Study Of Key Proteins Associated With Lnactivation Of Escherichia Coli By Dense Phase Carbon Dioxide

In this project, the Transcriptomics and Proteomics techniques were used to study and confirm thekey proteins of Escherichia coli during inactivation by dense phase carbon dioxide （DPCD）, providing atheoretical basis for exploring the sterilization mechanism. Firstly, a dense phase carbon dioxide-resistant mutant of Escherichia coli after rounds of DPCD domestication with gradually increasedpressure was obtained. Secondly, fatty acid composition and variations at protein expression level andgene expression level of mutant strain and parent strain before and after DPCD treatment were analyzed.Thirdly, the mutations in mutant strain were detected by high-throughput genome re-sequencing. Theresults were as follows:（1）The DPCD-tolerant mutant of Escherichia coli named M8₅was obtained from Escherichia coliMG1655after8–10cycles of exposure to DPCD when the pressure was gradually increased from8MPa to16MPa with2MPa interval. The survival rate of the mutant raised5.83magnitudes. The fattyacids of mutant strain and parent strain were analyzed by gas chromatographic. A significant increase ofpalmitic acid （C16:1）, cis-9,10-methylenehexadecanoic acid （C17:0）, oleic acid （C18:1） and linolenicacid（C18：3）content were observed in mutant strain and the total fatty acid content was alsosignificantly increased. The DPCD resistance of mutant strain may lead to the increased content of cis-9,10-methylenehexadecanoic acid. The total proteins and outer membrane proteins were analyzed bypolyacrylamide gel electrophoresis, and several differences were found between the mutant strain andparent strain.（2）The proteome of the mutant and parent strains were investigated by two-dimensionalelectrophoresis （2-DE） and isobaric tags for relative and absolute quantitation （iTRAQ） techniques. Theproteome of mutant and parent strains was separated by2-DE. Six proteins were obtained which wereexpressed in3.5-fold difference. Among these, DNA binding protein H-NS, manganese superoxidedismutase （MnSOD）, outer membrane protein F （OmpF） were up-regulated which were associated withthe increased DPCD-tolerance. The protein expression was detected using iTRAQ labeling technique. Atotal of420differentially expressed proteins were identified which significantly enriched in38KEGGpathways, such as ribosome, citric cycle, two-component system, oxidative phosphorylation andnucleotide excision repair. The analysis concluded that outer membrane protein Slp,gamma-aminobutyrate antiporter, phosphoenolpyruvate carboxylase and the differentially expressedproteins involved in ribosome and oxidative phosphorylation pathway, proteins involved in response toDNA damage and repair, response to heat stress played an important role in protecting the cell againstdifferent environment stress.（3）The gene expression before and after DPCD treatment were analyzed by transcriptome sequencingbetween mutant and parent strains. A total number of141differentially expressed genes （greater than2-fold difference） were detected which significantly enriched in flagellar assembly, bacterial chemotaxis,histidine metabolism, two-component system, alanine, aspartate and glutamate metabolism. The analysis found that the ibpA gene which coding a small heat shock protein, the asr gene which codingan acid shock protein and the genes involved in histidine metabolism all may be associated with theDPCD resistance.（4） The whole genome re-sequencing was used to test the mutant strain. The results were verified bychain termination method. Three small indels and10SNPs were detected. Among these mutations,1small indel and7SNPs located in exon regions, the relevant genes include ylbE （coding a putativeprotein）, fhuA （coding a outer membrane protein receptor for ferrichrome）, ybhJ（coding a putativehydratase）, mntP（coding a putative Mn（2+） efflux pump）, rpsD（coding30S ribosomal subunit proteinS4）, rpsL（coding30S ribosomal subunit protein S12）. The analysis concluded that fhuA, mntP, rpsD,rpsL may be associated with the DPCD resistance.