| Pseudomonas is a kind of common Gram-negative bacteria,which is widely distributed in plants,soils,oceans and other natural environments.A kind of secondary metabolite of cyclolipopeptide produced by Pseudomonas plays an important role in biosurfactant,antimicrobial activity and so on.So,the Pseudomonas can be used as plant rhizosphere-promoting bacteria to inhibit plant pathogens and promote plant growth.Therefore,Pseudomonas is widely used as a biocontrol strain in agricultural fields.In our laboratory,a strain of Pseudomonas sp.G4(hereinafter referred to as strain G4)which produces cyclolipopeptide,was isolated and screened in the early stage.And it was found to have good anti-life resistance through antibacterial activity and surface activity detection.The cyclolipopeptide produced by strain G4 may be a new cyclolipopeptides,which was preliminarily identified by mass spectrometry.However,the strain itself has a low production of cyclolipopeptide,and the regulatory network for cyclolipopeptides' biosynthesis is complex.It is not conducive to its function as a biocontrol strain.Studies has found that both the Gac/Rsm system and a global regulator Hfq can regulate the synthesis of a variety of cyclolipopeptides.Therefore,studying its regulation mechanism of synthesis of cyclolipopeptides can not only improve its yield,but also provide new thought and sufficient materials for the development of the targeted high-yield strains,the biocontrol strains and the new microbial agents.The present subject firstly confirmed the structure and synthetic pathway of cyclolipopeptide produced by strain G4 at the gene level and product level,and predicted the existence of Gac/Rsm system and Hfq protein;secondly,through a series of knockout,replenishment,real-time quantitative PCR and phenotypic experiments were performed to verify the regulation of the Gac/Rsm system and the global regulatory factor Hfq on the production of cyclolipopeptides by strain G4.(1)Analysis the structure and synthesis pathway of cyclosporin produced by strain G4 at genomic level and product level:first predicted the secondary metabolite produced by strain G4 by antiSMASH,and then analyzed the NRPS of Cluster 7 and constructed the phylogenetic tree by NRPS-A domain.The structural of the peptide moieties was determined at the genetic level.It is a novel cyclolipopeptide,which is temporarily named Arthro-Like.The biosynthetic pathway of Arthro-like was predicted according to its NRPS-encoding gene.At the product level,the structure of the cyclolipopeptide was analyzed by LC-MS,which was consistent with the results obtained by gene level analysis.And it was found that it also exists the homologue of Arthro-like.(2)Predicted the regulatory network of post-transcriptional levels of cyclic lipopeptide produced by strain G4 at genomic level:the existence of four core factors--GacS,GacA,RsmA and RsmE in Gac/Rsm systems and the global regulatory factor Hfq were determined by NCBI-blast homologous sequence alignment with G4 genomic information.Based on the genomic information of strain G4 and the secondary structure of sRNA,its GGA motif and its location in the MFOLD(37? folding)program,four sRNAs were predicted to participate the Gac/Rsm system.Two mRNA target genes were obtained which are located in the upstream and downstream of the Cluster 7 cyclolipopeptide synthesis gene cluster to encoding LuxR-type transcriptional regulatory factors,by the conserved motif 5'-WCANGGANGW-3' of the RsmA/E binding the target gene in the G4's genome.Through phylogenetic tree analysis,it was further determined that LuxR type transcriptional regulatory factors are two direct target genes of the Gac/Rsm system,which are temporarily named G4_LuxRU and G4_LuxRD,respectively.(3)Regulation of gacA gene on the synthesis of cyclolipopeptide in the strain G4:The G4-gacA gene mutant strain was obtained by the suicide vector pKnock-Gm,and the G4-gacA gene replenishing strain was obtained by the expression vector pME6032.Phenotypic experiments and HPLC tests showed that the production of cyclolipopeptide decreased after gacA gene mutation,and the production of cyclolipopeptide recovered after gacA gene replenishment.The results suggest that the GacA protein is a positive regulation in the regulatory pathway of synthesis of cyclolipopeptide in the strain G4.The results of real-time quantitative PCR showed that when the gacA gene mutated,the expression of RsmY was down-regulated but the expression of RsmZ was not changed,the expression of repressor proteins RsmA and RsmE were down-regulated and the expression levels of LuxRU and LuxRD were down-regulated.The results indicate that the GacA box binds to RsmY activates the synthesis of cyclolipopeptide in the Gac/Rsm regulatory pathway,and the GacA protein plays a positive regulatory role in this pathway.(4)Regulation of repressor protein RsmA on the synthesis of cyclolipopeptide in the strain G4:The G4-rsmA gene mutant strain was obtained by the suicide vector pKnock-Gm,and the G4-rsmA gene replenishing strain was obtained by the expression vector pME6032.The phenotypic experiment showed that after the rsmA gene mutated,the oil drainage activity and the swarming motility of the strain were weakened.HPLC detection showed that after the rsmA gene mutated,the species of Arthro-like and homologues' production were almost unchanged showed little change,but the yield increased slightly.After rsmA gene replenished,the oil drainage activity of the strain was not significantly different from the mutant strain,but the swarming motility of the strain was restored,the Arthro-like and homologues' production were decreased by HPLC tests.The results suggest that the rsmA gene don't regulate the synthesis of cyclolipopeptide in the strain G4.The results of real-time quantitative PCR showed that when the rsmA gene was mutated,the expression of another repressor protein RsmE was down-regulated,the expression of RsmY was up-regulated,the expression of RsmZ was down-regulated,the expression of LuxRU was not significantly changed and the expression of LuxRD was down-regulated.It is indicated that the repressor protein RsmA acts by binding to RsmY,positively regulates the expression of RsmE,RsmZ and LuxRD in strain G4.(5)Regulation of hfq gene on the synthesis of cyclolipopeptide in the strain G4:G4-hfq overexpression strain was obtained by the expression vector pME6032.The results of growth curve showed that after overexpression of hfq gene,the growth rate of the strain increased before entering the stationary phase,but the time to enter the stationary phase did not change.It indicated that the Hfq didn't the effect growth of the strain G4.Phenotypic experiments showed that the production of cyclolipopeptides was reduced after the hfq gene overexpressed in strain G4.This result indicates that the Hfq protein is a negative regulator in the regulatory pathway of synthesis of cyclolipopeptide in the strain G4.The results of real-time quantitative PCR showed that after the hfq gene overexpressed,the expression of RsmY was up-regulated and the expression of RsmZ was down-regulated,the expression of LuxRU and LuxRD were both down-regulated.The results indicate that the Hfq protein may bind to RsmY to increase its stability,and bind to the LuxRU and LuxRD to inhibit cyclolipopeptides'synthesis.Thus the Hfq protein acts as a negative regulator in the regulation of cyclolipopeptides' synthesis in the strain G4. |
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