Isolation Of Marine Invertebrate Symbiotic Microorganisms And Their Metabolite Quorum Effect Quenching

There is a huge difference between the marine ecological environment and the terrestrial ecological environment,resulting in biological species in marine varying from those on land.The abundant biological species in ocean and their metabolic products give us a way to fulfill the large demand of new natrual compounds.Up to now,the symbiotic microorganisms of invertetrates such as corals,sponges and sea anemones,and their metabolites have been studied a lot,but none is about the Homoiodoris japonic.Here,we have carried out the research on its symbiotic microorganisms and their quorum quenching activity.We have isolated 214 strains from Homoiodoris japonica,which fall in 87 species and 56genera,in particular 15 of them might be new species.The Proteobacteria is the most abundant phylum with 125 strains and the Planctomycetes is the rarest one with only one strain.The species composition suggests that the biodiversity of the microorganisms associated with Homoiodoris japonica is quite high and novel and warrants more in-detpth research.The 16S r RNA sequence of XY-359^T shares 96.1%similarity with F.flava DSM22638^Tand the phylogenetic trees shows that the XY-359^T clusters with the genus Flagellimonas and formed a monophyletic clade with F.flava DSM22638^T.The XY-359^T is Gram-negative and rod-shaped strain,and its optimum growth temperature is 30?,while optimum p H is 7.5-8.0and optimum Na Cl concentration is 1.5-2.0%.The XY-359^T is oxidase and catalase positive,and it can hydrolyze Tweens 20-80.The major polar lipids were phosphatidylethanolamine(PE),unidentified aminolipid(AL),unidentified phospholipid(PL)and unidentified polar lipid(L).The major fatty acids were iso-C_17:0 3-OH,iso-C_15:1 G and iso-C_15:0 3-OH.The respiratory quinone was MK-6.The genome size of strain XY-359T was 4,207,872 bp,and DNA G+C content is 39.1%.The combined phenotypic,chemotaxonomic and phylogenetic data suggest that strain XY-359^Trepresents a novel species of the genus Flagellimonas,for which the name Flagellimonas onchidii sp.nov.is proposed.We then screened the fermentation broth of the isolated microorganisms for AHL degradation activity,and in total we found 19 AHL-degrading bacteria,which fall into 15 genera.For 7 out of the 15 genera,this is the first time to report that their members have AHL degrading activity.We studied XY-85 in-deepth and found it could produce a lactonase which can degrade a series of AHL molecules and has strong resistance to high temperature and extreme p H.Through the whole gemone sequence analysis,we obtained a gene encoding a lactonase and then cloned it into the p ET28a vector.After induced expression and protein purification,the purified lactonase can inhibit biofilm formation of Pec,and the transformant can inhibit the bacterium from producing cell-wall degrading enzymes and soft-rot disease in protatoes.