Deg phenotype of Escherichia coli lon mutants.

Deg. one of the Escherichia coli systems for degrading abnormal polypeptides (e.g., nonsense fragments), is also involved in the degradation of some classes of missense proteins. Both missense proteins of beta-galactosidase and temperature-sensitive phage products appear to be degraded by the Deg system. Mutations in the Deg system are indistinguishable from mutations classically called lon or capR; all map near proC, all are mucoid, defective in protein degradation, sensitive to radiomimetic agents, and defective in P1 lysogenization. All are able to propagate temperature-sensitive phage better than lon+ parental strains. Mutations that suppress the radiation sensitivity of these strains (sul) also suppress the P1 lysogenization defect, but do not affect mucoidy or the degradation defect.     

Mini-F plasmid mutants able to replicate in Escherichia coli deficient in the DnaJ heat shock protein.

A subset of Escherichia coli heat shock proteins, DnaJ, DnaK, and GrpE, is required for mini-F plasmid replication, presumably at the step of functioning of the RepE initiator protein. We have isolated and characterized mini-F plasmid mutants that acquired the ability to replicate in the Escherichia coli dnaJ259. The mutant plasmids were found to replicate in any of dnaJ, dnaK, and grpE mutant hosts tested. In each case, the majority of the mutant plasmids carried a unique amino acid alteration in a localized region of repE coding sequence and showed an increased copy number, whereas the minority contained a common single base change (C to T) in the promoter/operator region and produced an increased amount of RepE. All RepE proteins with altered residues (between 92 and 134) exhibited increased initiator activities (hyperactive), and many showed reduced repressor activities as well, indicating that this region is important for the both major functions of RepE protein. These results together with evidence reported elsewhere indicate that the subset of heat shock proteins serves to activate RepE protein prior to or during its binding to the replication origin and that the mutant RepE proteins are active even in their absence. We also found that a C-terminal lesion (repE602) reduces the initiator activity particularly of some hyperactive mutant RepE proteins but does not affect the repressor activity. This finding suggests a functional interaction between the central and C-terminal regions of RepE in carrying out the initiator function.     

Lysogenic induction of lambdoid phages in lexA mutants of Escherichia coli

Summary UV irradiation of lexA3 mutants of E. coli caused lysogenic induction of prophage , _i21, _i434 and 80. Maximal induction in lexA3 lysogens needed less UV than in lexA ⁺ bacteria and gave 25–100% of the normal levels of infective centres induced. Assays of gene expression arising from derepression of a defective prophage showed at least 40% of the normal levels of induction by mitomycin C in lexA3 bacteria. The need for post-irradiation protein synthesis for lysogenic induction in lexA3 lysogens was reduced by increasing the basal level of recA protein with a recA ⁺ plasmid. It is concluded that in lexA E. coli some recA protein synthesis, too small to be detected by physical means, is needed for UV induced lysogenic induction.     

Abnormal induction of heat shock proteins in an Escherichia coli mutant deficient in adenosylmethionine synthetase activity.

Most prototrophic strains of Escherichia coli become restricted for methionine at 44 degrees C. A mutant strain (RG62 metK) in which the level of S-adenosylmethionine synthetase activity is only 10 to 20% of normal shows constitutive expression of one of the heat shock proteins, the lysU gene product, lysyl-tRNA synthetase form II, at 37 degrees C. These findings suggested a possible linkage between methionine metabolism and heat shock. We examined the induction of heat shock polypeptides in strain RG62 (metK) and in its parent, RG (metK+), from which it was derived by spontaneous mutation. Exponential-phase cultures of the two strains were pulse-labeled with [3H]leucine shortly after a shift from 37 to 44 degrees C, and the total cellular polypeptides were examined by two-dimensional electrophoresis. The results confirmed the constitutive production of the lysU gene product previously reported for strain RG62, but also revealed that the induction of 2 of the 17 heat shock polypeptides, C14.7 and G13.5, was markedly depressed. Otherwise the heat shock induction pattern was similar in timing and magnitude in the two strains. Transformation of the mutant strain with a plasmid, pK8, containing the metK coding sequence and promoter region as a 1.8-kilobase insert into pBR322 restored normal induction of C14.7 and G13.5, but did not prevent constitutive expression of the lysU gene product in the medium required for growth of this strain. The three heat shock polypeptides abnormally controlled in strain RG62 are the three polypeptides which are not induced when rapid synthesis of the htpR gene product is induced by isopropyl-beta-D-thiogalactopyranoside at 28 degree C (R. A. VanBogelen, M. A. Acton, and F. C. Neidhardt, Genes Dev. 1:525-531, 1987). We postulate that induction of these three polypeptides involves metabolic signals in addition to the synthesis of the htpR gene product and that strain RG62 (metK) fails to produce the signals involved in induction of C14.7 and G13.5 on a shift-up in temperature and produces the signal related to lysU induction even at 37 degree C.     

R-factor-mediated suppression of the galactose-sensitive phenotype of Escherichia coli K-12 galE mutants

Plasmids S-a and Rts1 suppress the galactose-sensitive phenotype of galE mutants of Escherichia coli K-12, giving rise to both galactose-fermenting and nonfermenting strains. Fermenting strains produce normal inducible UDP-galactose epimerase. Plasmids extracted from either a fermenting or a nonfermenting strain are indistinguishable when examined by either measurements of length of relaxed circular molecules by electron microscopy or electrophoretic pattern of restriction endonuclease digestion products. The phenomenon could be explained by reversible recombination between a plasmid-borne epimerase gene and homologous chromosomal sequences.     

Further characterization of SOS system induction in recBC mutants of Escherichia coli

Expression of several SOS functions such as induction of lambda prophage, inhibition of cell division and induction of both umuC and recA genes after UV-irradiation, nalidixic acid or mitomycin C addition was studied in an RecBC- mutant. UV-irradiation and mitomycin C induced all SOS functions studied in the RecBC- cells but at a lower level and delayed with respect to the wild-type strain. On the contrary, nalidixic acid was unable to trigger any of these SOS functions. In the RecBC- mutant, adenine only had a stimulating effect on the amplification of RecA protein synthesis following UV-irradiation. Nevertheless, in the wild-type strain the stimulating effect occurred in all SOS functions studied following UV-irradiation as well as in the amplification of RecA protein synthesis by nalidixic acid but not in the other SOS functions triggered by this compound. Furthermore, adenine produced a decrease in the mitomycin C-mediated induction of all SOS functions studied in both RecBC- and wild-type strains.     

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.     

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.     

Proteolysis in the Escherichia coli heat shock response: a player at many levels

Proteolysis is a fundamental process used by all forms of life to maintain homeostasis, as well as to remodel the proteome following environmental changes. Here, we explore recent advances in understanding the role of proteolysis during the heat shock response of Escherichia coli. Proteolysis both regulates and contributes directly to and the heat shock response at multiple different levels, from adjusting the levels of the master heat shock response regulator (σ(32)), to eliminating damaged cellular proteins, to altering the activity of chaperones that refold heat-denatured proteins. Recent results illustrate the complexity of the heat shock response and the pervasive role that proteolysis plays in both the cellular response to heat shock and the subsequent limiting of the response, as cells return to a more 'normal' physiological state.     

Analysis of streptomycin-resistance of Escherichia coli mutants.

We previously reported about Escherichia coli transformation experiments yielding streptomycin-resistant cells carrying a C912 to T transition in a plasmid-born 16S rRNA gene. These experiments were based on results obtained with streptomycin-resistant Euglena chloroplasts bearing an equivalent mutation in the single chloroplast 16S rRNA gene. We extended this study and transformed E. coli with plasmid constructs having a mutated 16S rRNA gene at position 914 (A to C) or a double mutation at positions 912 and 888 (C to T:G to A) or a mutation in the S12 gene (Lys-42 to Thr). We tested the transformed cells before and after a screening procedure in the presence of streptomycin. We find that the plasmid-born mutations protect colonies against a short streptomycin exposure, but ribosomes carrying mutated 16S rRNA do not significantly reduce codon misreading in vitro. However, ribosomes isolated from transformed cells after the screening procedure resist misreading. These ribosomes have acquired a second mutation in the S12 protein as shown in one case by sequencing and by transformation experiments. Furthermore, we show that the A914 to C mutation prevents (strongly reduces) base methylation in the central domain of 16S rRNA.     

Different sensitivity of autolytic deficient Escherichia coli mutants to the mode of induction

Abstract By a comparison of the rate øX174 gene E product (gpE)-induced autolysis of Escherichia coli RM4101 and its autolysis deficient mutant strains RK232, RK238 and RK316, it was shown that gpE-induced autolysis differs from autolysis induced by EDTA or moenomycin. Subclones of these strains which could no longer be lysed by gpE can be lysed by EDTA shock treatment or moenomycin at almost normal rates. GpE seems to induce only partially the activity of the autolytic system of E. coli.     

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.     

Thermoinduced radioresistance of Escherichia coli cells and heat shock proteins

Radioresistance of E. coli cells is slightly increased (dose modification factor (DMF) = 1.2) with temperature elevated from 4 degrees to 43 degrees C at the time of gamma-irradiation. However, an appreciable effect of the thermoinduced radioresistance (DMF = 1.7) was observed when the wild-type cells were exposed to gamma-radiation at 15-43 degrees C (but not at 4 degrees C) after 30-min preincubation at 43 degrees C. This effect was absent in htpR mutants, defective in induction of heat shock proteins, and coupled with the decreased post-irradiation DNA degradation in gamma-irradiated htpR+ cells. It is suggested that heat shock proteins are involved in the thermoinduced radioresistance.