微生物产生的冷休克蛋白研究进展

冷休克蛋白(cold shock protein,Csp)首先在大肠杆菌中发现,它与微生物对冷环境的适应及多种细胞功能有关。冷休克蛋白基因是一段编码70个左右氨基酸的DNA序列,在这段序列中有5′非翻译区(5′UTR)、冷盒及下游盒等特征。冷休克蛋白作为DNA或RNA结合蛋白在基因表达调控过程中起重要作用。冷休克蛋白在转录、mRNA稳定性及翻译等几个水平上被严格调控。     

异源表达古菌TRAM基因增强水稻的耐旱性

干旱会直接影响水稻的生长发育,导致其产量和品质下降。在水稻中异源表达细菌RNA分子伴侣Csp能够显著提高水稻的耐旱能力,并且不影响水稻的正常生长。古菌中也发现具有类似细菌分子伴侣Csp功能的TRAM (TRM2 and MiaB)蛋白,且古菌的DNA复制、转录和翻译等过程与真核生物有着更为相似的调控方式,然而,古菌中RNA分子伴侣蛋白能否调控植物耐旱能力还未见报道。我们选取了嗜冷甲烷古菌Methanolobus psychrophilus R15中两个TRAM蛋白在水稻中进行研究,发现在水稻中过量表达Mpsy_3066和Mpsy_0643两个TRAM蛋白均能显著提高水稻苗期和成株期时对干旱胁迫的耐受能力。同时,我们在水稻原生质体中验证了TRAM蛋白可以发挥其分子伴侣的功能消除RNA的错误折叠对翻译的影响,这可能是TRAM转基因植物发挥其耐旱能力的作用机制。该工作初步展示了异源表达古菌TRAMs可以作为提高水稻耐旱能力的一种有效手段。     

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.     

Functional similarities and differences of an archaeal Hsp70(DnaK) stress protein compared with its homologue from the bacterium Escherichia coli

Archaea are prokaryotes but some of their chaperoning systems resemble those of eukaryotes. Also, not all archaea possess the stress protein Hsp70(DnaK), in contrast with bacteria and eukaryotes, which possess it without any known exception. Further, the primary structure of the archaeal DnaK resembles more the bacterial than the eukaryotic homologues. The work reported here addresses two questions: Is the archaeal Hsp70 protein a chaperone, like its homologues in the other two phylogenetic domains? And, if so, is the chaperoning mechanism of bacterial or eukaryotic type? The data have shown that the DnaK protein of the archaeon Methanosarcina mazei functions efficiently as a chaperone in luciferase renaturation in vitro, and that it requires DnaJ, and the other bacterial-type chaperone, GrpE, to perform its function. The M. mazei DnaK chaperone activity was enhanced by interaction with the bacterial co-chaperone DnaJ, but not by the eukaryotic homologue HDJ-2. Both the bacterial GrpE and DnaJ stimulated the ATPase activity of the M. mazei DnaK. The M. mazei DnaK-dependent chaperoning pathway in vitro is similar to that of the bacterium Escherichia coli used for comparison. However, in vivo analyses indicate that there are also significant differences. The M. mazei dnaJ and grpE genes rescued E.coli mutants lacking these genes, but E.coli dnaK mutants were not complemented by the M. mazei dnaK gene. Thus, while the data from in vitro tests demonstrate functional similarities between the M. mazei and E.coli DnaK proteins, in vivo results indicate that, intracellularly, the chaperones from the two species differ.     

Cold stress proteins induced in Listeria monocytogenes in response to temperature downshock and growth at low temperatures.

Listeria monocytogenes is a food-borne pathogen with the ability to grow at refrigerator temperatures. Twelve cold shock proteins (Csps) with apparent M(r)s of 48,600, 41,000, 21,800, 21,100, 19,700, 19,200, 18,800, 18,800, 17,200, 15,500, 14,500, and 14,400 were induced by cold shocking L. monocytogenes 10403S from 37 to 5 degrees C, as revealed by labeling with L-[35S]methionine followed by two-dimensional gel electrophoresis. Strain SLCC53 showed a similar response. Cold acclimation proteins were observed in cultures of strain 10403S growing at 5 degrees C, and four of these proteins, with apparent M(r)s 48,000, 21,100, 19,700, and 18,800, were also Csps. Two cold-sensitive transposon-induced mutants were labeled less efficiently than the parent strain, but the Csp response of the mutant examined was very similar to that of the parent strain.     

Psychrotolerant methanogenic archaea: Diversity and cold adaptation mechanisms

Because of their diversity and abundance in a wide range of environments, particularly in cold regions, cold-adaptive archaea are expected to play a pivotal role in material recycling in cold environments. Methanogenic archaea are ubiquitous on earth and produce a large amount of methane (CH₄) as their main carbon metabolite. Methanogens are the most laboratory amendable archaea. The few psychrophilic archaea that have been cultured to date are mainly affiliated with methanogens, thus make them a good model for investigating mechanisms of archaeal cold adaptation. Studies of psychrotolerant methanogens have been ongoing since the 1990s. Using Methanococcoides burtonii, a methanogen isolated from Ace Lake in Antarctica, extensive studies on the genomic characteristics associated with cold adaptation have been carried out by the Cavicchioli laboratory. We recently analyzed the genome of another psychrophilic methanogen and identified the gene repertoire associated with cold adaptation. This review summarizes recent studies of psychroactive methanogens, particularly their diversity, the genomics and proteomics associated with their cold adaptation, and the cellular components and proteins likely involved in their cold protection.     

Complementation of cold shock proteins by translation initiation factor IF1 in vivo.

The cold shock response in both Escherichia coli and Bacillus subtilis is induced by an abrupt downshift in growth temperature and leads to a dramatic increase in the production of a homologous class of small, often highly acidic cold shock proteins. This protein family is the prototype of the cold shock domain (CSD) that is conserved from bacteria to humans. For B. subtilis it has been shown that at least one of the three resident cold shock proteins (CspB to D) is essential under optimal growth conditions as well as during cold shock. Analysis of the B. subtilis cspB cspC double deletion mutant revealed that removal of these csp genes results in pleiotropic alteration of protein synthesis, cell lysis during the entry of stationary growth phase, and the inability to differentiate into endospores. We show here that heterologous expression of the translation initiation factor IF1 from E. coli in a B. subtilis cspB cspC double deletion strain is able to cure both the growth and the sporulation defects observed for this mutant, suggesting that IF1 and cold shock proteins have at least in part overlapping cellular function(s). Two of the possible explanation models are discussed.     

Cloning, overexpression, purification, and physicochemical characterization of a cold shock protein homolog from the hyperthermophilic bacterium Thermotoga maritima

Thermotoga maritima (Tm) expresses a 7 kDa monomeric protein whose 18 N-terminal amino acids show 81% identity to N-terminal sequences of cold shock proteins (Csps) from Bacillus caldolyticus and Bacillus stearothermophilus. There were only trace amounts of the protein in Thermotoga cells grown at 80 degrees C. Therefore, to perform physicochemical experiments, the gene was cloned in Escherichia coli. A DNA probe was produced by PCR from genomic Tm DNA with degenerated primers developed from the known N-terminus of TmCsp and the known C-terminus of CspB from Bacillus subtilis. Southern blot analysis of genomic Tm DNA allowed to produce a partial gene library, which was used as a template for PCRs with gene- and vector-specific primers to identify the complete DNA sequence. As reported for other csp genes, the 5' untranslated region of the mRNA was anomalously long; it contained the putative Shine-Dalgarno sequence. The coding part of the gene contained 198 bp, i.e., 66 amino acids. The sequence showed 61% identity to CspB from B. caldolyticus and high similarity to all other known Csps. Computer-based homology modeling allowed the conclusion that TmCsp represents a beta-barrel similar to CspB from B. subtilis and CspA from E. coli. As indicated by spectroscopic analysis, analytical gel permeation chromatography, and mass spectrometry, overexpression of the recombinant protein yielded authentic TmCsp with a molecular weight of 7,474 Da. This was in agreement with the results of analytical ultracentrifugation confirming the monomeric state of the protein. The temperature-induced equilibrium transition at 87 degrees C exceeds the maximum growth temperature of Tm and represents the maximal Tm-value reported for Csps so far.     

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.     

Archaeal and bacterial SecD and SecF homologs exhibit striking structural and functional conservation

The majority of secretory proteins are translocated into and across hydrophobic membranes via the universally conserved Sec pore. Accessory proteins, including the SecDF-YajC Escherichia coli membrane complex, are required for efficient protein secretion. E. coli SecDF-YajC has been proposed to be involved in the membrane cycling of SecA, the cytoplasmic bacterial translocation ATPase, and in the stabilizing of SecG, a subunit of the Sec pore. While there are no identified archaeal homologs of either SecA or SecG, many archaea possess homologs of SecD and SecF. Here, we present the first study that addresses the function of archaeal SecD and SecF homologs. We show that the SecD and SecF components in the model archaeon Haloferax volcanii form a cytoplasmic membrane complex in the native host. Furthermore, as in E. coli, an H. volcanii deltasecFD mutant strain exhibits both severe cold sensitivity and a Sec-specific protein translocation defect. Taken together, these results demonstrate significant functional conservation among the prokaryotic SecD and SecF homologs despite the distinct composition of their translocation machineries.     

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.     

Cysteine-string protein: the chaperone at the synapse

Cysteine-string protein (Csp) is a major synaptic vesicle and secretory granule protein first discovered in Drosophila and Torpedo. Csps were subsequently identified from Xenopus, Caenorhabditis elegans, and mammalian species. It is clear from the study of a null mutant in Drosophila that Csp is required for viability of the organism and that it has a key role in neurotransmitter release. In addition, other studies have directly implicated Csp in regulated exocytosis in mammalian neuroendocrine and endocrine cell types, and its distribution suggests a general role in regulated exocytosis. An early hypothesis was that Csp functioned in the control of voltage-gated Ca2+ channels. Csp, however, must have an additional function as a direct regulator of the exocytotic machinery as changes in Csp expression modify the extent of exocytosis triggered directly by Ca2+ in permeabilised cells. Csps possess a cysteine-string domain that is highly palmitoylated and confers membrane targeting. In addition, Csps have a conserved "J" domain that mediates binding to an activation of the Hsp70/ Hsc70 chaperone ATPases. This and other evidence implicate Csps as molecular chaperones in the synapse that are likely to control the correct conformational folding of one or more components of the vesicular exocytotic machinery. Targets for Csp include the vesicle protein VAMP/synaptobrevin and the plasma membrane protein syntaxin 1, the significance of which is discussed in possible models to account for current knowledge of Csp function.     

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.     

Structural basis of the interspecies interaction between the chaperone DnaK(Hsp70) and the co-chaperone GrpE of archaea and bacteria

Hsp70s are chaperone proteins that are conserved in evolution and present in all prokaryotic and eukaryotic organisms. In the archaea, which form a distinct kingdom, the Hsp70 chaperones have been found in some species only, including Methanosarcina mazei. Both the bacterial and archaeal Hsp70(DnaK) chaperones cooperate with a GrpE co-chaperone which stimulates the ATPase activity of the DnaK protein. It is currently believed that the archaeal Hsp70 system was obtained by the lateral transfer of chaperone genes from bacteria. Our previous finding that the DnaK and GrpE proteins of M. mazei can functionally cooperate with the Escherichia coli GrpE and DnaK supported this hypothesis. However, the cooperation was surprising, considering the very low identity of the GrpE proteins (26%) and the relatively low identity of the DnaK proteins (56%). The aim of this work was to investigate the molecular basis of the observed interspecies chaperone interaction. Infrared resolution-enhanced spectra of the M. mazei and E. coli DnaK proteins were almost identical, indicating high similarity of their secondary structures, however, some small differences in band position and in the intensity of amide I' band components were observed and discussed. Profiles of thermal denaturation of both proteins were similar, although they indicated a higher thermostability of the M. mazei DnaK compared to the E. coli DnaK. Electrophoresis under non-denaturing conditions demonstrated that purified DnaK and GrpE of E. coli and M. mazei formed mixed complexes. Protein modeling revealed high similarity of the 3-dimensional structures of the archaeal and bacterial DnaK and GrpE proteins.     

Archaeal phospholipids: Structural properties and biosynthesis

Phospholipids are major components of the cellular membranes present in all living organisms. They typically form a lipid bilayer that embroiders the cell or cellular organelles, constitute a barrier for ions and small solutes and form a matrix that supports the function of membrane proteins. The chemical composition of the membrane phospholipids present in the two prokaryotic domains Archaea and Bacteria are vastly different. Archaeal lipids are composed of highly-methylated isoprenoid chains that are ether-linked to a glycerol-1-phosphate backbone while bacterial phospholipids consist of straight fatty acids bound by ester bonds to the enantiomeric glycerol-3-phosphate backbone. The chemical structure of the archaeal lipids and their compositional diversity ensures the required stability at extreme environmental conditions as many archaea thrive at such conditions including high or low temperature, high salinity and extreme acidic or alkaline pH values. However, not all archaea are extremophiles, and the presence of ether-linked phospholipids is a phylogenetic marker that distinguishes Archaea from other life forms. During the past decade, our understanding of the biosynthesis of archaeal lipids has progressed resulting in the characterization of the main biosynthetic steps of the pathway including the reconstitution of lipid biosynthesis in vitro. Here we describe the chemical and physical properties of archaeal lipids and membranes derived thereof, summarize the existing knowledge about the enzymology of the archaeal lipid biosynthetic pathway and discuss evolutionary theories associated with the “Lipid Divide” that resulted in the differentiation of bacterial and archaeal organisms. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.     

Recognition between tRNASer and archaeal seryl-tRNA synthetases monitored by suppression of bacterial amber mutations

Two dissimilar seryl-tRNA synthetases (SerRSs) exist in Methanosarcina barkeri : one of bacterial type (bMbSerRS) and the other resembling SerRSs present only in methanogenic archaea (mMbSerRS). While the expression of the archaeal bMbSerRS gene in Escherichia coli complements the function of thermolabile SerRS at a nonpermissive temperature, mMbSerRS does not. Our recent X-ray structural analysis of mMbSerRS revealed an idiosyncratic N-terminal domain and a catalytic zinc ion in the active site, identifying methanogenic-type SerRSs as atypical members of the SerRS family. To shed further light on substrate discrimination by methanogenic-type SerRS, we developed an in vivo system in E. coli to study tRNA serylation by mMbSerRS variants. We show that coexpression of the M. barkeri SerRS gene, encoding either bacterial- or methanogenic-type SerRS, with the gene for cognate archaeal suppressor tRNA leads to suppression of bacterial amber mutations, implying that the E. coli translation machinery can use serylated tRNA from methanogenic archaea as a substrate in protein synthesis. Furthermore, because serylation of M. barkeri serine-specific tRNA by endogenous E. coli SerRS is negligible, suppression is entirely dependent on recognition between archaeal partners (mMbSerRS/suppressor tRNA^Ser). Thus, the efficiency of suppression by mMbSerRS variants quantified in the described β-galactosidase-based reporter system, accurately reflects enzymes' serylation propensity obtained by in vitro kinetic measurements.     

Is there an en route folding intermediate for cold shock proteins?

Cold shock proteins (Csps) play an important role in cold shock response of a diverse number of organisms ranging from bacteria to humans. Numerous studies of the Csp from various species showed that a two-state folding mechanism is conserved and the transition state (TS) appears to be very compact. However, the atomic details of the folding mechanism of Csp remain unclear. This study presents the folding mechanism of Csp in atomic detail using an all-atom Go model-based simulations. Our simulations predict that there may exist an en route intermediate, in which β strands 1-2-3 are well ordered and the contacts between β1 and β4 are almost developed. Such an intermediate might be too unstable to be detected in the previous fluorescence energy transfer experiments. The transition state ensemble has been determined from the P(fold) analysis and the TS appears even more compact than the intermediate state.