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.     

In vitro effect of the Escherichia coli heat shock regulatory protein on expression of heat shock genes.

In Escherichia coli, the ability to elicit a heat shock response depends on the htpR gene product. Previous work has shown that the HtpR protein serves as a sigma factor (sigma 32) for RNA polymerase that specifically recognizes heat shock promoters (A.D. Grossman, J.W. Erickson, and C.A. Gross Cell 38:383-390, 1984). In the present study we showed that sigma 32 synthesized in vitro could stimulate the expression of heat shock genes. The in vitro-synthesized sigma 32 was found to be associated with RNA polymerase. In vivo-synthesized sigma 32 was also associated with RNA polymerase, and this polymerase (E sigma 32) could be isolated free of the standard polymerase (E sigma 70). E sigma 32 was more active than E sigma 70 with heat shock genes; however, non-heat-shock genes were not transcribed by E sigma 32. The in vitro expression of the htpR gene required E sigma 70 but did not require E sigma 32.     

Sensitization of Escherichia coli cells to oxidative stress by deletion of the rpoH gene, which encodes the heat shock sigma factor.

A deletion in the rpoH gene greatly increased the sensitivity of Escherichia coli sodA sodB mutants to oxidative stress. The effect of the rpoH deletion on sodA+ sodB+ cells was only marginal. Mutations in heat shock genes singly sensitized sodA sodB double mutant cells to plumbagin. sodA sodB double mutants were neither more sensitive nor more resistant to thermal stress than the wild type.     

Regulation of the alternative sigma factor sigma(E) during initiation,adaptation, and shutoff of the extracytoplasmic heat shock response in Escherichia coli

The alternative sigma factor sigma(E) is activated in response to stress in the extracytoplasmic compartment of Escherichia coli. Here we show that sigma(E) activity increases upon initiation of the stress response by a shift to an elevated temperature (43 degrees C) and remains at that level for the duration of the stress. When the stress is removed by a temperature downshift, sigma(E) activity is strongly repressed and then slowly returns to levels seen in unstressed cells. We provide evidence that information about the state of the cell envelope is communicated to sigma(E) primarily through the regulated proteolysis of the inner membrane anti-sigma factor RseA, as the degradation rate of RseA is correlated with the changes in sigma(E) activity throughout the stress response. However, the relationship between sigma(E) activity and the rate of degradation of RseA is complex, indicating that other factors may cooperate with RseA and serve to fine-tune the response.     

SigmaE is an essential sigma factor in Escherichia coli.

SigmaE is an alternative sigma factor that controls the extracytoplasmic stress response in Escherichia coli. SigmaE is essential at high temperatures but was previously thought to be nonessential at temperatures below 37 degrees C. We present evidence that sigmaE is an essential sigma factor at all temperatures. Cells lacking sigmaE are able to grow at low temperatures because of the presence of a frequently arising, unlinked suppressor mutation.     

Regulation of Escherichia coli starvation sigma factor (sigma s) by ClpXP protease.

In Escherichia coli, starvation (stationary-phase)-mediated differentiation involves 50 or more genes and is triggered by an increase in cellular sigma s levels. Western immunoblot analysis showed that in mutants lacking the protease ClpP or its cognate ATPase-containing subunit ClpX, sigma s levels of exponential-phase cells increased to those of stationary-phase wild-type cells. Lack of other potential partners of ClpP, i.e., ClpA or ClpB, or of Lon protease had no effect. In ClpXP-proficient cells, the stability of sigma s increased markedly in stationary-phase compared with exponential-phase cells, but in ClpP-deficient cells, sigma s became virtually completely stable in both phases. There was no decrease in ClpXP levels in stationary-phase wild-type cells. Thus, sigma s probably becomes more resistant to this protease in stationary phase. The reported sigma s-stabilizing effect of the hns mutation also was not due to decreased protease levels. Studies with translational fusions containing different lengths of sigma s coding region suggest that amino acid residues 173 to 188 of this sigma factor may directly or indirectly serve as at least part of the target for ClpXP protease.     

Structure-function analysis of the Escherichia coli GrpE heat shock protein.

We have isolated various missense mutations in the essential grpE gene of Escherichia coli based on the inability to propagate bacteriophage lambda. To better understand the biochemical mechanisms of GrpE action in various biological processes, six mutant proteins were overexpressed and purified. All of them, GrpE103, GrpE66, GrpE2/280, GrpE17, GrpE13a and GrpE25, have single amino acid substitutions located in highly conserved regions throughout the GrpE sequence. The biochemical defects of each mutant GrpE protein were identified by examining their abilities to: (i) support in vitro lambda DNA replication; (ii) stimulate the weak ATPase activity of DnaK; (iii) dimerize and oligomerize, as judged by glutaraldehyde crosslinking and HPLC size chromatography; (iv) interact with wild-type DnaK protein using either an ELISA assay, glutaraldehyde crosslinking or HPLC size chromatography. Our results suggest that GrpE can exist in a dimeric or oligomeric form, depending on its relative concentration, and that it dimerizes/oligomerizes through its N-terminal region, most likely through a computer predicted coiled-coil region. Analysis of several mutant GrpE proteins indicates that an oligomer of GrpE is the most active form that interacts stably with DnaK and that the interaction is vital for GrpE biological function. Our results also demonstrate that both the N-terminal and C-terminal regions are important for GrpE function in lambda DNA replication and its co-chaperone activity with DnaK.     

Control of the alternative sigma factor sigmaE in Escherichia coli

Signal transduction pathways that communicate information from the cell envelope to the cytoplasm of bacteria are crucial to maintain cell envelope homeostasis. In Escherichia coli, one of the key pathways that ensures the integrity of the cell envelope during stress and normal growth is controlled by the alternative sigma factor sigmaE. Recent studies have elucidated the signal transduction pathway that activates sigmaE in response to misfolded outer membrane porins. Unfolded porins trigger the degradation of the sigmaE-specific antisigma factor RseA by the sequential action of two inner membrane proteases, leading to release of sigmaE from RseA, and induction of the stress response. This mechanism of signal transduction, regulated intramembrane proteolysis, is used in transmembrane signaling pathways from bacteria to humans.     

RNA polymerase mutant with altered sigma factor in Escherichia coli.

Summary E. gracilis chloroplast DNA Bam fragments E and D, coding for rRNA were cloned separately using the plasmid pBR 322 as vector and E. coli as host. The newly constructed recombinant plasmids EgcKS 8 and EgcKS 11 (containing the Bam HI fragments E and D respectively) were analysed and characterized by gel electrophoresis, electronmicroscopy and analytical ultracentrifugation.Abbreviations Ap Ampicillin - Tc Tetracycline-hydrochloride - Bam HI endonuclease isolated from Bacillus amyloliquefaciens - Eco RI endonuclease isolated from E. coli RY13 - Bgl II endonuclease isolated from Bacillus globiggi - EDTA Ethylene-diamine-tetra-acetic-acid - ctDNA chloroplast DNA An abstract of this work was presented at the 10th annual meeting of the Union Schweizerischer Gesellschaften für Experimentelle Biologie, Davos 19th and 20th Mai, 1978. The recommendations of the Schweizerische Akademie für medizinische Wissenschaften for work with recombinant DNA-molecules were respected throughout this work.