Identification of an Iron-Binding Protein of the Dps Family Expressed by <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis>

Streptococcus thermophilus PB18 can grow between 20° and 52°C and is resistant to various stresses such as heat, acidic or cold shock. During cold shock, a protein of 21.5 kDa was previously shown to be induced in S. thermophilus. In addition to its cold-shock induction, 2D-PAGE revealed that the 21.5-kDa protein was also expressed during the stationary phase of growth. The recent access to the genome sequence of S. thermophilus LMG18311 allowed the identification of a 173-amino acid protein displaying a strong homology between the 21.5-kDa protein and members of the Dps family of proteins. Specific staining of non-denaturing polyacrylamide gel electrophoresis (ND-PAGE) followed by two-dimensional PAGE (2D-PAGE) showed that the 21.5-kDa protein was an iron-binding protein.     

Identification of a Cold Shock Gene in Lactic Acid Bacteria and the Effect of Cold Shock on Cryotolerance

When Lactic Acid Bacterial cultures were frozen at −20°C for 24 h, the cell viability decreased drastically, but when they were cold shocked at 10°C for 2 h prior to freezing, viability improved significantly for the Lactococcus lactis subsp. lactis strains (25–37%) and Pediococcus pentosaceus PO2 (18%), but not for the Lactococcus lactis subsp. cremoris strains tested or for one strain of Lactobacillus helveticus LB1 and Streptococcus thermophilus TS2. When the period for cold shock was extended to 5 h, the viability increased even further for those strains that displayed cold shock cryotolerance. Use of degenerate PCR primers based on the major cold shock protein (csp) of both Escherichia coli and Bacillus subtilis resulted in PCR products from all strains tested. The PCR product from Lactococcus lactis ssp. lactis M474 was cloned and sequenced, and the deduced amino acid sequence displayed a high sequence similarity to other csp's. Use of PCR primers based on the M474 sequence resulted in PCR products being produced only from the lactococcal strains studied and not from the Lactobacillus helveticus, Streptococcus thermophilus, or Pediococcus pentosaceus strains tested. Received: 18 October 1996 / Accepted: 28 January 1997     

Induction of cold shock proteins in Bacillus subtilis

The cold shock response in the Gram-positive soil bacterium Bacillus subtilis is described. Cells were exposed to sudden decreases in temperature from their optimal growth temperature of 37°C. The B. subtilis cells were cold shocked at 25°C, 20°C, 15°C, and 10°C. A total of 53 polypeptides were induced at the various cold shock temperatures and were revealed by two-dimensional gel electrophoresis. General stress proteins were identified by a comparative analysis with the heat shock response of B. subtilis. Some unique, prominent cold shock proteins such as the 115 kDa, 97 kDa, and 21 kDa polypeptides were microsequenced. Sequence comparison demonstrated that the 115-kDa protein had homology to the TCA cycle enzyme, aconitase.     

Adaptive Response to Cold Temperatures in Vibrio vulnificus

The effectiveness of rapid chilling or freezing of oysters to reduce Vibrio vulnificus levels in shellfish may be compromised by product handling procedures that permit cold adaptation. When a V. vulnificus culture was shifted from 35°C to 6°C conditions, it underwent transition to a non-culturable state. Cells adapted to 15°C prior to change to 6°C condition, however, remain viable and culturable. In addition, cultures adapted to 15°C were able to survive better upon freezing at −78°C compared with cultures frozen directly from 35°C. Inhibition of protein synthesis by addition of chloramphenicol in a V. vulnificus culture immediately prior to the exposure to the adaptive temperature eliminated inducible cold tolerance. These results suggest that cold-adaptive “protective” proteins may enhance survival and tolerance at cold temperatures. In addition, removal of iron from the growth medium by adding 2,2′-Dipyridyl prior to cold adaptation decreased the viability by approximately 2 logarithm levels. This suggests that iron plays an important role in adaptation at cold temperatures. Analysis of total cellular proteins on an SDS polyacrylamide gel electrophoresis, labeled with ³⁵S-methionine during exposure at 15°C, showed elevated expressions of a 6-kDa and a 40-kDa protein and decreased expression of an 80-kDa protein. These results suggest that, for V. vulnificus, survival and tolerance at cold temperatures could be due to the expression of cold-adaptive proteins other than previously documented major cold shock proteins such as CS7.4 and CsdA. In this study, for the first time we have shown that exposure to an intermediate cold temperature (15°C) causes a cold adaptive response, helping this pathogen remain in culturable state when exposed to a much colder temperature (6°C). This adaptive nature to cold temperatures could be important for shellfish industry efforts to reduce the risk of V. vulnificus infection from consuming raw oysters. Received: 30 July 1998 / Accepted: 1 October 1998     

Overexpression of Cold Shock Protein A of <Emphasis Type="Italic">Psychromonas arctica</Emphasis> KOPRI 22215 Confers Cold-Resistance

A polar bacterium was isolated from Arctic sea sediments and identified as Psychromonas artica, based on 16S rDNA sequence. Psychromonas artica KOPRI 22215 has an optimal growth temperature of 10 °C and a maximum growth temperature of 25 °C, suggesting this bacterium is a psychrophile. Cold shock proteins (Csps) are induced upon temperature downshift by more than 10 °C. Functional studies have researched mostly Csps of a mesophilic bacterium Escherichia coli, but not on those of psychrophilic bacteria. In an effort to understand the molecular mechanisms of psychrophilic bacteria that allow it withstand freezing environments, we cloned a gene encoding a cold shock protein from P. artica KOPRI 22215 (CspA_Pa) using the conserved sequences in csp genes. The 204 bp-long ORF encoded a protein of 68 amino acids, sharing 56% homology to previously reported E. coli CspA protein. When CspA_Pa was overexpressed in E. coli, it caused cell growth-retardation and morphological elongation. Interestingly, overexpression of CspA_Pa drastically increased the host’s cold-resistance by more than ten times, suggesting the protein aids survival in polar environments.     

Effect of Different Temperature Downshifts on Protein Synthesis by <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>

The psychrotrophic bacterium Aeromonas hydrophila 7966 was subjected to cold shocks from 30°C to 20°C, 15°C, 10°C, or 5°C, or were incubated at low temperature to determine its adaptative response. The cell protein patterns analyzed by two-dimensional electrophoresis revealed that only a few proteins were underexpressed, whereas numerous new proteins appeared with the decrease of temperature, and some others were overexpressed. Among them, a few constituted cold shock proteins because they were transiently induced, whereas others belong to the acclimatation family proteins. Two cold shock proteins of 11 kDa were synthesized at low level because they were visualized only after radiolabeling or silver staining. Moreover, under our experimental conditions, no major cold shock protein of a molecular mass similar to that of E. coli (7.4 kDa) could be identified.     

Heat Shock Protein Produced by Helicobacter pylori

The cells of Helicobacter pylori were suspended in the medium containing³⁵S-methionine. After a heat shock of the cells at 42 C for 5, 10, and 30 min, the production of proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Out of many proteins produced by the cells, only 66 kDa protein production was dramatically increased by heat treatment. The N-terminal amino acid sequence of 66 kDa protein was quite similar to that of 62 kDa and 54 kDa proteins previously suggested as heat shock protein (HSP) of H. pylori based on the reaction with polyclonal and monoclonal antibodies against HSP 60 family proteins produced by other bacteria. Therefore, it was concluded that H. pylori produces the 66 kDa protein as its major heat shock protein which belongs to HSP 60 family.     

Heat shock and cold shock in Deinococcus radiodurans

On the basis of acquired thermotolerance and cryotolerance, the optimal heat shock and cold shock temperatures have been determined for Deinococcus radiodurans. A heat shock at 42°C maximized survival at the lethal temperature of 52°C and a cold shock at 20°C maximized survival after repeated freeze-thawing. Enhanced survival from heat shock was found to be strongly dependent on growth stage, with its greatest effect shortly after phase. Increased synthesis of a total of 67 proteins during heat shock and 42 proteins during cold shock were observed by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and autoradiography. Eight of the most highly induced heat shock proteins shown by 2D PAGE were identified by MALDI-MS as Hsp20, GroEL, DnaK, SodA, Csp, Protease I and two proteins of unknown function.     

Ethylene Glycol Utilization,Cold and Ethylene Glycol Shockand Acclimation Proteins in a Psychrotrophic Bacterium

A psychrotrophic Pseudomonas fluorescens was isolated that utilizes ethylene glycol as a sole carbon source, with removal efficiencies of 98% and 96% in 20 and 55 days at 25° and 5°C, respectively. The response of the psychrotroph to environmental shifts was investigated using two-dimensional SDS-PAGE and computing scanning laser densitometry. During a 25°C to 5°C cold shock, the microorganism induced ten cold shock proteins. Under conditions of constant growth at 5°C, five cold acclimation proteins were synthesized. Ethylene glycol shock induced 14 ethylene glycol shock proteins. Ten ethylene glycol acclimation proteins were found. Similarities between the shock proteins and acclimation proteins for cold shock and acclimation and the ethylene glycol shock and acclimation may suggest that these proteins are of significance to both shock recovery as well as constant growth in a new environment.     

Expression of heat shock proteins in response to high and low temperature extremes in diapausing pharate larvae of the gypsy moth,Lymantria dispar

Diapausing pharate first instars of the gypsy moth, Lymantria dispar, respond to high temperature (37–41°C) by suppressing normal protein synthesis and synthesizing a set of seven heat shock proteins with M_rs of 90,000, 75,000, 73,000, 60,000, 42,000, 29,000, and 22,000 as determined by SDS-PAGE. During recovery at 25°C from heat shock, synthesis of the heat shock proteins gradually decreases over a period of 6 h, while normal protein synthesis is restored. A subset of these same heat shock proteins is also expressed during recovery at 4°C or 25°C from brief exposures to low temperature (-10 to 20°C), and its expression is more intense with increased severity of cold exposure. During recovery at 4°C after 24 h at ?20°C, both 90,000 and 75,000 M_r heat shock proteins are expressed for more than 96 h. While normal protein synthesis is suppressed during heat shock and recovery from heat shock, normal protein synthesis coincides with synthesis of the heat shock proteins during recovery from low temperatures, thus implying that expression of the heat shock proteins is not invariably linked to suppression of normal protein synthesis. Western transfer, using a monoclonal antibody that recognizes the inducible form of the human 70,000 M_r heat shock protein, demonstrates that immunologically related proteins in the gypsy moth are expressed at 4°C and during recovery from cold and heat shock.     

冷激蛋白的研究进展

冷激蛋白（CSPs）广泛存在于革兰氏阳性菌和阴性菌中,它是细胞在应对冷刺激时所产生的一系列7 ku左右的蛋白质,结构上富含芳香族氨基酸,起着重要的分子伴侣作用,能够增强细胞抵御冷激环境胁迫的能力.以大肠杆菌、枯草杆菌、嗜热链球菌、沙门氏杆菌等为例,介绍各种冷激蛋白在产生、结构、调控等方面的异同,以及它在生产、生活中的应用价值.     

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.     

Effect of Different Temperature Upshifts on Protein Synthesis by the Psychrotrophic Bacterium Pseudomonas fragi

Pseudomonas fragi, a psychrotroph bacterium involved in meat product spoilage, was shifted either from 5° to 20°C or 30°C and from 28° to 34°C. The heat-shocked cells in the mid-log phase rapidly reached the characteristic growth rate of the postshock temperature. The patterns of synthesized proteins were compared by autoradiography of two-dimensional gel electrophoregrams. The rates of synthesis, after transfer of cells from 5° to 30°C, 5° to 20°C, and 28° to 34°C, changed for 30, 26, and 21 proteins respectively, of which 19, 17, and 12 were increased respectively. Thirteen proteins changed similarly for the three treatments, and two of the seven overexpressed proteins were immunologically related to the Escherichia coli DnaK and GroEL heat shock proteins. From the four low-molecular-mass proteins, belonging to the family of DNA-binding cold shock proteins (CSPs) such as CS7.4, the major E. coli CSP [15], the amounts of C7.0 and C8.0 decreased rapidly after the upshifts, whereas that of E7.0 and E8.0 increased greatly. Received: 22 November 1995 / Accepted: 22 December 1995     

Growth, Survival and Characterization of cspA in Salmonella enteritidis Following Cold Shock

Salmonella enteritidis is a major foodborne microbial pathogen that can grow and survive at low temperatures for a considerable period of time. Increased survival was evidenced from a frozen S. enteritidis culture when treated at 10°C prior to freezing. Western blot analysis with Escherichia coli CspA antibody and analysis of radiolabeled proteins from S. enteritidis cultures after cold shock at 10°C and 5°C showed increased expression of a 7.4-kDa major cold shock protein, CS7.4, similar in size to that reported for E. coli. Cloning followed by nucleotide sequence analysis of the cspA gene from S. enteritidis showed a 100% nucleotide sequence identity in the promoter elements (−35 and −10) and the amino acid sequence encoded by the open reading frame (ORF) with the E. coli cspA gene. However, the differences in the nucleotide sequences between E. coli and S. enteritidis cspA genes in the putative repressor protein binding domain, the fragment 7, and in various segments throughout the upstream 0.642-kbp DNA may contribute to the expression of CS7.4 at less stringent temperatures in S. enteritidis. As in E. coli, the actual role of CS7.4 in protecting S. enteritidis from the damaging effects of cold or freezing temperatures is not yet understood. Received: 14 March 1997 / Accepted: 10 July 1997     

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°C, cryopreservation at -20°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°C). Also, the dissociation constants, KD (M) as the binding specificity for enolase, mutarotase, isocitrate dehydrogenase, and lactate dehydrogenase were 1.82×10^-10, 4.35×10^-9, 8.98×10^-12, and 3.05×10^-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.     

Protein Synthesis Related to Cold Temperature Stress in the Desiccation-Tolerant Moss Tortula ruralis

Incubation of hydrated Tortula ruralis (Hedw.) Gaertn., Meyer. Scherb. at temperatures down to 2°C resulted in an accumulation of polyribosomes and a decrease in single ribosomes. No changes in the levels of ribosomal subunits were detected. On rehydration of slowly dried moss, which contains no polyribosomes, these were reormed at 2, 8 and 20°C. Rapid incorporation of labelled leucine into protein was observed on reintroduction of the desiccated plant o water at 20°C and there was significant, but much reduced, ncorporation at 2°C. Previously undesiccated moss was also able o take up radioactive leucine and to synthesize protein at 2 and -2.5°C. Changes in the rate of protein synthesis at low temperature were not detected in cold hardened (winter collected or incubated at 2°C) T. ruralis. The moss appears to be adapted to survive freezing wear round and even summer-collected moss can conduct protein synthesis at low temperatures: seasonal cold hardiness changes do lot appear to take place.     

Involvement of Membrane Lipids in Cold Shock-induced Autolysis of Bacillus subtilis Cells

Exponentially growing Bacillus subtilis cells autolysed when exposed to cold shock treatment in minimal medium followed by incubation at 37°C. From characteristics of the lysis, it was suggested that the cold-shock-induced cell lysis resulted from the perturbation of membrane organization that is initiated by rapid changes in temperature, lipid phase transitions. For maximum lysis induction to occur, in addition to rapid cooling to 5°C or lower, retention at temperatures lower than 10°C for at least 20 min is required. The cell sensitivity to the autolysis induction by cold shock was different between cells grown at 25°C and cells grown at 37°C. Analyses of the fatty acid composition and the phase transition temperature of membrane lipids suggested that the membrane fluidity may affect the autolysis induction. Experiments to discover the effects of cerulenin treatment and lipid addition on autolysis induction and the autolysin activity level support the hypothesis that membrane lipids are involved in cold-shock-induced cell autolysis.     

Tetraploid Induction by Inhibiting Mitosis I with Heat Shock, Cold Shock, and Nocodazole in the Hard Clam Mercenaria mercenaria (Linnaeus, 1758)

Tetraploid induction by inhibiting mitosis I with heat shock (32, 35, and 38°C), cold shock (1, 4, and 7°C), and nocodazole (0.02 to 1.6 mg/L) was investigated in the hard clam Mercenaria mercenaria. All treatments were applied to fertilized eggs about 5 min before the first cell division at 22 to 23°C, and lasted for 10, 15, and 20 min. Three replicates were produced for each treatment with different parents. The ploidy of resultant larvae and juveniles was determined with flow cytometry. Heat shock of 35 and 38°C was effective in inhibiting mitosis I, producing 54% to 89% tetraploid larvae. Heat shock of 32°C accelerated embryonic development without inhibiting mitosis or producing tetraploids. In all heat-shock groups, the survival to D-stage larvae was lower than in controls, suggesting that heat-shock treatments and tetraploidy were detrimental to larval development. At the juvenile stage, survivors from heat-shock groups contained no tetraploids. Cold shocks suspended the first cell division during the treatment, but produced no tetraploids in the 4°C and 7°C treatment groups. Cold shock of 1°C produced 31% tetraploid larvae in one replicate, with none surviving to juvenile stage. Nocodazole inhibited mitosis I at concentrations of 0.04 mg/L or higher, but did not produce tetraploids. This study indicates that heat shock is most effective in inducing tetraploids through mitosis I inhibition, although none of the induced tetraploids survived to juvenile stage.     

Protein synthesis induced by heat in an Ixodes tick

We determined whether ambient temperature influences the proteins produced by Ixodes dammini ticks. Nonfed adult females were subjected to temperature pulses, and ³⁵S-labeled methionine was injected into the hemocoel or used as an incubation medium for excised salivary glands. Heat-induced protein synthesis was observed in both whole-body and excised salivary gland preparations from nonattached ticks solely when ambient temperature was raised to 42°C. The approximate M_r of each protein was 88 kilodaltons (kDa), 75 and 74 kDa, and between 21 and 27 kDa for a group of lower molecular weight proteins. In another experimental series, ticks were allowed to attach to rabbits and then were subjected to temperature pulses. The 88 and 74 kDa proteins were present in preparations from nonheated ticks that had attached for 1 h. Only small amounts of these proteins were evident in tissues prepared from ticks attached for 2 days or more. Heat-induced proteins became apparent in ticks that were incubated at 42°C, regardless of time of attachment.