Comparative Protein Modeling, Prediction of Conserved Residue and Active Sites in Cold Resistant Protein Isolated from CRPF1, A Cold Tolerant Mutant of Pseudomonas fluorescens

Proteins interacting with the biological information molecules DNA and RNA play important cellular roles in all organisms. One widespread super family of proteins implicated in such function(s) is cold shock protein (CSP) that contains the cold shock domain (CSD). This work is planned to study the three-dimensional structure, conserved residues, and different active sites in the structure of cold resistant protein (CRP) from CRPF₁, cold tolerant mutant of Pseudomonas fluorescence by comparative homology modeling. Here we tried to identify crucial residues that are involved in active sites or functional sites of the protein. The study reveals that CRP represent the prototype of the CSD and share a highly similar overall fold consisting of five antiparallel β-sheets forming a β-barrel structure with surface exposed aromatic and basic residues that were responsible for nucleic acid binding properties of variable binding affinities and sequence selectivity and harbors the nucleic acid binding motifs RNP1 and RNP2 that is highly conserved in CSP family.     

Structure and flexibility of the thermophilic cold-shock protein of Thermus aquaticus

The thermophilic bacterium Thermus aquaticus is a well-known source of Taq polymerase. Here, we studied the structure and dynamics of the T. aquaticus cold-shock protein (Ta-Csp) to better understand its thermostability using NMR spectroscopy. We found that Ta-Csp has a five-stranded β-barrel structure with five salt bridges which are important for more rigid structure and a higher melting temperature (76 °C) of Ta-Csp compared to mesophilic and psychrophilic Csps. Microsecond to millisecond time scale exchange processes occur only at the β1–β2 surface region of the nucleic acid binding site with an average conformational exchange rate constant of 674 s⁻¹. The results imply that thermophilic Ta-Csp has a more rigid structure and may not need high structural flexibility to accommodate nucleic acids upon cold shock compared to its mesophile and psychrophile counterparts.     

Rescue of a cold-sensitive mutant at low temperatures by cold shock proteins from <Emphasis Type="Italic">Polaribacter irgensii</Emphasis> KOPRI 22228

Exposure to low temperatures induces the biosynthesis of specific sets of proteins, including cold shock proteins (Csps). Since many of the specific functions of pychrophilic Csps are unknown, the roles of Csps from an Arctic bacterium, Polaribacter irgensii KOPRI 22228, were examined. The genes encoding CspA and CspC of P. irgensii were cloned in this study. Sequence analysis showed that these proteins have cold shock domains containing two RNA-binding motifs, RNP1 and RNP2. Both proteins bound oligo(dT)-cellulose resins, suggesting single-stranded nucleic acid-binding activity. When the P. irgensii Csps were overexpressed in Escherichia coli, the cold-resistance of the host was increased by more than five-fold. The P. irgensii Csps also rescued a cold-sensitive E. coli csp-quadruple deletion strain, BX04, at low temperatures. These results suggest that Csps from P. irgensii play a role in survival in polar environments.     

Adaptation to cold and proteomic responses of the psychrotrophic biopreservative Lactococcus piscium strain CNCM I-4031

There is considerable interest in the use of psychrotrophic bacteria for food biopreservation and in the understanding of cold adaptation mechanisms. The psychrotrophic biopreservative Lactococcus piscium strain CNCM I-4031 was studied for its growth behavior and proteomic responses after cold shock and during cold acclimation. Growth kinetics highlighted the absence of growth latency after cold shock, suggesting a very high promptness in cold adaptation, a behavior that has never been described before for lactic acid bacteria (LAB). A comparative proteomic analysis was applied with two-dimensional gel electrophoresis (2-DE), and upregulated proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Both cold shock and cold acclimation triggered the upregulation of proteins involved in general and oxidative stress responses and fatty acid and energetic metabolism. However, 2-DE profiles and upregulated proteins were different under both conditions, suggesting a sequence of steps in cold adaptation. In addition, the major 7-kDa Csp protein was identified in the L. piscium CNCM I-4031 genome but was not cold regulated. The implication of the identified cold shock proteins and cold acclimation proteins in efficient cold adaptation, the possible regulation of a histidyl phosphocarrier protein, and the roles of a constitutive major 7-kDa Csp are discussed.     

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.     

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°C to 10°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°C. These findings imply that cold-shocked cells are protected from HHP treatment, which may affect the efficiency of combined preservation techniques.     

Identification and purification of a family of dimeric major cold shock protein homologs from the psychrotrophic Bacillus cereus WSBC 10201.

Whole-cell protein patterns of a psychrotrophic Bacillus cereus strain from cultures grown at 7 and 30 degrees C were compared. This analysis revealed that at least three major proteins are expressed at a significantly higher rate at 7 degrees C than at 30 degrees C. The most abundant of these cold-induced proteins was a small polypeptide of 7.5 kDa, designated CspA, of B. cereus. In addition, four small proteins very similar in size to CspA were seen on both 7 degrees C and 30 degrees C two-dimensional protein gels. Immunoblot analysis using B. cereus anti-CspA antibodies indicated that the five proteins described above plus an additional sixth protein not visible on silver-stained two-dimensional gels are members of a B. cereus cold shock protein family. This hypothesis was corroborated by cloning and sequencing of the genes encoding five proteins of this family. The protein sequences deduced are highly similar and show homology to small procaryotic cold shock proteins and to the cold shock domain of eucaryotic Y-box proteins. Besides CspA, only one of the additional five CspA homologs was slightly cold inducible. In the presence of 100 mM NaCl, the two purified members of the protein family (CspA and CspE) elute as dimers at an apparent molecular mass of 15 kDa from a gel filtration column. At higher salt concentrations, they dissociate into their monomers. Their ability to bind to the ATTGG motif of single-stranded oligonucleotides was demonstrated by band shift analysis.     

Thermal stress responses in Antarctic yeast, Glaciozyma antarctica PI12, characterized by real-time quantitative PCR

Living organisms have some common and unique strategies to response to thermal stress. However, the amount of data on thermal stress response of certain organism is still lacking, especially psychrophilic yeast from the extreme habitat. Therefore, it is not known whether psychrophilic yeast shares the common responses of other organisms when exposed to thermal stresses. In this work, the cold shock and heat shock responses in Antarctic psychrophilic yeast Glaciozyma antarctica PI12 which had an optimal growth temperature of 12 °C were determined. The expression levels of 14 thermal stress-related genes were measured using real-time quantitative PCR (qPCR) when the yeast cells were exposed to cold shock (0 °C), mild cold shock (5 °C), and heat shock (22 °C) conditions. The expression profiles of the 14 genes at these three temperatures varied indicating that these genes had their specific roles to ensure the survival of the yeast. Under cold shock condition, the afp4 and fad genes were over-expressed possibly as a way for the G. antarctica PI12 to avoid ice crystallization in the cell and to maintain the membrane fluidity. Under the heat shock condition, hsp70 was significantly up-regulated possibly to ensure the proteins fold properly. Among the six oxidative stress-related genes, MnSOD and prx were up-regulated under cold shock and heat shock, respectively, possibly to reduce the negative effects caused by oxidative stress. Interestingly, it was found that the trehalase gene, nth1 that plays a role in degrading excess trehalose, was down-regulated under the heat shock condition possibly as an alternative way to accumulate trehalose in the cells to protecting them from being damaged.     

Discrimination of Psychrotrophic and Mesophilic Strains of the Bacillus cereus Group by PCR Targeting of Major Cold Shock Protein Genes

Detection of psychrotrophic strains (those able to grow at or below 7°C) of the Bacillus cereus group (Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides) in food products is at present extremely slow with conventional microbiology. This is due to an inability to discriminate these cold-adapted strains from their mesophilic counterparts (those able to grow only above 7°C) by means other than growth at low temperature, which takes 5 to 10 days for detection. Here we report the development of a single PCR assay that, using major cold shock protein-specific primers and appropriate annealing temperatures, is capable of both rapidly identifying bacteria of the B. cereus group and discriminating between psychrotrophic and mesophilic strains. It is intended that this development help to more accurately predict the shelf life of refrigerated pasteurized food and dairy products and to reduce the incidence of food poisoning by psychrotrophic strains of the B. cereus group.     

Heat shock response in psychrophilic and psychrotrophic yeast from Antarctica

The response to heat stress in six yeast species isolated from Antarctica was examined. The yeast were classified into two groups: one psychrophilic, with a maximum growth temperature of 20°C, and the other psychrotrophic, capable of growth at temperatures above 20°C. In addition to species-specific heat shock protein (hsp) profiles, a heat shock (15°C–25°C for 3 h) induced the synthesis of a 110-kDa protein common to the psychrophiles, Mrakia stokesii, M. frigida, and M. gelida, but not evident in Leucosporidium antarcticum. Immunoblot analyses revealed heat shock inducible proteins (hsps) corresponding to hsps 70 and 90. Interestingly, no proteins corresponding to hsps 60 and 104 were observed in any of the psychrophilic species examined. In the psychrotrophic yeast, Leucosporidium fellii and L. scottii, in addition to the presence of hsps 70 and 90, a protein corresponding to hsp 104 was observed. In psychrotrophic yeast, as observed in psychrophilic yeast, the absence of a protein corresponding to hsp 60 was noted. Relatively high endogenous levels of trehalose which were elevated upon a heat shock were exhibited by all species. A 10 Celsius degree increase in temperature above the growth temperature (15°C) of psychrophiles and psychrotrophs was optimal for heat shock induced thermotolerance. On the other hand, in psychrotrophic yeast grown at 25°C, only a 5 Celsius degree increase in temperature was necessary for heat shock induced thermotolerance. Induced thermotolerance in all yeast species was coincident with hsp synthesis and trehalose accumulation. It was concluded that psychrophilic and psychrotrophic yeast, although exhibiting a stress response similar to mesophilic Saccharomyces cerevisiae, nevertheless had distinctive stress protein profiles. Received: August 7, 1997 / Accepted: October 22, 1997     

Antibacterial metabolites synthesized by psychrotrophic bacteria isolated from cold-freshwater environments

The ability of three psychrotrophic Gram-negative bacilli isolated from Chilean Patagonian cold freshwater rivers to produce bioactive metabolites was evaluated. The strains were isolated from cold waters rivers and identified by their biochemical properties and 16S rRNA gene analysis. The metabolites fractions showing antibacterial activity were obtained by solvent extraction and partially characterized by gas–mass chromatography (GC-MS). Antibacterial activity of the fractions was evaluated by an agar-well diffusion test upon 14 bacterial strains, both Gram positive and Gram negative. Thermal and proteolytic resistances of the antibacterial metabolites fractions were also evaluated. Molecular analysis allows the identification of the three Patagonian strains as Pseudomonas sp. RG-6 (Pseudomonas brenneri 99.6 % identity), Pseudomonas sp. RG-8 (Pseudomonas trivialis 99.6 % identity) and Yersinia sp. RP-3 (Yersinia aldovae 99.5 % identity). These extracts were able to inhibit both Gram-positive and Gram-negative bacteria but not Listeria monocytogenes. The antibacterial activity of the filtrated supernatants was lost at temperatures ≥60 °C, and was not affected by proteinase K treatment. The chemical structure of the active molecule remains to be elucidated, although the GC-MS analysis of the filtrates suggests that compounds like sesquiterpenes derivatives from β-maaliene or δ-selinene could be responsible of this antibacterial activity. Pristine cold freshwater streams showed to be interesting sources of metabolites-producing microorganisms with antibacterial activity.     

Structure of single-stranded nucleic acids in the presence of ribosomal protein S1.

We have developed a new method for mounting nucleic acids and nucleic acidprotein complexes for high-resolution electron microscopy, and have used it to characterize the interaction between ribosomal protein S1 and single-stranded nucleic acids. We find that SI unwinds most, but not all of the secondary structure present in MS2 RNA and øX174 viral DNA. The binding of S1 to DNA and RNA is not highly co-operative, and has a stoichiometry of one S1 per 10 to 15 nucleotides. We have not observed any tendency for S1 nucleic acid complexes to form aggregates in either 0·01 m-Na⁺ or 0·1 m-Na⁺. An analogous protein isolated from the 30 S ribosomal subunit of Caulobacter crescentus is indistinguishable from Escherichia coli S1 in these studies. The mono-N-ethylmaleimide derivative of E. coli S1 will bind to both MS2 RNA and øX174 viral DNA with a stoichiometry of one N-ethylmaleimide-S1 per 10 to 15 nucleotides, but will not unwind the secondary structure of either of them.     

Structure prediction and docking-based molecular insights of human YB-1 and nucleic acid interaction

Y-box-binding protein 1 (YB-1), a cold shock domain protein, is one of the most conserved nucleic acid-binding proteins. The multifunctional human YB-1 is a member of a large family of proteins with an evolutionary ancient cold shock domain. The presence of a cold shock domain is a specific feature of Y-box-binding proteins and allows attributing them to a wider group of proteins containing a cold shock domain. This protein is involved in a number of cellular processes including proliferation, differentiation and stress response. The YB-1 performs its function both in the cytoplasm and in the cell nucleus. In this study, we present the structure of full-length human YB-1 protein along with investigation of their nucleic acid-binding preferential. The study also focuses on biases for particular purine and pyrimidine bases. The overall goal of this study was to model and validate full-length YB-1 protein and to compare its nucleic acid-binding studies with previous reports.