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

Two toluene-sensitive mutants were generated from Pseudomonas putida IH-2000, 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), cyclohexane (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 containing 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 showed no significant homology to any protein reported so far. The growth patterns of the Tn5 mutants with the inactivated cyo-like gene were similar to that of the wild-type strain in the absence of organic solvents, although the doubling times were slightly longer than that of the wild-type strain. Our findings indicate that cyo is an important gene for toluene tolerance, although its role is still unclear.