The Study Of The Phenotypic Variant From The Phenazine High-yielding Pseudomonas Chlororaphis HT66 And The Establishment Of High Throughput Screening Method For 2-hydroxyphenazine High-yielding Strain

Pseudomonas chlororaphis is a biologically diverse plant-beneficial bacterium that exhibits strong ability to adapt variant environments.P.chlororaphis strains are generally recognized to be eco-friendly biocontrol strains due to the presence of phenazine synthesis gene cluster and modifying genes that can produce phenazine derivatives,and they also synthesize other secondary metabolites(i.e.siderophore),can antagonize plant pathogenic bacteria and enhance colonizing capability to plant rhizosphere,which help them adapt living environment better.Phenazines,as a kind of antibiotics with broader spectrum,have essential applied and commercial value.The screening of high-producing phenazine strains and maintaining their genetic and yielding stability are also important.Pseudomonas chlororaphis HT66 and abnormal phenotypic variant HT66-FLUO,which cannot produce PCN but be fluorescent,are the first research objects of this paper.HT66-FLUO was isolated from the fermentation process of high-producing phenazine strain HT66.Exploring the course of phenotypic variant appearance is beneficial to keep genetic stability and continuous high-production of phenazines in the engineering strains.The RNA sequencing experiment was utilized to compare the transcriptional profile of HT66 and HT66-FLUO in logarithmic phase.It showed the significant difference between two strains on transcriptional levels.The whole-genome and gene engineering were performed to identify the reason of phenotypic variation in HT66-FLUO,why the production ability of PCN was lost and the fluorescence under UV light was enhanced as compared to the native HT66.P.chlororaphis GP72 that can produce PCA,2-OH-PCA,and 2-OH-PHZ is the second research object.The promoter position and rate-limiting enzymes of phenazine biosynthetic gene cluster which provide the fundamental basis for genetic engineering of high-producing phenazine strains were determined.High throughput screening method for 2-OH-PHZ was preliminary established.The main contents were described as follows:The RNA-sequencing experiment was utilized to compare and analyze the transcriptional profile of HT66 and HT66-FLUO in logarithmic phase,and study the causes of the phenotypic modulation observed in P.chlororaphis HT66 and HT66-FLUO.Compared with HT66 strain,the expression of 1,418 genes in HT66-FLUO changed,which represented approximately 22% of the open reading frames(ORFs).The expression of PCN biosynthetic operon and phzI/phzR genes which are part of quorum sensing system and directly regulate PCN biosynthesis were found to be significantly downregulated.It is consistent with the PCN disappearance in HT66-FLUO fermentation broth.The transcription level of genes associated with pyoverdine and achromobactin biosynthesis,regulation,and transport was significantly higher in HT66-FLUO than HT66.Gene deletion and complement results indicate that the fluorescent substance was pyoverdine,and the phenomenon of fluorescence in HT66-FLUO is related to the increased production of pyoverdine.Since the phenotype of HT66-FLUO was stable,genome sequencing was conducted to identify whether there exists Single-Nucleotide Polymorphisms(SNPs)or InDel in the genome of HT66-FLUO.The result indicated no gene alteration in HT66-FLUO as compared to HT66 according to the known reference sequence.Overexpressing gacS in HT66-FLUO could restore the biosynthesis of PCN,but overexpression of gacA could not restore it.On the other hand,the strains overexpressing exogenous gacS had no fluorescence under UV light.We also sequenced the entire GacS/GacA system of both HT66 and HT66-FLUO.These sequencing analyses showed no discrepancy between Gac/Rsm genes of the high-producing phenazine strain and the variant strain.We speculated that the genetic modification probably occurred at DNA-level or translational modification causing the deactivation of GacS protein that influences downstream gene expression(phenazine and siderophore biosynthesis),and the modification could not be detected by the genome re-sequencing and PCR-sequencing.We constructed pBB-rsmX/Y/Z-lacZ plasmids coupled with RT-PCR to detect the relative levels of rsmX/Y/Z,respectively,both in HT66 and HT66-FLUO.The gene expression of three sRNA was downregulated,and the expression of rsmX gene was most obviously decreased.This was the further evidence that deactivation of GacS protein that influences rsmX/Y/Z gene expression can regulate the biosynthesis of secondary metabolites.The HT66-FLUO colony morphology was restored to original strain to different extent by overexpressing rsmX/Y/Z,separately.The overexpression of rsmX,rsmY or rsmZ in HT66-FLUO resulted in PCN production recovery to some extent,among which the overexpressing rsmX in mutant showed the best efficiency.Accurate positioning the transcription start site(TSS)of phenazine gene cluster and phzR by 5'-RACE in P.chlororaphis GP72.The TSS of phz gene cluster is located at 112 bp upstream of phzA,and the TSS of phzR is located at 29 bp 5'-UTR of the gene.We also confirmed the results by constructing lacZ recombinant plasmid and analysis of the ?-galactosidase(LacZ)activity.Based on the comparson and analysis of the ?-galactosidase(LacZ)activity of 5'-UTR with different lengths,the result showed there exists no inhibition region that influences phenazine production.Overexpressing phzA/ phzB/ phzC/ phzD/ phzE/ phzF/ phzG/ phzO in GP72 showed that PhzE and PhzO are the rate-limiting enzymes related to 2-OH-PHZ production in GP72.2-OH-PHZ was separated and purified from the fermentation broth of GP72ND3.Then,the full wave scanning was conducted for 100 mg/L PCA in methanol solution or 100mg/L 2-OH-PHZ in methanol solution to show the discrepancy absorption wavelength was at 430 nm.Therefore,we selected 430 nm as test wavelength.A random mutagenesis library of phzO was made using error prone PCR with only 1 to 4 point substitutions introduced per phzO gene by using GeneMorph II random mutagenesis kit.The extraction and transformation of recombined plasmid is easy to realize by introducing TIANprep Rapid N96 Plasmid Kit and 96-well PCR tube.Initial screening is conducted by discarding the milk white mutants that cannot produce bolarious 2-OHPHZ.The other mutants were cultivated in 24 Deep-well Multiwell Plate,and the diluted fermentation broth was checked at 430 nm.High throughput screening method for 2-OH-PHZ production is also preliminarily established,the method provided a new thinking to improve 2-OH-PHZ yield.In this paper,we reveal the phenomenon discrepancy of abnormal phenotypic variant P.chlororaphis HT66-FLUO compared with the high-producing phenazine strain HT66,and the Whole Genome Resequencing result indicated no gene alteration in HT66-FLUO as compared to HT66 according to the known reference sequence.It is useful to intensively study the course of the phenotypic variant and maintain the genetic stability of phenazine high-producing strains.Determining the rate-limiting enzyme and accurate position of the transcription start site(TSS)of phenazine gene cluster and phzR by 5'-RACE lays a theoretical basis for gene engineering in P.chlororaphis GP72.The establishment of a high throughput screening method for strains of 2-OHPHZ high production provides a technical basis for improving 2-OH-PHZ yield.