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.     

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.     

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.     

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)     

Viability of rep recA mutants depends on their capacity to cope with spontaneous oxidative damage and on the DnaK chaperone protein

Replication arrests due to the lack or the inhibition of replicative helicases are processed by recombination proteins. Consequently, cells deficient in the Rep helicase, in which replication pauses are frequent, require the RecBCD recombination complex for growth. rep recA mutants are viable and display no growth defect at 37 or 42 degrees C. The putative role of chaperone proteins in rep and rep recA mutants was investigated by testing the effects of dnaK mutations. dnaK756 and dnaK306 mutations, which allow growth of otherwise wild-type Escherichia coli cells at 40 degrees C, are lethal in rep recA mutants at this temperature. Furthermore, they affect the growth of rep mutants, and to a lesser extent, that of recA mutants. We conclude that both rep and recA mutants require DnaK for optimal growth, leading to low viability of the triple (rep recA dnaK) mutant. rep recA mutant cells form colonies at low efficiency when grown to exponential phase at 30 degrees C. Although the plating defect is not observed at a high temperature, it is not suppressed by overexpression of heat shock proteins at 30 degrees C. The plating defect of rep recA mutant cells is suppressed by the presence of catalase in the plates. The cryosensitivity of rep recA mutants therefore results from an increased sensitivity to oxidative damage upon propagation at low temperatures.     

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.     

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.     

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.     

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.     

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.     

Roles of the Escherichia coli Small Heat Shock Proteins IbpA and IbpB in Thermal Stress Management: Comparison with ClpA, ClpB, and HtpG In Vivo

We have constructed an Escherichia coli strain lacking the small heat shock proteins IbpA and IbpB and compared its growth and viability at high temperatures to those of isogenic cells containing null mutations in the clpA, clpB, or htpG gene. All mutants exhibited growth defects at 46°C, but not at lower temperatures. However, the clpA, htpG, and ibp null mutations did not reduce cell viability at 50°C. When cultures were allowed to recover from transient exposure to 50°C, all mutations except Δibp led to suboptimal growth as the recovery temperature was raised. Deletion of the heat shock genes clpB and htpG resulted in growth defects at 42°C when combined with the dnaK756 or groES30 alleles, while the Δibp mutation had a detrimental effect only on the growth of dnaK756 mutants. Neither the overexpression of these heat shock proteins nor that of ClpA could restore the growth of dnaK756 or groES30 cells at high temperatures. Whereas increased levels of host protein aggregation were observed in dnaK756 and groES30 mutants at 46°C compared to wild-type cells, none of the null mutations had a similar effect. These results show that the highly conserved E. coli small heat shock proteins are dispensable and that their deletion results in only modest effects on growth and viability at high temperatures. Our data also suggest that ClpB, HtpG, and IbpA and -B cooperate with the major E. coli chaperone systems in vivo.     

Small heat shock proteins, ClpB and the DnaK system form a functional triade in reversing protein aggregation

Small heat shock proteins (sHsps) can efficiently prevent the aggregation of unfolded proteins in vitro. However, how this in vitro activity translates to function in vivo is poorly understood. We demonstrate that sHsps of Escherichia coli, IbpA and IbpB, co-operate with ClpB and the DnaK system in vitro and in vivo, forming a functional triade of chaperones. IbpA/IbpB and ClpB support independently and co-operatively the DnaK system in reversing protein aggregation. A delta ibpAB delta clpB double mutant exhibits strongly increased protein aggregation at 42 degrees C compared with the single mutants. sHsp and ClpB function become essential for cell viability at 37 degrees C if DnaK levels are reduced. The DnaK requirement for growth is increasingly higher for delta ibpAB, delta clpB, and the double delta ibpAB delta clpB mutant cells, establishing the positions of sHsps and ClpB in this chaperone triade.     

Escherichia coli Hsp31 functions as a holding chaperone that cooperates with the DnaK-DnaJ-GrpE system in the management of protein misfolding under severe stress conditions

Escherichia coli Hsp31 is a homodimeric protein that exhibits chaperone activity in vitro and is a representative member of a recently recognized family of heat shock proteins (Hsps). To gain insights on Hsp31 cellular function, we deleted the hchA gene from the MC4100 chromosome and combined the resulting null allele with lesions in other cytoplasmic chaperones. Although the hchA mutant only exhibited growth defects when cultivated at 48 degrees C, loss of Hsp31 had a strong deleterious effect on the ability of cells to survive and recover from transient exposure to 50 degrees C, and led to the enhanced aggregation of a subset of host proteins at this temperature. The absence of Hsp31 did not significantly affect the ability of the ClpB-DnaK-DnaJ-GrpE system to clear thermally aggregated proteins at 30 degrees C suggesting that Hsp31 does not possess disaggregase activity. Although it had no effect on the growth of groES30, Delta clpB or Delta ibpAB cells at high temperatures, the hchA deletion aggravated the temperature sensitive phenotype of dnaK756 and grpE280 mutants and led to increased aggregation in stressed dnaK756 cells. On the basis of biochemical, structural and genetic data, we propose that Hsp31 acts as a modified holding chaperone that captures early unfolding intermediates under prolonged conditions of severe stress and releases them when cells return to physiological conditions. This additional line of defence would complement the roles of DnaK-DnaJ-GrpE, ClpB and IbpB in the management of thermally induced cellular protein misfolding.     

Characterization of the dnaK multigene family in the Cyanobacterium Synechococcus sp. strain PCC7942

The cyanobacterium Synechococcus sp. strain PCC7942 has three dnaK homologues (dnaK1, dnaK2, and dnaK3), and a gene disruption experiment was carried out for each dnaK gene by inserting an antibiotic resistance marker. Our findings revealed that DnaK1 was not essential for normal growth, whereas DnaK2 and DnaK3 were essential. We also examined the effect of heat shock on the levels of these three DnaK and GroEL proteins and found a varied response to heat shock, with levels depending on each protein. The DnaK2 and GroEL proteins exhibited a typical heat shock response, that is, their synthesis increased upon temperature upshift. In contrast, the synthesis of DnaK1 and DnaK3 did not respond to heat shock; in fact, the level of DnaK1 protein decreased. We also analyzed the effect of overproduction of each DnaK protein in Escherichia coli cells using an inducible expression system. Overproduction of DnaK1 or DnaK2 resulted in defects in cell septation and formation of cell filaments. On the other hand, overproduction of DnaK3 did not result in filamentous cells; rather a swollen and twisted cell morphology was observed. When expressed in an E. coli dnaK756 mutant, dnaK2 could suppress the growth deficiency at the nonpermissive temperature, while dnaK1 and dnaK3 could not suppress this phenotype. On the contrary, overproduction of DnaK1 or DnaK3 resulted in growth inhibition at the permissive temperature. These results suggest that different types of Hsp70 in the same cellular compartment have specific functions in the cell.     

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.     

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

During a 3-day period, glucose starvation of wild-type Escherichia coli produced thermotolerant, H2O2-resistant, small cells with a round morphology. These cells contained elevated levels of the DnaK protein, adjusted either for total protein or on a per-cell basis. Immunoprecipitation of [35S]methionine-labeled protein produced by such starving cells demonstrated that DnaK underwent continuous synthesis but at decreasing rates throughout this time. Glucose resupplementation of starving cells resulted in rapid loss of thermotolerance, H2O2 resistance, and the elevated DnaK levels. A dnaK deletion mutant, but not an otherwise isogenic wild-type strain, failed to develop starvation-induced thermotolerance or H2O2 resistance. The filamentous phenotype associated with DnaK deficiency was suppressed by cultivation in a defined glucose medium. When starved for glucose, the nonfilamentous and rod-shaped dnaK mutant strain failed to convert into the small spherical form typical of starving wild-type cells. The dnaK mutant retained the ability to develop adaptive H2O2 resistance during growth but not adaptive resistance to heat. Complementation of DnaK deficiency by using Ptac-regulated dnaK+ and dnaK+J+ expression plasmids confirmed a specific role for the DnaK molecular chaperone in these starvation-induced phenotypes.     

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.