The Gac/Rsm Post-transcriptional Regulatory System And Hfq Global Regulatory Factors In Pseudomonas Extremaustralis YL19 Regulate The Synthesis Of Cycloaliphatic Peptides

Pseudomonas is widely found in the natural environment such as soil and ocean.Although some Pseudomonas can cause diseases in a variety of crops such as wheat,rice and tomatoes,most fluorescent Pseudomonas can act as plant growth promoting rhizobacteria to inhibit plant pathogens and promote plant growth.Studies have shown that Pseudomonas can produce a variety of antibiotic secondary metabolites,cyclic lipopeptides are a class of lipopeptides with stable physicochemical properties and a cyclic structure in the molecule.On the one hand,cyclic lipopeptides can be studied as biosurfactants,on the other hand,they can resist against bacteria,fungi,virus and antitumor activities,as well as immunosuppression.In our laboratory,a strain of extremaustralis YL19(hereinafter referred to as YL19)which was screened in the early stage has good antibiotic activities.A series of Massetolide homologues compounds in the Viscosin cyclic lipopeptides family were identified by mass spectrometry in YL19,but the amount of Viscosin synthesis is low,and the synthetic regulatory network is complex.Studies have shown that the Gac/Rsm post-transcriptional regulation system regulates the synthesis of various cyclic lipopeptides,and the Hfq protein acts as a global regulator to regulate the synthesis of various lipopeptides.In order to clarify the synthetic regulation mechanism of Viscosin in YL19,and to obtain a high-yield strain of Viscosin,in this study,we firstly predicted the type of cyclic lipopeptide produced by YL19,the synthetic pathway,the core factors of Gac/Rsm regulation system,and the presence of Hfq protein.And then we knocked out and replenished the genes gacA,rsmA,rsmE,overexpressed the gene hfq to verify the regulation mechanism of Gac/Rsm regulatory system and global regulatory factor Hfq of cyclic lipopeptides producted by YL19.(1)Prediction of cyclic lipopeptides producted by YL19 and its synthetic pathway at genomic level:11 kinds of secondary metabolites synthesized by YL19 were predicted by anti-SMASH online software.It was deterrmined that the cyclic lipopeptide synthesized by YL19 is the Viscosin family by analyzing the NRPS gene cluster and NRPS-A domain Amino acid sequence cluster analysis.Based on the identified PKS_KR and NRPS encoding genes,the pathway for Viscosin biosynthesis was deduced.(2)Prediction of regulatory network of cyclic lipopeptides producted by YL19:The existence of four core factors of GacS,GacA,RsmA and RsmE in Gac/Rsm regulatory system and the global regulatory factor Hfq were determined by NCBI-blast homologous sequence alignment and genomic information.It is predicted that the transcription of 10 sRNAs may be regulated by GacA protein in 17 sRNAs extracted from the genomic information of YL19.Combined with the prediction of the secondary structure of sRNA,GGA motif and its location in the MFOLD(37? folding)program,three sRNAs were further screened for possible involvement in the Gac/Rsm regulatory system.Through the conserved motif 5'-WCANGGANGW-3' of RsmA/E binding targe tgene,two mRNA target genes was located at upstream and downstream of the Viscosin synthetic gene cluster by searching the genome of the YL19.Developmental tree analysis further confirmed that these are direct target genes regulated by the Gac/Rsm system-LuxR type transcriptional regulators.(3)Regulation of GacA on the synthesis of cyclic lipopeptides producted by YL19:The gacA gene mutant was obtained by using the suicide vector pknock-Gm,and the gacA gene replenishing strain was obtained by using the expression vector pME6032.When the gacA gene was mutated,the expression of RsmY did not change,but the expression of RsmZ decreased,the expression of RsmA and RsmE increased significantly,the expression of LuxRU did not change and LuxRD increased significantly.It is preliminarily judged that the gacA gene plays a role in the Gac/Rsm regulatory system and regulates through sRNA RsmZ after activation in YL19.After gacA gene mutation,the production of cyclic lipopeptides decreased,and the production of cyclic lipopeptides after gacA gene replenishment recovered to some extent,indicating that GacA is a positive regulator in the regulation pathway of cyclic lipopeptides producted by YL19.(4)Regulation of repressor proteins RsmA and RsmE on the synthesis of cyclic lipopeptides producted by YL19:rsmA and rsmE gene mutants were obtained using the suicide vector pknock-Gm,and rsmA and rsmE gene replenishants were obtained using the expression vector pME6032.According to the real-time quantitative PCR results,when the rsmA or rsmE genes were mutated,the expression of another repressor protein was increased,indicating that RsmA and RsmE inhibited each other and coordinated with each other in the Gac/Rsm regulatory system.When the rsmA gene was mutated,the expression of LuxRU and LuxRD did not change.When the rsmE gene was mutated,the expression of LuxRU did not change and the expression of LuxRD was increased,further indicating that the cyclic lipopeptide gene cluster is regulated by the LuxR-type transcriptional regulator LuxRD.After rsmA gene was mutated,the expression of RsmE was increased,the production of cyclic lipopeptides was increased,and the expression of RsmA was increased after rsmE gene was mutated,but the production of cyclic lipopeptides was decreased.It was indicated that RsmA is a negative regulator in the regulation pathway of cyclic lipopeptides producted by YL19,while RsmE plays a positive regulatory role in the synthesis of cyclic lipopeptides produced by YL19 by inhibiting the expression of the repressor protein RsmA.(5)Regulation of Hfq on the synthesis of cyclic lipopeptides producted by YL19:The hfq gene overexpression strain was obtained using the expression vector pME6032.After overexpression of the hfq gene,RsmY expression was increased but RsmZ expression was decreased,suggesting that Hfq can regulate sRNA RsmY to increase its stability.After overexpression of the hfq gene,the expression of LuxRU was decreased,and the expression of LuxRD was increased.It is speculated that Hfq may regulate the cyclic lipopeptides producted by YL19 through the LuxR-type transcriptional regulators LuxRU and LuxRD.After overexpression of the hfq gene,the production of cyclic lipopeptides was decreased.It is speculated that the overexpression of the hfq gene will put off the time when the strain enters the stable phase,while the stationary phase is the best period for the harvest of cyclic lipopeptides,which leads to the accumulation of cyclic lipopeptides decreased.