Isolation And Phylogenetic Analysis Of Culturable Dimethyl Sulfide-Producing Bacteria From Mangrove Soil

The volatile dimethyl sulfide (DMS) is the most important biogenic sulfurous compound that is transferred from the oceans to the atmosphere, and its oxidation products in the atmosphere can affect the environment and global climate, thus it is becoming the highlight of studying on weather variations. Mangrove is the typical intertidal wetland ecosystem in tropical zone, and it plays an irreplaceable role in climate regulation, contributing greatly to the biological diversity of coastal wetland. As mangrove habitat is with strong reducibility, strong acidity, high salinity and rich nutrient, its microbial resource is abundant and characteristic. Mangrove soil is associated with large numbers of sulfur reduction bacteria, as well as its high sulfur content that make up good conditions for DMS production. However, little information is known about the group and diversity of DMS-producing bacteria in mangrove soil at the present time.Our research was focused on 29 soil samples collected from Hainan Wenchang mangrove section (land-high tide terrace-intertidal zone-low tide terrace). At first acrylate and betaine were used as sole carbon source, then via the selective medium added with DMSP as sole carbon source to verify for DMSP-degrading bacteria, finally 51 strains were isolated and purified. Screening for DMS-producing bacteria which was undertaken by gas chromatography equipped with FPD detector, it was found that 9 of them were capable of producing DMS. We also measured their ability of producing DMS quantitatively. The 16S rRNA gene analysis showed that, A26-1, A29-1, A18-3, B30-1 and B30-2 belong to Bacillus, B05-1, B05-2, B22-1 and B29-1 belong to Pseudomonas. Having designed the primers of reported ddd genes, we used the genomic DNA of DMS-producing strains as template for PCR, however, we did not get any expected product.16S rRNA gene phylogenetic analysis manifested that A26-1, A29-1, A18-3, B30-1 and B30-2 were far apart from other DMS-producing strains. It is worth noting that the reported DMS-producing bacteria are all from Proteobacteria phylum, and there has not been found a Gram-positive bacteria with Ddd+phenotype. Therefore, we considered these five Bacillus isolates as novel DMS-producing bacteria.Finally we selected strain A29-1 with highest yield of DMS production among the five Bacilli strains to construct genomic cosmid library, cloned in the wide-host-range cosmid pLAFR3, generating 2100 transfectants. This work has laid a good foundation for further cloning and identification of new functional genes involved in bacterial DMS production.