pregrown on LB medium with toluene supplied via the gas phase. The amount of ¹⁴C-labeled substrate in whole cells increased in competition assays in which toluene and xylenes were the unlabeled competitors, whereas this was not the case when benzene was the competitor. This finding suggests that the exclusion system works specifically with certain aromatic substrates. The mutation in P. putida DOT-T1E-18 was cloned, and the knockedout gene was sequenced and found to be homologous to the drug exclusion gene mexB, which belongs to the efflux pump family of the resistant nodulator division type.The sensitivity of microorganisms to toxic organic solvents is related to the logarithm of the partition coefficient of the solvent in a mixture of octanol and water (log P_ow). Aromatic hydrocarbons with a log P_ow of between 1.5 and 3.5 are extremely toxic to living organisms (47). These chemicals dissolve in the cytoplasmic membrane, disorganize it, and collapse the cell membrane potential; this, together with the induced loss of lipids and proteins, leads to irreversible damage resulting in the death of the cell (8, 47, 50).Independent laboratories have isolated Pseudomonas putida strains tolerant to different aromatic hydrocarbons such as toluene, styrene, and p-xylene (6, 15, 42, 48). All four isolated strains were able to grow in liquid culture medium to which a high concentration (1% [vol/vol]) of these aromatic hydrocarbons was added. Tolerance to organic solvents in these P. putida strains is achieved by a series of biochemical mechanisms that actively remove the organic solvent from cell membranes (16, 43) and by physical barriers that help the cell to become (to a certain degree) impermeable to the solvent (13, 37, 43, 48). The physical barriers involve the ordered organization of the cell surface lipopolysaccharides (37) together with modified phospholipids (4, 37, 43, 49). Modifications in phospholipids upon exposure to an organic solvent involve both a short-term response, in which the level of the trans isomers of unsaturated phospholipids increases, and a long-term response consisting of a modification of the polar head groups of phospholipids (4, 43, 49) and an increase in the total amount of phospholipids per dry weight (49). For P. putida DOT-T1, it was suggested that an energy-dependent exclusion system (such as an efflux pump) is critical for tolerance to solvents (43). This conclusion was based on the following findings: (i) P. putida DOT-T1 treated with the uncoupler carbonyl cyanide p-trifluoromethoxyphenyl hydrazone accumulated higher levels of 1,2,4-[¹⁴C]trichlorobenzene in cell membranes than did untreated cells, and (ii) P. putida DOT-T1 mutants which were sensitive to toluene, octanol, and other chemicals accumulated 5- to 20-fold-higher levels of 1,2,4-[¹⁴C]trichlorobenzene in cell membranes than did the wild-type strain. Similar observations have been reported for Pseudomonas sp. strain S12 (16).In this study, we report that P. putida DOT-T1 uses at least two efflux pumps for toluene exclusion, one that seems to be expressed constitutively and a second inducible one. A mini-Tn5′phoA-Km^r knocked out the constitutive efflux system of P. putida DOT-T1E. The mutant was shown to be hypersensitive to toluene but not to octanol. The Km^r marker of the mini-Tn5 and the 3′ adjacent chromosomal DNA were cloned, and the wild-type gene was rescued by colony screening hybridization and sequenced. Sequence analysis showed that the knocked-out gene in the mutant was a homolog of the mexB gene, which belongs to the efflux pump family of the resistant nodulator division type (34–36, 38–41).     

Sequence Diversity of the oprI Gene,Coding for Major Outer Membrane Lipoprotein I,among rRNA Group I Pseudomonads

The sequence of oprI, the gene coding for the major outer membrane lipoprotein I, was determined by PCR sequencing for representatives of 17 species of rRNA group I pseudomonads, with a special emphasis on Pseudomonas aeruginosa and Pseudomonas fluorescens. Within the P. aeruginosa species, oprI sequences for 25 independent isolates were found to be identical, except for one silent substitution at position 96. The oprI sequences diverged more for the other rRNA group I pseudomonads (85 to 91% similarity with P. aeruginosa oprI). An accumulation of silent and also (but to a much lesser extent) nonsilent substitutions in the different sequences was found. A clustering according to the respective presence and/or positions of the HaeIII, PvuII, and SphI sites could also be obtained. A sequence cluster analysis showed a rather widespread distribution of P. fluorescens isolates. All other rRNA group I pseudomonads clustered in a manner that was in agreement with other studies, showing that the oprI gene can be useful as a complementary phylogenetic marker for classification of rRNA group I pseudomonads.Pseudomonads are increasingly being recognized as important microorganisms in our biosphere, and Pseudomonas aeruginosa and Pseudomonas fluorescens are two important representatives of this genus. As a typical opportunist, P. aeruginosa is more and more involved in a variety of often fatal nosocomial infections, in which it accounts for more than 11% of all isolates recovered (29). In cystic fibrosis, one of the most common autosomal recessive genetic diseases, it is a characteristic pathogen responsible for most of the cases of morbidity and mortality (16, 38). In general, fluorescent pseudomonads, including P. aeruginosa, Pseudomonas putida, P. fluorescens, and other species, are frequently found as rhizosphere microorganisms, in some cases promoting plant growth (11, 19, 20).P. fluorescens and P. aeruginosa are also found as inherent flora of mineral water (14, 39). Identification of fluorescent pseudomonads is often tedious and not reliable. Indeed, the present taxonomy of this group is far from clear at the finer taxonomic level, as polyphasic investigations have demonstrated (4, 13, 18, 26). Ribosomal RNAs have been applied as molecular markers with great success to unravel the rough phylogenetic structure which, at the finer level, is not always in complete agreement with the genotypic and phenotypic similarities deduced from other parts of the genome. Horizontal gene transfer, chromosomal mutation hot spots, and internal genomic rearrangements are probably the bases of these discrepancies at the species and subspecies levels. These arguments, together with the importance of discriminating phenotypic tests in routine identifications, support a polyphasic approach in bacterial taxonomy (2, 8–10, 13, 37, 40). Additional phylogenetic information requires the identification of molecules, like the recA or the gyrB genes, that are widely distributed, large enough to contain a substantial amount of information, and conserved to an appropriate degree (24, 46). In the phylogenetic tree published by Woese (43), species with the same generic name were allocated in phylogenetically distant groups. This was the case for the “genus” Pseudomonas, which is known to be a dump of assemblages of distantly related species (3, 8–10, 17). Taxonomic rearrangements of the genus Pseudomonas sensu stricto resulted in the splitting of the genus and as a logical consequence, the present genus Pseudomonas is restricted to the rRNA group I organisms, with P. aeruginosa as the type species in this group (27, 28, 37, 42, 44, 45).The oprI gene, coding for the outer membrane lipoprotein I of P. aeruginosa (5), was found to be conserved among the fluorescent pseudomonads and was considered to be a possible phylogenetic marker (6, 31). In this study, we tested whether the oprI gene could be a useful detection and identification target molecule as well as a complementary phylogenetic marker for rRNA group I pseudomonads. Also, we examined to what extent the sequence variation of the oprI gene reflects the species diversity in P. aeruginosa and P. fluorescens.