超嗜热古菌整合性遗传元件的研究进展

细菌中整合性遗传元件与DNA修饰和防御、毒力因子传播以及次级代谢等生理功能存在关联,而相关研究在超嗜热古菌中尚处于起步阶段。本文综述了超嗜热古菌中整合性病毒、质粒及基因组岛等整合性遗传元件的分类、整合及维持机制。展示了整合性遗传元件参与的水平基因转移过程在超嗜热古菌基因组演化中扮演的重要角色。整合性遗传元件相关功能基因组学研究为理解超嗜热古菌的多样性及其环境适应性机制提供了新的视角。     

超嗜热古菌基因组的热稳定性

超嗜热古菌能够生活在80℃以上的高温环境中,它们的耐热性已经成为当前研究的热点之一。以往对超嗜热菌的认识多集中于蛋白质的耐热性,而很少有关于基因组热稳定性的综述文章。综述了当前对超嗜热古菌的基因组稳定性以及DNA损伤识别机制的研究进展,以期更好地了解超嗜热古菌的耐热机制。     

超嗜热古菌遗传操作系统研究进展

摘要：遗传操作系统,是研究基因和基因产物功能的一个极为重要的工具。超嗜热古菌遗传操作系统方面的研究落后于甲烷菌及嗜盐古菌中的研究,主要原因是选择标记的缺乏。然而,近十年来,在以硫化叶菌(Sulfolobus)为代表的超嗜热泉古菌和Thermococcus kodakaraensis为代表的超嗜热广古菌中,遗传操作系统研究取得了很大的进展。本文主要对这两种超嗜热古菌的遗传操作系统进展以及应用进行概述。     

极端微生物及其相关功能蛋白研究进展

极端微生物是一类能够适应特殊环境的微生物,相关功能蛋白在其适应极端环境过程中发挥着重要作用,探索极端微生物的特性及其相关的功能蛋白有助于深入了解生命的起源与进化,为蛋白酶在工业领域的应用提供一定理论依据。现概述耐辐射球菌、嗜盐菌、嗜热菌、嗜酸菌和嗜碱菌、嗜冷菌、嗜压菌的特性及其相关的功能蛋白质,从蛋白质水平阐述极端微生物对极端环境的适应机制。     

常温环境中产嗜热蛋白酶菌资源

通过计数、分离与筛选,对常温环境嗜热菌和产嗜热蛋白酶菌的分布及资源状况进行了研究。结果表明,常温环境中存在着一定数量的嗜热菌和产嗜热蛋白酶菌。土壤与水体相比,其嗜热菌资源相对丰富,且耕作肥沃的土壤中产嗜热蛋白酶菌多于贫瘠土壤;在水环境中,无论湖水、江水还是处理中的废水,在常温条件下均有一定比例的嗜热菌和产嗜热蛋白酶菌。在啤酒废水曝气阶段,产嗜热蛋白酶菌占嗜热菌的比例较大,达45％。本研究筛选的1株嗜热菌其产嗜热蛋白酶活性较高,该菌株在pH7．6、温度68℃条件下其蛋白酶活力达到642U·ml^-1。该项研究为开发产嗜热蛋白酶菌资源,在工业和环境治理等方面的应用提供重要科学依据。     

极端嗜热古菌的热休克蛋白

随着生物工程产业对于耐高温酶和菌体的需求, 极端嗜热古菌热休克蛋白(heat shock proteins, HSPs)的研究更受重视, 其热休克蛋白体系非常简洁, 不含HSP100s和HSP90s, 就是HSP70(DnaK)、HSP40、(DnaJ)和GrpE等嗜温古菌可能含有的在极端嗜热古菌中几乎不含有, 即仅包括HSP60, sHSP, prefoldin和AAA+蛋白四大类, 因此对其结构、功能和作用机制的研究在理论和实践上都特别有意义。系统地介绍了这四大类组分的结构、功能和作用机制和协同作用的研究进展, 论述了极端嗜热古菌热休克蛋白的系列研究难点和困惑, 展望了进一步的研究方向和重点。     

嗜盐古菌启动子的研究及应用

嗜盐古菌是古菌域的一个重要代表类群,在遗传与代谢、进化与适应、前沿生物技术及合成生物学领域都显示了其重要的研究价值。嗜盐古菌启动子的认识和利用,可以为嗜盐古菌的基础和应用研究提供必要的条件。本文从古菌启动子的结构与功能出发,就启动子的研究方法、嗜盐古菌启动子的特征及嗜盐古菌启动子的应用3个方面综述了嗜盐古菌启动子的研究现状,并对嗜盐古菌启动子未来研究的重点和方向进行了展望。     

极端嗜热古菌DNA修复核酸内切酶的研究进展

极端嗜热古菌由于生活在高温环境,其基因组DNA面临着严重的挑战,因此,它们如何维持其基因组稳定是本研究领域最为关注的科学问题之一。极端嗜热古菌具有与常温微生物相似的自发突变频率,暗示着它们比常温微生物具有更加有效的DNA修复体系进行修复高温所造成的基因组DNA损伤。目前,极端嗜热古菌DNA修复的分子机制尚不清楚。核酸内切酶在DNA修复途径中发挥着重要的作用。基因组序列显示极端嗜热古菌编码多种DNA修复核酸内切酶,但是其研究尚处于初期阶段。本文综述了极端嗜热古菌DNA修复核酸内切酶Nuc S、Endo V、Endo Q、XPF和Hjc的研究进展,并对今后的研究提出了展望。     

巴里坤湖和玛纳斯湖嗜盐菌的分离及功能酶的筛选

目的:了解新疆巴里坤湖与马纳斯湖中嗜盐菌及功能酶的多样性。方法:从两湖中采集水样进行菌种分离,采用PCR方法扩增出其16S rRNA基因(16S rDNA),并测定了基因的序列。对分离菌株进行了蛋白酶、淀粉酶、酯酶、脂肪酶、以及纤维素酶的筛选。结果:从两湖水样共分离得到51株嗜盐菌。基于16SrDNA序列的同源性比较和系统发育学分析,发现从两湖分离获得的中度嗜盐菌分别属于Planococcaceae、Bacillacea、Staphylococcus、Halomonadaceae、Salicolaceae以及Pseudomonadacaeae 6个属。分离得到的极端嗜盐古菌属于Halobacteriaceae属。功能酶筛选结果表明产蛋白酶的嗜盐菌共有15株,产酯酶的共有23株,产淀粉酶的共有8株,未获得产脂肪酶和纤维素酶的嗜盐菌。结论:新疆巴里坤湖和马纳斯湖中有丰富的嗜盐微生物资源及酶资源,有重要的研究意义和应用前景。     

极端嗜热微生物及其高温适应机制的研究进展

极端嗜热微生物在高温条件下生长繁殖,其必然具有适应高温环境的特殊细胞结构、基因类型以及生理生化机制。极端嗜热微生物的研究对探索生命的起源以及极端嗜热微生物的开发和应用具有重要意义。对极端嗜热微生物中细胞膜、核酸分子、蛋白质分子、代谢产物和辅酶的高温适应机制的研究进展进行了概述,旨为极端嗜热微生物以及来源于极端嗜热微生物的各种生物分子的开发和应用提供理论依据。     

Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability.

Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of > 80 degrees C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described.     

Type I signal peptidase: an overview

The signal hypothesis suggests that proteins contain information within their amino acid sequences for protein targeting to the membrane. These distinct targeting sequences are cleaved by specific enzymes known as signal peptidases. There are various type of signal peptidases known such as type I, type II, and type IV. Type I signal peptidases are indispensable enzymes, which catalyze the cleavage of the amino-terminal signal-peptide sequences from preproteins, which are translocated across biological membranes. These enzymes belong to a novel group of serine proteases, which generally utilize a Ser-Lys or Ser-His catalytic dyad instead of the prototypical Ser-His-Asp triad. Despite having no distinct consensus sequence other than a commonly found 'Ala-X-Ala' motif preceding the cleavage site, signal sequences are recognized by type I signal peptidase with high fidelity. Type I signal peptidases have been found in bacteria, archaea, fungi, plants, and animals. In this review, I present an overview of bacterial type I signal peptidases and describe some of their properties in detail.     

Stability and repair of DNA in hyperthermophilic Archaea

Evolutionary and physiological considerations argue that study of hyperthermophilic archaea should reveal new molecular aspects of DNA stabilization and repair. So far, these unusual prokaryotes have yielded a number of genes and enzymatic activities consistent with known mechanisms of excision repair, photo-reversal, and trans-lesion synthesis. However, other DNA enzymes of hyperthermophilic archaea show novel biochemical properties which may be related to DNA stability or repair at extremely high temperature but which remain difficult to evaluate rigorously in vivo. Perhaps the most striking feature of the hyperthermophilic archaea is that all of them whose genomes have been sequenced lack key genes of both the nucleotide excision repair and DNA mismatch repair pathways, which are otherwise highly conserved in biology. Although the growth properties of these micro-organisms hinder experimentation, there is evidence that some systems of excision repair and mutation avoidance operate in Sulfolobus spp. It will therefore be of strategic significance in the next few years to formulate and test hypotheses in Sulfolobus spp. and other hyperthermophilic archaea regarding mechanisms and gene products involved in the repair of UV photoproducts and DNA mismatches.     

Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of >80°C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described.     

极端嗜热古菌尿嘧啶DNA糖苷酶的研究进展

高温会加快碱基脱氨基反应形成损伤碱基的速率,进一步对脱氨基的碱基进行复制会导致突变。因此,极端嗜热古菌基因组的稳定性面临着其生存高温环境的挑战。胞嘧啶脱氨基形成尿嘧啶,是常见的脱碱基类型,复制DNA中尿嘧啶会造成GC→AT的突变。尿嘧啶DNA糖苷酶(Uracil DNA glycosylase,UDG)是修复DNA中尿嘧啶的关键酶。基于识别底物的特异性,UDG分为6个家族,广泛分布在细菌、古菌、真核生物以及一些病毒中。基因组序列显示,极端嗜热古菌至少编码一种UDG。目前,对于细菌和真核生物的UDG已进行了大量的研究,但是关于极端嗜热古菌UDG的研究相对较少,尚处于初期阶段。本文综述了极端嗜热古菌UDG的研究进展,并对今后的研究提出了展望。     

Identification and characterization of thioredoxin and thioredoxin reductase from Aeropyrum pernix K1.

We have identified and characterized a thermostable thioredoxin system in the aerobic hyperthermophilic archaeon Aeropyrum pernix K1. The gene (Accession no. APE0641) of A. pernix encoding a 37 kDa protein contains a redox active site motif (CPHC) but its N-terminal extension region (about 200 residues) shows no homology within the genome database. A second gene (Accession no. APE1061) has high homology to thioredoxin reductase and encodes a 37 kDa protein with the active site motif (CSVC), and binding sites for FAD and NADPH. We cloned the two genes and expressed both proteins in E. coli. It was observed that the recombinant proteins could act as an NADPH-dependent protein disulfide reductase system in the insulin reduction. In addition, the APE0641 protein and thioredoxin reductase from E. coli could also catalyze the disulfide reduction. These indicated that APE1061 and APE0641 express thioredoxin (ApTrx) and thioredoxin reductase (ApTR) of A. pernix, respectively. ApTR is expressed as an active homodimeric flavoprotein in the E. coli system. The optimum temperature was above 90 degrees C, and the half-life of heat inactivation was about 4 min at 110 degrees C. The heat stability of ApTR was enhanced in the presence of excess FAD. ApTR could reduce both thioredoxins from A. pernix and E. coli and showed a similar molar specific activity for both proteins. The standard state redox potential of ApTrx was about -262 mV, which was slightly higher than that of Trx from E. coli (-270 mV). These results indicate that a lower redox potential of thioredoxin is not necessary for keeping catalytic disulfide bonds reduced and thereby coping with oxidative stress in an aerobic hyperthermophilic archaea. Furthermore, the thioredoxin system of aerobic hyperthermophilic archaea is biochemically close to that of the bacteria.     

Hyperthermophiles and the problem of DNA instability

Rates of chemical decomposition of DNA at the optimal growth temperatures of hyperthermophiles seem incongruent with the requirements of accurate genome replication. The peculiar physiology, ecology and phylogeny of hyperthermophiles combine to suggest that these prokaryotes have solved a molecular problem (spontaneous loss of native DNA structure) of a magnitude that well-studied microorganisms do not face. The failure of DNA base composition to correlate with optimal growth temperature among hyperthermophiles provides indirect evidence that other mechanisms maintain their chromosomal DNA in the duplex form. Studies in vitro indicate that DNA primary structure is more difficult to maintain at extremely high temperature than is secondary structure, yet hyperthermophiles exhibit only modest levels of spontaneous mutation. Radiation sensitivity studies also indicate that hyperthermophiles repair their DNA efficiently in vivo , and underlying mechanisms are beginning to be examined. Several enzymes of DNA metabolism from hyperthermophilic archaea exhibit unusual biochemical features that may ultimately prove relevant to DNA repair. However, genomic sequencing results suggest that many DNA repair genes of hyperthermophilic archaea may not be recognized because they are not sufficiently related to those of well-studied organisms.