The Diversity Of Lipopeptide Antibiotics Secreted From Bacillus Spp. And The Preliminary Exploration Of Key Ragul Ation Factors Of Locillomycin Promoter Ploc

Bacillus spp.strains are important biological resources which can produce rich lipopeptide antibiotics with high utilization value in bioengineering.Lipopeptide antibiotics from Bacillus which were researched in detail wer surfactins,iturins and fengycins.Bacillus spp.lipopeptide antibiotics were the main components in inhibiting plant pathogenic fungi.To clear the inhibition effect of 55 Bacillus spp.strains to pathogenic fungi,Rhizoctonia cerealis,Fusarium graminearum and Fusarium oxysporum f.sp.niveum,and the qualitative and quantitative relationship with lipopeptide antibiotics produced by the Bacillus strains.Inhibition rates of the 55 Bacillus spp.strains and their crude lipopeptides extracts against three pathogenic fungi were tested respectively by dual culture on plate.Three pairs of primers designed based on the known lipopeptide genes were used to amplify the corresponding genes,srfA,ituA and fenA,from the genome of the 55 strains.The lipopeptides antibiotics were identified by HPLC and MS techniques.The results showed that the lipopeptides antibiotics were the main components to inhibit plant pathogenic fung.The structure of the fengycins and surfactins were simple,while the iturins had various types of structure.According to the time and type of chromatographic peak,iturins were divided into 4 types,named as types 1,2,3,and 4.The antifungal activities intensity of 4 types of iturins were type 1>type 2>type 3>type 4.There were two Unknownn lipopeptides(named as Unknowns:Unknownl,2)which were speculated as new lipopeptides were detected.Lipopeptides antibiotics produced by Bacillus spp.were the main composition against plant pathogenic fungi and had diversity characteristics.The structure changes and secretion of iturins had certain relevance to antifungal activities.There were some unknown new resisted resources in lipopeptides compounds from Bacillus spp..For further analyzing the antifungal activities ofthree lipopeptide antibiotics,we chose Bacillus a my lo liq ue fac ie ns 1619(B1619)as the representative strain to separate and identify of three lipopeptide antibiotics.Bacillus amyloliquefaciens,biocontrol strain B1619,can effectively prevent and control the occurrence and damage of tomato fusarium wilt.In order to study and analyze the antifungal substances produced by them preliminarily,the lipopeptide antibiotics were isolated from the supernatant of fermented broth.Three primers designed based on the known lipopeptide genes were used to amplify the corresponding genes from the genome of B1619 strain.The crude lipopeptides extracts were extracted by acid precipitation and methanol.The supelcleanTM LC-1b column and PHASE HF-NH2 column were used to separate lipopeptide antibiotics.All the samples were analyzed by HPLC and MALDI-TOF-MS techniques.Inhibition rates of the three lipopeptide antibiotics against Fusarium oxysporum f.sp.lycopersici were tested by dual culture on plate.The PCR products analysis showed that the sfp,ituA or fenB genes existed in the genome of B1619.The results showed that bacillomycin L,fengycins and surfatins were isolated from 60%,70%,100%methanol eluent.The mycostatic tests showed that the antifungal activities of fengycins was higher than bacillomycin L,and the surfactins had weakest effect on the F.oxysporum B1619 strain secreted the three kinds of lipopeptide antibiotics in which fengycins and bacillomycin Lplayed a imporment role in the inhibitory effect on F.oxysporum,while the surfactins had no obvious effect on it.Bacillus subtilis was the biological control mode strain.Locillomycin was a new kind of lipopeptide antibiotic which was found in Bacillus subtilis 916(Bs916).The transcriptional level of the ploc promoter was subjected to various protein regulation factors.The report gene xynB(GenBank number:AY340789)encoding extremely thermostable xylanase from Thermotoga maritima was cloned.We fused it with the ploc promoter,built the fusion plasmid vector pXYNB2.And then the ploc promoter region of fusion gene was truncated to 15 different length,the fusion plasmid vector ApXYNB2 were built.We transformated the vectors to Bs916,then the recombinant strain(Bs916/pXYNB2)and 15 truncated gene recombinant strains(Bs916/?pXYNB2)was obtained.By conparing the xylanase activity of the recombinant strain(Bs916/pXYNB2),15 truncated gene recombinant strains(Bs916/?pXYNB2)and control strain(Bs916/pRP22),we found that when the two regulatory factors 1 ResD and 9 Hpr were removed one by one the xylanase activity decreased obviously.Howere,when we removed 2 spo?D and 8 spA?regulatory factors,the xylanase activity increased obviously.So we speculated that the 1resD and 9 hprregulatory factors played a positive regulatory role in ploe promoter,While 2Spo?D and 8 spoA?regulatory factors played a negative regulatory role in the ploc promoter.