Cloning And Functional Identification Of New Genes Involved In Dimethylsulfoniopropionae Degradation

Dimethylsulfoniopropionae (DMSP) is a widespread biogenic sulfur compound in the surface ocean water, which not only plays an important role in the process of marine ecosystems and biogeochemical cycles, but also its degradation products affects environmental acidification and climate change. DMSP degradation process has become one of the hot spots of the field research. Pseudomonas sp. B22-1was isolated from mangrove soil in our early work; B22-1could utilize DMSP as a sole carbon source and emit DMS. Genomic cosmid library construction and Tn5transposon insertion mutagenesis techniques were used to isolate the novel genes involved in DMSP catabolic pathway of B22-1, the main results were obtained as following:1. A genomic cosmid library of B22-1, with a total number of2,200clones, was constructed, but no functional genes were isolated in this study.2. A genomic Tn5insertion mutagenesis library of B22-1, with a total number of10,000, was constructed. Three mutants, namely Mu-1-B10, Mu-20-B7and Mu-38-H11, which could not utilize DMSP or acetate as a sole carbon source, were screened. These two mutants could grow on glucose or betaine-containing medium; the DMS producing ability of Mu-1-B10and Mu-20-B7were similar with that of wild type strain B22-1.3. Tn5transposon flanking sequences were amplified by TAIL-PCR, it was found that the Tn5transposons in Mu-1-B10and Mu-20-B7were both inserted in same site of mqo gene, and the the Tn5transposons in Mu-38-H11was inserted in the alcohol dehydrogenase gene.4. Functional complementation restored the mutants’ DMSP catabolic ability.5. mqo gene expression levels at different carbon sources were comparatively determined by real-time PCR. It was found the expression of mqo gene was significantly induced by DMSP. The mqo expression level was7times higher in DMSP than in glucose as the sole carbon source. The above results indicate mqo gene of B22-1is essential for DMSP metabolism. It is the first functional gene identified from carbon central metabolism, which has been proved to participate the DMSP metabolic process. The present study provides direct evidences and deeper understanding on the biochemical pathways for DMSP degradation in bacteria.