Effects Of BcsG And PTS Genes On Cronobacter Spp. Biofilm

Cronobacter is a kind of food-borne pathogenic bacteria that seriously harms the life and health of infants and young children.Infection of Cronobacter can lead to necrotizing enterocolitis,bacteremia and meningitis.Due to the threat to infants,the elderly and other specific populations,the prevention and treatment of Cronobacter infection is very important.The formation of Cronobacter biofilm can resistant to fungicides and other agents.It is difficult to remove Cronobacter biofilm completely,which resulting in increased harm.In order to understand the mechanism of the formation of Cronobacter biofilm and reduce the risk of infection in infants and potential population,Cronobacter dublinense DSM 18707 was selected as the experimental object,and the key biofilm forming gene bcsG was screened out in this study.The changes of biofilm forming ability,extracellular polysaccharide and membrane-related gene expression level of the bcsG gene deletion strain were detected.Furthermore,UOO＿RS0109670(PTS)gene was screened out by transcriptome analysis in the knockout experiment,to explore the influence of PTS gene on the biofilm forming ability of Cronobacter,and the regulation of PTS gene on bcsG gene.The main research contents and conclusions are as follows:1.Effect of bcsG gene on biofilm formation of Cronobacter(1)DSM 18707 with the strongest biofilm-forming ability was screened out from 58 strains of Cronobacter.The effects of culture time,temperature and medium on biofilm formation ability,extracellular polymer content and gene expression levels of bcs A,bcs B,bcs C and bcsG were detected.The results showed that Cronobacter had the strongest biofilm formation ability when cultured at 37℃ for 3days in skim milk powder medium.The expression level of bcsG gene was basically consistent with the biofilm-forming ability of Cronobacter and the content of extracellular polysaccharide(EPS),which suggested that bcsG gene may be related to the formation of EPS and biofilm.(2)The bcsG gene knockout strain(△bcsG)was constructed by homologous recombination method.Firstly,the target sequence was constructed.Secondly,the successfully constructed target sequence was transferred into Escherichia coli and conjugated with Cronobacter DSM 18707.PCR identification and sequencing showed that the bcsG gene deletion strain was successfully constructed.On the basis of that,the bcsG gene complement strain was constructed.The bcsG gene was cloned into plasmid PRK415 to obtain the complement plasmid,and the complement plasmid was transferred into competent cells with bcsG gene deletion by the method of electric transformation.Combined with PCR and sequencing technology,the complement strain was successfully constructed.(3)The biofilm formation ability of △bcsG and the expression levels of membrane related genes were detected.The results showed that the biofilm formation ability of △bcsG and the content of EPS was decreased significantly.The decreased tendency indicated that bcsG gene played a role in regulating the biofilm formation of Cronobacter by promoting the synthesis of EPS.(4)After heat,drying and acid treatment,the morphology of △bcsG strain was observed by fluorescence microscopy and scanning electron microscopy(SEM),respectively.The results showed that the number of damaged cells increased with the increase of temperature,the prolonging of drying time and the decrease of p H.Besieds,flow cytometry was used to quantitatively detect the proportion of damaged and dead cells.The results showed that △bcsG was significantly less resistant to heat,dryness and acid than WT.2 Effect of PTS gene on biofilm formation of Cronobacter(1)Biofilm RNA(DSM＿M)was extracted from DSM 18707 cultured for 3days and free bacteria(DSM＿S)RNA was extracted from overnight culture.Transcriptome analysis was performed with DSM＿M as the experimental group and DSM＿S as the control group.Through differential gene expression,GO and KEGG enrichment analysis,our results showed that PTS gene expression may be related to the formation of Cronobacter biofilm.(2)Gene knockout and complement were used to construct △PTS and cp PTS strains.PCR identification and sequencing showed that the knockout and complement strains were successfully constructed.On this basis,the influence of PTS gene on the biofilm formation ability of DSM 1870 was further explored.The results showed that the PTS gene promoted the synthesis of EPS by regulating the expression of cellulose synthesis gene bcsG,and thus made Cronobacter form more biofilm.(3)Fluorescence microscopy,SEM and flow cytometry were used to detect the effects of heat,dryness and p H on PTS gene knockout Cronobacter.The experimental results showed that the resistance of heat,dryness and acid of △PTS strain decreased significantly.