Many bacteria living in soil have developed the ability to use a wide variety of organosulfur compounds. Pseudomonas putida strain DS1 is able to utilize dimethyl sulfide as a sulfur source via a series of oxidation reactions that sequentially produce dimethyl sulfoxide, dimethyl sulfone (DMSO2), methanesulfonate, and sulfite. To isolate novel genes involved in DMSO2 utilization, a transposon-based mutagenesis of DS1 was performed. Of c. 10 000 strains containing mini-Tn5 inserts, 11 mutants lacked the ability to utilize DMSO2, and their insertion sites were determined. In addition to the cysNC, cysH, and cysM genes involved in sulfate assimilation, the ptsP gene encoding the phosphoenolpyruvate:sugar phosphotransferase system (PTS) family protein EINtr was identified, which is necessary for DMSO2 utilization. Using quantitative reverse transcriptase-polymerase chain reaction analysis, it was demonstrated that the expression of the sfn genes, necessary for DMSO2 utilization, was impaired in the ptsP disruptant. To the authors' knowledge, this is the first report of a PTS protein that is involved in bacterial assimilation of organosulfur compounds.