The CLO3403/CLO3404 Two-Component System of Clostridium botulinum E1 Beluga Is Important for Cold Shock Response and Growth at Low Temperatures

In order to survive a temperature downshift, bacteria have to sense the changing environment and adjust their metabolism and structure. Two-component signal transduction systems (TCSs) play a central role in sensing and responding to many different environmental stimuli. Although the nonproteolytic (group II) Clostridium botulinum represents a major hazard in chilled foods, the cold adaption mechanisms of group II C. botulinum organisms are not known. Here, we show that the CLO3403/CLO3404 TCS of C. botulinum E1 Beluga is involved in the cold shock response and growth at 12°C. Cold shock induced the expression of the genes encoding the histidine kinase (clo3403) and the response regulator (clo3404) by more than 100-fold after 5 h relative to their expression in a nonshocked culture at the corresponding time point. The involvement of CLO3403/CLO3404 in growth at low temperature was demonstrated by impaired growth of the insertional clo3403 and clo3404 knockout mutants at 12°C compared to the growth of the wild-type culture. Additionally, the inactivation of clo3403 had a negative effect on motility. The growth efficiency at 12°C of the TCS mutants and the motility of the kinase mutants were restored by introducing a plasmid harboring the operon of the CLO3403/CLO3404 TCS. The results suggest that the CLO3403/CLO3404 TCS is important for the cold tolerance of C. botulinum E1 Beluga.     

Two-Component-System Histidine Kinases Involved in Growth of Listeria monocytogenes EGD-e at Low Temperatures

Two-component systems (TCSs) aid bacteria in adapting to a wide variety of stress conditions. While the role of TCS response regulators in the cold tolerance of the psychrotrophic foodborne pathogen Listeria monocytogenes has been demonstrated previously, no comprehensive studies showing the role of TCS histidine kinases of L. monocytogenes at low temperature have been performed. We compared the expression levels of each histidine kinase-encoding gene of L. monocytogenes EGD-e in logarithmic growth phase at 3°C and 37°C, as well as the expression levels 30 min, 3 h, and 7 h after cold shock at 5°C and preceding cold shock (at 37°C). We constructed a deletion mutation in each TCS histidine kinase gene, monitored the growth of the EGD-e wild-type and mutant strains at 3°C and 37°C, and measured the minimum growth temperature of each strain. Two genes, yycG and lisK, proved significant in regard to induced relative expression levels under cold conditions and cold-sensitive mutant phenotypes. Moreover, the ΔresE mutant showed a lower growth rate than that of wild-type EGD-e at 3°C. Eleven other genes showed upregulated gene expression but revealed no cold-sensitive phenotypes. The results show that the histidine kinases encoded by yycG and lisK are important for the growth and adaptation of L. monocytogenes EGD-e at low temperature.     

Control of ion transport by mitochondrion-rich chloride cells of eurythermic teleost fish: Cold shock vs. cold acclimation

Seawater-acclimated eurythermic mummichogs (Fundulus heteroclitus L.) were acclimated to cold and warm conditions (5 and 20 °C, 4 weeks). Opercular epithelia (OE) from 20 °C-acclimated animals, containing numerous mitochondrion-rich chloride cells were mounted in Ussing-style membrane chambers, cooled to 16, 13, 10, 5 and 2.5 °C, then subjected to hypotonic shock that normally inhibits Cl(-) secretion (as short-circuit current, I(sc)). Cold exposure to 10 °C slowed Cl(-) secretion (Q(10)=1.62 ± 0.204 95% CI) and OEs responded rapidly and reversibly to hypotonic shock, but below 8.0 °C a sharp decrease (Q(10)=5.63 ± 0.736) occurred and the tissue was unresponsive to hypotonicity. By immunocytochemistry, Focal Adhesion Kinase (FAK) phosphorylated at tyrosine-407 (pY(407)) colocalized with CFTR in apical membrane and dephosphorylated with hypotonic shock at 20 °C but failed to dephosphorylate at 5 °C, while opercular epithelia from cold-acclimated fish at 5 and 20 °C responded normally to hypotonic shock. Cold-shock of warm-acclimated OEs also stimulated covering over of mitochondrion- rich cell apical crypts, detected by SEM. Cold-acclimation increased C18:1 and decreased C18:0 fatty acids in liver, indicating homeoviscous adaptation. Eurythermic fish acclimate osmoregulatory systems to cold by maintaining membrane fluidity and preserving complex transport regulation pathways.     

Protection of Bacillus subtilis against cold stress via compatible-solute acquisition

Accumulation of compatible solutes is a strategy widely employed by bacteria to achieve cellular protection against high osmolarity. These compounds are also used in some microorganisms as thermostress protectants. We found that Bacillus subtilis uses the compatible solute glycine betaine as an effective cold stress protectant. Glycine betaine strongly stimulated growth at 15°C and permitted cell proliferation at the growth-inhibiting temperature of 13°C. Initial uptake of glycine betaine at 15°C was low but led eventually to the buildup of an intracellular pool whose size was double that found in cells grown at 35°C. Each of the three glycine betaine transporters (OpuA, OpuC, and OpuD) contributed to glycine betaine accumulation in the cold. Protection against cold stress was also accomplished when glycine betaine was synthesized from its precursor choline. Growth of a mutant defective in the osmoadaptive biosynthesis for the compatible solute proline was not impaired at low temperature (15°C). In addition to glycine betaine, the compatible solutes and osmoprotectants l-carnitine, crotonobetaine, butyrobetaine, homobetaine, dimethylsulfonioactetate, and proline betaine all served as cold stress protectants as well and were accumulated via known Opu transport systems. In contrast, the compatible solutes and osmoprotectants choline-O-sulfate, ectoine, proline, and glutamate were not cold protective. Our data highlight an underappreciated facet of the acclimatization of B. subtilis to cold environments and allow a comparison of the characteristics of compatible solutes with respect to their osmotic, heat, and cold stress-protective properties for B. subtilis cells.     

Expression of a New Cold Shock Protein of 21.5 kDa and of the Major Cold Shock Protein by Streptococcus thermophilus After Cold Shock

Streptococcus thermophilus is widely used in food fermentations; it commonly suffers diverse stress challenges during manufacturing. This study investigated the cold shock response of S. thermophilus when the cell culture temperature shifted from 42°C to 15°C or 20°C. The growth of cells was affected more drastically after cold shock at 15°C than at 20°C. The generation time was increased by a factor of 19 when the temperature was lowered from 42° to 20°C, and by a factor of 72 after a cold shock at 15°C. The two-dimensional electrophoretic protein patterns of S. thermophilus under cold shock conditions were compared with the reference protein pattern when cells were grown at optimal temperature. Two proteins of 21.5 and 7.5 kDa synthesized in response to cold shock were characterized. N-terminal sequencing and sequence homology searches have shown that the 7.5-kDa protein belonged to the family of the major cold shock proteins, while no homology was found for the new cold shock protein of 21.5 kDa. Received: 4 June 1999 / Accepted: 6 July 1999     

Sequential temperature control of multi-phasic dormancy release and germination of Paeonia corsica seeds

Aims The physiological responses during dormancy removal and multi-phasic germination were investigated in seeds of Paeonia corsica (Paeoniaceae).Methods Seeds of P. corsica were incubated in the light at a range of temperatures (10–25 and 25/10°C), without any pre-treatment, after W (3 months at 25°C), C (3 months at 5°C) and W + C (3 months at 25°C followed by 3 months at 5°C) stratification, and a GA 3 treatment (250 mg·l^-1 in the germination substrate). Embryo growth, time from testa to endosperm rupture and radicle emergence were assessed as separate phases. Epicotyl–plumule emergence was evaluated incubating the germinated seeds at 15°C for 2 weeks, at 5 and 25°C for 2 months on agar water before transplanting to the soil substrate at 10, 15 and 20°C and at 15°C for 2 months on the surface agar water with GA 3 .Important findings Embryo growth, testa rupture, endosperm rupture (radicle emergence) and growth of the epicotyl were identified as four sequential steps in seeds of P. corsica. Gibberellic acid alone and warm stratification followed by 15°C promoted embryo growth and subsequent seed germination. Cold stratification induced secondary dormancy, even when applied after warm stratification. After radicle emergence, epicotyl–plumule emergence was delayed for ca. 3 months. Mean time of epicotyl–plumule emergence was positively affected by cold stratification (2 months at 5°C) and GA 3. P. corsica seeds exhibited differential temperature sensitivity for the four sequential steps in the removal of dormancy and germination processes that resulted in the precise and optimal timing of seedling emergence.     

Some aspects of respiration and respiration inhibitors in low temperature effects of the cotton plant

Respiration of leaf tissue from cotlon plants in the nine leaf stage was found to be severely reduced at temperatures below 15°C. In another study, young cotton plants were exposed to chilling temperature (2.8°C) for 72 hours and the ability of the plant tissues to recover respiration at normal temperatures (15 and 25°C) was examined at periodic intervals. Chilling exposures of 12 hours injured cotton tissues as indicated by increased respiratiou of leaves and roots at 25°C. Further chilling malerially reduced the capacity of the leaves to re-estahlish normal respiration rales at higher temperatures. Picolinic acid, Dexon and matonate were used to study the influence of respiratory inhibitors on the chill damage of the collon plants. The chemicals were applied six hours before cold exposure aud fhe growth and development of cold injured plants was studied to indicate the effectiveness of the treatments. Tissue weights at the final harvests indicated that picolinic acid and Dexon treated plants recovered better after cold injury than the untreated plauts. The results suggest thai 15°C may be a critical temperaliire for many physiological processes of the cotton plant because the supply of energy needed for plant reactionsis restricted due to inadequate respiration. They also indicate that the disturhances in respiration are among the early effects of chilling colton plants and may be the cause of delayed growth and development of cotton plants subjccled to non-lethal chilling exposures. It is concluded that chemicals like picolinie acid and Dexon may he effective through protection of specific systems rather than a general reduction of respiration.     

Structure and function of a cold shock domain fold protein, CspD, in Janthinobacterium sp. Ant5-2 from East Antarctica

A cold shock domain (CSD)-containing protein, CspD, of molecular mass ~7.28 kDa in a psychrotolerant Antarctic Janthinobacterium sp. Ant5-2 (ATCC BAA-2154) exhibited constitutive expression at 37, 22, 15, 4 and -1°C. The cspD gene encoding the CspD protein of Ant5-2 was cloned, sequenced and analyzed. The deduced protein sequence was highly similar to the conserved domains of the cold shock proteins (Csps) from bacteria belonging to the class Betaproteobacteria. Its expression was both time- and growth phase-dependent and increased when exposed to 37°C and UV radiation (UVC, dose: 1.8 and 2.8 mJ cm(-2)). The results from the electrophoretic mobility shift and subcellular localization study confirmed its single-stranded DNA-binding property. In silico analysis of the deduced tertiary structure of CspD from Ant5-2 showed a highly stable domain-swapped dimer, forming two similar monomeric Csp folds. This study established an overall framework of the structure, function and phylogenetic analysis of CspD from an Antarctic Janthinobacterium sp. Ant5-2, which may facilitate and stimulate the study of CSD fold proteins in the class Betaproteobacteria.     

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.     

Draft Genome Sequence of a Psychrotolerant Sulfur-Oxidizing Bacterium, Sulfuricella denitrificans skB26, and Proteomic Insights into Cold Adaptation

Except for several conspicuous cases, very little is known about sulfur oxidizers living in natural freshwater environments. Sulfuricella denitrificans skB26 is a psychrotolerant sulfur oxidizer recently isolated from a freshwater lake as a representative of a new genus in the class Betaproteobacteria. In this study, an approximately 3.2-Mb draft genome sequence of strain skB26 was obtained. In the draft genome, consisting of 23 contigs, a single rRNA operon, 43 tRNA genes, and 3,133 coding sequences were identified. The identified genes include those required for sulfur oxidation, denitrification, and carbon fixation. Comparative proteomic analysis was conducted to assess cold adaptation mechanisms of this organism. From cells grown at 22°C and 5°C, proteins were extracted for analysis by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry. In the cells cultured at 5°C, relative abundances of ribosomal proteins, cold shock proteins, and DEAD/DEAH box RNA helicases were increased in comparison to those at 22°C. These results suggest that maintenance of proper translation is critical for growth under low-temperature conditions, similar to the case for other cold-adapted prokaryotes.     

Proteomics of early and late cold shock stress on thermophilic bacterium, Thermus sp. GH5

Thermus sp. GH5 is an aerobic thermophilic bacterium with optimal growth at 70-75°C isolated from a hot spring in Ardabil, North West province of Iran. Due to industrial and biotechnological applications of thermophils, it is very important to know more about their proteomes and metabolomes. Since thermophils live in stressful environments it will be very useful to study their survival mechanisms. There are many reports on stress induced proteins, particularly the well characterized heat shock proteins, but little is known about the functions of proteins induced after a decrease in temperature. In this study, the proteomes of the thermophilic bacterium after a temperature down shift from 75°C to 45°C for 2h and 5h were investigated. We also compared protein profiles of early and late cold shock processes to that of cells grown at 75°C and identified a set of proteins, some of which are involved in metabolic processes such as fatty acid synthesis, pentose phosphate pathway, aromatic component degradation and signal transduction. Our data showed this organism could be tolerating the stress conditions by changing its metabolism and physiology.     

Restart of exponential growth of cold-shocked Yersinia enterocolitica occurs after down-regulation of cspA1/A2 mRNA

The cellular content of major cold shock protein (MCSP) mRNA transcribed from the tandem gene duplication cspA1/A2 and growth of Yersinia enterocolitica were compared when exponentially growing cultures of this bacterium were cold shocked from 30 to 20, 15, 10, 5, or 0 degrees C, respectively. A clear correlation between the time point when exponential growth resumes after cold shock and the degradation of cspA1/A2 mRNA was found. A polynucleotide phosphorylase-deficient mutant was unable to degrade cspA1/A2 mRNA properly and showed a delay, as well as a lower rate, of growth after cold shock. For this mutant, a correlation between decreasing cspA1/A2 mRNA and restart of growth after cold shock was also observed. For both wild-type and mutant cells, no correlation of restart of growth with the cellular content of MCSPs was found. We suggest that, after synthesis of cold shock proteins and cold adaptation of the cells, MCSP mRNAs must be degraded; otherwise, they trap ribosomes, prevent translation of bulk mRNA, and thus inhibit growth of this bacterium at low temperatures.     

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.     

Identification and characterization of novel low-temperature-inducible promoters of Escherichia coli.

Escherichia coli promoters that are more active at low temperature (15 to 20 degrees C) than at 37 degrees C were identified by using the transposon Tn5-lac to generate promoter fusions expressing beta-galactosidase (beta-Gal). Tn5-lac insertions that resulted in low-temperature-regulated beta-Gal expression were isolated by selecting kanamycin-resistant mutants capable of growth on lactose minimal medium at 15 degrees C but which grew poorly at 37 degrees C on this medium. Seven independent mutants were selected for further studies. In one such strain, designated WQ11, a temperature shift from 37 degrees C to either 20 or 15 degrees C resulted in a 15- to 24-fold induction of beta-Gal expression. Extended growth at 20 or 15 degrees C resulted in 36- to 42-fold-higher beta-Gal expression over that of cells grown at 37 degrees C. Treatment of WQ11 with streptomycin, reported to induce a response similar to heat shock, failed to induce beta-Gal expression. In contrast, treatment with either chloramphenicol or tetracycline, which mimics a cold shock response, resulted in a fourfold induction of beta-Gal expression in strain WQ11. Hfr genetic mapping studies complemented by physical mapping indicated that in at least three mutants (WQ3, WQ6, and WQ11), Tn5-lac insertions mapped at unique sites where no known cold shock genes have been reported. The Tn5-lac insertions of these mutants mapped to 81, 12, and 34 min on the E. coli chromosome, respectively. The cold-inducible promoters from two of the mutants (WQ3 and WQ11) were cloned and sequenced, and their temperature regulation was examined. Comparison of the nucleotide sequences of these two promoters with the regulatory elements of other known cold shock genes identified the sequence CCAAT as a putative conserved motif.     

Engineering trehalose synthesis in Lactococcus lactis for improved stress tolerance

Trehalose accumulation is a common cell defense strategy against a variety of stressful conditions. In particular, our team detected high levels of trehalose in Propionibacterium freudenreichii in response to acid stress, a result that led to the idea that endowing Lactococcus lactis with the capacity to synthesize trehalose could improve the acid tolerance of this organism. To this end, we took advantage of the endogenous genes involved in the trehalose catabolic pathway of L. lactis, i.e., trePP and pgmB, encoding trehalose 6-phosphate phosphorylase and β-phosphoglucomutase, respectively, which enabled the synthesis of trehalose 6-phosphate. Given that L. lactis lacks trehalose 6-phosphate phosphatase, the respective gene, otsB, from the food-grade organism P. freudenreichii was used to provide the required activity. The trehalose yield was approximately 15% in resting cells and in mid-exponential-phase cells grown without pH control. The intracellular concentration of trehalose reached maximal values of approximately 170 mM, but at least 67% of the trehalose produced was found in the growth medium. The viability of mutant and control strains was examined after exposure to heat, cold or acid shock, and freeze-drying. The trehalose-producing strains showed improved tolerance (5- to 10-fold-higher survivability) to acid (pH 3) and cold shock (4°C); there was also a strong improvement in cell survival in response to heat shock (45°C), and no protection was rendered against dehydration. The insight provided by this work may help the design of food-grade strains optimized for the dairy industry as well as for oral drug delivery.     

Overexpression of Hsp90 from grass carp (Ctenopharyngodon idella) increases thermal protection against heat stress

With homologous DNA probes, we had screened a grass carp heat shock protein 90 gene (CiHsp90). The full sequence of CiHsp90 cDNA was 2793 bp, which could code a 798 amino acids peptide. The phylogenetic analysis demonstrated that CiHsp90 shared the high homology with Zebrafish Grp94. Quantitative RT-PCR analysis showed that CiHsp90 was ubiquitously expressed at lower levels in all detected tissues and up-regulated after heat shock at 34 °C or cold stress at 4 °C. To understand the function of CiHsp90 involving in thermal protection, an expression vector containing coding region cDNA was expressed in E. coli BL21 (DE3) plysS. Upon transfer from 37 °C to 42 °C, these cells that accumulated CiHsp90 peptides displayed greater thermoresistance than the control cells. While incubated at 4°C for different periods, it could also improve the cell viability. After transient transfected recombinant plasmid pcDNA3.1/CiHsp90 into mouse myeloma cell line SP2/0, we found that CiHsp90 could contribute to protecting cells against both thermal and cold extremes. On the contrary, the mutant construct ΔN-CiHsp90 (256-798aa) could abolish the protection activity both in prokaryotic cells and eukaryotic cells. Additionally, both CiHsp90 and ΔN-CiHsp90 peptides could reduce the level of citrate synthase aggregation at the high temperature.     

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.