Identification And Regulation Mechanism Of Antimicrobial Cyclic Dipeptide CFP In Burkholderia Seminalis R456

Cyclic L-phenylalanine L-proline(cyclo-(L-pro-L-phe),cFP)is a very important natural product and has been reported in the secondary metabolites of a variety of bacteria and fungi.With cFP antifungal,antibacterial,antiviral and induction of quorum sensing and many other biological activities,cFP has become the research focus in biological control of plant diseases field.From the application prospects,the effective biological synthetic of cFP is of importance,however,the current regulatory mechanism about the biosynthesis of cFP is still not entirely clear.Recently,some domestic studies have found that Burkholderia cepacia CF-66 can produce cFP,which show significant inhibitory effect on the growth of Fusarium semitectum.Interestingly,our study also found that,Burkholderia seminalis R456 and its metabolites can significantly inhibit the mycelium growth of Fusarium graminearum Fg.As an antagonistic bacterium,R456 was isolated from paddy soil earlier by our laboratory.It is reasonable to speculate that cFP may be one of the active substances of antagonizing bacteria R456 to inhibit Fg.Accordingly,the following work is carried out in this study:To make clear whether the Fg R456 antagonism is partly caused by the secretion of antimicrobial substances cFP,firstly,the antagonism effect was determined by using plate confrontation method.As a result,997 of Tn5 inserted mutant strains of Fg R456 was constructed.Compared to the wild type,8 mutants(no.88,189,117,113,885,495,268 and 255)indicate improved antagonism effect(increase by more than 20%),and 5 mutants(no.216,123,85,224 and 225)show reduced antagonism effect(decreased more than 20%).Then,the supernatant filtrate of these 13 mutants was analyzed by high performance liquid chromatography(HPLC)technique.Compare to other mutants and wild type,a unique peak was observed at 20 min for mutant 88,which is consist with the peak time of synthesized cFP standard under similar experimental conditions.Further LC-MS(mass spectrometry)analysis showed relative molecular mass of 245.12,which is close to the relative molecular mass of cFP.The results of the secondary mass spectrometry showed that there were 7 debris peaks with relative molecular mass of 65.04,70.06,92.05,154.07,172.11,217.13 and 245.12,which were close to the chemical structure of cFP.Finally,the results of the plate antagonism experiment showed that both the synthetic cFP and biosynthetic cFP in strain R456 could strongly inhibit the growth of Fg mycelium,and the antagonistic effect was enhanced with the increased concentration within the range of 0.1-1000 ppm.To further explore the synthesis and regulatory mechanism of cFP in antagonistic bacteria R456,first of all,a reverse PCR amplification was applied for the above mentioned 13 mutants with antagonism effects and insertional locus was detected through blast against the genome sequence of R456.The insertional locus for mutant 88 was successfully identified for gene DctB,which is composed of Dct transfer system(Dct-A,-B,-D),and is response for the transport of four carbon dicarboxylic acid C4-dicarboxylates(dCAs).Moreover,single Dct-A,-B,or-D gene knockout mutant and complement strain was constructed,and cFP content was determined for each supernatant filtrate by using HPLC.Compared with wild type,significantly increased cFP content was observed for Dct(-A,-B or-D)mutant and no significant differences was detected for the corresponding complementary strain.It is therefore reasonable to infer that cFP synthesis of R456 is closely related to the Dct transfer system.Finally,the effect of substrate dCAs for Dct transfer system on cFP production is analyzed for R456 and its mutants by using HPLC.Significant decreased cFP production was observed for wild type strain with the presence of either tested dCAs including oxaloacetic acid,malic acid,aspartic acid and succinic acid.By contrast,there is no difference on cFP production for either of Dct-A,-B-D gene deletion mutant.Taken together,dCAs may negatively control the synthesis of cFP in strains R456.In summary,this study clearly shows that cFP is one of the main active substances of strain R456 antagonized against Fg,and its biosynthesis is negatively regulated by dCAs.