DnaK mutants defective in ATPase activity are defective in negative regulation of the heat shock response: expression of mutant DnaK proteins results in filamentation

Abstract

Site-directed mutagenesis has previously been used to construct Escherichia coli dnaK mutants encoding proteins that are altered at the site of in vitro phosphorylation (J. S. McCarty and G. C. Walker, Proc. Natl. Acad. Sci. USA 88:9513-9517, 1991 Escherichia coli, in common with many organisms, responds to sudden shifts in temperature by transiently inducing members of the heat shock regulon, which consists of genes and operons that are under the transcriptional regulation of the heat shock sigma factor 32 (42). By genetic analysis, this regulon has been shown to be negatively regulated by three of these heat shock proteins, DnaK, DnaJ, and GrpE. dnaK, dnaJ, and grpE mutants have the phenotype of overproducing the proteins of the heat shock regulon (50). The primary mechanisms for this negative regulation appear to be effects on the level of synthesis, stability, and activity of c32 (19,(49)(50)(51)53

An essential role for the Escherichia coli DnaK protein in starvation-induced thermotolerance, H2O2 resistance, and reductive division

“…Complementation analysis of the ⌬dnaK52 mutant with a dnaJ ϩ plasmid did not indicate a role for DnaJ in the starvation-induced dnaK mutant phenotypes other than a modest effect on reductive division. However, the effect of DnaJ deficiency on the starvation-induced phenotypes studied here may not be as readily apparent at the reduced levels reported to be present in ⌬dnaK52 mutant strains (40). A requirement for DnaK during carbon starvation is also consistent with previous reports on the inability of an htpR mutant to develop starvation-induced thermotolerance or H 2 O 2 resistance (24).…”

“…It shares DNA sequence and certain antigenic epitopes with DnaK and may also be expressed during growth (40). However, monoclonal antibody 2G5 did not detect any proteins, such as Hsc, in the ⌬dnaK52 mutant during either growth or starvation.…”

“…Cultures were exposed to 15 mM H 2 O 2 for the times indicated and then plated for survivors. The strains and culture conditions are as indicated for panel A. promoter(s) may also be responsible for a reduction in DnaJ levels in strains containing the ⌬dnaK52 mutation (40). It was therefore necessary to determine to what extent DnaK deficiency specifically was responsible for the starvation-related ⌬dnaK52 mutant phenotypes.…”

“…Plasmid-bearing derivatives of PBL500 or PBL501 were constructed by transformation at 30ЊC. Complementation of the chromosomal ⌬dnaK52 mutation was required for efficient transduction of the ⌬dnaK52 mutant allele (40). A pBN15 transformant of GW4813 was used as the donor to transfer the ⌬dnaK52::Cm r mutation into the PBL500 background by phage P1 vir transduction.…”

“…Plasmids used in these studies were pBN13 (P tac ::dnaK ϩ lacI ϩ bla ϩ ), pBN15 (P tac :: dnaK ϩ dnaJ ϩ lacI ϩ bla ϩ ), pBN16 (P tac ::dnaJ ϩ lacI ϩ bla ϩ ), and pBN8 (lacI ϩ bla ϩ ) (3). These plasmids utilize a ColE2 replicon and are therefore DnaK independent for replication (15,40). Plasmid-bearing derivatives of PBL500 or PBL501 were constructed by transformation at 30ЊC.…”