Identification Of A Marine Bacterium Strain And Cloning Of Related Functional Gene Cluster

A marine bacterium MWYL1, originally isolated from the roots of Spartina anglicagrowing in a salt marsh near seaside, was studied via morphology characterization,physiological test and 16S rDNA sequencing and Blast analysis . It's proved that thestrain tested belongs to Marinomonas. The result showed that it is short, rod,gram-negative, aerobic, preferring growing at 28℃. The analysis of 16S rDNA sequencesuggests that the sequence similarity values are 97% and 95% with Marinomonas ponticaand Marinomonas dokdonensis, respectively. It is necessary to further research andexperiments related to identification for its precise species classification status.The isolates MWYL1 could degrade DMSP (DMSP is dimethylsulfoniopropionateas an osmoprotectant produced and stored by marine phytoplankton, macroalgae, cyanob-acteria, and coastal vascular plants, which primarily exists in the ocean. Gaseous dimethy1 sulfide (DMS) was produced by bacterial degradation of DMSP, then released into theatmosphere from the ocean, which plays an important role in driving the sulfur cyclebetween the ocean and the atmosphere). Besides, it can uitilize nitrate or nitrite as solenitrogen source, which may be involved in the assimilation of nitrate reduction(controlled by nas gene) or aerobic denitrification (controlled by nitrate reductase, napgene or nitrite reductase, nir gene). For cloning and researching related functional gene,thereby further clariflng its metabolic pathway, Genomic fosmid library of MWYL1 wasconstructed.Genomic DNA was prepared and purified using Genomic-tip 100/G kit andthe library Construction using Copy Control Fosmid Library Production Kit. The resultshow that exogenous insert DNA is average 35-40 kb and genomic fosmid librarycontains about 1.5-2.0 X 10~6 clones. One fosmid clone of producing melanin was directlyisolated by plating from the genomic library of Marinomonas sp. MWYL1. The novelfunctional gene cluster involved in melanin biosynthesis was discovered after subcloningand sequencing of the 14kb insert in pUC18, further more, the putative functional geneswas preminary analyzed in the method of bioinformatics. However, positive fosmid doneutilizing nitrate or nitrite as sole nitrogen source wasn't screened.Because positive clones utilizing nitrate or nitrite as sole nitrogen source weren'tdirectly isolated from the genomic library, random miniTn5 insertion mutant library wasconstructed via biparental conjugational transfer between E. coli S17-1 (with pUT-miniTn5 Tc) and spontaneous mutant of Marinomonas resistant to rifampin. Eight mutants ofabout 10000 transconjugants can not use nitrate as as sole nitrogen source were isolated, and flanking sequence of Tn5 insertion was analyzed by Tail-PCR or Genomic walkingPCR, Furthermore the system established lay the foundation of functional genomics ofMWYL1.