Characterization of the heat shock response inEnterococcus faecalis

We have characterized the general properties of the heat shock response of the Gram-positive hardy bacteriumEnterococcus faecalis. The heat resistance (60°C or 62.5°C, 30 min) of log phase cells ofE. faecalis grown at 37°C was enhanced by exposing cells to a prior heat shock at 45°C or 50°C for 30 min. These conditioning temperatures also induced ethanol (22%, v/v) tolerance. The onset of thermotolerance was accompanied by the synthesis of a number of heat shock proteins. The most prominent bands had molecular weights in the range of 48 to 94kDa. By Western blot analysis two of them were found to be immunologically related to the well known DnaK (72 kDa) and GroEL (63 kDa) heat shock proteins ofEscherichia coli. Four other proteins showing little or no variations after exposure to heat are related to DnaJ, GrpE and Lon (La)E. coli proteins and to theBacillus subtilis ⁴³ factor. Ethanol (2% or 4%, v/v) treatments elicited a similar response although there was a weaker induction of heat shock proteins than with heat shock.     

Production of hydrogen peroxide by Lactobacillus hilgardii and its effect on Leuconostoc oenos growth

In mixed culture of Lactobacillus hilgardii X₁B and Leuconostoc oenos X₂L, isolated from Argentinian wines, an amensalistic growth response was observed: Leuconostoc oenos did not grow, and after 24 h of incubation at 30°C no viable cells were detected. In pure and mixed cultures, Lactobacillus hilgardii produced hydrogen peroxide early in the growth cycle, reaching the maximum at 24 h. The values of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for the action of hydrogen peroxide on the growth of Leuconostoc oenos were: 4.08g ml^-1 and 17.00 g ml^-1 respectively.     

Acid tolerance inLeuconostoc oenos. Isolation and characterization of an acid-resistant mutant

The acid tolerance ofLeuconostoc oenos was examined in cells surviving at pH 2.6, which is lower than the acid limit of growth (about pH 3.0). Acid-adapted cells survived better than non-adapted cells. Tolerance to acid stress was found to be dependent upon the adaptive pH. Acid resistance was increased by an order of magnitude for cultures adapted to a pH of about 2.9. Inhibiting protein synthesis with chloramphenicol prior to acid shock revealed that acid adaptation may involve two separate systems, one of which appears to be independent of protein synthesis. The acid-resistant mutant LoV8413, isolated during a long-term survival screen at pH 2.6, was found to be able to grow in acidic media and was characterized by a high H⁺-ATPase activity at low pH. The data from electrophoretic analysis of total proteins labeled with [³⁵S] methionine indicate that large amounts of a protein of 42 kDa molecular mass were produced within this acid-resistant mutant.     

Assessment of the tolerance of Lactococcus lactis cells at elevated temperatures

When Lactococcus lactis strains were exposed directly to the lethal temperature of 50 C for 30 ;min, 0.1–31% of the cells survived. However, when pre-exposed to 40 °C, prior to exposure at 50 °C, 4–61% of the cells survived. A plasmid carrying a unique heat shock gene from the thermophile Streptococcus thermophilus was cloned into L. ;lactis. When the transformed cells were cultivated at 30 °C the introduction of the plasmid had no obvious effect on the growth of L. ;lactis. However, when the temperature was abruptly shifted from 30 °C to 42 °C at mid-growth phase the growth decreased by 50%.     

The heat shock response inLocusta migratoria

Summary Locusta migratoria adults reared at 27–30°C die after 2 h at 50°C, but they survive this temperature stress if first exposed to 45°C for 0.5 to 4.5 h. Fat bodies from adult females produce a set of at least six specific polypeptides with molecular weights of 81, 73, 68, 42, 28, and 24×10³ in reponse to heat shock (39–47°C for 1.5 h). These molecular weights closely match those of the heat shock proteins (hsps) observed inDrosophila, with the possible exception of the 42 kd protein of locusts. The optimal temperature for induction of hsps in locusts is 45°C, which is one of the highest heat shock temperatures reported in metazoans. The correspondence between the optimal temperature for hsp induction and the temperature at which enhanced heat tolerance is acquired (both 45 °C) suggests that the hsps may be associated with thermal protection in these insects.There appears to be no substantial translational control in the locust heat shock response, since other proteins are produced, albeit with some reduction, under heat shock conditions. Vitellogenin synthesis in fat bodies at 45°C is 55% of that observed at 30°C. The high optimal heat shock induction temperature and the continued synthesis of non-heat shock proteins may be adaptive to the locust's natural environment.     

Isolation and characterization of immunity temperature sensitive mutants of Enterobacter cloacae harbouring the cloacinogenic factor DF13

Summary Enterobacter cloacae cells, harbouring the cloacinogenic factor DF13 (Clo DF13) are immune to the cloacin they produce. We describe the isolation of eleven Enterobacter cloacae (Clo DF13) mutants, which are immune at 30°C, but lose their immunity at 42°C. The temperature sensitive immunity (Imm^ts) of these mutants appeared not to be transferable together with the Clo DF13 factor to non-cloacinogenic acceptor strains. Apparently host mutations are involved in the Imm^ts phenotype. Two different groups of Imm^ts mutants could be identified. Imm^tsC6 and Imm^tsC8, representatives of each group, have been compared with the parent strain. Imm^tsC6 as well as Imm^tsC8 is sensitive to crude cloacin at 42°C. Imm^ts mutants appeared to be also sensitive to cell components other than cloacin, indicating that the Imm^ts mutations may result in pleiotropic changes of cell properties.The Imm^tsC6 mutant is sensitive to deoxycholate and osmotic shock at 42°C. Spheroplasts of Imm^tsC6 cells incubated at 42°C are sensitive to DOC at 42°C and 30°C. The pleiotrophic changes of the Imm^tsC6 mutant may be attributed to a defect in the cell membrane.The Imm^tsC8, incubated at 42°C, is sensitive to deoxycholate, osmotic shock, ethylene-diaminetetraacetic acid, dyes, drugs and UV. Furthermore they form filaments. Imm^tsC8 spheroplasts are as sensitive to deoxycholate as the parent strain at 42°C. The pleiotropic changes in the phenotype of Imm^tsC8 are considered to be the result of a defect in the outer layers of the cell envelope, most likely the lipopolysaccharide layer.The possible relationship between the observed structural defects in the cell envelope of Imm^ts mutants and the phenomenon of immunity have been discussed.     

Heat and cold shock responses in different strains of Thiobacillus ferrooxidans

Different strains of Thiobacillus ferrooxidans were examined for their ability to produce a heat shock and a cold shock response. Strain A1, heat shocked from 20° to 35°C, acquired thermotolerance, as it showed a 1000-fold reduction in cell mortality when exposed to the supermaximum temperature of 42°C, as compared to a non-heat-shocked control. A heat shock from 25° to 35°C yielded similar results, although a higher degree of thermotolerance was achieved for the shorter exposure times. Cultures heat shocked for 5 h showed a five-log reduction in viable counts after 41 h at 42°C, whereas non-heat-shocked cultures showed a similar reduction in viability in 28 h. Conferred thermotolerance was immediate and sustained for the duration of the exposure to 42°C. Heat-shocked cultures were not significantly protected against loss of viability due to freezing (-15°C for 24 h). Strain S2, cold shocked from 25° to 10°C, and strain D6, cold shocked from 25° to 5°C, were not protected against freezing at-15°C. An analysis of proteins extracted from heat-shocked cells of strain A1 showed the presence of at least one newly induced protein and eight hyper-induced proteins. The molecular weights of the heat shock proteins were in the range of 15–80.3 kDa.     

Basic features of the Streptococcus thermophilus heat shock response

The heat shock response was investigated in the thermophilic acid bacterium Streptococcus thermophilus. The heat resistance (58°C, 30 min) of log-phase cells grown at 42°C was enhanced by pretreatment at 52°C for 15 or 30 min. Concurrently to this acquired thermotolerance, two-dimensional gel electrophoresis indicates that the cells induced the synthesis of at least 22 heat shock proteins after temperature upshift. Furthermore, following SDS-PAGE, Western blotting, and immunological analysis, six proteins were found to be antigenically related to the Escherichia coli heat shock proteins DnaK, DnaJ, GroEL, GrpE, and La and to the Bacillus subtilis ⁴³ factor Among these six proteins, two related to DnaK and GroEL, are clearly overexpressed during this stress. It is concluded that S. thermophilus possesses a heat shock response similar to that known to occur in mesophilic microorganisms.     

Effects of heat shock and ethanol stress on the viability of aSaccharomyces uvarum (carlsbergensis) brewing yeast strain during fermentation of high gravity wort

Summary The effects of heat shock and ethanol stress on the viability of a lager brewing yeast strain during fermentation of high gravity wort were studied. These stress effects resulted in reduced cell viability and inhibition of cell growth during fermentation. Cells were observed to be less tolerant to heat shock during the fermentation of 25°P (degree Plato) wort than cells fermenting 16°P wort. Degree Plato (^oP) is the weight of extract (sugar) equivalent to the weight of sucrose in a 100 g solution at 20°C. Relieving the stress effects of ethanol by washing the cells free of culture medium, improved their tolerance to heat shock. Cellular changes in yeast protein composition were observed after 24 h of fermentation at which time more than 2% (v/v) ethanol was present in the growth medium. The synthesis of these proteins was either induced by ethanol or was the result of the transition of cells from exponential phase to stationary phase of growth. No differences were observed in the protein composition of cells fermenting 16°P wort compared to those fermenting 25°P wort. Thus, the differences in the tolerance of these cells to heat shock may be due to the higher ethanol concentration produced in 25°P wort which enhanced their sensitivity to heat shock.     

Intracellular multiplication ofLegionella pneumophila in amoebae isolated from hospital hot water tanks

We studied the ability ofLegionella to multiply in potable water samples obtained from investigations of nosocomial legionellosis. AutochthonousLegionella multiplied in three of 14 hospital water samples after incubation at 35°C and 42°C. All three samples were from hot water tanks. Multiplication did not occur when a selected sample was filtered through a 0.45-m membrane and reinoculated with indigenousLegionella. We isolated bothLegionella pneumophila and one or more species of free-living amoebae, primarity members of theHartmannellidae, from each of these hot water tank samples. Amoebae from a total of six hot water tank samples were used for cocultivation studies withL. pneumophila. All amoebae supported multiplication ofLegionella in coculture at 35°C. Four of six isolates of amoebae supported multiplication oflegionella at 42°C, while none supported multiplication at 45°C. Gimenez staining and electron microscopy showed thatLegionella multiplied intracellularly in amoebae. Control of these amoebae in potable water may prevent colonization and multiplication ofLegionella in domestic hot water systems.     

Polymorphic behavior of gram-negative bacteria membranes

Summary Freeze-fracture and ultrathin section electron microscopy as well as³¹P-NMR spectroscopy and light scattering ofEscherichia coli andPseudomonas putida cells under conditions promoting the ability of cells to take up exogenous DNA's (high concentrations of divalent cations and a specific temperature regime) reveal the extensive polymorphic changes and the formation of various structural defects in cellular membranes. Polymorphic changes occur during the heat shock at 42 to 44°C of the cells preincubated at 0°C in the presence of high concentration of Ca²⁺ or Ba²⁺ cations and include the formation of various vesicle- and tube-like structures, intermembrane and intercellular contacts followed by membrane fusion and sometimes even by cell fusion. The results obtained suggest the occurrence of phospholipid-enriched zones in the outer leaflet ofE. coli outer membrane. This suggestion is verified and confirmed with the help of phospholipase C, a specific phospholipid binding and digesting enzyme. The presented experimental evidence directly supports the suggestion of Ahkong et al. (Nature 253:194–195, 1975) on the identity of the mechanisms of membrane contact formation and membrane fusion in model and cellular membranes. The biological relevance of the polymorphic changes observed is shortly discussed.     

Effects of cadmium on gene expression in cadmium-tolerant and cadmium-sensitiveDatura innoxia cells

The effect of Cd on gene expression in suspension cultures of twoDatura innoxia cell lines with differing Cd tolerance was studied.In vivo labeling experiments using [³H] leucine showed that Cd induced the synthesis of a similar range of proteins in both cell lines at a concentration which will kill the sensitive but not the tolerant cells. Corresponding changes in levels of translatable mRNA were also observed. The induction of the synthesis of proteins by Cd was transient since Cd-tolerant cells growing continuously in 250 M CdCl₂ contained a similar set ofin vitro translation products to cells growing in the absence of Cd. Although Cd had a similar effect on gene expression in both cell lines, Cd-tolerant cells possess two abundant mRNAs which are constitutively produced. These mRNAs encode proteins of low molecular weight (about 11 kDa) and are either absent or present at a low level in Cd-sensitive cells. The functions of these proteins are not known but they may be involved in the tolerance mechanism. Two-dimensional gel electrophoresis ofin vitro translation products showed that many of the Cd-induced proteins are also induced by heat shock. A 42°C heat shock resulted in agreater range and more intense induction of translatable mRNAs than 4 h exposure to 250 M CdCl₂. However a subset of mRNAs were induced specifically by Cd while other mRNAs were heat shock-specific. There was no difference in the ability of the two cell lines to tolerate heat shock. This was also reflected by the same pattern of major proteins induced by heat shock in the two cell lines.     

Initiation of chromosomal DNA replication which is stimulated without oversupply of DnaA protein in Escherichia coli

Summary The temperature-sensitive dnaA46 mutation in Escherichia coli can be phenotypically suppressed at 42° C by oversupply of GroELS proteins, and the suppressed cells grow extremely slowly at 30° C. We found that the phenotype of dnaA46 showing this cold sensitivity was dominant over the phenotype of dnaA ⁺, and could not be rescued by introduction of oriC-independent replication systems. These results suggest that the cold sensitivity was not caused by a simple defect in replication. When a growing culture of a dnaA46 strain with a GroELS-overproducing plasmid was shifted from 42° to 30° C in the presence of chloramphenicol, the chromosomal DNA replicated excessively. Initiation of replication occurred at the site of oriC repeatedly four or five times during a 4 h incubation period without concomitant protein synthesis, indicating an excessive capacity for initiation. Such overreplication did not take place at 42° C in the suppressed dnaA46 strain, or at either temperature in GroELS-oversupplied dnaA ⁺ cells. No significant difference was detected between the cellular content of DnaA protein in suppressed cells where the initiation capacity was abnormally high, and that in wild-type cells in which the initiation capacity was normal. Thus, DnaA protein might function in vivo through some phase control mechanism for initiation, apart from a simple regulation by its total amount. A possible mechanism is proposed based on the participation of GroELS proteins in protein folding.A preliminary account of this work was presented at the Annual Meeting of the Molecular Biology Society of Japan in 1989.     

Bioconversion of acid-hydrolyzed poplar hemicellulose to acetic acid byClostridium thermoaceticum

Summary Clostridium thermoaceticum was used to ferment carbohydrate released from pretreated oat splet xylan and hemicellulose isolated from hybrid poplar. Hydrolysis with dilute sulfuric acid (2.5% (v/v) for oat spelt xylan and 4.0% (v/v) for poplar hemicellulose) at 100°C for 60 min was found to release the highest concentration of fermentable substrate.C. thermoaceticum, when grown in non-pH controlled batch culture at 55°C under a headspace of 100% CO₂, typically produced 14gl^–1 acetic acid during a 48 h fermentation in medium containing 2% xylose. In fed-batch fermentations this organism was able to produce 42gl^–1 acetic acid after 116h when the concentration of xylose was maintained at approximately 2% and the pH was controlled at 7.0.     

Gene affecting longevity of messenger RNA: a mutant of Escherichia coli with altered mRNA stability.

Summary we have screened 897 temperature sensitive growth mutants ofE. coli for mutant strains showing longer mRNA half-life. The fate of pulse-labelled RNA was examined at 42° C after cessation of RNA synthesis and with prior exposure to nonpermissive temperature (42° C). Eight stains showed altered turn-over of RNA (presumably mRNA), and further analysis on mutant strain JE15144 indicated that the stability of pulse-labeled RNA as well as of tryptophan (trp) mRNA increased four to seven fold over its parental strain at 42° C. At 4 min or 10 min after addition of rifampicin, some 70 to 80% of polyribosome in the growing cells could still be conserved in JE15144 cultured at the nonpermissive temperature while little, if any, polyribosomes remained in its parental strain (PA3092) under the same condition. Two generation times were required for complete stoppage of growth of this mutant strain after shifting to 42° C, and protein synthesis continued at a significant, but slightly reduced, rate at 42° C. However, functional decay of mRNA in the mutant strain, with respect to the capacity for producing peptides, appeared to be similar to the parent strain, with half-lives of 3.5 min in PA3092 and 4.7 min in JE15144.     

Heat shock protein induction and the acquisition of thermotolerance in the psychrotrophic yeastTrichosporon pullulans

Two-dimensional gel electrophoretic analysis of the heat shock response in the psychrotrophic yeastTrichosporon pullulans revealed the induction of 11 heat shock proteins (hsps) after a 5° to 21°C heat shock, 12 hsps after a 5° to 26°C heat shock, and 12 hsps after a 5° to 29°C heat shock. Heat shock from 5° to 26° or 29°C resulted in a statistically significant increase in thermotolerance to a lethal heat challenge at 45°C for 5 min. When the protein synthesis inhibitor, cycloheximide, was added prior to the heat shock, no statistically significant thermotolerance was acquired. To confirm the correlation between the synthesis of hsps and the acquisition of thermotolerance, protein extracts of cells that had been heat shocked in the presence or absence of cycloheximide were electrophoretically analyzed. Addition of the same concentration of cycloheximide that prevented the acquisition of thermotolerance also inhibited the synthesis of any hsps.     

A mutant in a major heat shock protein of Escherichia coli continues to show inducible thermotolerance

Summary Escherichia coli cells carrying the dnaK756 mutation, were inactivated at 52°C faster than control cells. This suggests that the intact dnaK gene product plays a role in protecting the cell from lethal damage at 52°C. The effect of the dnaK mutation on induced thermotolerance was examined. Prior heat shock at 42°C greatly lowered the subsequent inactivation rate in both mutant and control cells. This result suggests that, although produced in large amounts in response to thermal stress, mutation in the DnaK protein has little or no effect on induced thermotolerance.     

Synthesis of heat shock proteins inThermoanaerobacterium thermosulfurigenes EM1 (Clostridium thermosulfurogenes EM1)

The response to heat stress was examined inThermoanaerobacterium thermosulfurigenes EM1. Upon a temperature shift-up from 50° to 62°C, four heat shock proteins (hsps) were synthesized at an elevated level. Two proteins were found to be immunologically related to theEscherichia coli GroEL protein and theMycobacterium tuberculosis hsp71 (DnaK similar protein), and the correspondinggroE anddnaK homologous sequences were detected in the chromosome ofT. thermosulfurigenes EM1. The heat shock response in this thermophile was transient, with a maximum synthesis of hsps between 10 and 15 min after the shock. The enhanced synthesis of DnaK and GroEL was consistent with increased mRNA levels of the genes, which reached a maximum 15 min after heat treatment.     

Effects of acetic acid on the temperature profile of ethanol tolerance inSaccharomyces cerevisiae

Summary In a strain ofSaccharomyces cerevisiae, acetic acid at concentrations up to 1% (v/v) depressed the tolerance to added ethanol, from 11% (v/v) down to zero, and simultaneously narrowed the temperature range of growth from 3–42°C to 19–26°C. In addition, acetic acid shifted the associative temperature profile of growthand death to lower temperatures, and depressed the growth yield on glucose.     

Erythromycin resistant mutations in Bacillus subtilis cause temperature sensitive sporulation.

Summary All of several hundred erythromycin resistant (ery^R) single site mutants ofBacillus subtilis W168 are temperature sensitive for sporulation (spo^ts). The mutants and wild type cells grow vegetatively at essentially the same rates at both permissive (30° C) and nonpermissive (47° C) temperatures. In addition, cellular protein synthesis, cell mass increases and cell viabilities are similar in mutant and wild type strains for several hours after the end of vegetative growth (47° C). In the mutants examined, the temperature sensitive periods begin when the sporulation process is approximately 40% completed, and end when the process is 90% complete. At nonpermissive temperatures, the mutants produce serine and metal proteases at 50% of the wild type rate, accumulate serine esterase at 16% of the wild type rate, and do not demonstrate a sporulation related increase in alkaline phosphatase activity.The ery^R and spo^ts phenotypes cotransform 100%, and cotransduce 100% using phage PBS1. Revertants selected for ability to sporulate normally at 47° C (spo⁺), simultaneously regain parental sensitivity to erythromycin. No second site revertants are found.Ribosomes from ery^R spo^ts strains bind erythromycin at less than 1% of the wild type rate. A single 50S protein (L17) from mutant ribosomes shows an altered electrophoretic mobility. Ribosomes from spo⁺ revertants bind erythromycin like parental ribosomes and their proteins are electrophoretically identical to wild type. These data indicate that the L17 protein of the 50S ribosomal subunit fromBacillus subtilis may participate specifically in the sporulation process.