Identification And Functional Studies Of 5-Methylcytosine-Binding Proteins In Escherichia Coli

5-methylcytosine(5mC)is an important epigenetic modification,and its binding proteins play significant roles in various biological processes such as regulation of gene expression,chromosome remodeling and maintenance of epigenetic memory in eukaryotes.However,little is known about the 5mC-binding proteins in prokaryotes.To understand the 5mC-binding proteins and their biological functions in prokaryotes,we used Escherichia coli(E.coli)as the research model to identify the potential 5mCbinding proteins by dimethyl labeling coupled with mass spectrometry.The main contents and results of the thesis are summarized as follows:(1)The potential 5mC-binding proteins in E.coli were identified by stableisotope dimethyl labeling-based quantitative proteomics method.First,the potential 5mC-binding proteins were enriched on the 5mC-containing double-stranded DNA probe.Then,the trypsin-digested peptides were lightly or heavily labeled with dimethyl labeling reagents,and the mass difference was used for the detection and analysis of the peptide samples with mass spectrometry.Finally,we identified robA(MDR efflux pump AcrAB transcriptional activator RobA)and hupA(DNA-binding protein HU-alpha)as the potential binding proteins for 5mC.(2)The binding relationships of 5mC and its potential binding proteins(robA and hupA)were confirmed in vitro.Firstly,the recombinant plasmids for protein expression were constructed and transferred into competent E.coli cells for overexpression.Next,we performed a Western blot combined with DNA pull-down assay and found that the robA protein can directly interact with 5mC in DNA,while the hupA protein can’t directly bind to the 5mC-bearing probe.(3)The robA gene knockout strain was constructed and the effect of the robA on DNA transcription was investigated.First,we used the single-plasmid strategy system and the Golden Gate assembly to construct a plasmid containing the guide DNA and donor DNA of robA gene,which was transferred into competent E.coli AB1157 to initiate genome editing.We obtained a knockout strain of robA(ΔrobA strain)by sequencing analysis of single-clonal strains,and we also verified the successful construction of ΔrobA strain by real-time quantitative reverse transcription PCR(qRT-PCR)and n-butanol tolerance experiments.Finally,we further studied the mRNA transcription levels of 5mC motif-containing genes in ΔrobA strain by using qRT-PCR technology combined with the analysis of previously published transcriptome sequencing data.The results uncovered that depletion of robA can significantly affect the expression levels of these genes.In summary,we have identified the E.coli protein robA as a novel 5mC-binding protein by using dimethyl labeling-based quantitative proteomics approach combined with in vitro DNA-protein interaction assay.In addition,we revealed that the robA protein may play an important role in regulation of expression of 5mC motifcontaining genes by multiple methods,including CRISPR/Cas9 genome editing technology,qRT-PCR analysis of gene expression as well as re-analysis of previously published transcriptome-wide sequencing data.These findings would be helpful for a better understanding of the biological effects of DNA methylation in E.coli cells.