| GroEL belongs to the chaperonin family of molecular chaperone. GroEL is named heat shock protein for either the structure or the function of which is almost identical with Hsp60 which is ubiquitously distributed in eukaryote. The function of GroEL is versatile in the cell by assisting folding, assembly, transport and degradation of many different kind of proteins. In E.coli, about 10%-15% of the total protein in the cell are dependent on GroEL to fold properly. There are three major characteristics of GroEL:firstly, the sequence of GroEL is conserved. GroEL is widely distributed in prokaryote, eukaryote and archaea, the sequence of them are highly conserved at lower taxonomic levels and the sequence similarity between different genuses is relatively high. For example, at amino acid level, the similarity of GroEL between E.coli, chlamydia pneumoniae, mycobacterium tuberculosis and other bacteria is higher than 95%, even the similarity of HSP60 of human and GroEL of bacteria is higher than 55%. This has allowed their use in phylogenetic analysis of the evolutionary relationships of eubacterial lineages. Secondly, multi-copied groELs gene in a genome are common. In the genomes of bacteria which had been sequence, as more as 20% of which possess more than one copy of groELs. The characteristic of multi-copy of groEL derived from either duplication or horizontal gene transfer, leading to the divergence of the function of GroEL which is the third trait of GroEL. The products of duplicated groEL genes were reported to play divergent roles in some different bacteria. For example, in Mycobacterium which possesses duplicated groELs, GroEL2 protein was essential for cell growth, while inactivating the groELl gene did not affect the growth of cells, but prevented the formation of mature biofilms and the biosynthesis of mycolic acid. Sinorhizobium meliloti has as many as five copies of groEL gene in its genome, but only the product encoded by groELl is the house-keeping chaperonin. It seems that the divergent function of GroEL is somewhat species-specific.GroEL has a barrel like structure which is composed of two stack-to-stack rings, and each of the ring comprise seven identical 60 kDa subunits. The chamber encapsulated by the fourteen subunits is the "machining center" for the substrate protein of GroEL. GroES is the co-chaperone of GroEL which is composed of seven homo-subunits and form a dome like structure. In the cycle of folding, GroES just likes a lid which encloses the barrel of GroEL. The binding of GroES to GroEL not only enlarges the cavity for folding of substrate protein, but also triggers a series of allosteric effect which eventually lead to the release of folded protein. So in the classical mechanism of GroEL-assisted protein folding, GroES is indispensable.In most cases, groEL is arranged on with an upstream co-chaperone gene groES, forming a complete bicistronic groESL operon. In the genome which possesses multi-copied groELs, some of the groEL copy stand alone losing the accompanying groES gene. It is speculated that groEL has evolved groES-independent functional manner which lead to the losing of groES gene. In fact, there is another possibility that several groELs might share a groES to act, which has yet not been verified by experiment.Previously, we used groELs as classification scale to compensate the low resolution of 16S rDNA at species level and found that there were two copies of groELs in the genome of M. xanthus, when used to classify respectively, they would not interfered with each other, which suggested that the duplicate groELs were relatively independent during the evolution, and there were potential divergence of function of them. Further bioinformatics analysis shows that there are extensive differences between the two genes:first of all, in the composition structure, groELl is arranged on in a groESL operon, which is not the case for groEL2, and instead of groES which has been lost, the response regulation elements are distributed downstream and upstream instead. The second, there are some difference in the sequence of them. The similarity of nucleotide sequence and amino acid sequence is 78.94% and 83% respectively. The third, for the potential regulation mechanism, two conserved CIRCE(Controlling Inverted Repeat for Chaperone Expression) element are distributed in the promoter of groELl which suggest the existence of negative regulation, while for groEL2, the element has lost probably at the same time with groES, instead of which response regulation elements were evolved.In this study, we focus on the duplicate groELs of M. xanthus, by deletion and complementary inactivation of specific gene and construction of lacZ-fused report strains, the basic biological characteristics of growth and stress responses as well as the social behaviors of gliding motility, predation feeding and development were investigated. Our results revealed the function and relation ship of the two copies of groELs in the social lifestyle of M. xanthus. Furthermore, GroEL-assisted protein soluble expression system of M. xanthus and S.cellulosum 0157-2 were developed by using the combined method of compatible plasmids and tandem gene expression strategy. Myxobacteria originated HrcA protein were then selected as model substrate for GroEL-assisted protein folding assay in E.coli, which showed that duplicate groELs of M. xanthus share a groES gene to function and there is substrate specificity between the two GroELs.The two copies of groELs cooperated with each other in growth and stress responses. Either one of the duplicate groELs could be inactivated without affecting cell viability, but deleting both groELs caused cell death unless a complemented groELl or groEL2 copy was present. It is thus proved that the existence of one groEL gene is indispensable for the survival of M. xanthus, and either of the duplicate groELs one will suffice. The two genes expresses throughout the growth stage, during which the expression level of groELl is higher then groEL2 except in the late exponential stage. Results of quantitative PCR further showed that deletion of groELl would trigger the up-regulated expression of groEL2, and in the strain whose groEL2 gene were inactivated, the down-regulated expression of groELl would lead to the significant delay of growth. The above results indicate that the two copied groELs is complementary in the growth of M. xanthus. The two genes are all involve in the stress responses of cold, heat, alcohol, NaCl and osmotic pressure, during which both of the genes are up-regulated, though at different levels. Under heat shock conditions, the groELl gene was essential for cell survival, while mutating groEL2 caused cells to be more sensitive to higher temperatures than the wild type strain.The function divergence of groELs are present in the social behaviors of M. xanthus. For development, the groELl deletion mutant disorderly aggregated and was seriously defected in fruiting body development Conversely, the groEL2 deletion mutant had a similar phenotype of fruiting body structures to the wild type strain. The expression level of groELl was higher than groEL2 throughout the development stage, forming two expression summits, while the expression level of groEL2 is relatively stable. Over-expression of groEL2 in the groELl-deleted strain could partially reverse the development defect but the development of the over-expression strain were not so well as wild type strain, which suggested that the difference of expression level of groELs are just one possible reason for their divergent function. For motility, deletion of groELl reduced the A motility ability of M. xanthus, but both of them were not involved in the S motility. For predation feeding, deletion of groEL2 severely decreased the predation capacity of cells, both on the plate using living E. coli as substrate and in the liquid culture using casein as the sole nutriment. Altogether, the two copies of groELs are function-divergent in the social behaviors of M. xanthus, of which groELl take part in A motility and is dispensable for the development, while groEL2 is essential for predation.In the attempts of construction the GroEL-assisted protein folding system, three strategies of gene co-expression were compared. Our results showed that incompatible plasmids carrying different antibiotic resistance marker were unstable, and the tandem gene expression drived by a promoter is effective. The combined method of compatible plasmids and tandem gene expression is the best choice for co-expression of more then two genes. Using this method, we constructed the GroEL-assisted protein in vivo folding system of both of M. xanthus DK1622 and S.cellulosum 0157-2, and succeeded in the soluble expression of HrcA protein, the divalent metal ions sensitivity of which was overcame at the same time. Furthermore, taken the results of in vivo soluble expression of HrcA, in vitro MDH refolding assay, native PAGE, and the gene deletion attempts together, we verified that in the strain which possess multi-copy groELs, two groELs shares a groES to act is the case and there is substrate specificity between the two GroEL proteins.
|
PDF Full Text Request