The Quorum Sensing Regulation And Over-expression Of Phenazine Biosynthetic Gene Cluster In Pseudomonas Chlororaphis GP72

Pseudomonas chlororaphis GP72 is a biocontrol rhizospherepromoting bacterium isolated from the rhizosphere of green peppers.It secretes phenazine compounds such as phenazine-1-carboxylic acid(PCA)and 2-hydroxyphenazine(2-OH-PHZ),which have a strong inhibitory effect on phytopathogenic fungi.Therefore it is of great significance to study the regulatory mechanism of phenazine biosynthesis and to increase the production of phenazines by molecular biological methods.Previous studies had shown that the biosynthesis of phenazines is regulated by the phzI/phzR regulatory system,and the phzI gene produces the acylhomoserine lactones(AHLs),which bind to PhzR to regulate the expression of phenazine biosynthetic genes.Based on the whole genome sequence analysis of GP72,it was found that there are three other genes aurI,csaI and hdtS,which were reported to be able to synthesize AHLs,and the regulatory system aurI/aurR,csaI/csaR may regulate the production of phenazines.Firstly,the four genes aurI,csaI,hdtS and phzI in GP72 were studied by constructing overexpression plasmids pBbB5k-aurI,pBbB5k-csaI,pBbB5k-hdtS and pBbB5k-phzI and transforming into E.coli DH5?.pBbB5 k is an expression vector containing a strong promoter that can initiate downstream gene expression under the induction of IPTG.Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NTL4 can be used as indicator bacteria to detect AHLs.While CV026 is sensitive to short-chain signal molecules,NTL4 is sensitive to long-chain signal molecules.The signal molecules encoded by aurI,csaI and phzI made CV026 purple and NTL4 blue,while the signal molecules encoded by hdtS only make NTL4 blue,indicating that aurI,csaI and phzI can simultaneously produce long-chain and short-chain signal molecules,hdtS can only produce long-chain signal molecules.The four strains were fermented,and the fermentation broth were used to prepare LC-MS samples to determine signal molecular species.Next,deletion mutants of each genes,GP72?aurI,GP72?csaI,GP72?hdtS and GP72?phzI,were constructed,and the growth curve and phenazine compounds production were determined.Compared with the wild-type GP72,the phenazine production of GP72?phzI was reduced about half.The phenazine production of GP72?aurI was about 4-fold higher than that of the wild-type.The phenazine production of GP72?csaI and GP72?hdtS mutant strains did not change,and the growth curves of the four mutant strains were consistent with the wild-type,indicating that phzI positively regulates phenazine production,aurI negatively regulates phenazine production,and csaI and hdtS genes produce signal molecules but may not be involved in phenazine production.In order to study the effect of the aurI/aurR regulatory system on phenazine production,we constructed the deletion mutants GP72?aurR and GP72?aurI?aurR.The results of fermentation showed that the phenazine production of GP72?aurI,GP72?aurR and GP72?aurI?aurR were about 4 times higher than those of the wild-type,indicating that aurI and aurR genes co-operate to regulate phenazines production.The transcriptional fusion plasmid fused with the promoter of phenazine biosynthetic genes was constructed and ?-galactosidase activity can reflect the transcriptional level of phenazine biosynthetic promoter.The enzyme activity of the mutant strain was about 4 times as high as that of the wild-type enzyme activity,thus demonstrating that the aurI/aurR regulatory system negatively regulate the phenazine biosynthesis.Finally,in order to study the effect of expression of the phenazine biosynthetic gene cluster on phenazine production,the phenazine biosynthetic gene cluster was overexpressed in GP72 wild-type strain.After 48 hours of fermentation,the yield of phenazine compounds reached 407 mg/L,a 23-fold increase,demonstrating that increasing the copy numbers of gene clusters can increase the yield of phenazine compounds.Based on pUC18-mini-Tn7T-Gm vector and site-specific recombination,the gene cluster was integrated at the downstream of the particular glms gene of Pseudomonas.Compared with the wild-type strain,however,the production of phenazines in the strain containing two copies of phenazine biosynthetic gene cluster did not increase significantly,and the gene cluster integrated at the downstream of the glms gene did not express well.Pseudomonas chlororaphis GP72 has a good prospect for biocontrol application.This research explains the regultory phenomenon in GP72 to a certain extent,and provides a theoretical basis for a comprehensive understanding of the quorum sensing regulatory system and phenazine biosynthesis in GP72.On this basis,we can further design genetically engineered targets and construct high-yield phenazine producing strains.