The Regulatory Effects Of RpoN On Salmonella Typhimurium Biofilm Formation

Salmonella can infect many hosts and cause septicemia,gastroenteritis,and diarrhea.It can also cause human disease through food poisoning.Foodborne diseases in general and salmonellosis in particular,are one of the most serious health problems affecting public health and development.Salmonella alternate between two distinct modes of growth:as free-living cells or as members of surface-attached and exopolymer-embedded communities known as biofilms in nature.Salmonella form a biofilm on an abiotic or living surface,with high resistance to disinfectants,environmental stresses,antibiotics and the host immune system,consequently promoting bacterial dispersal and survival and enhancing its virulence.Thus,biofilm formation is associated with the outbreak of salmonellosis and persistent infections in patients.In order to better control salmonellosis in humans and animals,it is necessary to clarify the regulatory mechanisms of Salmonella biofilm formation.Two S.Typhimurium strains with strong biofilm-formation ability were selected for construction of deletion mutants with deficiency of gene rpoN using the Red recombination system.The biofilm-forming ability,and biofilm components of the wild-type,rpoN mutant and rpoN complemented strains were compared.The rpoN gene was identified as one of the associated sigma factors in biofilm formation in S.Typhimurium,A quantitative Real-time PCR method was established to compare the expression of downstream genes between wild-type and rpoN mutant strains during the process of biofilm formation.Significantly up-regulated genes of transcription were deleted respectively in both wild-type and rpoN mutant strains,and then their biofilm-forming abilities were determined by crystal violet assay.This study may help to elucidate the network of rpoN gene regulates S.Typhimurium biofilm formation.1.Construction and biological characterization of rpoN gene deletion mutants of Salmonella TyphimuriumTwo S.Typhimurium strains with strong biofilm-formation ability were selected for construction of deletion mutants with deficiency of gene rpoN by using the Red recombination system,and complemented strain was constructed by using prokaryotic expression Vector.The biofilm-forming ability,and resistance to environmental stress of the wild-type,rpoN mutant,and rpoN complemented strains were compared.A quantitative Real-time PCR method based on csgA and bcsA genes was established to compare the expression of their biofilm components.The results showed that the biofilm formation was enhanced significantly in the rpoN gene deletion mutations when compared with the wild-type strains,which was mainly contributed by increased expression of curli.The biofilm formation of revertants was similar to that of the wild-type strains.Also,deletion of rpoN gene of S.Typhimurium strains resulted in their increased resistance to acid and alkali environment.The rpoN gene was identified as one of the associated sigma factors in biofilm formation in S.Typhiimurium.2.The regulatory effects of RpoN on the downstream genes during the biofilm formation of Salmonella TyphimuriumReverse transcription real-time PCR was used to quantify the level of expression of csgD,csgA,bcsA,adrA,gcpA,lpfE,and hfq genes.The real-time PCR data were represented relative to a housekeeping gene,and gyrB gene was used as the housekeeping gene in this study.Once rpoN gene was deleted,the expression of csgD and csgA in the rpoN mutant increased significantly at 4,8,and 24 h during the process of biofilm formation,and the expression of gcpA,fimA,lpfE,and hfq were found significantly increased at 8 h of biofilm formation.However,no significant change was found in expression of bcsA and adrA.The genes adrA,gcpA,fimA,and lpfE were then deleted by using the Red recombination system in both wild-type and rpoN mutant strains,respectively.Crystal violet assay was used to determine all the mutants’ biofilm-forming abilities.The results showed that knocking out the adrA gene in wild type strain impaired the ability of bacteria to form biofilm,while losing gcpA,fimA,and lpfE gene had no apparent impact on the bacterial biofilm-forming ability.Compared to the rpoN mutants,loss of adrA or gcpA gene in rpoN mutants could damage the mutants,biofilm formation,and the biofilm formation ability of the gcpA and rpoN double deletion mutant was similar as that of the wild type strain.But deficieny of fimA or lpfE gene had little effect on biofilm formation of S.Typhimurium.Gene expression of rpoN in rpoS-deletion strain and rpoS in rpoN-deletion strain were measured by real-time PCR,no significant differences were found,suggesting that there was no obvious interaction between sigma factors RpoN and RpoS.Therefore,the rpoN gene negatively may regulate the biofilm formation by gcpA gene.