| Pseudomonas protegens is a kind of environmental microorganism with biological control function.Pseudomonas protegens have a variety of host species,which widely exist in soil,plants,water and other natural environments.It can inhibit the growth of pathogens by producing a wide range of antibiotics.Moreover,the colonization ability of rhizosphere and the function of secretion of ferritin are also important reasons for its biological control ability.However,in practical application,the bottleneck problem of P.protegens SN15-2 are the weak stress resistance,easy inactivation under high temperature and short shelf life of preparation.At present,there is little research on the regulatory mechanism of P.protegens against adversity,so it has great academic value and practical significance to explore this.In this study,SN15-2 whole genome was sequenced and analyzed.it was found that there were 6433 protein coding genes(88.31%of the total length of the whole genome),208 non coding RNA,71 tRNA,16 Microsatellite DNA and 6 CRISPER sequences.5210,3259 and 5277 genes were annotated in COG,KEGG and Go databases respectively.Through the establishment of protein evolutionary development tree,it was found that there are multiple conserved amino acid sequences in the SpoT protein of SN15-2,and these conserved domains largely affect the protease activity.The homology between SpoT protein and homologous proteins in Pseudomonas fluorescens and Pseudomonas aeruginosa reached 99.57%and 90.46%,respectively.Then,spoT deletion mutant strain ΔspoT,which mediates stringent response in P.protegens SN15-2,was successfully constructed by traceless gene knockout technology.It was also found that spoT had no effect on growth rate and was not involved in the antagonistic effect of Ralstonia solanacearum,but could regulate the swimming ability,high temperature tolerance and acid stress tolerance of SN15-2.The growth rate of ΔspoT strain was not different from that of wild strain,and it did not change the antagonistic ability of SN15-2 against Ralstonia solanacearum.The swimming ability of the ΔspoT strain was always stronger than the wild strain on the swimming ability test plate,and there was a significant difference between the diameters of the swimming circles of the two strains.After 6 hours of culture in 450 mmol salt stress environment,the survival rate of wild strain was 4.58 times that of mutant strain,which showed obvious difference(p<0.05);after 2 hours of high temperature stress at 45 C,the survival rate of wild strain was 57.83 times that of ΔspoT;while in alkaline environment,there was no significant difference between them.Therefore,spoT-mediated stringent response responds to the stress response of bacteria,and it is speculated that spoT may be involved in the regulation of genes related to the defense against high temperature and hyperosmotic pressure in P.protegens SN15-2.Finally,the effects of spoT gene deletion on biofilm formation,quorum sensing signaling molecules and gene expression related to biofilm synthesis were studied by HPLC analysis and RT-PCR.It was found that the ability of wild bacteria to synthesize biofilm was significantly stronger than that of the deleted strain.At 6 and 24 hours of culture,the biofilm quantities of SN15-2 and spoT were significantly different(p<0.01).Mediated stringent reaction have a positive regulatory effect on strain biofilm synthesis.Detection of quorum sensing signaling molecules(AHLs)showed a significant decrease in C8-HSL and C10-HSL after spoT deletion.ΔspoT was down-regulated in 8 genes related to biofilm synthesis at 12 h of culture,among which four genes,csrA,wepI,hcpl,anoR,were down-regulated by 4.51,9.35,25.4 and 24.8 times,respectively.It can be seen that spoT gene not only regulates the biofilm of strain amount,but also regulates the synthesis of quorum sensing signaling molecules,and regulates quorum sensing and biofilm formation by downregulating the expression of multiple genes such as hcpl,anoR,qseC and luxQ.Therefore,spoT plays an important role in maintaining biofilm formation,stress resistance and quorum sensing mechanism in SN15-2,revealing the regulatory role of stringent response as a global regulatory mechanism on the physiological activity of SN15-2.Based on the analysis of genomics and bioinformatics technology,this paper reveals the evolution and conserved sequence of SpoT protein in the P.protegens SN15-2.By using traceless gene knockout technology and other systems,it was found that the stringent response mediated by spoT could effectively regulate the swimming ability,high temperature tolerance and acid stress tolerance of SN15-2.Analysis by HPLC and RT-PCR revealed the regulatory mechanism between stringent reaction,biofilm formation and quorum sensing in SN15-2.It enriches the theory of rigorous response regulation of biofilm formation and quorum sensing signaling molecule synthesis,and lays a theoretical foundation for the study of stress resistance mechanism of P.protegens SN15-2 and the application of strain. |
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