Isolation of Toluene-Sensitive Mutants from Pseudomonas putida Strains and a Concomitant Loss of Resistance to Organic Solvents and Antibiotics
Hisako HIRAYAMA*, Fumiyasu FUKUMORI, Hideto TAKAMI, Akira INOUE, and Koki HORIKOSHI
The DEEPSTAR group, Japan Marine Science and Technology Center, 2-15 Natsushima- cho, Yokosuka 237, Japan
Organic solvents are generally toxic to microorganisms because they readily permeate into lipid cell membranes and disrupt their structures and function. Nevertheless, many bacteria showing organic solvent-tolerance (OST) have been reported in the past few years and many aspects of their mechanisms for OST have been studied (1,2). Mechanisms of bacterial OST are generally ascribed to cell surface structures and processes, including conformational adaptation of fatty acids, enzymatic degradation of solvents, and regulation of transmembrane fluxes (2,3).
Pseudomonas putida strain IH-2000, which is intrinsically toluene-tolerant, and KT2442, which is p-xylene-tolerant but toluene-sensitive, have been used to study the OST mechanisms. We obtained a toluene-tolerant mutant TOL by adaptation of KT2442 to toluene. From these toluene-tolerant strains, we obtained seven OST- deficient (OST-) mutants by using the transposon Tn5. Southern hybridization experiments suggest that Tn5 could have been inserted into chromosomal DNAs at a different site in each mutant. That infers participation of various genes in OST. Furthermore the mutants showed varying levels of organic solvent-sensitivity, which suggests that the expression and degree of OST is regulated in a complex way. One of the OST- mutants of KT2442 illustrated a considerable loss of OST and resistance against certain antibiotics simultaneously, compared with the other OST- mutants. This suggests that OST in KT2442 could be associated with the antibiotic resistance. Recently this association was reported in E. coli (4) but not in Pseudomonas Spp.. A plausible explanation is that the antibiotic resistance system affects the transmembrane fluxes of organic solvents. Further experiments are needed to assess the effect of the antibiotic resistance on OST loss and transmembrane flux changes.
1. Inoue, A. and Horikoshi, K. (1989) Nature, 338, 264-266.
2. Ramos, J.L. et al. (1997) J. Biol. Chem., 272, 3887-3890.
3. Yen, K.-M. et al. (1991) J. Bacteriol., 173, 5315-5327.
4. White, D.G. et al. (1997) J. Bacteriol., 179, 6122-6126.
