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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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