Promoter Analysis And Transcription Factors Screening Of Translation Elongation Factor P Gene In Staphylococcus Aureus

Translation elongation factor P is ubiquitous in bacteria and has a high degree of conservation,although it is not a necessary protein for bacterial survival,but it is closely related to bacterial growth metabolism,virulence and environmental stress regulation,which may provide new targets for finding new antibiotics or developing new antibacterial treatments methods.However,the current research on bacterial EF-P is mainly about its source,distribution,crystal structure,posttranslational modification,mechanism of action,function and biological significance,and this paper takes Staphylococcus aureus subsp.aureus NCTC8325 as the experimental object to study the influence of different environmental factors on EF-P expression under culture conditions,and analyze the promoter structure of efp gene.The specific contents are as follows:1.Through the Quantitative Real-time PCR experiment,the relative expression change of S.aureus efp gene under the action of high temperature,p H,antibiotics and other environmental factors was investigated.The results showed that the expression of S.aureus efp was upregulated under high temperature and acid-base environment,while under the action of antibiotics,its expression was upregulated first and then downregulated;This result also showed that the expression of efp in S.aureus was adjustable.2.Based on the prediction and analysis of the upstream sequence of the efp gene of S.aureus by bioinformatics software,the promoter probe vector was constructed,and the predicted efp promoter fragment Pro was connected with the fluorescent protein AcGFP1.The qualitative and quantitative analysis of the expression of the fluorescent protein Ac GFP1 showed that the Pro fragment had strong promoter activity.3.Three regions of the Pro sequence of the efp promoter of S.aureus were mutated,and the mutated promoter was cloned into the promoter probe vector.The expression of the fluorescent protein AcGFP1 was analyzed by fluorescence microscope,enzyme marker,Quantitative Real-time PCR experiments to determine the effect of mutations at different sites on the activity of the promoter.At the same time,combined with the analysis of bioinformatics,the sequence of the-10 element of the efp promoter is: CCTTATAGT,and the sequence of the-35 element is: TTTACT.4.Using the efp promoter sequence of S.aureus as probe,two suspected transcription factors were screened by DNA pull down assay combined with protein mass spectrometry analysis,and Sar S protein was expressed and purified by protein expression technique in vitro.The specificity of Sar S protein binding to efp promoter was investigated by gel blockade experiment in vitro,and the sequence specificity of Sar S protein binding to efp promoter was verified by unlabeled mutated competitive promoter probe reaction.The above results provide experimental basis for revealing the regulation mechanism of EF-P expression in S.aureus,and lay a foundation for further elucidating the biological characteristics of EF-P,such as its adaptability to bacterial environment,toxicity effect and stress.