Heat and cold shock protein synthesis in arctic and temperate strains of rhizobia.

We compared heat shock proteins (HSPs) and cold shock proteins (CSPs) produced by different species of Rhizobium having different growth temperature ranges. Several HSPs and CSPs were induced when cells of three arctic (psychrotrophic) and three temperate (mesophilic) strains of rhizobia were shifted from their optimal growth temperatures (arctic, 25 degrees C; temperate, 30 degrees C) to shock temperatures outside their growth temperature ranges. At heat shock temperatures, three major HSPs of high molecular weight (106,900, 83,100, and 59,500) were present in all strains for all shock treatments (29, 32, 36.4, 38.4, 40.7, 41.4, and 46.4 degrees C), with the exception of temperate strains exposed to 46.4 degrees C, in which no protein synthesis was detected. Cell survival of arctic and temperate strains decreased markedly with the increase of shock temperature and was only 1% at 46.4 degrees C. Under cold shock conditions, five proteins (52.0, 38.0, 23.4, 22.7, and 11.1 kDa) were always present for all treatments (-2, -5, and -10 degrees C) in arctic strains. Among temperate strains, five CSPs (56.1, 37.1, 34.4, 17.3, and 11.1 kDa) were present at temperatures down to 0 degrees C. The 34.4- and the 11.1-kDa components were present in all temperate strains at -5 degrees C and in one strain at -10 degrees C. Survival of all strains decreased with cold shock temperatures but was always higher than 50%. These results show that rhizobia can synthesize proteins at temperatures not permissive for growth. In all shock treatments, no correspondence between the number of HSPs or CSPs produced and rhizobial survival was found.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heat and cold shock protein synthesis in arctic and temperate strains of rhizobia.

We compared heat shock proteins (HSPs) and cold shock proteins (CSPs) produced by different species of Rhizobium having different growth temperature ranges. Several HSPs and CSPs were induced when cells of three arctic (psychrotrophic) and three temperate (mesophilic) strains of rhizobia were shifted from their optimal growth temperatures (arctic, 25 degrees C; temperate, 30 degrees C) to shock temperatures outside their growth temperature ranges. At heat shock temperatures, three major HSPs of high molecular weight (106,900, 83,100, and 59,500) were present in all strains for all shock treatments (29, 32, 36.4, 38.4, 40.7, 41.4, and 46.4 degrees C), with the exception of temperate strains exposed to 46.4 degrees C, in which no protein synthesis was detected. Cell survival of arctic and temperate strains decreased markedly with the increase of shock temperature and was only 1% at 46.4 degrees C. Under cold shock conditions, five proteins (52.0, 38.0, 23.4, 22.7, and 11.1 kDa) were always present for all treatments (-2, -5, and -10 degrees C) in arctic strains. Among temperate strains, five CSPs (56.1, 37.1, 34.4, 17.3, and 11.1 kDa) were present at temperatures down to 0 degrees C. The 34.4- and the 11.1-kDa components were present in all temperate strains at -5 degrees C and in one strain at -10 degrees C. Survival of all strains decreased with cold shock temperatures but was always higher than 50%. These results show that rhizobia can synthesize proteins at temperatures not permissive for growth. In all shock treatments, no correspondence between the number of HSPs or CSPs produced and rhizobial survival was found.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced levels of cold shock proteins in Listeria monocytogenes LO28 upon exposure to low temperature and high hydrostatic pressure

Listeria monocytogenes is a psychrotrophic food-borne pathogen that is problematic for the food industry because of its ubiquitous distribution in nature and its ability to grow at low temperatures and in the presence of high salt concentrations. Here we demonstrate that the process of adaptation to low temperature after cold shock includes elevated levels of cold shock proteins (CSPs) and that the levels of CSPs are also elevated after treatment with high hydrostatic pressure (HHP). Two-dimensional gel electrophoresis combined with Western blotting performed with anti-CspB of Bacillus subtilis was used to identify four 7-kDa proteins, designated Csp1, Csp2, Csp3, and Csp4. In addition, Southern blotting revealed four chromosomal DNA fragments that reacted with a csp probe, which also indicated that a CSP family is present in L. monocytogenes LO28. After a cold shock in which the temperature was decreased from 37 degrees C to 10 degrees C the levels of Csp1 and Csp3 increased 10- and 3.5-fold, respectively, but the levels of Csp2 and Csp4 were not elevated. Pressurization of L. monocytogenes LO28 cells resulted in 3.5- and 2-fold increases in the levels of Csp1 and Csp2, respectively. Strikingly, the level of survival after pressurization of cold-shocked cells was 100-fold higher than that of cells growing exponentially at 37 degrees C. These findings imply that cold-shocked cells are protected from HHP treatment, which may affect the efficiency of combined preservation techniques.     

Cold shock and cold acclimation proteins in the psychrotrophic bacterium Arthrobacter globiformis SI55.

The psychrotrophic bacterium Arthrobacter globiformis SI55 was grown at 4 and 25 degrees C, and the cell protein contents were analyzed by two-dimensional electrophoresis. Cells subjected to cold shocks of increasing magnitude were also analyzed. Correspondence analysis of protein appearance distinguished four groups of physiological significance. Group I contained cold shock proteins (Csps) overexpressed only after a large temperature downshift. Group II contained Csps with optimal expression after mild shocks. Group III contained proteins overexpressed after all cold shocks. These last proteins were also overexpressed in cells growing at 4 degrees C and were considered to be early cold acclimation proteins (Caps). Group IV contained proteins which were present at high concentrations only in 4 degrees C steady-state cells and appeared to be late Caps. A portion of a gene very similar to the Escherichia coli cspA gene (encoding protein CS7.4) was identified. A synthetic peptide was used to produce an antibody which detected a CS7.4-like protein (A9) by immunoblotting two-dimensional electrophoresis gels of A. globiformis SI55 total proteins. Unlike mesophilic microorganisms, this CS7.4-like protein was still produced during prolonged growth at low temperature, and it might have a particular adaptive function needed for balanced growth under harsh conditions. However, A9 was induced at high temperature by chloramphenicol, suggesting that CS7.4-like proteins have a more general role than their sole implication in cold acclimation processes.     

Unfolding and double-stranded DNA binding of the cold shock protein homologue Cla h 8 from Cladosporium herbarum

The cloning, purification, and biophysical characterization of the first eukaryotic cold shock protein homologue, Cla h 8, expressed as single functional polypeptide is reported here. It was discovered as a minor allergen of the mold Cladosporium herbarum by phage display using a library selectively enriched for IgE-binding proteins. Based on the sequence homology of Cla h 8 with bacterial cold shock proteins (CSPs), a homology-based computer model of the allergen was computed indicating an all-beta structure of Cla h 8. This major structural feature was confirmed by CD spectroscopy. Despite the structural similarities with bacterial CSPs, the DNA-binding and unfolding behavior of Cla h 8 exhibited unique and previously undescribed characteristics. High affinities of Cla h 8 for single-stranded DNA as well as for double-stranded DNA corresponding to the human Y-box were detected. The affinity for double-stranded DNA increased significantly with decreasing temperature, which was paralleled by an increase in the beta sheet content of the protein. Temperature-dependent fluorescence anisotropy and far-UV CD measurements revealed different unfolding transitions at 28 and at 35.7 degrees C, respectively, indicating a multistate transition, which is uncommon for CSPs. The enhanced affinity for DNA at low temperatures together with the low unfolding transition refer to the functional significance of Cla h 8 at reduced temperatures.     

Protein synthesis during cold shock in barley tissues

Summary In barley (Hordeum vulgare L.) seedlings, a temperature step-down from 24 °C to 6°C (cold shock) determined a reduction in the incorporation of labeled aminoacids and modified the electrophoretic pattern of total proteins. At 6 °C some new proteins appeared and others were intensified (cold shock-induced proteins= CSPs); meantime, few proteins disappeared or were curtailed (cold-repressed proteins=CRPs). The majority of the proteins of the seedlings were labeled at about the same rate both at 6 °C and 24 °C, whereas at 0 °C only the cold shock proteins and a few others were detectable. The cold shock-induced variations of the protein profile differed in roots and in seed remnants which showed only some of the CSPs detected in roots. Total protein synthesis of barley genotypes Onice and Georgie, which have respectively a winter and spring growth habit, were similarly inhibited by a temperature drop. The two genotypes, however, showed some differences in the CSPs and CRPs pattern. Because Onice and Georgie have also a different thermotolerance, the hypothesis can be made that in barley specific CSPs are involved in conferring various degrees of cold resistance.     

The synthesis of cold shock proteins and cold acclimation proteins in the psychrophilic bacteriumAquaspirillum arcticum

The synthesis of cold shock proteins (csps) in response to cold shock, and of cold acclimation proteins (caps) in response to continuous growth at low temperature, in the psychrophileAquaspirillum arcticum was investigated. With two-dimensional gel electrophoresis and computing scanning laser densitometry, cold shock treatments (10° to 0°C, 5° to 0°C, and 10° to 5°C) induced a total of 14 csps, 6 of which were induced by all three cold shocks. The production of caps in response to continuous growth at 0°C was also found. Five of the 8 caps produced were also csps which suggests that these proteins may share a common involvement in cold adaptation.     

Selective mRNA degradation by polynucleotide phosphorylase in cold shock adaptation in Escherichia coli

Upon cold shock, Escherichia coli cell growth transiently stops. During this acclimation phase, specific cold shock proteins (CSPs) are highly induced. At the end of the acclimation phase, their synthesis is reduced to new basal levels, while the non-cold shock protein synthesis is resumed, resulting in cell growth reinitiation. Here, we report that polynucleotide phosphorylase (PNPase) is required to repress CSP production at the end of the acclimation phase. A pnp mutant, upon cold shock, maintained a high level of CSPs even after 24 h. PNPase was found to be essential for selective degradation of CSP mRNAs at 15 degrees C. In a poly(A) polymerase mutant and a CsdA RNA helicase mutant, CSP expression upon cold shock was significantly prolonged, indicating that PNPase in concert with poly(A) polymerase and CsdA RNA helicase plays a critical role in cold shock adaptation.     

Cold shock domain proteins and glycine-rich RNA-binding proteins from Arabidopsis thaliana can promote the cold adaptation process in Escherichia coli

Despite the fact that cold shock domain proteins (CSDPs) and glycine-rich RNA-binding proteins (GRPs) have been implicated to play a role during the cold adaptation process, their importance and function in eukaryotes, including plants, are largely unknown. To understand the functional role of plant CSDPs and GRPs in the cold response, two CSDPs (CSDP1 and CSDP2) and three GRPs (GRP2, GRP4 and GRP7) from Arabidopsis thaliana were investigated. Heterologous expression of CSDP1 or GRP7 complemented the cold sensitivity of BX04 mutant Escherichia coli that lack four cold shock proteins (CSPs) and is highly sensitive to cold stress, and resulted in better survival rate than control cells during incubation at low temperature. In contrast, CSDP2 and GRP4 had very little ability. Selective evolution of ligand by exponential enrichment (SELEX) revealed that GRP7 does not recognize specific RNAs but binds preferentially to G-rich RNA sequences. CSDP1 and GRP7 had DNA melting activity, and enhanced RNase activity. In contrast, CSDP2 and GRP4 had no DNA melting activity and did not enhance RNAase activity. Together, these results indicate that CSDPs and GRPs help E.coli grow and survive better during cold shock, and strongly imply that CSDP1 and GRP7 exhibit RNA chaperone activity during the cold adaptation process.     

Influence of cooling temperature and duration on cold adaptation of Lactobacillus acidophilus RD758

The effect of different cooling temperatures and durations on resistance to freezing and to frozen storage at -20 degrees C in Lactobacillus acidophilus RD758 was studied, by using a central composite rotatable design. A cold adaptation was observed when the cells were maintained at moderate temperature (26 degrees C) for a long time (8h) before being cooled to the final temperature of 15 degrees C. These conditions led to a low rate of loss in acidification activity during frozen storage (0.64 minday(-1)) and a high residual acidification activity after 180 days of frozen storage (1011 min). The experimental design allowed us to determine optimal cooling conditions, which were established at 28 degrees C during 8h. Adaptation to cold temperatures was related to an increase in the unsaturated to saturated fatty acid ratio and in the relative cycC19:0 fatty acid concentration. Moreover, an increased synthesis of four specific proteins was observed as an adaptive response to the optimal cooling conditions. They included the stress protein ATP-dependent ClpP and two cold induced proteins: pyruvate kinase and a putative glycoprotein endopeptidase.     

Expression of heat shock and cold shock proteins in the gorgonian Leptogorgia virgulata

In this study, we analyzed the response of the temperate, shallow-water gorgonian, Leptogorgia virgulata, to temperature stress. Proteins were pulse labeled with (35)S-methionine/cysteine for 1 h to 2 h at 22 degrees C (control), or 38 degrees C, or for 4 h at 12.5 degrees C. Heat shock induced synthesis of unique proteins of 112, 89, and 74 kDa, with 102, 98 and 56 kDa proteins present in the control as well. Cold shock from 22 degrees C-12.5 degrees C induced the synthesis of a 25 kDa protein, with a 44 kDa protein present in the control as well. Control samples expressed unique proteins of 38, and 33 kDa. Non-radioactive proteins expressed under the same conditions as above, as well as natural field conditions, were tested for reactivity with antibodies to heat shock proteins (HSPs). HSP60 was the major protein found in L. virgulata. Although HSP47, HSP60, and HSP104 were present in all samples, the expression of HSP60 was enhanced in heat stressed colonies, while HSP47 and HSP104 expression were greatest in cold shocked samples. Inducible HSP70 was expressed in cold-shocked, heat-shocked, and field samples. Constitutively expressed HSP70 was absent from all samples. The expression of HSP90 was limited to heat shocked colonies. The expression of both HSP70 and HSP104 suggests that the organism may also develop a stress tolerance response.     

CspA, CspB, and CspG, major cold shock proteins of Escherichia coli, are induced at low temperature under conditions that completely block protein synthesis

CspA, CspB, and CspG, the major cold shock proteins of Escherichia coli, are dramatically induced upon temperature downshift. In this report, we examined the effects of kanamycin and chloramphenicol, inhibitors of protein synthesis, on cold shock inducibility of these proteins. Cell growth was completely blocked at 37 degrees C in the presence of kanamycin (100 microgram/ml) or chloramphenicol (200 microgram/ml). After 10 min of incubation with the antibiotics at 37 degrees C, cells were cold shocked at 15 degrees C and labeled with [35S]methionine at 30 min after the cold shock. Surprisingly, the synthesis of all these cold shock proteins was induced at a significantly high level virtually in the absence of synthesis of any other protein, indicating that the cold shock proteins are able to bypass the inhibitory effect of the antibiotics. Possible bypass mechanisms are discussed. The levels of cspA and cspB mRNAs for the first hour at 15 degrees C were hardly affected in the absence of new protein synthesis caused either by antibiotics or by amino acid starvation.     

A cold acclimation protein with refolding activity on frozen denatured enzymes

We found that a cold acclimation protein from an ice-nucleating bacterium, Patoea ananas KUIN-3, has refolding activity on frozen denatured protein. Based on a SDS-PAGE analysis, we confirmed that the cold shock-treated cells of strain KUIN-3 could produce some cold acclimation proteins that inhibit their syntheses by the addition of chloramphenicol during the cold acclimation. Among such proteins, Hsc25 had refolding activity similar to GroELS. Hsc25 was purified to apparent homogeneity by (NH4)2SO4 precipitation and some chromatographies. The purified Hsc25 was composed of 8 subunits of 25,000 each with a molecular mass of 200,000 and had refolding activity against denatured enzymes, which were denatured by heat-treatment at 100 degrees C, cryopreservation at -20 degrees C, or guanidine hydrochloride, in a manner similar to GroELS. The N-terminal sequence of Hsc25 was Met-Arg-Ala-Ser-Thr-Tyr-His-Ala-Ala-Arg-. Furthermore, Hsc25 had a high level of activity at low temperature (12 degrees C). Also, the dissociation constants, KD (M) as the binding specificity for enolase, mutarotase, isocitrate dehydrogenase, and lactate dehydrogenase were 1.82x10(-10), 4.35x10(-9), 8.98x10(-12), and 3.05x10(-11), respectively. The affinity of Hsc25 for frozen danatured enzymes was higher than the affinity for heat denatured enzymes when compared with the affinity of GroEL. These results are the first report on the characterization of a purified chaperon that was induced by cold acclimation.     

Thermoregulation in the cold after physical training at different ambient air temperatures

Since human thermoregulation at rest is altered by cold exposure, it was hypothesized that physical training under cold conditions would alter thermoregulation. Three groups (n = 8) of male subjects (mean age 24.3 +/- 0.9 years) were evaluated: group T (interval training at 21 degrees C), group CT (interval training at 1 degrees C), and group C (no training, equivalent exposure to 1 degrees C). Each group was submitted, before and after 4 weeks of interval training (5 d/week), to a cold air test at rest (SCAT) (dry bulb temperature (Tdb) = 1 degrees C) for a 2-h period for evaluation of the thermoregulatory responses. During SCAT, after the training/acclimation period, group T exhibited a higher rectal temperature (Tre) (P < 0.05) without significant change in mean skin temperature (Tsk) whereas metabolic heat production (M) was higher at the beginning of the SCAT (P < 0.05). For group CT, no thermoregulatory change was observed. Group C showed a lower Tre (P < 0.05) without significant change in either Tsk or in M, suggesting the development of a hypothermic general cold adaptation. This study showed, first, that the cold thermoregulatory responses induced by an interval training differed following the climatic conditions of the training and, second, that this training performed in the cold prevented the development of a general cold adaptation.