Identification And Validation Of Acetylation Proteins In Salmonella Typhimurium And Characrterization Of The Role Of Acetylation On Lrp(Leucine-responsive Protein)

Post-translational modifications include phosphorylation,glycosylation,ubiqu itination,acetylation,etc.Lysine acetylation is an important post-translational m odification.The presence of acetyl groups on histones was first discovered in 1964.Since then,more and more acetylated proteins were identified in both eu karyotes and prokaryotes.Dynamic K-acetylation is important for gene regulatio n,metabolism and other cell processes.Research of acetylation in prokaryotes mainly focused on E.coli and Salm onella.In the two strains,a lot of acetylated proteins were identified by immu noblotting with monoclonal anti-AcK antibodies to locate acetylated protein ban ds combined with mass spectrometry(MS).In salmonella,CobB and Pat are r esponsible for deacetylation and acetylation respectively.In addition,AcP could chemically acetylate lysine residues.But the substrates of AcP are not specific ity.Therefore,we identified proteins interacted with CobB and Pat by bacteria two-hybrid system,to discover more acetylated proteins.In this study,we firstly constructed Salmonella enerica serovar Typhimuriu m's genome DNA library.In this library,we used bacteria two-hybrid system to identified proteins interact with CobB and Pat.Western blot was used to det ect the acetylation level and regulation by Pat/CobB/AcP.Our results showed 12 proteins could interact with CobB or Pat.These fi ve proteins(Lrp?NrdF?RhaR?1074?FliT)could be detected acetylation exce pt FliT.Interestingl-y,these four proteins showed different regulated mechanism by Pat/CobB/AcP.Lrp(leucine-responsive protein)is a global transcription regu lator in Salmonella and it could positive and negative regulate a number of ge ne and operon expression including Amino acid metabolism and transport,fimb riae synthesis and Salmonella virulence,etc.To further study the acetylation function of Lrp.Because K36 locate HTH domain ofLrp N-terminus,so we mutant K36 to Q(minic acetylation lysine)and to R(minic deacetylation lysine).EMSA was used to detect ability of mut ants bind to fimZ's promoter.Complement Lrp WT and Lrp(K36Q)in ?lrp a nd detect the downstream gene expression by qPCR.EMSA results showed tha t ability of Lrp(K36Q)bind to fimZ's promoter was decrease.Complement exp eriment showed that Lrp(K36Q)repress downstream gene's expression.In summary,we identified 12 proteins interact with Pat or CobB.Acetylati on of Lrp K36 repress Lrp bind to fimZ's promoter and then repress downstrea m gene's expression.