Isolation and Characterization of Toluene-Sensitive Mutants from the Toluene-Resistant Bacterium <i>Pseudomonas putida</i> GM73

<jats:title>ABSTRACT</jats:title> <jats:p> To understand the mechanism underlying toluene resistance of a toluene-tolerant bacterium, <jats:italic>Pseudomonas putida</jats:italic> GM73, we carried out Tn <jats:italic>5</jats:italic> mutagenesis and isolated eight toluene-sensitive mutants. None of the mutants grew in the presence of 20% (vol/vol) toluene in growth medium but exhibited differential sensitivity to toluene. When wild-type cells were treated with toluene (1% [vol/vol]) for 5 min, about 2% of the cells could form colonies. In the mutants Ttg1, Ttg2, Ttg3, and Ttg8, the same treatment killed more than 99.9999% of cells (survival rate, <10 <jats:sup>−6</jats:sup> ). In Ttg4, Ttg5, Ttg6, and Ttg7, about 0.02% of cells formed colonies. We cloned the Tn <jats:italic>5</jats:italic> -inserted genes, and the DNA sequence flanking Tn <jats:italic>5</jats:italic> was determined. From comparison with a sequence database, putative protein products encoded by <jats:italic>ttg</jats:italic> genes were identified as follows. Ttg1 and Ttg2 are ATP binding cassette (ABC) transporter homologs; Ttg3 is a periplasmic linker protein of a toluene efflux pump; both Ttg4 and Ttg7 are pyruvate dehydrogenase; Ttg5 is a dihydrolipoamide acetyltransferase; and Ttg7 is the negative regulator of the phosphate regulon. The sequences deduced from <jats:italic>ttg8</jats:italic> did not show a significant similarity to any DNA or proteins in sequence databases. Characterization of these mutants and identification of mutant genes suggested that active efflux mechanism and efficient repair of damaged membranes were important in toluene resistance. </jats:p>