Isolation And Identification Of Serratia Marcescens YD25 And The Study Of Its Antibiotic Secondary Metabolic Components

Serratia is one of the most important biocontrol bacterium.The previous studies have shown the antagonistic mechanisms included antibiosis and induced systemic resistance.A number of inhibitory molecules are produced by Serratia spp.including prodigiosin,oocydin A,pyrrolnitrin,carbapenem,and the surfactant serrawettin as well as chitinase and other cell-wall and cell-membrane degrading enzymes.Here,we isolated a pigment-produced bacterial srain from plant rhizosphere soils.Interestingly,when it co-cultivated with the phytopathogenic fungus,the pigment-produced strain suppressed the growth of Rhizoctonia solani,Fusarium oxysporum,Bipolaris sorokiniana and Colletotrichum capsici.This indicated some antimicrobial substance had been produced.The objective of this study including the identification of the pigment-produced bacterial and the purification,structural elucidation of the bioactive substances and the antibacterial spectrum,cytotoxicity of serrawettin W2.(1)Strain identification:Based on the 16S rRNA gene and rpoB gene sequences analysis,strain YD25 should be positioned within the genus Serratia,being related most closely to S.marcescens.The 16S rRNA phylogentic analysis showed strain YD25 formed an out group to the clade containing S.marcescens subsp.sakuensis KRED and S.marcescens subsp.marcescens DSM 30121,while the rpoB gene tree showed that strain YD25 formed a cluster with S.marescens subsp.marcescens Db11.For strain YD25,it couldn’t use L-Arabinose and D-Xylose as the carbon source,which was different from S.marcescens.Interestingly,the major fatty acids components of strain YD25 were different from the other Serratia species,it had a 71.8%match with Cedecea davisae in the MIDI database.However,Cedecea was unable to hydrolyze gelatin.On the basis of the data presented,strain YD25 was confirmed to be a new subspecies of S.marcescens.(2)The bioactive substances isolated and identification:The crude bioactive substances was obtained using ethanol extracting from mycetome and purified by flash chromatography employing the 300-400 mesh silica gel.Firstly,the crude extract was eluted with ethyl acetate and petroleum ether(1:1,v/v).A pink,an orange-red fraction and a red fraction named comonent 1,2,and 3,respectively,were collected.A dark-red fraction named component 4 was collected eluted with ethanol later.The component 2 was identified prodigiosin by MS and MS/MS.The component 4 was purified by preparative HPLC further.Eight compounds named sw-1 to sw-8 was obtained.By ESI-MS,sw-1,sw-2,sw-6,sw-7,and sw-8 had the molecular weight of 703.3,717.3,745.4,757.2 and 759.3 Da respectively.sw-3,sw-4,sw-5 shared a same molecular weight of 731.2 Da.Sw-5 was identical to Serrawettin W2 when it was analyzed by NMR((’H,13CNMR,HMQC,HMBC,ROESY)in depth.(3)Biological activity assays:To further examine the antibacterial spectrum of sw-5(serrawettin W2),an expanded indicator panel composed of Gram-negative and Gram-positive bacteria were tested.The result indicated serrawettin W2 had a relative narrow antibacterial spectrum as it only showed activities to R.rhodochrous CGMCC 4.1815,S.dysenteriae CGMCC 1.1869,M.luteus CGMCC 1.2299 and P.aeruginosa A47.But it was noteworthy that sw-5 could inhibit some clinically-relevant pathogens S.aureuss.Among thirty S.aureuss clinical isolates,we found serrawettin W2 exhibited evident inhibitory activities to nine strains and slight inhibition to twelve strains.The cytotoxicity of sw-5(serrawettin W2)was also examined.sw-5 could inhibite growth of Hela significantly,while caco2 seemed to be less affected.For the normal cell lines,sw-5 did not show a marked decrease in viability of Vero cells and HEK293 cells at concentrations of up to 30 μM.From previous studies we confirmed that it was the first report that strain YD25 produce the pigment prodigiosin and serrawettin W2 concurrently.The main antibacterial compounds produced by strain YD25 were prodigiosin and the members of serrawettin W2 family.These indicate that strain YD25 can be a promising candidate that yield prodigiosin and serrawettin W2 family simultaneously in the future.For strain YD25,further researches were required to reveal the biosyntheses mechanisms of prodigiosin and serrawettins.Also,it might help to improve the production of the prodigiosin and serrawettins by elucidation of regulatory mechanisms of these compounds in strain YD25.