Escherichia coli heat shock protein DnaK: production and consequences in terms of monitoring cooking

Through use of commercially available DnaK proteins and anti-DnaK monoclonal antibodies, a competitive enzyme-linked immunosorbent assay was developed to quantify this heat shock protein in Escherichia coli ATCC 25922 subjected to various heating regimens. For a given process lethality (F(70)(10) of 1, 3, and 5 min), the intracellular concentration of DnaK in E. coli varied with the heating temperature (50 or 55 degrees C). In fact, the highest DnaK concentrations were found after treatments at the lower temperature (50 degrees C) applied for a longer time. Residual DnaK after heating was found to be necessary for cell recovery, and additional DnaK was produced during the recovery process. Overall, higher intracellular concentrations of DnaK tended to enhance cell resistance to a subsequent lethal stress. Indeed, E. coli cells that had undergone a sublethal heat shock (105 min at 55 degrees C, F(70)(10) = 3 min) accompanied by a 12-h recovery (containing 76,786 +/- 25,230 molecules/cell) resisted better than exponentially growing cells (38,500 +/- 6,056 molecules/cell) when later heated to 60 degrees C for 50 min (F(70)(10) = 5 min). Results reported here suggest that using stress protein to determine cell adaptation and survival, rather than cell counts alone, may lead to more efficient heat treatment.     

Elevated serine catabolism is associated with the heat shock response in Escherichia coli.

The biochemical events associated with the heat shock response are not well understood in any organism, nor have the signals that initiate the induction of heat shock protein synthesis been identified. In this work, we demonstrate that the rate of serine catabolism of Escherichia coli cells grown in glucose minimal medium supplemented with serine is elevated three- to sevenfold when the growth temperature is shifted from 37 to 44 degrees C. Elevations in growth temperature and mutations or treatments that lead to elevated basal rates of serine catabolism at 37 degrees C result in the excretion into the culture medium of acetate derived from exogenous serine. Increases in the basal level of serine catabolism at 37 degrees C do not per se induce a heat shock response but are associated with abnormalities in the pattern of induction of heat shock polypeptides following a temperature shift. We postulate that the events responsible for or resulting from the elevation in serine catabolism associated with a shift-up in temperature modulate the induction of 3 of the 17 heat shock polypeptides identified in E. coli. These observations suggest that heat shock diverts serine away from the production of glycine and C1 units, which are required for initiation of protein synthesis and for nucleotide biosynthesis, and towards acetyl coenzyme A and acetate.     

Acquired thermotolerance and heat shock proteins in thermophiles from the three phylogenetic domains.

Thermophilic organisms from each of the three phylogenetic domains (Bacteria, Archaea, and Eucarya) acquired thermotolerance after heat shock. Bacillus caldolyticus grown at 60 degrees C and heat shocked at 69 degrees C for 10 min showed thermotolerance at 74 degrees C, Sulfolobus shibatae grown at 70 degrees C and heat shocked at 88 degrees C for 60 min showed thermotolerance at 95 degrees C, and Thermomyces lanuginosus grown at 50 degrees C and heat shocked at 55 degrees C for 60 min showed thermotolerance at 58 degrees C. Determinations of protein synthesis during heat shock revealed differences in the dominant heat shock proteins for each species. For B. caldolyticus, a 70-kDa protein dominated while for S. shibatae, a 55-kDa protein dominated and for T. lanuginosus, 31- to 33-kDa proteins dominated. Reagents that disrupted normal protein synthesis during heat shock prevented the enhanced thermotolerance.     

甜椒细胞质小分子量热激蛋白基因(CaHSP18)的cDNA克隆与表达

用RT-PCR和RACE-PCR技术,从热激处理的甜椒叶片总RNA中扩增出了细胞质小分子量热激蛋白(sHSP)全长779 bp的cDNA基因序列,包含一个480 bp开放阅读框,编码159个氨基酸.Southern杂交结果表明在甜椒基因组中有该基因的小的多基因家族.Northern结果显示该基因在甜椒根、茎、叶中的表达受热激和低温的诱导.原核表达分析表明该基因在高温以及低温条件下可以提高大肠杆菌的生存能力.     

Effect of heat shock on acetylcholinesterase activity in chick muscle cultures

The effect of heat shock was studied on the acetylcholinesterase activity of chick muscle primary cultures. In cultures transferred from 37 degrees C to 45 degrees C, a sharp drop in activity was followed by rapid spontaneous recovery. The time of onset of recovery resembled the time needed for expression of heat shock proteins. In cultures exposed to heat shock at 45 degrees C and allowed to recover at 37 degrees C, reappearance of acetylcholinesterase activity did not involve de novo protein synthesis since it was not prevented by cycloheximide. Our data raise the possibility of a role for heat shock proteins as molecular chaperones in rescuing heat-denaturing acetylcholinesterase.     

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.     

Induction of the heat shock regulon of Escherichia coli markedly increases production of bacterial viruses at high temperatures.

Production of bacteriophages T2, T4, and T6 at 42.8 to 44 degrees C was increased from 8- to 260-fold by adapting the Escherichia coli host (grown at 30 degrees C) to growth at the high temperature for 8 min before infection; this increase was abolished if the host htpR (rpoH) gene was inactive. Others have shown that the htpR protein increases or activates the synthesis of at least 17 E. coli heat shock proteins upon raising the growth temperature above a certain level. At 43.8 to 44 degrees C in T4-infected, unadapted cells, the rates of RNA, DNA, and protein synthesis were about 100, 70, and 70%, respectively, of those in T4-infected, adapted cells. Production of the major processed capsid protein, gp23, was reduced significantly more than that of most other T4 proteins in unadapted cells relative to adapted cells. Only 4.6% of the T4 DNA made in unadapted cells was resistant to micrococcal nuclease, versus 50% in adapted cells. Thus, defective maturation of T4 heads appears to explain the failure of phage production in unadapted cells. Overproduction of the heat shock protein GroEL from plasmids restored T4 production in unadapted cells to about 50% of that seen in adapted cells. T4-infected, adapted E. coli B at around 44 degrees C exhibited a partial tryptophan deficiency; this correlated with reduced uptake of uracil that is probably caused by partial induction of stringency. Production of bacteriophage T7 at 44 degrees C was increased two- to fourfold by adapting the host to 44 degrees C before infection; evidence against involvement of the htpR (rpoH) gene is presented. This work and recent work with bacteriophage lambda (C. Waghorne and C.R. Fuerst, Virology 141:51-64, 1985) appear to represent the first demonstrations for any virus that expression of the heat shock regulon of a host is necessary for virus production at high temperature.     

Heat shock response of Pseudomonas aeruginosa.

The general properties of the heat shock response in Pseudomonas aeruginosa were characterized. The transfer of cells from 30 to 45 degrees C repressed the synthesis of many cellular proteins and led to the enhanced production of 17 proteins. With antibodies raised against the Escherichia coli proteins, two polypeptides of P. aeruginosa with apparent molecular weights of 76,000 and 61,000 (76K and 61K proteins) were shown to be analogous to the DnaK and GroEL heat shock proteins of E. coli due to their immunologic cross-reactivity. The major sigma factor (sigma 87) of P. aeruginosa was shown to be a heat shock protein that was immunologically related to the sigma 70 of E. coli by using polyclonal antisera. A hybridoma was produced, and the monoclonal antibody MP-S-1 was specific for the sigma 87 and did not cross-react with sigma 70 of E. coli. A smaller 40K protein was immunoprecipitated with RNA polymerase antisera from cells that had been heat shocked. The 40K protein was also associated with RNA polymerase which had been purified from heat-shocked cells and may be the heat shock sigma factor of P. aeruginosa. Exposure to ethanol resulted in the production of seven new proteins, three of which appeared to be heat shock proteins.     

A cytosolic class I small heat shock protein, RcHSP17.8, of Rosa chinensis confers resistance to a variety of stresses to Escherichia coli, yeast and Arabidopsis thaliana

Among the heat shock proteins (HSPs) of higher plants, those belonging to the small HSP (sHSP) family remain the least characterized in functional terms. To improve our understanding of sHSPs, we have characterized RcHSP17.8 from Rosa chinensis . Sequence alignments and phylogenetic analysis reveal this to be a cytosolic class I sHSP. RcHSP17.8 expression in R. chinensis was induced by heat, cold, salt, drought, osmotic and oxidative stresses. Recombinant RcHSP17.8 was overexpressed in Escherichia coli and yeast to study its possible function under stress conditions. The recombinant E. coli and yeast cells that accumulated RcHSP17.8 showed improved viability under thermal, salt and oxidative stress conditions compared with control cultures. We also produced transgenic Arabidopsis thaliana that constitutively expressed RcHSP17.8. These plants exhibited increased tolerance to heat, salt, osmotic and drought stresses. These results suggest that R. chinensis cytosolic class I sHSP (RcHSP17.8) has the ability to confer stress resistance not only to E. coli and yeast but also to plants grown under a wide variety of unfavorable environmental conditions.     

Role of peptide chain elongation factor G in guanosine 5'-diphosphate 3'-diphosphate synthesis.

In a wild-type strain (relA+) of Escherichia coli, starvation of amino acid led to an immediate cessation of the synthesis of stable ribonucleic acids, together with the accumulation of an unusual nucleotide, guanosine 5'-diphosphate 3'-diphosphate, commonly known as ppGpp. This compound also accumulated during heat shock. When temperature-sensitive protein synthesis elongation factor G (EF-G) was introduced into E. coli NF859, a relA+ strain, the synthesis of ppGpp was reduced to approximately one-half that of wild-type EF-G+ cells at a nonpermissive temperature of 40 degrees C. Furthermore, fusidic acid, an inhibitor of protein synthesis which specifically inactivates EF-G, prevented any accumulation of ppGpp during the heat shock. We suggest that a functional EF-G protein is necessary for ppGpp accumulation under temperature shift conditions, possibly by mediating changes in the function of another protein, the relA gene product. However, EF-G is probably not required for the synthesis of ppGpp during the stringent response, since its inactivation did not prevent ppGpp accumulation during amino acid starvation.     

Lysis of Escherichia coli by the bacteriophage phi X174 E protein: inhibition of lysis by heat shock proteins.

Lysis of Escherichia coli by the cloned E protein of bacteriophage phi X174 was more rapid than expected when bacteria were shifted from 30 to 42 degrees C at the time of E induction. Since such treatment also induces the heat shock response, we investigated the effect of heat shock proteins on lysis. An rpoH mutant was more sensitive to lysis by E, but a secondary suppressor mutation restored lysis resistance to parental levels, which suggests that the sigma 32 subunit itself did not directly increase lysis resistance. At 30 degrees C, mutants in five heat shock genes (dnaK, dnaJ, groEL, groES, and grpE) were more sensitive to lysis than were their wild-type parents. The magnitude of lysis sensitivity varied with mutation and strain background, with dnaK, dnaJ, and groES mutants consistently exhibiting the greatest sensitivities. Extended protection against lysis occurred when overproduction of heat shock proteins was induced artificially in cells that contained a plasmid with the rpoH+ gene under control of the tac promoter. This protective effect was completely abolished by mutations in dnaK, dnaJ, or groES but not by grpE or groEL mutations. Altered membrane behavior probably explains the contradiction whereby an actual temperature shift sensitized cells to lysis, but production of heat shock proteins exhibited protective effects. The results demonstrate that E-induced lysis can be divided into two distinct operations which may now be studied separately. They also emphasize a role for heat shock proteins under non-heat-shock conditions and suggest cautious interpretation of lysis phenomena in systems where E protein production is under control of a temperature-sensitive repressor.     

Over-expression and characterization of the recombinant small heat shock protein from Pyrococcus furiosus

The gene encoding a small heat shock protein (sHSP) from Pyrococcus furiosus was redesigned and chemically synthesized by using bacteria-preferred codons. The gene product was over-expressed in Escherichia coli BL21(DE)₃ and purified to homogeneity. In the presence of this protein, the activities of Taq DNA polymerase, DNA restriction endonuclease HindIII and lysozyme were protected at elevated temperature, and also, thermal aggregation of lysozyme was prevented by this purified recombinant sHSP.Huayou Chen, Zhongmei Chu, Contributed equally to this work     

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)     

Germ cell-specific heat shock protein 105 binds to p53 in a temperature-sensitive manner in rat testis.

Heat shock protein (HSP)105 is a testis-specific and HSP90-related protein. The aim of this study was to explore the functions of HSP105 in the rat testis. Signals of HSP105 were detected immunohistochemically in the germ cells and translocated from the cytoplasm to the nucleus at 2 days after experimental induction of cryptorchidism. In cultured testicular germ cells, a significant increase in the expression of HSP105 in response to heat stress (37 degrees C) was detected in the insoluble protein fractions. Several binding proteins were isolated from rat testis using a HSP105 antibody immunoaffinity column, and p53, the tumor suppressor gene product, was copurified with these. Furthermore, immunoprecipitation using antibodies to p53 led to coprecipitation of HSP105 together with p53 after culturing germ cells at 32.5 degrees C, but not at 37 or 42 degrees C. In conclusion, HSP105 is specifically localized in the germ cells and may translocate into the nucleus after heat shock. HSP105 is suggested to form a complex with p53 at the scrotal temperature, and dissociate from it at suprascrotal temperatures. At scrotal temperature, HSP105 may thus contribute to the stabilization of p53 proteins in the cytoplasm of the germ cells, preventing the potential induction of apoptosis by p53.     

Compatible solutes contribute to heat resistance and ribosome stability in Escherichia coli AW1.7

This study investigated the mechanisms of heat resistance in Escherichia coli AW1.7 by quantification of cytoplasmic solutes, determination of ribosome denaturation, and by determination of protein denaturation. To assess the contribution of heat shock proteins and compatible solutes, experiments were conducted after exposure to sublethal heat shock, and with cultures grown at NaCl concentrations ranging from 0 to 6%. Heat resistance of E. coli AW1.7 was compared to the heat sensitive E. coli GGG10 and a plasmid-cured, heat sensitive derivative of E. coli AW1.7 named E. coli AW1.7ΔpHR1. Sublethal heat shock improved survival at 60°C of E. coli GGG10 and AW1.7ΔpHR1 but not of E. coli AW1.7. Addition of NaCl increased the heat resistance of all three strains, but only E. coli AW1.7 exhibited high heat resistance when grown in NaCl concentrations ranging from 2 to 6%. E. coli AW1.7 and GGG10 accumulated 16.1±0.8 and 8.8±0.8mmolL(-1) amino acids when grown at 0% NaCl, and 1.47±0.07 and 0.78±0.06mmolL(-1) carbohydrates when grown at 6% NaCl, respectively. Ribosome denaturation was determined by differential scanning calorimetry. After growth in the presence of 0% NaCl, the 30S subunit denatured at 63.7±0.8°C and 60.7±0.3°C in E. coli AW1.7 and GGG10, respectively. Fourier-transformed-infrared-spectroscopy did not indicate differences in protein denaturation between the strains during heating. In conclusion, heat resistance in E. coli AW1.7 correlates to ribosome stability at 60°C and is dependent on accumulation of cytoplasmic solutes.     

Proteome analysis on differentially expressed proteins of the fat body of two silkworm breeds, Bombyx mori, exposed to heat shock exposure

Proteomes of heat tolerant (multivoltine) and heat susceptible (bivoltine) silkworms (Bombyx mori) in response to heat shock were studied. Detected proteins from fat body were identified by using MALDI-TOF/TOF spectrometer, MS/MS, and MS analysis. Eight proteins, including small heat shock proteins (sHSPs) and HSP70, were expressed similarly in both breeds, while 4 protein spots were expressed specifically in the bivoltine breed and 12 protein spots were expressed specifically in the multivoltine breed. In the present proteomics approach, 5 separate spots of sHSP proteins (HSP19.9, HSP20.1, HSP20.4, HSP20.8, and HSP21.4) were identified. Protein spot intensity of sHSPs was lower in the multivoltine breed than in the bivoltine breed after the 45°C heat shock treatment, while the difference between two breeds was not significant after the 41°C heat shock treatment. These results indicated that some other mechanisms might be engaged in thermal tolerance of multivotine breed except for the expression of sHSP and HSP70. There were visible differences in the intensity of heat shock protein expression between male and female, however, differences were not statistically significant.