Isolation and characterization of toluene-sensitive mutants from Pseudomonas putida IH-2000

Two toluene-sensitive mutants were generated from Pseudomonas putida IH-200 0, the first known toluene-tolerant isolate, by Tn5 transposon mutagenesis. These mutants were unable to grow in the presence of toluene (log P-ow 2.8 ) but they could grow in medium overlaid with organic solvents having a log P-ow value higher than that of toluene such as p-xylene (log P-ow 3.1), cy clohexane (log P-ow 3.4) and n-hexane (log P-ow 3.9). The Tn5 transposable element knocked out a cyoB-like gene in one mutant and a cyoC-like gene in the other mutant. Seven open reading frames were found in a 5.5-kb region c ontaining the cyoB- and cyoC-like genes of strain IH-2000. ORFs 3-7 showed significant identity to the cyoABCDE gene products of Escherichia coli, but ORFs 1 and 2 shove-ed no significant homology to any protein reported so f ar. The growth patterns of the Tn5 mutants with the inactivated cyo-like ge ne were similar to that of the wild-type strain in the absence of organic s olvents, although the doubling times were slightly longer than that of the wild-type strain. Our findings indicate that cyo is an important gene for t oluene tolerance, although its role is still unclear. (C) 1998 Federation o f European Microbiological Societies. Published by Elsevier Science B.V. Al l rights reserved.