The role of DnaK/DnaJ and GroEL/GroES systems in the removal of endogenous proteins aggregated by heat-shock from Escherichia coli cells

The submission of Escherichia coli cells to heat-shock (45 degrees C, 15 min) caused the intracellular aggregation of endogenous proteins. In the wt cells the aggregates (the S fraction) disappeared 10 min after transfer to 37 degrees C. In contrast, the S fraction in the dnaK and dnaJ mutant strains was stable during approximately one generation time (45 min). This demonstrated that neither the renaturation nor the degradation of the denatured proteins was possible in the absence of DnaK and DnaJ. The groEL44 and groES619 mutations stabilised the aggregates to a lesser extent. It was shown by the use of cloned genes, dnaK/dnaJ or groEL/groES, producing the corresponding proteins in about 4-fold excess, that the appearance of the S fraction in the wt strain resulted from a transiently insufficient supply of the heat-shock proteins. Overproduction of the GroEL/GroES proteins in dnaK756 or dnaJ259 background prevented the aggregation, however, overproduction of the DnaK/DnaJ proteins did not prevent the aggregation in the groEL44 or groES619 mutant cells although it accelerated the disappearance of the aggregates. The properties of the aggregated proteins are discussed from the point of view of their competence to renaturation/degradation by the heat-shock system.     

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.     

Biosynthesis and secretion of several enzymes in Escherichia coli dnaK and dnaJ mutants

Escherichia coli null dnaJ and dnaKdnaJ mutants were defective in the biosynthesis and secretion of several enzymes. The synthesis of beta-galactosidase induced in delta dnaJ and delta dnaKdnaJ mutants was abolished at 42 degrees C and significantly decreased at 30 and 37 degrees C. The activity of alkaline phosphatase in the periplasm in both mutant strains at high temperature was lower than in the wild-type strain. The synthesis of b-type cytochromes was defective in two deletion mutants while the synthesis of nitrate reductase-A at 42 degrees C was influenced by dnaK mutation only. The lack of DnaK and DnaJ does not impair the activity of catechol 2,3-dioxygenase irrespective of growth temperature.     

Participation of the dnaK and dnaJ gene products in phosphorylation of glutaminyl-tRNA synthetase and threonyl-tRNA synthetase of Escherichia coli K-12.

The heat shock proteins DnaK and DnaJ of Escherichia coli participate in phosphorylation of both glutaminyl-tRNA synthetase and threonyl-tRNA synthetase. When cellular proteins extracted from the dnaK7(Ts) and dnaJ259(Ts) mutant cells labeled with 32Pi at 42 degrees C were analyzed by two-dimensional gel electrophoresis, no phosphorylation of these proteins was observed when they were compared with those from wild-type cells.     

Phosphorylation of glutaminyl-tRNA synthetase and threonyl-tRNA synthetase by the gene products of dnaK and dnaJ in Escherichia coli K-12 cells

In Escherichia coli K-12, the heat shock protein DnaK and DnaJ participate in phosphorylation of both glutaminyl-tRNA synthetase and threonyl-tRNA synthetase since when cellular proteins extracted from the dnaK7(Ts), dnaK756(Ts) and dnaJ259(Ts) mutant cells labeled with 32Pi at 42 degrees C were analyzed by two-dimensional gel electrophoresis, no phosphorylation of glutaminyl-tRNA synthetase and threonyl-tRNA synthetase was observed while phosphorylation of both aminoacyl-tRNA synthetases was detected in the samples extracted from wild-type cells.     

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.     

Cloning and characterization of the Brucella ovis heat shock protein DnaK functionally expressed in Escherichia coli.

The Brucella ovis dnaK gene, homolog to the eukaryotic hsp70 genes, was cloned by using a Drosophila melanogaster probe. Comparison of B. ovis and Escherichia coli sequences revealed a similar organization for the dnaK and dnaJ genes and putative regulatory signals. In E. coli transfected with the cloned fragment, B. ovis hsp70 was expressed at 30 and 50 degrees C apparently under the control of its own promoter. The recombinant protein and a B. ovis native protein displaying the same molecular weight were both recognized by anti-E. coli DnaK serum. Native B. ovis protein was also recognized by sera of sheep either infected or vaccinated with an attenuated Brucella strain, suggesting that Brucella hsp70 could be up-regulated during host colonization. A thermosensitive E. coli dnaK mutant transfected with the cloned fragment recovered colony-forming ability at 42 degrees C, showing that the B. ovis DnaK protein could behave as a functional heat shock protein in E. coli.     

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.     

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)     

The DnaK chaperone is necessary for alpha-complementation of beta-galactosidase in Escherichia coli

We show here the involvement of the molecular chaperone DnaK from Escherichia coli in the in vivo alpha-complementation of the beta-galactosidase. In the dnaK756(Ts) mutant, alpha-complementation occurs when the organisms are grown at 30 degrees C but not at 37 or 40 degrees C, although these temperatures are permissive for bacterial growth. Plasmid-driven expression of wild-type dnaK restores the alpha-complementation in the mutant but also stimulates it in a dnaK(+) strain. In a mutant which contains a disrupted dnaK gene (DeltadnaK52::Cm(r)), alpha-complementation is also impaired, even at 30 degrees C. This observation provides an easy and original phenotype to detect subtle functional changes in a protein such as the DnaK756 chaperone, within the physiologically relevant temperature.     

Role of heat shock protein DnaK in osmotic adaptation of Escherichia coli.

Escherichia coli can adapt and recover growth at high osmolarity. Adaptation requires the deplasmolysis of cells previously plasmolyzed by the fast efflux of water promoted by osmotic upshift. Deplasmolysis is essentially ensured by a net osmo-dependent influx of K+. The cellular content of the heat shock protein DnaK is increased in response to osmotic upshift and does not decrease as long as osmolarity is high. The dnaK756(Ts) mutant, which fails to deplasmolyze and recover growth, does not take up K+ at high osmolarity; DnaK protein is required directly or indirectly for the maintenance of K+ transport at high osmolarity. The temperature-sensitive mutations dnaJ259 and grpE280 do not affect the osmoadaptation of E. coli at 30 degrees C.     

Gene structure and transcriptional regulation of <Emphasis Type="Italic">dnaK</Emphasis> and <Emphasis Type="Italic">dnaJ</Emphasis> genes from a psychrophilic bacterium, <Emphasis Type="Italic">Colwellia maris</Emphasis>

The dnaK and dnaJ genes, encoding heat shock proteins, were cloned from a psychrophilic bacterium, Colwellia maris. Significant homology was evident comparing DnaK and DnaJ of the psychrophilile with the counterparts of mesophilic and thermophilic bacteria. In the DnaJ protein, three conserved regions of the Hsp40 family were observed. A putative promoter similar to the sigma32 consensus sequence was found upstream of the dnaK gene. The G+C content in the 5'-untranslated region of the dnaK gene was much lower than that in the corresponding region of mesophilic bacteria. Northern-blot analysis and primer-extension analysis showed that both genes were transcribed separately as monocistronic mRNAs. Following several temperature upshifts from 10 to 26 degrees C, maximum induction of the dnaK and dnaJ mRNAs was detected at 20 degrees C, suggesting that this temperature induces the heat shock response in this bacterium. In addition, the level of the induction of the dnaJ gene was much lower than that of the dnaK gene. These findings together revealed several specific features of the heat shock response at a relatively low temperature in psychrophiles.     

Participation of Escherichia coli heat shock proteins DnaJ, DnaK, and GrpE in P1 plasmid replication.

Low-copy-number plasmids, such as P1 prophage and the fertility factor F, require a plasmid-encoded replication protein and several host products for replication. Stable maintenance also depends on active partitioning of plasmids into daughter cells. Mini-P1 par+ and par plasmids were found to be destabilized by mutations in the dnaJ, dnaK, and grpE genes of Escherichia coli. The transformation efficiency and stability of mini-F plasmids were also reduced in the mutant strains. These results indicate that heat shock proteins DnaJ, DnaK, and GrpE play roles in the replication of plasmid P1 and probably also in of F.     

Effect of DnaK and DnaJ proteins deprivation on Escherichia coli response to starvation

E. coli defects in response to nutritional starvation caused by DnaK and DnaJ proteins deprivation are examined. The ability of delta dnaKdnaJ mutant to survive carbon, nitrogen and phosphorus starvation is highly impaired while delta dnaJ mutant is characterized by the diminished survival of phosphorus starvation only. delta dnaKdnaJ mutant grows slowly utilizing maltose and glycerol and delta dnaJ mutant utilizes glycerol inefficiently. The growth on alternate nitrogen sources is comparable to wild-type strain.     

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.     

Isolation and characterization of dnaJ null mutants of Escherichia coli.

Bacteriophage lambda requires the lambda O and P proteins for its DNA replication. The rest of the replication proteins are provided by the Escherichia coli host. Some of these host proteins, such as DnaK, DnaJ, and GrpE, are heat shock proteins. Certain mutations in the dnaK, dnaJ, or grpE gene block lambda growth at all temperatures and E. coli growth above 43 degrees C. We have isolated bacterial mutants that were shown by Southern analysis to contain a defective, mini-Tn10 transposon inserted into either of two locations and in both orientations within the dnaJ gene. We have shown that these dnaJ-insertion mutants did not grow as well as the wild type at temperatures above 30 degrees C, although they blocked lambda DNA replication at all temperatures. The dnaJ-insertion mutants formed progressively smaller colonies at higher temperatures, up to 42 degrees C, and did not form colonies at 43 degrees C. The accumulation of frequent, uncharacterized suppressor mutations allowed these insertion mutants to grow better at all temperatures and to form colonies at 43 degrees C. None of these suppressor mutations restored the ability of the host to propagate phage lambda. Radioactive labeling of proteins synthesized in vivo followed by immunoprecipitation or immunoblotting with anti-DnaJ antibodies demonstrated that no DnaJ protein could be detected in these mutants. Labeling studies at different temperatures demonstrated that these dnaJ-insertion mutations resulted in altered kinetics of heat shock protein synthesis. An additional eight dnaJ mutant isolates, selected spontaneously on the basis of blocking phage lambda growth at 42 degrees C, were shown not to synthesize DnaJ protein as well. Three of these eight spontaneous mutants had gross DNA alterations in the dnaJ gene. Our data provide evidence that the DnaJ protein is not absolutely essential for E. coli growth at temperatures up to 42 degrees C under standard laboratory conditions but is essential for growth at 43 degrees C. However, the accumulation of extragenic suppressors is necessary for rapid bacterial growth at higher temperatures.     

Physiological consequences of mutations in Escherichia coli heat shock dnaK and dnaJ genes.

Mutations in the heat shock genes, dnaK and dnaJ, cause severe defects of several cellular functions. Null dnaJ and dnaKdnaJ mutations can be transduced in a restricted range of temperature. The efficiency of transformation with three unrelated plasmids, viz pACYC184, pBR322 and pSC101, is two times lower in dnaK mutants while the dnaJ mutant is characterized by slightly impaired transformation with pSC101 only. The lack of DnaJ function negatively influences the stability of pSC101 at 42 degrees C, and this plasmid cannot be stably maintained at 30 degrees C in the delta dnaKdnaJ mutant. The double deletion mutant, delta dbaKdnaJ, is characterized by impaired osmoadaptation. The galactokinase content is lower in both mutants tested compared with wild-type strains even at 30 degrees C. The efficient complementation of some of these defects by the wild-type alleles present on low-copy number plasmid was achieved.     

Isolation and characterization of a temperature-sensitive dnaK mutant of Escherichia coli B.

A temperature-sensitive dnaK mutant (strain MT112) was isolated from Escherichia coli B strain H/r30RT by thymineless death selection at 43 degrees C. By genetic mapping, the mutation [dnaK7(Ts)] was located near the thr gene (approximately 0.2 min on the may). E. coli K-12 transductants of the mutation to temperature sensitivity were assayed for their susceptibility to transducing phage lambda carrying the dnaK and/or the dnaJ gene. All of the transductants were able to propagate phage lambda carrying the dnaK gene. When macromolecular synthesis of the mutant was assayed at 43 degrees C, it was observed that both deoxyribonucleic acid and ribonucleic acid syntheses were severely inhibited. Thus, it was suggested that the conditionally defective dnaK mutation affects both cellular deoxyribonucleic acid and ribonucleic acid syntheses at the nonpermissive temperature in addition to inability to propagate phage lambda at permissive temperature.