The Effect Of Heat Shock Protein DnaK And DnaJ On The Escherichia Coli Cell Cycle

Fine regulation of the cell cycle is very important for life activities of the cell,but the effect of heat shock proteins on the cell cycle has not been studied throughly.Therefore,the effect of heat shock protein DnaK and DnaJ on the cell cycle was investigated in the study.The cell cycle parameters of AdnaK and AdnaJ cells were measured.It was found that deletion of the dnaK gene caused a delay in initiation of replication while absence of the dnaJ gene led to an early initiation of replication.In addition,the cell size and growth rate of the ?dnaK and ?dnaJ cells were different from that of wild-type cells.Epitopic expression of DnaK or DnaJ in AdnaK or AdnaJ cells can restore defects found in AdnaK or?dnaJ cells.Overexpression of DnaK or DnaJ in the wild-type cells led to the opposite trends in parameters of the cell cycle relative to that of the?dnaK or ?dnaJ mutant.The total protein amount and the DnaA amount per cell were found to be lower in the ?dnaK cell than those in the wild-type cell.The low amount of total protein and DnaA in the?dnaK was restored by epitopic expression of DnaK.However,overexpression of DnaK in the wild type cell increased the amount of total protein and DnaA per cell.The amount of total protein and DnaA in the AdnaJ cell was not changed by overexpression of DnaJ compared with the wild-type cell.Those results suggest that DnaK may affect the initiation of chromosome replication by affecting the amount of total protein and DnaA per cell while DnaJ may affect the replication initiation in an unknown way.The interaction between DnaK and YfiA,DnaJ and YfiA or DnaK and DnaJ was found by the bacterial two hybrid system.The YfiA protein is a ribosome hibernation factor,which cause ribosome hibernation during the stationary phase.Site-directed mutagenesis showed that lysine,histidine and arginine in nucleotide binding domain of DnaK are key amino acids for function of DnaK in regard with the interation with YfiA.Histidine and aspartic acid in the DnaK interaction domain of DnaJ are essential for interaction of DnaJ with YfiA.The incomplete replication in exponentially growing ?yfiA?dnaK or ?yfiAAdnaJ cell was found after treatment of rifampicin and cephelexin,which may be due to DNA damage or asynchronous initiation.These double mutant cells were fat and spherical in shape.It was found that DNA replication and sporulation occurred during stationary phase of the ?yfiA?dnaK or ?yfiA ?dnaJ cell.