Functional Divergence And Regulation Mechanism Of Duplicate GroELs Of Myxococcus Xanthus DK1622

GroEL,which has typical characteristic of Hsp60family, belongs to chaperonin family of molecular chaperones. and is named heat shock protein. GroEL takes part in many important cellular bioactivities widely, helping immature or damaged protein to depolymerize and refold. groELs can express greatly to help cells resist poor environment like heat shock or salt stress. According to the biological analyze, nearly one-fifth of sequencing bacterial genome harbors two or more copies of groELs These copies derive from horizontal gene transfer or gene duplication, and are speculated to be related with differentiation of gene functions. The typical structure of GroELs comprises two back-to-back nng structures and one lid-like GroES. GroEL is a14-mer, and each ring is made up of7same subunits. Changing from a hydrophobic cavity to a hydrophilic one. GroEL and GroES create an environment which is beneficial to polypeptides folding, in order to help proteins complete folding themselves.There are two copies of groELs in Myxococcus xanthns DK1622:groELl comprised groEL（MXAN₄895） and a cofactor groES （MXAN₄894） in the front; but the other one. gruEL2（MXAN₄467）. had lost the groES. The gene sequence identity of these two copies is81.36%, and the amino acid sequence identity of them is78.94%. The high homology of these two genes implied that while their functions were different to a certain extent, their partial functions might have something in common. According to the early studies in laboratory, the expression of both groEL1and groEL2would increase when cells were under adverse situation like heat shock, and both these two genes had a great influence on the development and predation of M.xanthus DK1622. The deletion of groEL1led to developmental deficiency of M. xanthus DK1622. This mutant strain couldn’t form mature black spherical fruiting body, and its ratio of spore germination decreased to4%-7%of the wild type. But the group predation behavior of this mutant strain was very similar with wild type. However, the knock-out of gene groEL2resulted in a severe delay of predation behavior. During predation. both the wild type and the groEL1knock-out mutant strain formed a visible degradation regions in12hours, while the groEL2knock-out mutant strain wasn’t able to form such a degradation regions until48hours later. Besides, in macromolecular feeding behavior, the damage of liquid predation ability of groEL2knock-out mutant strain （the generation time of mutant strain delays for about one hour compared with the wild type） had no effect upon the development of the strain（Li el al.,2010）.This study based on the different effects of groELs on the social behavior of M. xanihus DK.1622and the function of groEL, as a molecular chaperone, which helped protein to fold By using the method of co-immunoprecipitation of YL0301（AgroEL1） and YLO3O2（AgroEL2） during the stage of development and macromolecular feeding, we found out the substrate differences between GroEL1and GroEL2. in order to certify the reason of GroELs functional differentiation. In addition, we had also probed the regulatory mechanisms between GroELs and HrcA-CIRCE? and we had a preliminary explanation of the acting mechanism of groEL.The co-immunoprecipitation substrates of the two copies of GroELs had both similarities and differences to a certain extent. Both two copies’ substrates participated in the most essential metabolic activities in cells, like pyruvate metabolism, tncarboxylic acid cycle, redox,and activities related to genetic information. The most important respect was in the differences of social behavior, such as matrix-associated zinc metalloprotease FibA; which was reported as a protein related to development and movement of myxobactena. and it was the same substrate of GroEL1and GroEL2. Besides, the methyiation of the specific substrate of GroEL1, frizzy aggregation protein FrzCD, had a close link with development process. It impacted cell motion, and prohibited formation of cellular aggregation （fruiting body）. As for the specific substrate of GroEL2. for instance, putative adventurous gliding motility protein, putative chemotaxis MotB protein, effected cell movements, and some were secondary metabolite relevant proteins, like non-ribosomal peptide synthase/polyketide synthase Tal. which was related to predation, as reported. All of these results indicated that GroELs played a great role in the growth of M. xanthus DK1622. and thev also have functional differentiation.We had found out the transcriptional start sites of groEL12, by using fluorescence labeling primers to amplify promoter area fragment of groEL12and primer extension experiment. We also assayed that in heat shock condition （42℃）. there was another transcriptional start site in front of the promoter area of groEI.2. This discovery was the most direct proof which indicated that the expression level of groEL1and groEL2increased in a heat shock situation.In the study of regulatory mechanism of groEL,through bioinformatics analyze, we found two CIRCE elements in the promoter area of groKL1and one CIRCE element in groEL2. We expressed the myxobacterial HrcA protein in Escherichia coli. got enough soluble protein, and obtained electrophoretically pure HrcA protein bv FPL.C purification. Band-shift experiment indicated that the promoter area of both groEL1and groEL2could combine with HrcA protein, but neither of these two combinations inhibited the fragments harboring CIRCE element utterly.