Unique and overlapping pollutant stress proteins of Escherichia coli.

Exposure of growing batch cultures of Escherichia coli to nine different "model micropollutants" (benzene, cadmium chloride, chlorpyrivos, 2,4-dichloroaniline, dioctylphtalate, hexachlorobenzene, pentachlorophenol, trichloroethylene, and tetrapropylbenzosulfonate) led to the induction of 13 to 39 proteins, as analyzed by two-dimensional gel electrophoresis. Some of these proteins overlapped with heat shock and carbon starvation proteins, but at least 50% were unique to a given chemical. The stress protein induction showed a temporal pattern, indicating sequential gene expression. Chemical stress protein synthesis occurred even at concentrations that had no effect on growth. Thus, the synthesis of these proteins can be a sensitive index of stress and the nature of environmental pollution.     

Stress Protein and Crystallin Synthesis During Heat Shock and Transdifferentiation of Embryonic Chick Neural Retina Cells

Embryonic chick neural retina responds to heat shock by the synthesis of "stress" polypeptides with molecular weights of 85 and 70 kd. Both stress proteins are synthesised from newly-transcribed messenger RNA. Sodium arsenite induces an additional stress protein of MW 25 kd. The heat shock response does not change during culture and subsequent transdifferentiation, and crystallin synthesis is not coinducible with the heat-shock proteins. We have also examined the pattern of protein synthesis at various stages of culture in both monolayer and aggregate systems; although changes in the protein synthetic profine are evident, there is no stress protein induction above basal levels at any time. Whilst mammalian α crystallin (B₂ chain) exhibits considerable homology to four small Drosophila heat-shock proteins, no significant antigenic similarity is apparent between δ crystallin and the major avian heat shock proteins. Thus during transdifferentiation, (a) the crystallin proteins do not behave in a manner analogous to stress proteins; moreover (b) crystallin production is not mediated by stress proteins resulting from a culture-induced stress response.     

Characterization of the thermotolerant cell. I. Effects on protein synthesis activity and the regulation of heat-shock protein 70 expression

Exposure of mammalian cells to a nonlethal heat-shock treatment, followed by a recovery period at 37 degrees C, results in increased cell survival after a subsequent and otherwise lethal heat-shock treatment. Here we characterize this phenomenon, termed acquired thermotolerance, at the level of translation. In a number of different mammalian cell lines given a severe 45 degrees C/30-min shock and then returned to 37 degrees C, protein synthesis was completely inhibited for as long as 5 h. Upon resumption of translational activity, there was a marked induction of heat-shock (or stress) protein synthesis, which continued for several hours. In contrast, cells first made thermotolerant (by a pretreatment consisting of a 43 degrees C/1.5-h shock and further recovery at 37 degrees C) and then presented with the 45 degrees C/30-min shock exhibited considerably less translational inhibition and an overall reduction in the amount of subsequent stress protein synthesis. The acquisition and duration of such "translational tolerance" was correlated with the expression, accumulation, and relative half-lives of the major stress proteins of 72 and 73 kD. Other agents that induce the synthesis of the stress proteins, such as sodium arsenite, similarly resulted in the acquisition of translational tolerance. The probable role of the stress proteins in the acquisition of translational tolerance was further indicated by the inability of the amino acid analogue, L-azetidine 2-carboxylic acid, an inducer of nonfunctional stress proteins, to render cells translationally tolerant. If, however, analogue-treated cells were allowed to recover in normal medium, and hence produce functional stress proteins, full translational tolerance was observed. Finally, we present data indicating that the 72- and 73-kD stress proteins, in contrast to the other major stress proteins (of 110, 90, and 28 kD), are subject to strict regulation in the stressed cell. Quantitation of 72- and 73-kD synthesis after heat-shock treatment under a number of conditions revealed that "titration" of 72/73-kD synthesis in response to stress may represent a mechanism by which the cell monitors its local growth environment.     

Salinity stress proteins in Eurytemora affinis

Seasonal densities of Eurytemora affinis, a calanoid copepod in the Chesapeake Bay, seem to be controlled by temperature and salinity. To examine the role of osmotic stress we analyzed protein synthesis under various conditions of temperature and osmotic stress. Adult females were exposed in groups for 5 hours to different temperature and salinity regimes in the presence of isotope-labelled amino acid. Newly synthesized (stress) proteins could be separated and identified using polyacrylamide gel electrophoresis and autoradiography. The protein profiles occurring in copepods experiencing osmotic shock alone were different from those of control animals. Copepods transferred to lower (2 and 5) and higher (15 and 20) salinities showed differences in the up- and down-regulation of specific proteins. Concurrent heat stress changed these protein patterns. Animals experiencing osmotic and heat shock at the same time exhibited enhanced expression of another set of proteins. Variation in induced proteins occurred among individuals.     

Aureolib — A Proteome Signature Library: Towards an Understanding of Staphylococcus aureus Pathophysiology

Gel-based proteomics is a powerful approach to study the physiology of Staphylococcus aureus under various growth restricting conditions. We analyzed 679 protein spots from a reference 2-dimensional gel of cytosolic proteins of S. aureus COL by mass spectrometry resulting in 521 different proteins. 4,692 time dependent protein synthesis profiles were generated by exposing S. aureus to nine infection-related stress and starvation stimuli (H₂O₂, diamide, paraquat, NO, fermentation, nitrate respiration, heat shock, puromycin, mupirocin). These expression profiles are stored in an online resource called Aureolib (http://www.aureolib.de). Moreover, information on target genes of 75 regulators and regulatory elements were included in the database. Cross-comparisons of this extensive data collection of protein synthesis profiles using the tools implemented in Aureolib lead to the identification of stress and starvation specific marker proteins. Altogether, 226 protein synthesis profiles showed induction ratios of 2.5-fold or higher under at least one of the tested conditions with 157 protein synthesis profiles specifically induced in response to a single stimulus. The respective proteins might serve as marker proteins for the corresponding stimulus. By contrast, proteins whose synthesis was increased or repressed in response to more than four stimuli are rather exceptional. The only protein that was induced by six stimuli is the universal stress protein SACOL1759. Most strikingly, cluster analyses of synthesis profiles of proteins differentially synthesized under at least one condition revealed only in rare cases a grouping that correlated with known regulon structures. The most prominent examples are the GapR, Rex, and CtsR regulon. In contrast, protein synthesis profiles of proteins belonging to the CodY and σ^B regulon are widely distributed. In summary, Aureolib is by far the most comprehensive protein expression database for S. aureus and provides an essential tool to decipher more complex adaptation processes in S. aureus during host pathogen interaction.     

Developmental control of stress stimulons in Streptomyces coelicolor revealed by statistical analyses of global gene expression patterns

Stress-induced regulatory networks coordinated with a procaryotic developmental program were revealed by two-dimensional gel analyses of global gene expression. Four developmental stages were identified by their distinctive protein synthesis patterns using principal component analysis. Statistical analyses focused on five stress stimulons (induced by heat, cold, salt, ethanol, or antibiotic shock) and their synthesis during development. Unlike other bacteria, for which various stresses induce expression of similar sets of protein spots, in Streptomyces coelicolor heat, salt, and ethanol stimulons were composed of independent sets of proteins. This suggested independent control by different physiological stress signals and their corresponding regulatory systems. These stress proteins were also under developmental control. Cluster analysis of stress protein synthesis profiles identified 10 different developmental patterns or "synexpression groups." Proteins induced by cold, heat, or salt shock were enriched in three developmental synexpression groups. In addition, certain proteins belonging to the heat and salt shock stimulons were coregulated during development. Thus, stress regulatory systems controlling these stimulons were implicated as integral parts of the developmental program. This correlation suggested that thermal shock and salt shock stress response regulatory systems either allow the cell to adapt to stresses associated with development or directly control the developmental program.     

Newcastle disease virus stimulates the cellular accumulation of stress (heat shock) mRNAs and proteins.

A biological agent, Newcastle disease virus, stimulated the synthesis of stress proteins in cultured chicken embryo cells. Previously, only physical and chemical agents were known to induce these proteins. The levels of translatable stress mRNAs were elevated in cells infected with avirulent or virulent strains; however, stress protein synthesis was stimulated strongly only in cells infected by avirulent strains. As did several other paramyxoviruses, avirulent strains of Newcastle disease virus stimulated the synthesis of glucose-regulated proteins as well as stress proteins. Possible stimuli of the synthesis of these two sets of proteins in paramyxovirus-infected cells are considered.     

Proteomic analysis of differentially expressed proteins in Lactobacillus brevis NCL912 under acid stress

Protein expression of Lactobacillus brevis NCL912 under acid stress was analysed by two-dimensional gel electrophoresis and MS. Twenty-five proteins were differentially expressed under acid stress. Among them, eight protein spots were identified by matrix-assisted laser desorption/ionization time-of-flight MS, of which seven were upregulated and one was downregulated. The function of the downregulated protein was unknown and the putative functions of the upregulated proteins were categorized as stress response, DNA repair, protein synthesis and glycolysis. Quantitative real-time PCR was used to further validate these differentially expressed proteins at the mRNA level and a positive correlation between the content of the proteins and their mRNA levels was found. The results suggest that these proteins are involved in the acid stress response mechanisms of this bacterium.     

Major E. coli heat-stress protein do not translocate: implications for cell survival

When Escherichia coli are exposed to heat stress, the majority of proteins in the process of synthesis at the time of heat stress are rapidly translocated to the outer membrane of the bacterium. The synthesis of most of these proteins appears to take place on membrane-bound polyribosomes. With the temperature shift, overall protein synthesis is inhibited while the synthesis of a small group of proteins is initiated. These proteins are not translocated, but remain in the cytosolic compartment, and they are identifiable as heat-stress proteins. Both the translocation phenomenon and the retention of heat-stress proteins in the cytosolic compartment in proximity to the nucleoid could counteract the effects of heat stress. The translocated proteins may operate by stabilizing the outer membrane prior to the induction of heat-stress proteins and the latter, which are confined to the cytoplasmic compartment, may serve to protect the integrity of the nucleoid structures.     

Alterations in Glial Cell Metabolism During Recovery from Chronic Osmotic Stress

NMR spectroscopy of F98 glioma cell extracts showed that chronic hypertonic conditions largely increased the intracellular content of small, osmotically active molecules. Moreover, hypertonic stress decreased the incorporation of ¹³C-labeled amino acids into the cellular proteins albeit their cytosolic concentrations were increased, which reflects an inhibition of protein synthesis under these conditions. Reincubation with isotonic medium restored almost completely the control values for the cytosolic metabolites but not for amino acid incorporation into the protein. An increased amount of ¹³C label was found in the phospholipids, which indicates stimulation of membrane synthesis processes due to the recovery-induced cell swelling. On the other hand, chronic hypotonic conditions largely decreased the steady state concentration and synthesis of small, cytosolic molecules, whereas the effect on the incorporation of ¹³C-labeled amino acids into the cellular proteins was variable. Reincubation with isotonic medium partially restored the depressed cytosolic metabolite content and also the incorporation of labeled amino acids into cellular protein, but induced an inhibition of phospholipid synthesis. The results verify that readaptation of glial cell metabolism during recovery from chronic osmotic stress is impaired or at least seriously retarded.     

Yeast thermotolerance does not require protein synthesis.

Heat shock at 37 degrees C induces synthesis of stress (heat shock) proteins in Saccharomyces cerevisiae and also induces thermotolerance. Amino acid analogs that are powerful inducers of stress protein synthesis failed to induce thermotolerance, suggesting that the stress proteins do not play a causal role in acquired thermotolerance at 37 degrees C. This suggestion was confirmed by the observation that protein synthesis was not required for the induction of thermotolerance at 37 degrees C.     

Patterns of protein synthesis and tolerance of anoxia in root tips of maize seedlings acclimated to a low-oxygen environment, and identification of proteins by mass spectrometry

Tolerance of anoxia in maize root tips is greatly improved when seedlings are pretreated with 2 to 4 h of hypoxia. We describe the patterns of protein synthesis during hypoxic acclimation and anoxia. We quantified the incorporation of [(35)S]methionine into total protein and 262 individual proteins under different oxygen tensions. Proteins synthesized most rapidly under normoxic conditions continued to account for most of the proteins synthesized during hypoxic acclimation, while the production of a very few proteins was selectively enhanced. When acclimated root tips were placed under anoxia, protein synthesis was depressed and no "new" proteins were detected. We present evidence that protein synthesis during acclimation, but not during subsequent anoxia, is crucial for acclimation. The complex and quantitative changes in protein synthesis during acclimation necessitate identification of large numbers of individual proteins. We show that mass spectrometry can be effectively used to identify plant proteins arrayed by two-dimensional gel electrophoresis. Of the 48 protein spots analyzed, 46 were identified by matching to the protein database. We describe the expression of proteins involved in a wide range of cellular functions, including previously reported anaerobic proteins, and discuss their possible roles in adaptation of plants to low-oxygen stress.     

The effect of heat shock on primary cultures of brain capillary endothelium: inhibition of assembly of zonulae occludentes and the synthesis of heat-shock proteins

Subjecting primary cultures of bovine brain microvessel endothelial cells to thermal stress (heat shock) results in: (1) an inhibition of further tight junction assembly, (2) the disappearance and/or disassembly of tight junctions, (3) a 30-fold increase in the number of plasmic fracture (PF)-face intramembrane particles, and (4) the new and/or enhanced synthesis of at least three heat-shock polypeptides (HSPs) with molecular masses of approximately 100,000, 90,000 and 70,000. Endothelial cells which are heat-shocked and allowed to recover at 37 degrees C exhibit, within the first 2 h, a marked depression in the synthesis of HSPs and the new and/or enhanced synthesis of a 47,000 dalton "recovery" polypeptide. In later periods of recovery (2-4 h), the synthesis of this polypeptide is even more pronounced and is accompanied by the new and/or enhanced synthesis of a polypeptide(s) with a molecular mass of 35 to 37,000. The appearance of these "recovery protein(s)" in the endothelial cells is concomitant with a decrease in the number of PF-face intramembrane particles and the resumption of tight junction assembly. Results of this study suggest that some of the HSPs synthesized by thermally-stressed cultures of brain endothelial cells may activate or be directly involved in a mechanism(s) to ensure survival of these cells by decreasing membrane fluidity and stabilizing the plasma membrane of these cells. Moreover, our results also suggest that the recovery of these cells from the stress of heat shock is accompanied by the synthesis of "recovery" proteins which, in some manner, may be directly involved in, or necessary for, rapidly reversing the membrane-stabilizing effect of heat shock by promoting membrane fluidity and the apparent amplified synthesis and assembly and/or reassembly of tight junctions.     

Heat shock induced changes in the gene expression of terminally differentiating avian red blood cells

Reticulocytes, purified from the blood of quail and chickens recovering from anaemia, respond to heat shock by the new and (or) enhanced synthesis of heat-shock protein (HSPs) with relative molecular masses of greater than 400,000, 90,000, 70,000, and 26,000 (quail) or 24,000 (chicken) and the depressed synthesis of many proteins normally produced at a control temperature. The synthesis of these HSPs is noncoordinate since the expression of each protein depends upon the particular temperature and duration of the time at that temperature. Separation of proteins from quail reticulocytes into Triton X-100 soluble and insoluble fractions demonstrates that the 70,000 and 26,000 Da HSPs are found in both fractions, whereas the greater than 400,000 and 90,000 Da HSPs are located only in the detergent-soluble fraction. Triton X-100 fractionation also reveals that there are three isoelectric variants of the 70,000 Da HSP and that they are constitutively synthesized and selectively partitioned between cellular compartments. Heat shock induced synthesis of the 90,000, 70,000, and 26,000 Da quail HSPs is prevented by actinomycin D, while enhanced synthesis of the greater than 400,000 Da HSP is unaffected by this inhibitor. These results demonstrate that nucleated, terminally differentiating avian red blood cells are capable of responding to heat stress by rapid changes in their highly restricted "program" of gene expression.     

Stress- and Growth Phase-Associated Proteins of Clostridium acetobutylicum

The response of Clostridium acetobutylicum ATCC 4259 to the stresses produced by a temperature upshift from 28°C to 45°C and by exposure of the organisms to 0.1% n-butanol or to air was examined by analysis of pulse-labeled proteins. The stress response was the induction of the synthesis of a number of proteins, some of which were elicited by the three forms of stress. Eleven heat shock proteins were identified by two-dimensional electrophoresis, as were two proteins whose synthesis was heat sensitive. In the absence of applied stress, the synthesis of four proteins was found to be associated with the growth phase in batch culture; three of these proteins had a higher rate of de novo synthesis when the cells entered the solvent production phase. One of the stress-induced proteins, hsp74, was partially purified an found to be immunologically related to Escherichia coli heat shock protein Dnak. The similarities of the proteins induced at the onset of solventogenesis and by stress suggest a relationship between the two processes.     

Inactivation of the stress- and starvation-inducible gls24 operon has a pleiotrophic effect on cell morphology, stress sensitivity, and gene expression in Enterococcus faecalis

Enterococcus faecalis induces the synthesis of at least 42 proteins during 24 h of glucose starvation. Because of its induction during carbohydrate and complete starvation (incubation in tap water) and CdCl(2) and bile salts stresses, one of these proteins (Gls24) was qualified as a "general stress protein" and was analyzed at the molecular level. Its corresponding gene, gls24, seems to be the penultimate gene of an operon composed, altogether, of six open reading frames (ORFs). The ORF preceding gls24 (orf4) showed very strong identity with gls24. The deduced polypeptides of these two genes showed similarity with a 20-kDa hypothetical protein from Lactococcus lactis and an alkaline stress protein from Staphylococcus aureus with no previously known biological significance. Data from the operon sequence and Northern analysis led to the conclusions that (i) gls24 possesses its own promoter which is especially induced at the onset of starvation and (ii) the operon promoter is stress inducible in exponential-phase cells. A mutation in the gls24 gene led to a severe reduction of growth rate and reduction of survival against 0.3% bile salts in the 24-h-starved cells compared to the wild-type strain. Moreover, the chain length of the mutant is significantly reduced during growth. These results argue strongly for a role of the protein Gls24 and/or GlsB in morphological changes and in stress tolerance in E. faecalis. Comparison of two-dimensional protein gels from wild-type cells with those from gls24 mutant cells revealed a pleiotropic effect of the mutation on gene expression. At least nine proteins were present in larger amounts in the mutant. For six of them, the corresponding N-terminal microsequence has been obtained. Three of these sequences map in genes coding for L-lactate dehydrogenase, lipoamide dehydrogenase, and pyruvate decarboxylase, all involved in pyruvate metabolism.     

Stress induced biosynthesis of a 31 kd-glycoprotein in goldfish brain

The de novo protein synthesis in goldfish brain has been studied under defined stress conditions and after training in an active swimming task. One- and two-dimensional gel electrophoresis of the brain proteins previously labelled in vivo with 35S-methionine indicated that the synthesis of one distinct protein increases after stress but not after successful training. This protein is a glycoprotein with an apparent molecular weight of 31 kd and an isoelectric point of pH 5. It is discussed that the protein may be the ACTH precursor "pro-opiomelanocortin".