The Establishment And Polymorphism Study Of Pseudomonas Fluorescens Strain Library Carrying The Cyclic Lipopeptide NRPS Gene

Cyclic lipopeptides amphiphilic antibiotics,is a form of biological surfactants.This feature leads to a cyclic lipopeptide in chemical structure and mechanisms of action,with all kinds of antibiotics previously found are different.Significant advantage cyclic lipopeptide antibiotic family of products in clinical,as well as its new structure type,makes cyclic lipopeptide antibiotic compounds become the new hot spot screening.Fluorescens Pseudomonas is an excellent source of cyclic lipopeptide antibiotic.Fluorescens Pseudomonas cyclic lipopeptide produced thiolated module using a non-ribosomal peptide synthetase(NRPS)synthesis.Common feature cyclic lipopeptide synthesis linear NRPS module is:the first module contains C,A and PCP structure,and C1 domain who is responsible for the first amino acid acylation,and so oligopeptide chain aliphatic chains are linked;epimerization(E)domain by its unique C/E domain is responsible for the L-amino acid is converted to D-amino acids;C has two terminal’thioesterase(thioesterase,Te)domain,catalyzed cyclization and the mature peptide chain release.In this paper,we designed a pair of primers at NRPS C1 conservative district;two pairs of primers at Te conserved region.Using the method of PCR,we found 90 strains of Fluorescens Pseudomonas carrying cyclic lipopeptide genes from soil samples of Shaanxi and Fujian.Then cyclic lipopeptide PCR gene polymorphism analysis were made by SSCP and other methods.Meanwhile,the DGGE method was used to the 16S V3 diversity analysis.The main contents and results are as follows:1,Over 1000 strains of Fluorescens Pseudomonas of which 48 strains carrying cyclic lipopeptide genes were isolated from soil samples of Shaanxi;More than 400 strains of Fluorescens Pseudomonas of which 42 strains carrying cyclic lipopeptide genes were isolated from soil samples of Fujian.2,ARDRA analysis shows that the Te area from different places had its own diversity.3,The diversity of cyclic lipopeptide gene was studied by SSCP.Fluorescens Pseudomonas strains isolateed from the same place may carry different cycloaliphatic peptide gene,while Fluorescens Pseudomonas strains isolateed from the different place may carry the same cycloaliphatic peptide gene.4,The strain diversity of different area were analyzed based on 16S rDNA PCR-DGGE fingerprint.The results showed that abundant Pseudomonas fluorescens strains were existed in the soil samples of Shaanxi and Fujian.The study found that,PCR method established in this experiment can isolate Fluorescens Pseudomonas strains carrying cyclic lipopeptide genes efficiently and economically.The Fluorescens Pseudomonas strains carrying cyclic lipopeptide genes isolated in this study can be established like a library,and this library can be foundation for the discovery of new kind of cyclic lipopeptide.The following work will be the isolation and identification of active substances.By the HPLC method,we can classify the biosurfactants produced by Fluorescens Pseudomonas.Then we can determine the structure of cyclic lipopeptide antibiotic by NMR and amino acid composition analysis and other methods.In summary,the present study was helpful to finding strains that produce new lipopeptide antibiotic.