Alteration of cell morphology and viability in a recA mutant of Streptococcus thermophilus upon induction of heat shock and nutrient starvation

We identified the recA gene of the moderately thermophilic bacterium Streptococcus thermophilus and investigated the role of its product in the adaptation to heat shock and nutrient starvation. Expression of recA was required for optimal viability and normal cell morphology upon induction of both stresses. Normal induction of GroEL and ClpL in a recA knock-out mutant suggests that the RecA role in heat shock and nutrient starvation response of S. thermophilus is independent from the intracellular accumulation of these stress-specific chaperones.     

Effect of different carbon sources and cold shock on protein synthesis by a psychrotrophic Acinetobacter sp

The induction of proteins after a 25 to 5 degrees C cold shock in the psychrotrophic Acinetobacter HH1-l was examined using two-dimensional polyacrylamide gel electrophoresis. In addition, effects of various carbon sources (acetate, Tween 80, and olive oil) on protein synthesis after cold shock were assessed. HH1-1 responded to cold shock by synthesizing both cold shock proteins (csps) and cold acclimation proteins (caps). The synthesis of two csps (89 and 18) was increased 2 h after cold shock by the cells, regardless of the carbon source provided. An additional csp (csp 12), with an estimated molecular mass of 12 kDa, was observed in cells grown in olive oil only. Csp 12 was also synthesized when cells were incubated at 30 degrees C, suggesting that this protein may serve as a general stress protein. In addition to csps, caps were observed post cold shock at 72 h in acetate-grown cells and at 140 h in cells grown in Tween 80 and olive oil. Induction of cold-acclimated periplasmic proteins was observed for cells grown in olive oil only, suggesting cells grown in olive oil may be stressed by low temperatures to a greater extent than cells grown in either acetate or Tween 80.     

Impaired temperature stress response of a Streptococcus thermophilus deoD mutant

An insertional deoD mutant of Streptococcus thermophilus strain SFi39 had a reduced growth rate at 20 degrees C and an enhanced survival capacity to heat shock compared to the wild type, indicating that the deoD product is involved in temperature shock adaptation. We report evidence that ppGpp is implicated in this dual response.     

Proteomic study of cold shock protein in Bacillus stearothermophilus P1: Comparison of temperature downshifts

The thermophilic bacterium Bacillus stearothermophilus P1 is unique in its ability to thrive in extreme environments such as high temperatures or high pH conditions. The study of cold shock response is very interesting and interpreted as a shock response to express the genes involved in synthesis of specific proteins. This study investigated the study of cold shock protein of B. stearothermophilus P1 when the cell culture temperature shifted from 65 degrees C to 37 degrees C and 25 degrees C. Cell growth at 37 degrees C weakly increased in the previous 3 h and then slowly decreased. In contrast, cell growth at 25 degrees C was slowly decreased. The protein contents after temperature downshifts were analyzed by proteomic techniques using protein chip and two-dimensional (2-D) electrophoresis that are highly effective and useful for protein separation and identification. The different proteins after a temperature decrease from 65 degrees C to 37 degrees C and 25 degrees C were expressed on 2-D gel patterns and the cold shock protein was detected in the acidic area with the isoelectric point and molecular mass approximately 4.5 and 7.3 kDa, respectively. The NH(2)-terminal sequence of a major cold shock protein from B. stearothermophilus P1 was MQRGKVKWFNNEKGFGFIEVEGGSD, similar to other cold shock proteins from Bacillus sp. up to 96% identity, but different from the other bacteria with homology less than 80% identity.     

Metabolic control of the membrane fluidity in Bacillus subtilis during cold adaptation

Membrane fluidity adaptation to the low growth temperature in Bacillus subtilis involves two distinct mechanisms: (1) long-term adaptation accomplished by increasing the ratio of anteiso- to iso-branched fatty acids and (2) rapid desaturation of fatty acid chains in existing phospholipids by induction of fatty acid desaturase after cold shock. In this work we studied the effect of medium composition on cold adaptation of membrane fluidity. Bacillus subtilis was cultivated at optimum (40 degrees C) and low (20 degrees C) temperatures in complex medium with glucose or in mineral medium with either glucose or glycerol. Cold adaptation was characterized by fatty acid analysis and by measuring the midpoint of phospholipid phase transition T(m) (differential scanning calorimetry) and membrane fluidity (DPH fluorescence polarization). Cells cultured and measured at 40 degrees C displayed the same membrane fluidity in all three media despite a markedly different fatty acid composition. The T(m) was surprisingly the highest in the case of a culture grown in complex medium. On the contrary, cultivation at 20 degrees C in the complex medium gave rise to the highest membrane fluidity with concomitant decrease of T(m) by 10.5 degrees C. In mineral media at 20 degrees C the corresponding changes of T(m) were almost negligible. After a temperature shift from 40 to 20 degrees C, the cultures from all three media displayed the same adaptive induction of fatty acid desaturase despite their different membrane fluidity values immediately after cold shock.     

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.     

The cold shock response of the psychrotrophic bacterium Pseudomonas fragi involves four low-molecular-mass nucleic acid-binding proteins.

The psychrotrophic bacterium Pseudomonas fragi was subjected to cold shocks from 30 or 20 to 5 degrees C. The downshifts were followed by a lag phase before growth resumed at a characteristic 5 degrees C growth rate. The analysis of protein patterns by two-dimentional gel electrophoresis revealed overexpression of 25 or 17 proteins and underexpression of 12 proteins following the 30- or 20-to-5 degrees C shift, respectively. The two downshifts shared similar variations of synthesis of 20 proteins. The kinetic analysis distinguished the induced proteins into cold shock proteins (Csps), which were rapidly but transiently overexpressed, and cold acclimation proteins (Caps), which were more or less rapidly induced but still overexpressed several hours after the downshifts. Among the cold-induced proteins, four low-molecular-mass proteins, two of them previously characterized as Caps (CapA and CapB), and heat acclimation proteins (Haps) as well as heat shock proteins (Hsps) for the two others (TapA and TapB) displayed higher levels of induction. Partial amino acid sequences, obtained by microsequencing, were used to design primers to amplify by PCR the four genes and then determine their nucleotide sequences. A BamHI-EcoRI restriction fragment of 1.9 kb, containing the complete coding sequence for capB, was cloned and sequenced. The four peptides belong to the family of small nucleic acid-binding proteins as CspA, the major Escherichia coli Csp. They are likely to play a major role in the adaptative response of P. fragi to environmental temperature changes.     

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.     

Gene structure and transcriptional regulation of <Emphasis Type="Italic">dnaK</Emphasis> and <Emphasis Type="Italic">dnaJ</Emphasis> genes from a psychrophilic bacterium, <Emphasis Type="Italic">Colwellia maris</Emphasis>

The dnaK and dnaJ genes, encoding heat shock proteins, were cloned from a psychrophilic bacterium, Colwellia maris. Significant homology was evident comparing DnaK and DnaJ of the psychrophilile with the counterparts of mesophilic and thermophilic bacteria. In the DnaJ protein, three conserved regions of the Hsp40 family were observed. A putative promoter similar to the sigma32 consensus sequence was found upstream of the dnaK gene. The G+C content in the 5'-untranslated region of the dnaK gene was much lower than that in the corresponding region of mesophilic bacteria. Northern-blot analysis and primer-extension analysis showed that both genes were transcribed separately as monocistronic mRNAs. Following several temperature upshifts from 10 to 26 degrees C, maximum induction of the dnaK and dnaJ mRNAs was detected at 20 degrees C, suggesting that this temperature induces the heat shock response in this bacterium. In addition, the level of the induction of the dnaJ gene was much lower than that of the dnaK gene. These findings together revealed several specific features of the heat shock response at a relatively low temperature in psychrophiles.     

Expression of cspH, Encoding the Cold Shock Protein in Salmonella enterica Serovar Typhimurium UK-1

Both Salmonella enterica serovar Typhimurium and Escherichia coli contain the cspH gene encoding CspH, one of the cold shock proteins (CSPs). In this study, we investigated the expression of cspH in S. enterica serovar Typhimurium and found that it was induced in response to a temperature downshift during exponential phase. The cspH promoter was activated at 37 degrees C, and its mRNA was more stable than the other csp mRNAs at 37 degrees C. Moreover, lacZ expression of the translational cspH-lacZ fusion was induced at that temperature. Interestingly, the cspH mRNA had a much shorter 5'-untranslated region than those in the other cold-shock-inducible genes, and the promoter sequence, which was only 55 bp, was sufficient for cspH expression. The 14-base downstream box located 12 bases downstream of the initiation codon of cspH mRNA was essential for its cold shock activation.     

CspI, the ninth member of the CspA family of Escherichia coli, is induced upon cold shock

Escherichia coli contains the CspA family, consisting of nine proteins (CspA to CspI), in which CspA, CspB, and CspG have been shown to be cold shock inducible and CspD has been shown to be stationary-phase inducible. The cspI gene is located at 35.2 min on the E. coli chromosome map, and CspI shows 70, 70, and 79% identity to CspA, CspB, and CspG, respectively. Analyses of cspI-lacZ fusion constructs and the cspI mRNA revealed that cspI is cold shock inducible. The 5'-untranslated region of the cspI mRNA consists of 145 bases and causes a negative effect on cspI expression at 37 degrees C. The cspI mRNA was very unstable at 37 degrees C but was stabilized upon cold shock. Analyses of the CspI protein on two-dimensional gel electrophoresis revealed that CspI production is maximal at or below 15 degrees C. Taking these results together, E. coli possesses a total of four cold shock-inducible proteins in the CspA family. Interestingly, the optimal temperature ranges for their induction are different: CspA induction occurs over the broadest temperature range (30 to 10 degrees C), CspI induction occurs over the narrowest and lowest temperature range (15 to 10 degrees C), and CspB and CspG occurs at temperatures between the above extremes (20 to 10 degrees C).     

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     

A chaperonin from a thermophilic bacterium, Thermus thermophilus, that controls refoldings of several thermophilic enzymes.

A chaperonin has been purified from a thermophilic bacterium, Thermus thermophilus. It consists of two kinds of proteins with approximate Mr 58,000 and 10,000 and shows a 7-fold rotational symmetry from the top view and a "football"-like shape from the side view under the electron microscopic view. Its weak ATPase activity is inhibited by sulfite and activated by bicarbonate. ATP causes change of its mobility in nondenaturating polyacrylamide gel electrophoresis. The T. thermophilus chaperonin can promote in vitro refolding of several guanidine HCl-denatured enzymes from thermophilic bacteria. At high temperatures above 60 degrees C, where the native enzymes are stable but their spontaneous refoldings upon dilution of guanidine HCl fail, the chaperonin induces productive refolding in an ATP-dependent manner. No or very poor refolding is induced when the chaperonin is added to the solution aged after dilution. An excess amount of the chaperonin is inhibitory for refolding. At middle temperatures (30-50 degrees C), where spontaneous refoldings of the enzymes occur, the chaperonin arrests refolding in the absence of ATP and refolding is induced when ATP is supplemented. At temperatures below 20 degrees C, where spontaneous refoldings also occur, the chaperonin arrests the refolding but ATP does not induce refolding.     

Impact of the metabolic activity of Streptococcus thermophilus on the colon epithelium of gnotobiotic rats

The thermophilic lactic acid bacterium Streptococcus thermophilus is widely and traditionally used in the dairy industry. Despite the vast level of consumption of S. thermophilus through yogurt or probiotic functional food, very few data are available about its physiology in the gastrointestinal tract (GIT). The objective of the present work was to explore both the metabolic activity and host response of S. thermophilus in vivo. Our study profiles the protein expression of S. thermophilus after its adaptation to the GIT of gnotobiotic rats and describes the impact of S. thermophilus colonization on the colonic epithelium. S. thermophilus colonized progressively the GIT of germ-free rats to reach a stable population in 30 days (10(8) cfu/g of feces). This progressive colonization suggested that S. thermophilus undergoes an adaptation process within GIT. Indeed, we showed that the main response of S. thermophilus in the rat's GIT was the massive induction of the glycolysis pathway, leading to formation of lactate in the cecum. At the level of the colonic epithelium, the abundance of monocarboxylic acid transporter mRNAs (SLC16A1 and SLC5A8) and a protein involved in the cell cycle arrest (p27(kip1)) increased in the presence of S. thermophilus compared with germ-free rats. Based on different mono-associated rats harboring two different strains of S. thermophilus (LMD-9 or LMG18311) or weak lactate-producing commensal bacteria (Bacteroides thetaiotaomicron and Ruminococcus gnavus), we propose that lactate could be a signal produced by S. thermophilus and modulating the colon epithelium.     

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.