Cytoplasmic Proteins And Protein Complexes Related With Heat-regulation In Escherichia Coli K-12

The mechanism of bacteria's thermal adaptation,which is one of the most important parts on the evolution of bacteria and its adaptation to various environments, has been a research focus for a long time.Previous researches were mainly on the heat shock response.But it's still unclear that how bacteria can sustain its adaptation to heat stress after heat shock response.Herein,by the functional proteomics technology we studied the cytoplasmic proteins of E.coli K-12 and its complexes which played regulatory roles after heat shock response.We aimed to elucidate the mechanism of thermal adaptation and the protein regulatory networks under heat stress in bacteria.The cytoplasmic proteins of E.coli K-12 cultured in different temperature were analyzed by proteomics methods based on 2-D native/SDS-PAGE and MALDI-TOF. Six differentially expressed proteins(GroL,ClpB,DnaK,Mdh,MalE and LysU) were identified.And after preparing polyclonal antibodies to these proteins by genetic engineering technology,western blotting was performed to validate the differential expression.The protein complexes of the heat-regulation proteins were investigated by 2-D native/SDS-PAGE,MALDI-TOF and the searching of databases.And the interactions among GroL-Tig-RpsA,DnaK-AspS,GapA-AnsB were verified by His-pull down method.Then the effect of six heat-regulation proteins on the normal growth and the growth under heat stress was evaluated by the use of gene knock-out strains and gene over-expression strains.The protein expression profiles of gene knock-out strains cultured in different temperature were analyzed to study the protein regulatory networks.And thirty-six differentially expressed proteins including the six above were identified.All the differential proteins were parts of the components of the heat regulation networks in bacteria and they played important roles during the process of many significant physiological activities.By searching the IntAct database,we found most of these proteins not only associated in the function,but also interacted in the physiology.The results of this study can not only facilitate us have a better understanding to the mechanism of thermal adaptation and the protein regulatory networks in bacteria but also provide valuable references for the research of bacteria's adaptation to other stress.