| Staphylococcus aureus,Gram-positive,is usually symbiotic with humans or animals and is an opportunistic pathogen.It is widely distributed in nature,is a common food contamination bacteria,in many countries in the food poisoning incident reports ranked second,only after Salmonella.In the dairy industry,S.aureus can cause mastitis in cows,and milk produced by infected cows can also be contaminated with S.aureus.However,there are many adverse environments in the processing of dairy products,including high temperature,high pressure and dryness.Therefore,the study on the molecular mechanism of S.aureus stress resistance has important research value for pollution prevention and control.The Mfd protein was screened by a series of stress experiments,such as high temperature,osmotic pressure and drying.It is usually described as a DNA repair protein and a transcription repair coupling factor.There are few studies on Mfd in S.aureus.Currently,only Mfd is known to be involved in the prevention of oxidative stress,immune response and drug-induced DNA damage,as well as the reduction of Mfd expression in S.aureus can lead to reduced biofilm formation,and the specific mechanism remains to be further analyzed.This study will take this as the breakthrough point to conduct relevant experiments and studies to explore the molecular mechanism of Mfd regulating S.aureus stress resistance.In this study,we study the stress-related regulatory mechanism of S.aureus RMSA 24.Through gene backup,growth curve experiment,stress resistance experiment(high temperature experiment,acid base experiment),biofilm-related experiment(biofilm composition analysis experiment,extracellular polysaccharide experiment,protein content determination experiment,Triton X-100 autolysis experiment,hemolysis experiment),transcriptome sequencing and real-time fluorescence quantitative PCR experiment,To explore the regulation of transcription repair coupling factor Mfd on the molecular mechanism of S.aureus stress resistance.The purpose of this study is to provide a basis for the anti-stress mechanism of lactogenic S.aureus,to help prevent the contamination of dairy products caused by the bacteria,and to provide a new idea for the treatment of S.aureus infection.1.The successful construction of C-mfd complement strain was verified by PCR.Stress resistance of wild,mutant and back-up strains were tested.The results showed that the strain’s resistance to heat stress and p H tolerance were weakened after the mutation of mfd gene.2.Biofilm-related experiments were performed on the wild strains,mutant strains and complement strains.The results showed that the biofilm of RMSA 24 was mainly composed of protein.The mutation of mfd gene promoted the synthesis of PIA and e DNA,inhibited the synthesis of protein,and reduced the biofilm content.At the same time,the mfd mutation decreased the hemolytic ability of S.aureus.3.The wild type strain RMSA 24 and M-mfd mutant were sequenced by transcriptome.A total of 2649 genes were quantified by sequencing results,among which 173 gene transcripts were defined as DEGs.12 genes related to biofilm and hemolysis were selected for real-time fluorescence quantitative PCR.The results showed that the transcription level of the Ia-related gene sar X in M-mfd mutant was up-regulated by a factor of 4 compared with the wild type strain.The transcription levels of autolysis-related genes yyc I,wal K and cid A were up-regulated by 1.3 times,1.2 times and 2.6 times,respectively,while the transcription levels of lrg A were down-regulated by 2 times.Transcription levels of the protein-related gene clf A were down-regulated by a factor of two;The transcription levels of hemolysis-related genes hld,hlg B and hlg C were also down-regulated by 1.7 times,5times and 2.5 times,which were consistent with the phenotypic results. |
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