Effect Of Deletion Of Trigger Factor Domain On Escherichia Coli

In Escherichia coli,ribosome-binding chaperone TF is the first chaperone encountered by the nascent peptide chains,and it plays an important role in the folding of the nascent peptide chain on the ribosome.It was found that all domains of TF have important functions.The N domain contains the sites binding to ribosomes,the C domain contains the sites binding to newborn peptide chains,and the P domain is also an indispensable active domain,which has peptide prolyl cis-trans isomerase activity and auxiliary binding sites.However,the effect of the functions of domains on the physiological status of E.coli is not clear.Therefore,in order to study the function of each domain of TF,here,we constructed the mutant strains deleting single domain of TF,and analyzed the effect of the deletion of single domain of TF on the physiological status of E.coli at the transcriptional and phenotypic levels.In this study,the Red positive and negative selection system was used to carry out gene knockout and knockin in the tig locus of wild-type Escherichia coli BW25113 strain,and TF single domain deletion mutant strains tig::nc,tig::pc,tig::np were obtained.The changes in gene expression in these mutant strains were analyzed by high-throughput sequencing technology,and the results showed that: in tig::nc strain,the levels of 96 genes were significantly changed(88 genes were up-regulated,8 genes were down-regulated);in tig::pc strain,the levels of 183 genes were significantly changed(85 genes were up-regulated,98 genes were down-regulated),the levels of142 genes were significantly altered in tig::np strains(100 genes were up-regulated and 42 genes were down-regulated).By analyzing the properties of proteins encoded by the significantly regulated genes,we found that the molecular weight of proteins encoded by the significantly changed genes accounted for the largest proportion in the range of 25-35 k D,but the distribution profiles of isoelectric point and hydrophobicity of proteins encoded by the significantly changed genes was similar to those of proteome of E.coli.KEGG annotation results showed that the genes with significant changes in expression were mainly located in metabolic pathways,transcription and translation pathways,and membrane lipid pathways.By cultivating mutant strains under different conditions,the phenotype analysis of mutant strains showed that the mutant strains lacking of single domain of TF could normal grow under the condition of 42 ? heat shock as same as wild type strain,but mutant strains was more sensitive than wild type strain to oxidative phosphorylation inhibitors DNP and dibucaine,protein synthesis inhibitors kanamycin,detergent SDS and cell wall synthesis inhibitors ampicillin.The results of this study will lay a foundation for further elucidating the functions of different domains of TF,contributing to the application of TF.