Study On The Effect Of RfaC Gene Deletion On Sensitivity Of Salmonella Typhimurium To Blue Light

Salmonella is a kind of Gram-negative pathogenic bacteria widely existing in nature,and the frequent occurrence of drug-resistant pathogens requires the development of new sterilization methods.Blue light(400-470 nm)is a broad-spectrum sterilization method developed in recent years.Blue light can induce endogenous photosensitizers in bacteria to produce reactive oxygen species,and then damage intracellular biological macromolecules.Our lab previously revealed that the outer membrane is an important target.At present,the research on sterilization mechanism of blue light needs to be further deepened.Lipopolysaccharide is one of the main components of the outer membrane of Gram-negative bacteria,which has diversified structural modifications.The structure of lipopolysaccharide affects the stability of the outer membrane.Therefore,the structural modifications of lipopolysaccharide may affect the sensitivity of bacteria to blue light.Based on this,this paper constructs a mutant of lipopolysaccharide core polysaccharide,and explores its function and possible mechanism for the first time.In the present study,Salmonella Typhimurium is taken as the research object.Firstly,the inactivation effect of blue light on S.Typhimurium was explored,and the intracellular superoxide anion O₂^-,total antioxidant capacity and extracellular K⁺content of the bacteria were investigated.Secondly,by knocking out the key gene rfaC in lipopolysaccharide synthesis pathway,the influence of this gene on blue light sensitivity was studied,and the cell characteristics of rfaC gene-deleted strains were investigated.Finally,the reasons of different blue light sensitivity caused by this gene were further analyzed through the lipid groups of bacteria before and after this gene knock-out.The main conclusions were as follows:1)By measuring the inactivation curve of blue light to S.Typhimurium.Blue light irradiation can kill S.Typhimurium very well.When the irradiation dose was 164 J×cm^-2,the survival number of S.Typhimurium decreased by nearly 2 log₁₀ CFU×m L^-1,and when the irradiation dose reached 273 J×cm^-2,the survival number of bacteria decreased by 3 log₁₀CFU×m L^-1,and the blue light irradiation dose continued to increase to 383 J×cm^-2,the bacterial biomass decreased by 4.43 log₁₀ CFU×m L^-1,and the mortality rate reached 99.99.Then,through the determination of superoxide anion O₂^-and total antioxidant capacity extracellular K⁺of S.Typhimurium under sublethal blue light,it was confirmed that blue light irradiation would damage the bacterial cell membrane.In the sublethal dose,a large amount of ROS was produced in bacteria,and reactive oxygen species would attack the macromolecules of bacterial cell membrane,which was an important cause of bacterial death.2)rfaC,the key gene of lipopolysaccharide synthesis pathway in S.Typhimurium,was knocked out to construct rfaC deletion strain by using Red homologous recombination method.there was no obvious difference between the growth of original strain and deletion strain,which indicated that the deletion of this gene did not affect the growth of bacteria.it was confirmed by extraction of lipopolysaccharide from original strain and deletion strain and reactive oxygen species that rfaC gene was the key gene encoding glycosylase in lipopolysaccharide synthesis pathway.Then,through the analysis of cell surface hydrophobicity and cell membrane fatty acids by gas chromatography-mass spectrometry,the influence of lipopolysaccharide heptulose chain deletion on bacterial cell membrane was discussed.3)By measuring the inactivation curve of blue light to rfaC deletion strain,it can be seen that the original strain and rfaC mutant strain have different sensitivity to blue light.When the irradiation energy was 164 J×cm^-2,the survival rate of rfaC deletion strain was 4.5%,while the original strain,and the survival rate of replanted plants was 22.4%.When the irradiation dose reached 383 J×cm^-2,the cell logarithm of the original strain SL1344 decreased by nearly 4log₁₀ CFU×m L^-1,while the survival logarithm of the rfaC deletion strain decreased by more than 8 log₁₀ CFU×m L^-1,which was significantly higher than the former.The reason why the deletion strain is sensitive to blue light is analyzed by measuring the outer membrane permeability of the deletion strain under sublethal blue light.Before blue light irradiation,the permeability of the deletion strain is 2.1-fold that of the original strain,and during blue light irradiation,the permeability of the rfaC deletion strain at 20 J×cm^-2 is increased by 2.7-fold,so the improvement of permeability may be the main reason for the sensitivity improvement under blue light.Through the extraction of lipid from original strain and deletion strain,it was confirmed that the truncation of lipopolysaccharide caused the relative content of membrane phospholipids such as phosphatidylethanolamine,phosphatidylcholine,phosphatidylglycerol and cardiolipin in lipid to decrease,and the content of sphingomyelin also decreased.As an important target of cell membrane,the structural change of lipopolysaccharide affects the stability of cell outer membrane,and finally leads to a large increase in blue light sensitive type of bacteria.In the current work,the effect of lipopolysaccharide on sensitivity to blue light was conducted for the first time,and the mechanism of the increased sensitivity to blue light in the deletion strain was studied from the perspective of lipidomics,which is of great significance for exploring the mechanism of blue light sterilization and controlling food-borne pathogens.