Effects Of RpoS And RpoN On Important Biological Characteristics Of Salmonella Typhimurium And Their Regulation Role

Objectives: 1.To study the role of the regulatory factor RpoS in the biofilm formation ability,toxicity,environmental tolerance and other aspects of Salmonella Typhimurium,and find the regulatory role of RpoS and RpoN in the above aspects,and support for further antimicrobial treatment of Salmonella Typhimurium.2.To compare the difference in proteome expression under stress resistance conditions,explore the pathway relationship between the two regulatory factors,rpo S and rpo N,and provide reference for studying the regulatory relationship between the σ factors family.Methord: 1.To select Salmonella Typhimurium SH15SF175 as the research object,and construct ?rpo S and ?rpo Srpo N by CRISPR,which are verified by PCR and m RNA expression level.2.To evaluated the growth of the strain under different p H,osmotic pressure,oxygen stress,low water molecular activity and drying conditions;to evaluate of biofilm forming ability by crystal violet staining,laser confocal microscopy,and Congo red medium;to evaluate the changes of virulence characteristics of the strains before and after knockout evaluated by the m RNA expression level of flagellar-related genes;to measure the mobility test and the adhesion and invasion test of Hela cells to bacteria,to compare by Biolog biochemical identification.2.The protein products of WT,SF175?rpo S and SF175?rpo Srpo N strain were identified by DIA proteomics.The differential protein functions were characterized by domain analysis and GO functional analysis and find the main differential protein pathways by KEGG pathway analysis.Results: 1.The successful construction of SF175?rpo S and SF175?rpo Srpo N were verified by PCR and m RNA expression level.2.Measurement of biofilm by crystal violet staining the experimental results showed that both SF175?rpo S and SF175?rpo Srpo N had attenuated biofilm formation ability compared to WT strain,indicating that bacterial biofilm formation ability was decreased after rpo S gene knockdown.Biofilm formation ability of SF175?rpo Srpo N was a little restored compared to SF175?rpo S at 28℃.3.Congo red assay found that the cellulose content of SF175?rpo S and SF175?rpo Srpo N was significantly decreased compared to that of WT strain at 28℃,but there was no obvious difference in morphology between SF175?rpo S and SF175?rpo Srpo N.4.The results of virulence assay showed increased adhesion rate and decreased invasion rate of SF175?rpo S and decreased adhesion rate and invasion rate of SF175?rpo Srpo N.5.Biochemical identification results showed that 10 biochemical reaction values changed,of which 8 biochemical reactions showed that the reaction values of two knockout strains were lower than those of wild strains,and 2biochemical reactions showed that the reaction values of knockout strains were higher than those of wild strains,and the biochemical reaction values of SF175?rpo Srpo N were higher than SF175?rpo S.6.DIA proteomic data showed that the expression of trehalose6-phosphate synthetase(Ots A)in SF175?rpo S and ?rpo N was significantly downregulated compared with wild strains in the face of hypertonic environment.Domain prediction analysis showed that the differential proteins were mainly in ABC transporters and responsive regulatory receptors.GO functional analysis showed that the most significant difference between SF175?rpo Srpo N and ?rpo N proteins under 28℃hypertonic condition was the biosynthesis of glutamine family amino acids.KEGG pathway analysis showed that the most significant difference between SF175?rpo S and SF175?rpo Srpo N protein pathway was arginine synthesis pathway under 28℃hypertonic condition.Conclusion: 1.The study has confirmed that the knock-out of rpo S could weaken the growth and biofilm formation ability of bacteria under acid-base,high osmotic stress,low water molecular activity and pressure of dry environment,which shows that rpo S plays an important regulatory role in response to environmental stress,biofilm formation and virulence.2.We found that rpo S and rpo N play an important regulatory role in different environmental stress,biofilm formation and virulence,which shows different regulatory directions.3.The proteomic analysis showed that rpo S and rpo N are involved in the response to hypertonic stress through ots A and hpf,and the arginine synthesis pathway may be one of the important mechanisms involved in the regulation of rpo S and rpo N to response to 28℃ hypertonic stress.