Functional Analysis Of Heat Shock Proteins Hsp33 And Hsp40 And Transcriptional Regulator RpoS Gene In Pseudomonas Protegens SN15-2

Pseudomonas protegens SN15-2,a highly efficient biocontrol bacterium isolated from tomato roots,can colonize the plant rhizosphere and inhibit the plant pathogens by producing secondary metabolites such as antibiotics and siderophores and inducing plant systemic resistance,which has promising applications in sustainable agriculture.As a living microorganism,P.protegens is susceptible to various environmental factors such as temperature,pH,osmotic pressure and oxidative stress during preparation processing and field application.These factors lead to a decrease in the number of viable bacteria and thus affect the performance of the maximum biocontrol effect.Heat shock proteins are a class of highly conserved proteins that are closely related to bacterial stress resistance and maintain normal cellular homeostasis.The transcriptional regulator RpoS can regulate the production of secondary metabolites in bacteria stable phase and plays an important role in protecting the strain from external harsh environmental stresses.In this paper,the heat shock proteins Hsp33 and Hsp40 and the transcriptional regulator RpoS in potential biocontrol agent P.protegens SN15-2 were investigated for their functions in the mechanisms of biological control and stress resistance.First,three related gene mutants,ΔhslO,ΔdnaJ and ΔrpoS,were constructed by suicide plasmids and double-swap homologous recombination and their effects on the phenotypes related to the function of P.protegens SN152 were characterized.The results showed that the growth rate of the hslO gene deletion strain was significantly lower than that of the wild-type strain in the logarithmic phase,while the deletion of the dnaJ and rpoS genes had no significant effect on growth,indicating that the hslO gene could regulate the growth of SN15-2.In terms of biofilm formation ability,the wild type at 6 h and 24 h of incubation was 1.5-fold and 1.31-fold higher than the mutant ΔrpoS with significant differences(P<0.01,below),while the mutant ΔhslO and ΔdnaJ were not significantly different,indicating a positive regulation of biofilm formation in SN15-2 by the rpoS gene.The deletion of the hslO gene reduced the production of siderophore by SN15-2,while the siderophore-forming ability of the mutant strain ΔhslO and ΔdnaJ did not change significantly compared with the wild strain;the deletion of the dnaJ gene weakened the motility of the strain,while the deletion of the rpoS gene enhanced the motility of the strain,indicating that dnaJ and rpoS positively and inversely regulated the motility of SN15-2,respectively;Separate deletion of hslO,dnaJ and rpoS genes did not affect the antagonistic effect of SN152 against Ralstonia solanacearum.Secondly,the roles of and rpoS genes in P.protegens SN15-2 in response to adversity were analyzed.The results showed that the survival rate of the wild strain under high temperature stress at 47℃ and 49℃ for 30 min was 1.84 and 21.27 times that of ΔhslO;the survival rate of the wild strain reached 8.57 and 2.39 times that of the mutant ΔrpoS after freezing treatment(-20℃)and H2O2(30 mM)oxidation treatment for 60 min,respectively;the survival rate of the wild strain under hypertonic stress(500 mM NaCI)and acid The survival rates of ΔhslO,ΔdnaJ and ΔrpoS under hypertonic stress(500 mM NaCI)and acid stress(pH=5.0)were not significantly different from those of the wild strain.It was observed that the deletion of hslO gene led to the reduced tolerance of P.protegens SN15-2 to high temperature and the deletion of rpoS gene led to the reduced tolerance of P.protegens SN15-2 to low temperature and oxidative stress.Finally,the roles of hslO,dnaJ and rpoS genes in the synthesis of antibiotic 2,4-DAPG and Quorum sensing molecules(QSMs)were analyzed by high performance liquid chromatography HPLC and qRT-PCR.The results showed that the production and synthesis-related gene expression of 2,4-DAPG and signaling molecule C8HSL were significantly lower in the Δ rpoS mutant strain than wild-type strain SN15-2,while the deletion of hslO and dnaJ genes had no significant effect on the biosynthesis of antibiotic 2,4-DAPG and QSMs.Therefore,rpoS may positively regulate the expression of antibiotic 2,4DAPG and QSMs synthesis gene clusters in P.protegens SN15-2.In conclusion,the hslO gene in P.protegens SN15-2 regulates the ability to form iron phagocytosis and resistance to high temperature;the heat shock protein gene dnaJ can affect the motility of the strain;the global regulator rpoS not only regulates motility,biofilm formation ability and tolerance to adverse environments such as low temperature and oxidation,but also the antibiotic 2,4-DAPG and the synthesis of population-sensing signaling molecules.This paper analyzes the potential regulatory mechanisms of stress genes such as hslO,dnaJ and rpoS,which are important for improving the biocontrol ability and stress resistance of P.protegens SN15-2.