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J Bacteriol, July 1998, p. 3692-3696, Vol. 180, No. 14
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Isolation and Characterization of Toluene-Sensitive Mutants from the Toluene-Resistant Bacterium Pseudomonas putida GM73

Kwang Kim, Sungjin Lee, Kyunghee Lee, and Dongbin Lim*

Department of Microbiology, Gyeongsang National University, Gazwadong 900, Chinju 660-701, Korea

Received 17 November 1997/Accepted 14 May 1998

To understand the mechanism underlying toluene resistance of a toluene-tolerant bacterium, Pseudomonas putida GM73, we carried out Tn5 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, <10-6). In Ttg4, Ttg5, Ttg6, and Ttg7, about 0.02% of cells formed colonies. We cloned the Tn5-inserted genes, and the DNA sequence flanking Tn5 was determined. From comparison with a sequence database, putative protein products encoded by ttg 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 ttg8 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.

* Corresponding author. Mailing address: Department of Microbiology, Gyeongsang National University, Gazwadong, Chinju 660-701, Korea. Phone: 82-591-751-5946. Fax: 82-591-759-0187. E-mail: dblim{at}nongae.gsnu.ac.kr.

J Bacteriol, July 1998, p. 3692-3696, Vol. 180, No. 14
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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