The role of the Escherichia coli heat shock protein, grpE, in Escherichia coli growth and [1ambda] DNA replication.

The Escherichia coli grpE gene was first recognized as one of several host functions (termed groP or grp) required by bacteriophage X for replication of its DNA. Through a combination of genetic and biochemical analysis, its effects on E. coli growth and initiation of X DNA replication have been examined. The E. coli grpE2S0 mutant is temperature-sensitive for growth above 43°C and both RNA and DNA syntheses are affected in the mutant at the nonpermissive temperature. The grpE gene product has been identified as the heat shock protein, B25.3, whose expression is regulated at the transcriptional level by the htpR gene product, a32. It interacts with the dnaK protein, which is the bacterial analog of the eukaryotic heat shock protein Hsp 70; this complex is specifically disrupted in the presence of ATP. Attempts to delete the grpE gene in a wild type background were unsuccessful, demonstrating that the grpE gene product is essential for E. coli growth at all temperatures, and not only at high temperatures. Surprisingly, the deletion was viable at 30°C in a strain with a dnaK' mutation. This particular dnaK' strain was shown to have extragenic suppressor(s) which allow loss of both dnaK and grpE functions, suggesting that the two proteins function in the same pathway. To study the interaction between the grpE and dnaK proteins in greater detail, wild type grpE and mutant grpE280 proteins were purified. Wild type grpE and dnaK form a complex which is stable in glycerol. In contrast, under similar conditions, grpE280 and dnaK do not form an isolatable complex. Unexpectedly, in the presence of glutaraldehyde, grpE280 is crosslinked to dnaK more efficiently than wild type grpE. Further experiments, however, suggest that the grpE2S0 mutation results in a more transient interaction with dnaK. The interaction also appears to be more hydrophobic in nature. Others have shown that inhibition of X DNA replication in groP- mutants occurs at the level of initiation. DnaK and grpE are required in initiation at the step of unwinding of the DNA at oriX. Addition of grpE to the purified in vitro X DNA replication assay and the Xdv DNA unwinding assay allows a ten-fold reduction in the amount of dnaK added. This reduction might be due to interaction of grpE with either dnaK and/or XP.