Cellular defects caused by deletion of the Escherichia coli dnaK gene indicate roles for heat shock protein in normal metabolism.

DnaK is a major heat shock protein of Escherichia coli and has been previously reported to be essential for growth at high temperatures. We systematically investigated the role of DnaK in cellular metabolism at a wide range of growth temperatures by analyzing cellular defects caused by deletion of the dnaK gene (delta dnaK52). At intermediate temperatures (30 degrees C), introduction of the delta dnaK52 allele into wild-type cells caused severe defects in cell division, slow growth, and poor viability of the cells. delta dnaK52 mutants were genetically unstable at 30 degrees C and frequently acquired secondary mutations. At high (42 degrees C) and low (11 and 16 degrees C) temperatures the delta dnaK52 allele could only be introduced into the subpopulation of wild-type cells that had duplicated the dnaK region of their chromosome. delta dnaK52 mutants isolated at 30 degrees C were cold sensitive as well as temperature sensitive for growth. Cell division defects of delta dnaK52 mutants at 30 degrees C were largely suppressed by overproduction of the FtsZ protein, which is normally required for septation during cell division; however, slow growth and poor viability at 30 degrees C and cold sensitivity and temperature sensitivity of growth were not suppressed, indicating that delta dnaK52 mutants had additional defective cellular functions besides cell division.     

Escherichia coli dnaK null mutants are inviable at high temperature.

DnaK, a major Escherichia coli heat shock protein, is homologous to major heat shock proteins (Hsp70s) of Drosophila melanogaster and humans. Null mutations of the dnaK gene, both insertions and a deletion, were constructed in vitro and substituted for dnaK+ in the E. coli genome by homologous recombination in a recB recC sbcB strain. Cells carrying these dnaK null mutations grew slowly at low temperatures (30 and 37 degrees C) and could not form colonies at a high temperature (42 degrees C); furthermore, they also formed long filaments at 42 degrees C. The shift of the mutants to a high temperature evidently resulted in a loss of cell viability rather than simply an inhibition of growth since cells that had been incubated at 42 degrees C for 2 h were no longer capable of forming colonies at 30 degrees C. The introduction of a plasmid carrying the dnaK+ gene into these mutants restored normal cell growth and cell division at 42 degrees C. These null mutants showed a high basal level of synthesis of heat shock proteins except for DnaK, which was completely absent. In addition, the synthesis of heat shock proteins after induction in these dnaK null mutants was prolonged compared with that in a dnaK+ strain. The well-characterized dnaK756 mutation causes similar phenotypes, suggesting that they are caused by a loss rather than an alteration of DnaK function. The filamentation observed when dnaK mutations were incubated at a high temperature was not suppressed by sulA or sulB mutations, which suppress SOS-induced filamentation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Escherichia coli DnaK and GrpE heat shock proteins interact both in vivo and in vitro.

Previous studies have demonstrated that the Escherichia coli dnaK and grpE genes code for heat shock proteins. Both the Dnak and GrpE proteins are necessary for bacteriophage lambda DNA replication and for E. coli growth at all temperatures. Through a series of genetic and biochemical experiments, we have shown that these heat shock proteins functionally interact both in vivo and in vitro. The genetic evidence is based on the isolation of mutations in the dnaK gene, such as dnaK9 and dnaK90, which suppress the Tr- phenotype of bacteria carrying the grpE280 mutation. Coimmunoprecipitation of DnaK+ and GrpE+ proteins from cell lysates with anti-DnaK antibodies demonstrated their interaction in vitro. In addition, the DnaK756 and GrpE280 mutant proteins did not coimmunoprecipitate efficiently with the GrpE+ and DnaK+ proteins, respectively, suggesting that interaction between the DnaK and GrpE proteins is necessary for E. coli growth, at least at temperatures above 43 degrees C. Using this assay, we found that one of the dnaK suppressor mutations, dnaK9, reinstated a protein-protein interaction between the suppressor DnaK9 and GrpE280 proteins.     

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

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). These mutants are unable to autophosphorylate and are severely defective in ATP hydrolysis. These mutant dnaK genes were placed under the control of the lac promoter and were found not to complement the deficiencies of a delta dnaK mutant in negative regulation of the heat shock response. A decrease in the expression of DnaK and DnaJ below their normal levels at 30 degrees C was found to result in increased expression of GroEL. The implications of these results for DnaK's role in the negative regulation of the heat shock response are discussed. Evidence is also presented indicating the existence of a 70-kDa protein present in a delta dnaK52 mutant that cross-reacts with antibodies raised against DnaK. Derivatives of the dnaK+ E. coli strain MC4100 expressing the mutant DnaK proteins filamented severely at temperatures equal to or greater than 34 degrees C. In the dnaK+ E. coli strain W3110, expression of these mutant proteins caused extreme filamentation even at 30 degrees C. Together with other observations, these results suggest that DnaK may play a direct role in the septation pathway, perhaps via an interaction with FtsZ. Although delta dnaK52 derivatives of strain MC4100 filament extensively, a level of underexpression of DnaK and DnaJ that results in increased expression of the other heat shock proteins did not result in filamentation. The delta dnaK52 allele could be transduced successfully, at temperatures of up to 45 degrees C, into strains carrying a plasmid expressing dnaK+ dnaJ+, although the yield of transductants decreased above 37 degrees C. In contrast, with a strain that did not carry a plasmid expressing dnaK+ dnaJ+, the yield of delta dnaK52 transductants decreased extremely sharply between 39 and 40 degrees C, suggesting that DnaK and DnaJ play one or more roles critical for growth at temperatures of 40 degrees C or greater.     

Only one dnaK homolog,dnaK2, is active transcriptionally and is essential in Synechocystis

We examined the expression and the function of the DnaK chaperone family in the photoautotrophic cyanobacterium, Synechocystis PCC 6803. Surprisingly, only one of the three dnaK genes was transcribed either under normal or heat shock conditions. Their predicted cochaperones (four dnaJs and one grpE) proved to be uninducible under our experimental conditions. Attempts to inactivate the active dnaK2 has failed, indicating that the gene is essential. The partial mutant displayed lower inducibility of chaperones (especially GroEL and HSP17) both at mRNA and protein levels upon heat shock. The mutant showed temperature sensitive phenotype, but was able to acquire thermotolerance.     

Low temperature of GroEL/ES overproduction permits growth of Escherichia coli cells lacking trigger factor DnaK

Escherichia coli trigger factor (TF) and DnaK cooperate in the folding of newly synthesized proteins. The combined deletion of the TF-encoding tig gene and the dnaK gene causes protein aggregation and synthetic lethality at 30 degrees C. Here we show that the synthetic lethality of deltatigdeltadnaK52 cells is abrogated either by growth below 30 degrees C or by overproduction of GroEL/GroES. At 23 degrees C deltatigdeltadnaK52 cells were viable and showed only minor protein aggregation. Overproduction of GroEL/GroES, but not of other chaperones, restored growth of deltatigdeltadnaK52 cells at 30 degrees C and suppressed protein aggregation including proteins >/= 60 kDa, which normally require TF and DnaK for folding. GroEL/GroES thus influences the folding of proteins previously identified as DnaK/TF substrates.     

Low temperature or GroEL/ES overproduction permits growth of Escherichia coli cells lacking trigger factor and DnaK

Escherichia coli trigger factor (TF) and DnaK cooperate in the folding of newly synthesized proteins. The combined deletion of the TF-encoding tig gene and the dnaK gene causes protein aggregation and synthetic lethality at 30 degrees C. Here we show that the synthetic lethality of DeltatigDeltadnaK52 cells is abrogated either by growth below 30 degrees C or by overproduction of GroEL/GroES. At 23 degrees C DeltatigDeltadnaK52 cells were viable and showed only minor protein aggregation. Overproduction of GroEL/GroES, but not of other chaperones, restored growth of DeltatigDeltadnaK52 cells at 30 degrees C and suppressed protein aggregation including proteins >/=60 kDa, which normally require TF and DnaK for folding. GroEL/GroES thus influences the folding of proteins previously identified as DnaK/TF substrates.     

Molecular cloning, purification and immunological responses of recombinants GroEL and DnaK from Streptococcus pyogenes

To better understand the roles of heat shock proteins in streptococcal diseases, the groEL and dnaK genes from Streptococcus pyogenes were cloned and their products (GroEL and DnaK) and derivatives (F2GroEL, F3GroEL and C1DnaK) purified as His-tagged fusion proteins. Western blot analysis of the purified proteins with sera from individuals with streptococcal diseases demonstrated that 29 out of 36 sera tested were reactive with GroEL and eight recognized DnaK. Rabbit antiserum against myosin recognized both GroEL and DnaK. Antibodies raised against purified F2GroEL and DnaK reacted with myosin in the ELISA but not in a Western immunoblot. These data indicate that the S. pyogenes GroEL and DnaK may be important immunogens during streptococcal infections. Furthermore, we provide evidence of an immunogenic relatedness of the GroEL and DnaK proteins with myosin that could play a role in the pathogenesis of streptococcal non-suppurative sequelae.     

Delta dnaK52 mutants of Escherichia coli have defects in chromosome segregation and plasmid maintenance at normal growth temperatures.

Major heat shock proteins, such as the Escherichia coli DnaK protein, not only are required for cell growth after heat shock but seem to possess important functions in cellular metabolism at normal growth temperatures as well. E. coli delta dnaK52 mutants have severe cellular defects at 30 degrees C, one of which is in cell division (B. Bukau and G. C. Walker, J. Bacteriol, 171:2337-2346, 1989). Here we show that at 30 degrees C, delta dnaK52 mutants have defects in chromosome segregation and in maintenance of low-copy-number plasmids. Fluorescence microscopic analysis revealed that chromosomes were frequently lacking at peripheries of cell filaments of delta dnaK52 mutants and clustered at other locations. In other parts of the cell filaments, chromosomes were apparently normally distributed and they were also present in most of the small cells found in populations of delta dnaK52 cells. These defects might be at the level of DNA replication, since delta dnaK52 mutants have a threshold lower rate of DNA synthesis than wild-type cells. Chromosome segregation defects of delta dnaK52 mutants were also observed in an rnh dnaA mutant background, in which initiation of DNA replication is DnaA-oriC independent. We also found that low-copy-number P1 miniplasmids could not be stably maintained in delta dnaK52 mutants at 30 degrees C. delta par P1 miniplasmids that carry the P1-encoded rep functions required for their replication but lack the P1-encoded par functions required for faithful partitioning of the plasmids during cell division were also unstable in delta dnaK52 mutants. Taken together, our results indicate important, although not absolutely essential, functions for DnaK at 30 degrees C in one or more processes necessary for correct replication and/or partitioning of chromosomes and P1 miniplasmids. Furthermore, we found that P1 miniplasmids were also highly unstable in dnaJ259 mutants, indicating a role for the DnaJ heat shock protein in maintenance of these plasmids.     

Heat shock response of the archaebacterium Methanococcus voltae.

The general properties of the heat shock response of the archaebacterium Methanococcus voltae were characterized. The induction of 11 heat shock proteins, with apparent molecular weights ranging from 18,000 to 90,000, occurred optimally at 40 to 50 degrees C. Some of the heat shock proteins were preferentially enriched in either the soluble (cytoplasm) or particulate (membrane) fraction. Alternative stresses (ethanol, hydrogen peroxide, NaCl) stimulated the synthesis of subsets of the heat shock proteins as well as unique proteins. Western blot (immunoblot) analysis, in which antisera to Escherichia coli heat shock proteins (DnaK and GroEL) were used, did not detect any immunologically cross-reactive proteins. In addition, Southern blot analysis did not reveal any homology between M. voltae and four highly conserved heat shock genes, mopB and dnaK from E. coli and hsp70 genes from Drosophila species and Saccharomyces cerevisiae.     

Role of the DnaK and HscA homologs of Hsp70 chaperones in protein folding in E.coli.

Folding of newly synthesized cytosolic proteins has been proposed to require assistance by Hsp70 chaperones. We investigated whether two Hsp70 homologs of Escherichia coli, DnaK and HscA, have this role in vivo. Double mutants lacking dnaK and hscA were viable and lacked defects in protein folding at intermediate temperature. After heat shock, a subpopulation of pre-existing proteins slowly aggregated in mutants lacking DnaK, but not HscA, whereas the bulk of newly synthesized proteins displayed wild-type solubility. For thermolabile firefly luciferase, DnaK was dispensable for de novo folding at 30 degrees C, but essential for aggregation prevention during heat shock and subsequent refolding. DnaK and HscA are thus not strictly essential for folding of newly synthesized proteins. DnaK instead has functions in refolding of misfolded proteins that are essential under stress.     

Physiological consequences of DnaK and DnaJ overproduction in Escherichia coli.

The physiological consequences of molecular chaperone overproduction in Escherichia coli are presented. Constitutive overproduction of DnaK from a multicopy plasmid containing large chromosomal fragments spanning the dnaK region resulted in plasmid instability. Co-overproduction of DnaJ with DnaK stabilized plasmid levels. To examine the effects of altered levels of DnaK and DnaJ in a more specific manner, an inducible expression system for dnaK and dnaJ was constructed and characterized. Differential rates of DnaK synthesis were determined by quantitative Western blot (immunoblot) analysis. Moderate levels of DnaK overproduction resulted in a defect in cell septation and formation of cell filaments, but co-overproduction of DnaJ overcame this effect. Further increases in the level of DnaK terminated culture growth despite increased levels of DnaJ. DnaK overproduction was found to be bacteriocidal, and this effect was also partially suppressed by DnaJ. The bacteriocidal effect was apparent only with cultures which were allowed to enter stationary phase, indicating that DnaK toxicity is growth phase dependent.     

Mutations altering heat shock specific subunit of RNA polymerase suppress major cellular defects of E. coli mutants lacking the DnaK chaperone.

An Escherichia coli mutant lacking HSP70 function, delta dnaK52, is unable to grow at both high and low temperatures and, at intermediate temperature (30 degrees C), displays defects in major cellular processes such as cell division, chromosome segregation and regulation of heat shock gene expression that lead to poor growth and genetic instability of the cells. In an effort to understand the roles of molecular chaperones such as DnaK in cellular metabolism, we analyzed secondary mutations (sid) that suppress the growth defects of delta dnaK52 mutants at 30 degrees C and also permit growth at low temperature. Of the five suppressors we analyzed, four were of the sidB class and mapped within rpoH, which encodes the heat shock specific sigma subunit (sigma 32) of RNA polymerase. The sidB mutations affected four different regions of the sigma 32 protein and, in one case, resulted in a several fold reduction in the cellular concentration of sigma 32. Presence of any of the sidB mutations in delta dnaK52 mutants as well as in dnaK+ cells caused down-regulation of heat shock gene expression at 30 degrees C and decreased induction of the heat shock response after shift to 43.5 degrees C. These findings suggest that the physiologically most significant function of DnaK in the metabolism of unstressed cells is its function in heat shock gene regulation.     

Examination of the Tn5 transposase overproduction phenotype in Escherichia coli and localization of a suppressor of transposase overproduction killing that is an allele of rpoH.

Tn5 transposase (Tnp) overproduction is lethal to Escherichia coli. Tnp overproduction causes cell filamentation, abnormal chromosome segregation, and an increase in anucleated cell formation. There are two simple explanations for the observed phenotype: induction of the SOS response or of the heat shock response. The data presented here show that overproduction of Tnp neither induces an SOS response nor a strong heat shock response. However, our experiments do indicate that induction of some sigma32-programmed function(s) (either due to an rpoH mutation, a deletion of dnaK, or overproduction of sigma32) suppresses Tnp overproduction killing. This effect is not due to overproduction of DnaK, DnaJ, or GroELS. In addition, Tnp but not deltall Tnp (whose overproduction does not kill the host cells) associates with the inner cell membrane, suggesting a possible correlation between cell killing and Tnp membrane association. These observations will be discussed in the context of a model proposing that Tnp overproduction titrates an essential host factor(s) involved in an early cell division step and/or chromosome segregation.