The Preliminary Study On The Properties And Functions Of The Electron-bifucating NfnAB From Clostridium Difficile

The study of intestinal microbes in recent years suggests that it plays an important role in a series of physiological processes that are critical to the health of the host,including energy balance,metabolism,intestinal epithelial health,immune activity,and neurodevelopment.The intestinal microbes is dynamic and subject to important changes during the life of the host inresponse to a variety of factors including diet,environment,medical interventions,and disease states.The intestinal environment is relatively oxygen free,which is more conducive to the growth of anaerobic bacteria.It is well-known that antibiotics have a clear and continuous inhibitory effect on the intestinal flora,which results in a decrease in the colonization resistance of the intestinal microflora against certain pathogens and make the original dominant flora in a weak state,and the balance of intestinal flora is broken.In the same time,the conditional pathogens that settled in the intestines will enter and cause various diseases,including Clostridium difficile.C.difficileis a Gram-positive,obligate anaerobic Clostridium bacillus,it is a conditional pathogen and present in the human intestinal tractnormally.When the balance of intestinal flora is broken,C.difficilewill hyperproliferates and releases large amounts of toxins,causing various intestinal diseases.In the past ten years,C.difficile infection have caused great concern.due to the high morbidity and health care costs.Hospitalized patients with advanced age and antibiotic treatment are the major risk factors for C.difficile infection.Therefore,C.difficile is also considered to be a common pathogen that causes medically relevant infections.However,so far,there are no ideal methods for the diagnosis and treatment of C.difficile infections that meet the clinical needs.Therefore,it is necessary to strengthen basic research on the physiological metabolism of C.difficile and clinical infections,and to speed up the development of C.difficile detection methods and prevention methods.We do Blast with the protein sequence and found that the C.difficile genome contains the NfnAB coding gene.Flavin-based electron bifurcationinvolved in this enzyme is a recently discovered mechanism found in Clostridia.It is considered to be the third energy conservation mode of microbial removal of substrate phosphorylation and oxidative phosphorylation.Therefore,in this study,we heterologously expressed and purified NfnAB of C.difficile,and test its biochemical reaction.The results showed that the recombinant protein NfnAB was highly expressed in E.coli C41(DE3)?.SDS-PAGE analysis showed that the obtained protein was highly pure and was composed of two subunits with the sizes as 51 kDa and 33 kDa,respectively.The purified protein presented characteristic absorption at390 nm and 430 nm,which is consistent with the idea that C.difficile NfnAB contains FAD and Fe-S cluster cofactors.The specific NAD~+-dependent TTC and Fd reduction activity with NADPH.These biochemical properties are consistent with those of the corresponding enzymes from other anaerobes,indicating that they perform the same biochemical function.Ferredoxin has been reported as a reaction substrate for all of the specific reactions catalyzed by electron-bifurcating enzymes.So far,there is no method for the preparation of Fd that is suitable for the determination of biochemical reactions,with high yield,good stability,easy accessibility,and easy preservation.Therefore,there is an urgent need to find such a method to prepare stable Fd,which was to meet the needs of the research on the capacity mechanism involved in the novel electron bifurcation mechanism.Therefore,we constructed the heterogeneous expression vector of Fd from T.maritima.We expressed and purified of Fd inE.coli C41(DE3)?,and the wavelength scanning detection was performed.The results showed that recombinant Fd was highly expressed in E.coli.Only heat treatment can remove60%of the contaminated protein.After affinity purification with Ni column,Fd with90%purity can be obtained.The results of spectroscopic scanning show that it presented characteristic absorption of[4Fe4S]cluster.The results of determination of iron content of recombinant Fd showed that the molar ratio of Fe to protein was about 4,which was consistent with the expected results.Using the heat-treated recombinant Fd as an electron acceptor,the activity of NADPH reduced Fd catalyzed by NfnAB from T.maritima at45?was approximately 4.6 U/mg.This indicates that the recombinant Fd prepared in this paper can serve as a substrate for electron bifurcation.The fourth chapter of this study analyzed the genomic information of C.difficile in further.By literature investigating and genomic analysing according to the reported genomic information of anaerobes,and the bioinformatics analysis was performed through sequence comparison.The results showed that the proteins related substances and energy metabolism in C.difficile have high consistency with anaerobic bacteria that have been reported,indicating that those proteins are highly conserved in anaerobic bacteria.Based on the above data of genomic analysing,we mapped the metabolic pathways of C.difficile under autotrophic and heterotrophic growth conditions and at least three electron dismutases were found in the entire metabolic pathway of C.difficile,containing NfnAB,Bcd/Etf and Hyd,showed that the electron bifurcation mechanism does play an important role in the metabolism of anaerobic bacteria.