Study On The Strategy Of Thermoactinospora Rubra YIM 77501~T For Utilizing Cellulose At Different Temperatures And Its Thermal Adoptation

Temperature as one of the most important environmental factors,can affect life processes of microorganisms,including utilizing cellulose as a carbon source from microenvironment.However,the potential strategy of carbon source acquisition(such as cellulose)at different temperatures remains unclear for microorganisms.Thermoactinospora rubra YIM 775011 is an aerobic,Gram-positive,spore-forming and cellulose degrading thermophilic actinomycete isolated from a sandy soil sample of a volcano.Its growth temperature range is 28 to 60?.The genomic sequence of this strain revealed that there are 27 cellulase genes belonging to six glycoside hydrolase families.To understand the strategy that this strain uses to utilize carbon sources such as cellulose at different temperatures,comparative transcriptomics analysis of T.rubra YIM 77501T was performed by growing it with cellulose(CMC)and without cellulose(replaced with glucose)at 30?,40? and 50?,respectively.Transcriptomic analyses showed four cellulase genes(TrBG2,TrBG3,TrBG4 and ThrCel6B)were up-regulated at 30?,40? and 50?.The rate of gene expression of TrBG2,TrBG3,TrBG4 and ThrCel6B were 50?>30?>40?.One cellulase gene(TrBG1)and two cellulase genes(TrBG5 and ThrCel6A)were up-regulated only at 30? and 50?,respectively.These up-regulated cellulase genes were cloned and expressed in Escherichia coli.The enzymatic properties of up-regulated cellulases showed a variety of responses to temperature.Special up-regulated cellulases TrBG1 and ThrCel6A displayed temperature acclimation for each growth condition.These expression patterns revealed that a hybrid strategy was used by T.rubra to utilize carbon sources at different temperatures.Under the high temperature,heat shock protein DnaK is an important way for strain T.rubra YIM77501T to response the high temperature.Namely,strains can up-express some molecular chaperones(e.g.,DnaK,GrpE,DnaJI and ClpB),like DnaK molecular chaperone systems,to prevent the protein denaturation and to repair damaged protein after heat shock.The survival rate of Escherichia coli BL21 DE3 was enhanced after expression of genes DnaK and N-DnaK(N-terminal part of DnaK)in E.coli BL21 DE3 under the stress of high temperature.This protective ability of N-DnaK is significantly higher than DnaK.The protective ability of N-DnaK for strain E.coli BL21 DE3,is no significant correlation with enzyme activity of ATPase.It means the protective mechanism of N-DnaK for E.coli is quite different from DnaK.Base on study of N-DnaK mutants,we found that the protective ability of N-DnaK for E.coli was affected by the change of its structure flexible.This study provides genomic,transcriptomics and experimental data useful for understanding how microorganisms respond to environmental changes and their application in enhancing cellulose hydrolysis for animal feed and bioenergy production.The protective ability of N-DnaK is good for us to understanding the biological mechanism of thermal adoptation.These given us enlightenment in terms of engineering resistance of strains,culturing resistant animals and plants.