Screening Of High-Salt Adaptability Related Genes In Salmonella

Salmonella is one of the most important foodborne pathogens,and causes disease in human through food of animal origin,which seriously threatens public health and food safety.Salmonella can use different strategies to overcome unfavorable environments,which poses a great challenge to the prevention and control of Salmonella.High-salt treatment is a commonly used food processing method to inhibit the growth and reproduction of pathogenic bacteria.According to the surveillance data of foodborne disease outbreaks,the main cause of Salmonella contamination in China was sauce and brine meat,and the resistance of Salmonella to high salt is worthy of attention.A well-studied osmotic stress response of Salmonella is interaction of two-component EnvZ/OmpR system,pore proteins OmpC and OmpF,and compatible solutes,but the regulatory network of Salmonella high-salt adaptation system remains unclear.In this study,we assessed the salt tolerance of Salmonella strains and analyzed the similarity among strains with different salt tolerance.Using transposon mutant library and RNA-seq transcriptomic analysis,we screened the genes related to the high-salt adaptability of Salmonella.The mechanism of high-salt adaptation in Salmonella was analyzed from the aspects of cell membrane properties,sodium transportation,signaling transduction,which will contribute to the enrichment of regulatory network of high-salt adaptation in Salmonella,and lay the foundation for further research on the mechanism of high-salt adaptation of Salmonella.The main research contents and results are shown as follows:1.Assessment of salt tolerance of Salmonella strainsSalt tolerance of 110 Salmonella strains isolated from the samples collected during pig breeding and slaughtering process in Hubei Province was tested by microwell method,and 98.18%were found to grow well in LB medium containing 5.0%(w/v)of NaCl.The ST19 and ST34 strains were more tolerant to high-salt than the ST40 and ST469 strains.Salmonella strain SH16SP46 recovered from pork sample in pig slaughterhouse grew well in LB medium containing 7.5%of NaCl,and the survival test showed that the viable bacterial number decreased from 5.96×10~7CFU/mL to 1.16×10~2CFU/mL after one week in 8.5%NaCl.The results suggested that Salmonella had adaptablity to high-salt treatment.By comparing the cell membrane properties of different salt-tolerant strains,it was found that cell membrane stability was closely related to high-salt adaptation.2.Analysis of differentially expressed genes of Salmonella strain SH16SP46 under high-salt environmentRNA-seq transcriptomics was used to investigate the genetic transcription level in Salmonella strain SH16SP46 under high-salt environment,and analyze the differential expression of genes in significantly enriched pathways.The results showed that the genes related to ABC transporter system,two-component system,flagella and chemotaxis,phage shock protein,SPI-1,and glycerophospholipid metabolism were up-regulated under high-salt environment,and genes related to SPI-2,LuxS/AI-2 quorum-sensing system,lipopolysaccharide and peptidoglycan biosynthesis pathway,and biofilm formation were down-regulated.3.Screening of genes related to high-salt adaptability of Salmonella from transposon mutant libraryFrom the Mariner transposon mutant library of 13 000 mutants with Salmonella strain SH16SP46 as the parental,strains with significantly decreased salt tolerance were screened using the microwell method.A total of 12 genes related to high-salt adaptability were identified by two rounds of semi-PCR sequencing,including lsrD,proP,cpdA,cysC,wzxC,helD,thiI,mnmE,rho,nlpD,cyoB,and the putative protein ysg D,and the transcriptional level of these genes varied by time under the exposure of 8.5%NaCl.Five genes related to bacterial growth were identified,including polA,xerD,recC,rffA and yigA.4.Construction of the regulatory network for high-salt adaptation in SalmonellaInteraction network analysis of genes screened by RNA-seq and transposon mutant library using STRING database and Cytoscape revealed that TorR/TorS ranked high in the interactions network and interacted closely with other genes,and it was hypothesized that the two-component TorR/TorS system plays an important role in high-salt adaption of Salmonella.Combined with the changes of cell membrane properties of Salmonella under high-salt environment,the screened genes and pathways from RNA-seq and transposon mutant library,we constructed the regulatory network of high-salt adaptation of Salmonella.Our results suggested that Salmonella might tolerant to high-salt environment through the interaction of genes related to two-component system,stress response,transcriptional regulators,amino acid synthesis and transport,motility and chemotaxis,and cell membrane composition,which comprehensively enriches the network of high-salt adaption mechanism in Salmonella.This study will provide a new insight to systematacially disclose the molecular mechanism of Salmonella tolerance to high-salt environment at the gene level.