An alkaline shift induces the heat shock response in Escherichia coli.

Activation of heat shock response was observed after an alkaline shift of extracellular pH: it peaked at 5 to 10 min, as was previously reported for the heat-induced response, and was dependent on a functional rpoH gene, which is the positive regulator of the heat shock response. An induction of over sixfold was observed for dnaK and groE. The response was induced by the alkalization of extracellular pH but not by the alkalization of intracellular pH. An acidic shift of extracellular pH failed to activate the heat shock response, showing that the response is specific to the alkaline shift.     

Modulation of the heat shock response by one-carbon metabolism in Escherichia coli.

A genetic screen designed to isolate mutants of Escherichia coli W3110 altered in the ability to induce the heat shock response identified a strain unable to induce the heat shock proteins in a rich, defined medium lacking methionine after exposure to 2,4-dinitrophenol. This strain also grew slowly at 28 degrees C and linearly at 42 degrees C in this medium. The abnormal induction of the heat shock proteins and abnormal growth at both high and low temperatures were reversed when methionine was included in the growth medium. The mutation responsible for these phenotypes mapped to the glyA gene, a biosynthetic gene encoding the enzyme that converts serine and tetrahydrofolate to glycine and 5,10-methylenetetrahydrofolate. This reaction is the major source of glycine and one-carbon units in the cell. Because fixed one-carbon units, in the form of methionine, allowed mutant cells to induce the heat shock response after exposure to 2,4-dinitrophenol, a one-carbon restriction may be responsible for the phenotypes described above.     

Topoisomerase activity during the heat shock response in Escherichia coli K-12.

During the upshift of temperature from 30 to 42, 45, 47, or 50 degrees C, an increase in the level of supercoiling of a reporter plasmid was observed. This increase was present in groE and dnaK mutants but was inhibited in cells treated with chloramphenicol and novobiocin. The intracellular [ATP]/[ADP] ratio increased rapidly after an upshift in temperature from 30 to 47 degrees C and then decreased to reach a level above that observed at 30 degrees C. These results suggest that gyrase and proteins synthesized during heat shock are responsible for the changes seen in plasmid supercoiling. Proteins GroE and DnaK are probably not involved in this phenomenon.     

The role of antioxidant systems in response of bacteria Escherichia coli to heat shock]

Shifting the temperature from 30 to 45 degrees C in an aerobic Escherichia coli culture inhibited the expression of the antioxidant genes katG, katE, sodA, and gor. The expression was evaluated by measuring beta-galactosidase activity in E. coli strains that contained fusions of the antioxidant gene promoters with the lacZ operon. Heat shock inhibited catalase and glutathione reductase, lowered the intracellular level of glutathione, and increased its extracellular level. It also suppressed the growth of mutants deficient in the katG-encoded catalase HPI, whereas the sensitivity of the wild-type and sodA sodB mutant cells to heat shock was almost the same. In the E. coli culture adapted to growth at 42 degrees C, the content of both intracellular and extracellular glutathione was two times higher than in the culture grown at 30 degrees C. The temperature-adapted cells grown aerobically at 42 degrees C showed an increased ability to express the fused katG-lacZ genes.     

The role of heat shock proteins in the osmoregulation of Escherichia coli

E. coli has a number of biochemical systems which protect cells from different chemical and physical damages. The aim of this work is to characterize the interaction between two of these: the osmoregulation system and the heat shock system. It is shown that exposure of E. coli to 42 degrees C to induce hsps synthesis, abolish the growth inhibition by high (0.45 M) NaCl concentration. Also, transient pretreatment of cells with high NaCl protect them from heat damage. It is shown that osmotic shock induces the hsps synthesis. The cell growth restoration after the complete inhibition by high (0.6 M) NaCl concentration correlates with the hsps accumulation. Moreover the heat shock treatment reduces the adaptation time.     

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.     

A new Escherichia coli heat shock gene, htrC, whose product is essential for viability only at high temperatures.

We identified and characterized a new Escherichia coli gene, htrC. Inactivation of the htrC gene results in the inability to form colonies at 42 degrees C. An identical bacterial phenotype is found whether the htrC gene is inactivated either by Tn5 insertions or by a deletion spanning the entire gene. The htrC gene has been localized at 90 min, immediately downstream of the rpoC gene, and has been previously sequenced. It codes for a basic polypeptide with an Mr of 21,130. The htrC gene is under heat shock regulation, since it is transcribed actively only in bacteria possessing functional sigma 32. Inactivation of htrC results in (i) bacterial filamentation at intermediate temperatures, (ii) cell lysis at temperatures above 42 degrees C, (iii) overproduction of sigma 32-dependent heat shock proteins at all temperatures, (iv) overproduction of a few additional polypeptides, (v) underproduction of many polypeptides, and (vi) an overall defect in cellular proteolysis as judged by the reduced rate of puromycyl polypeptide degradation. In addition, the presence of an htrC mutation eliminates the UV sensitivity normally exhibited by lon mutant bacteria.     

The Escherichia coli heat shock protease HtrA participates in defense against oxidative stress

The serine protease HtrA (DegP), which is indispensable for cell survival at elevated temperatures, is a peripheral membrane protein, localized on the periplasmic side of the inner membrane in Escherichia coli, and the biochemical and genetic evidence indicates that the physiological role of HtrA is to degrade denatured proteins formed in the cellular envelope during heat shock. The aim of this study was to find out if the HtrA protease contributes to protection of the cell against oxidative stress. We compared the influence of various oxidizing agents on htrA mutant cells with their effects on wild-type bacteria, and found that the htrA mutation did not increase sensitivity to hydrogen peroxide or paraquat but made the cell extremely sensitive to ferrous [Fe(II)] ions, which are known to enhance oxidation of proteins. Treatment with ferrous ions caused a larger increase in the level of protein carbonyl groups in the membrane fraction of the cell than in the periplasm and cytoplasm. Iron-induced oxidation of membrane proteins was enhanced in the htrA mutant relative to wild-type cells. Inhibition of the growth of the htrA mutant by iron could be alleviated more efficiently by a nitroxide antioxidant that localizes in the membranes (A-TEMPO) than by a derivative (4OH-TEMPO) that acts mainly in the soluble fraction of the cell. Inhibition of the growth of the htrA mutant was more pronounced following treatment with cumene hydroperoxide, which partitions into membranes, than with t-butyl hydroperoxide, which forms radical mainly in the cytosol. Both ferrous ions and cumene hydroperoxide, but not hydrogen peroxide, paraquat or t-butyl hydroperoxide, induced synthesis of HtrA. Our results show that HtrA plays a role in defense against oxidative shock and support the hypothesis that HtrA participates in the degradation of oxidatively damaged proteins localized in the cell envelope, especially those associated with the membranes.     

Selective degradation of unfolded proteins by the self-compartmentalizing HtrA protease, a periplasmic heat shock protein in Escherichia coli

HtrA, which has a high molecular mass of about 500 kDa, is a periplasmic heat shock protein whose proteolytic activity is essential for the survival of Escherichia coli at high temperatures. To determine the structural organization of HtrA, we have used electron microscopy and chemical cross-linking analysis. The averaged image of HtrA with end-on orientation revealed a six-membered, ring-shaped structure with a central cavity, and its side-on view showed a two-layered structure. Thus, HtrA behaves as a dodecamer consisting of two stacks of hexameric ring. HtrA can degrade thermally unfolded citrate synthase and malate dehydrogenase but cannot when in their native form. HtrA degraded partially unfolded casein more rapidly upon increasing the incubation temperature. However, it hydrolyzed oxidized insulin B-chain, which is fully unfolded, at nearly the same rate at all of the temperatures tested. HtrA also rapidly degraded reduced insulin B-chain generated by treatment of insulin with dithiothreitol but not A-chain or intact insulin. Moreover, HtrA degraded fully unfolded alpha-lactalbumin, of which all four disulfide bonds were reduced, but not the native alpha-lactalbumin and its unfolded intermediates containing two or three disulfide bonds. These results indicate that unfolding of the protein substrates, such as by exposure to high temperatures or reduction of disulfide bonds, is essential for their access into the inner chamber of the double ring-shaped HtrA, where cleavage of peptide bonds may occur. Thus, HtrA with a self-compartmentalizing structure may play an important role in elimination of unfolded proteins in the periplasm of Escherichia coli.     

Protein substrates and heat shock reduce the DNA-binding ability of Escherichia coli Lon protease

Interaction between the fusion protein MBP-Lon, formed by maltose-binding protein and Lon protease, and the plasmid pBR322 was studied to clarify the DNA-binding behavior of the Lon protease. Since the MBP-Lon fusion protein that was bound to the plasmid was strongly adsorbed by amylose resin, complex formation and dissociation were determined by quantifying the unadsorbed plasmid using agarose gel electrophoresis. The autolysis of MBP-Lon fusion protein was suppressed when the protein was bound to the plasmid. The plasmid was completely dissociated from MBP-Lon fusion protein by the addition of the protein substrates of Lon protease (i.e. -casein and denatured bovine serum albumin). In addition, at high temperatures, MBP-Lon fusion protein lost its plasmid-binding ability, although it fully retained ATP-dependent protease activity. These results suggest that Lon protease loses DNA-binding ability when cells are exposed to abnormal conditions and the amount of damaged proteins increases. On the other hand, DNA probably plays an important role in controlling the Lon protease activity in cells under normal conditions by entrapping the enzyme.     

A model for heterooligomer formation in the heat shock response of Escherichia coli

Small heat shock proteins (sHsp) are widely distributed molecular chaperones that bind to misfolded proteins to prevent irreversible aggregation and aid in refolding to a competent state. The sHsps characterized thus far all contain a conserved α-crystallin, and variable N- and C-termini critical for chaperone activity and oligomerization. The Escherichia coli sHsps IbpA and IbpB share 48% sequence homology, are induced by heat shock and oxidative stress, and each requires the presence of the other to effect protein protection. Molecular Dynamics (MD) simulations of homology-modeled monomers and heterooligomers of these sHsps identify a possible mechanism for cooperation between IbpA and IbpB.     

Proteome analysis of the Escherichia coli heat shock response under steady-state conditions

In this study a proteomic approach was used to investigate the steady-state response of Escherichia coli to temperature up-shifts in a cascade of two continuously operated bioreactors. The first reactor served as cell source with optimal settings for microbial growth, while in the second chemostat the cells were exposed to elevated temperatures. By using this reactor configuration, which has not been reported to be used for the study of bacterial stress responses so far, it is possible to study temperature stress under well-defined, steady-state conditions. Specifically the effect on the cellular adaption to temperature stress using two-dimensional gel electrophoresis was examined and compared at the cultivation temperatures of 37°C and 47.5°C. As expected, the steady-state study with the double bioreactor configuration delivered a different protein spectrum compared to that obtained with standard batch experiments in shaking flasks and bioreactors. Setting a high cut-out spot-to-spot size ratio of 5, proteins involved in defence against oxygen stress, functional cell envelope proteins, chaperones and proteins involved in protein biosynthesis, the energy metabolism and the amino acid biosynthesis were found to be differently expressed at high cultivation temperatures. The results demonstrate the complexity of the stress response in a steady-state culture not reported elsewhere to date.     

Biogenesis of inner membrane proteins in Escherichia coli

The inner membrane proteome of the model organism Escherichia coli is composed of inner membrane proteins, lipoproteins and peripherally attached soluble proteins. Our knowledge of the biogenesis of inner membrane proteins is rapidly increasing. This is in particular true for the early steps of biogenesis - protein targeting to and insertion into the membrane. However, our knowledge of inner membrane protein folding and quality control is still fragmentary. Furthering our knowledge in these areas will bring us closer to understand the biogenesis of individual inner membrane proteins in the context of the biogenesis of the inner membrane proteome of Escherichia coli as a whole. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.