Anabaena sensory rhodopsin: a photochromic color sensor at 2.0 A

Microbial sensory rhodopsins are a family of membrane-embedded photoreceptors in prokaryotic and eukaryotic organisms. Structures of archaeal rhodopsins, which function as light-driven ion pumps or photosensors, have been reported. We present the structure of a eubacterial rhodopsin, which differs from those of previously characterized archaeal rhodopsins in its chromophore and cytoplasmic-side portions. Anabaena sensory rhodopsin exhibits light-induced interconversion between stable 13-cis and all-trans states of the retinylidene protein. The ratio of its cis and trans chromophore forms depends on the wavelength of illumination, thus providing a mechanism for a single protein to signal the color of light, for example, to regulate color-sensitive processes such as chromatic adaptation in photosynthesis. Its cytoplasmic half channel, highly hydrophobic in the archaeal rhodopsins, contains numerous hydrophilic residues networked by water molecules, providing a connection from the photoactive site to the cytoplasmic surface believed to interact with the receptor's soluble 14-kilodalton transducer.     

Structure of the light-driven chloride pump halorhodopsin at 1.8 A resolution

Halorhodopsin, an archaeal rhodopsin ubiquitous in Haloarchaea, uses light energy to pump chloride through biological membranes. Halorhodopsin crystals were grown in a cubic lipidic phase, which allowed the x-ray structure determination of this anion pump at 1.8 angstrom resolution. Halorhodopsin assembles to trimers around a central patch consisting of palmitic acid. Next to the protonated Schiff base between Lys(242) and the isomerizable retinal chromophore, a single chloride ion occupies the transport site. Energetic calculations on chloride binding reveal a combination of ion-ion and ion-dipole interactions for stabilizing the anion 18 angstroms below the membrane surface. Ion dragging across the protonated Schiff base explains why chloride and proton translocation modes are mechanistically equivalent in archaeal rhodopsins.     

Human rhodopsin

Human rhodopsin in aqueous solution has lambda(max). of 493 mmicro and is lower in the spectrum than the rhodopsins of all other known vertebrates, with the exception of certain deep-sea fishes. Its molar extinction is 40,000 +/- 800. Like other rhodopsins, it bleaches to a mixture of opsin and all-trans retinene and is resynthesized by incubating opsin with neo-b (11-cis) retinene. The regenerated rhodopsin has the same lambda(max). as the extracted pigment; this is due, therefore, not to an unusual retinene but to a characteristic human opsin. The regeneration in solution from opsin and neo-b retinene is a second-order reaction with a half-time, at 29.5 degrees C, of about 2.5 minutes. This is much faster than the synthesis of rhodopsin in the living human eye, and faster than human rod dark-adaptation; the rate of both processes in vivo must be limited by reactions which precede the union of neo-b retinene with opsin, the final step in rhodopsin synthesis. In the rods, rhodopsin is virtually in the solid state-highly oriented in close relation with other highly oriented molecules. In this situation its spectrum is displaced toward the red (lambda(max) 500 mmicro) and is narrower than in solution. For light entering the rods axially, rhodopsin has also a considerably increased extinction, some 1.5 times higher than when randomly oriented. The spectrum of rhodopsin in rods agrees well in form and position with the spectral sensitivity of human rod vision, measured at the retinal surface.     

乌鲁木齐地震断裂带泉水古菌群落对水文地球化学元素的响应

[目的]揭示乌鲁木齐地震断裂带泉水中古菌群落对水文地球化学元素变化的响应.[方法]采用微孔滤膜法收集泉水中菌体,直接提取环境总DNA,嵌套式PCR扩增古菌16S rDNA基因V3区,变性梯度凝胶电泳(DGCE)检测古菌群落结构的变化,所得信息与监测的地球化学指标进行典型相关分析.[结果]DGGE分析及切胶测序共得到古菌14个主要类群,分属于广域古菌和泉古菌两个门,全部为不可培养类群,其中前者占绝对优势.典型相关性分析发现类群B-11和B-12与He以及CH<,4>含量呈正相关,与硫化物呈负相关;类群B-2、B-3和B-9与F<'->离子含量成正相关而类群B-13与它呈负相关;而仅有的泉古菌B-7与CH呈负相关.另外,所得绝大部分古菌类群与其它冷泉或者低温环境中发现的古菌类群具有95;左右的相似性.[结论]乌鲁木齐断裂带泉水古菌群落能够对泉水中的某些地球化学元素变化产生一定的响应,并且可能存在大量嗜冷新类群.     

Bleaching of rhabdoms in eyes of intact butterflies

The photochemistry of butterfly rhabdoms has properties that had been associated exclusively with the photoreceptor organelles of vertebrates. Noninvasive measurements of the absorbance spectra of rhabdoms in intact butterflies show that their rhodopsins are converted by light to metarhodopsins that decay from the rhabdom in the dark. A total bleach is possible because the first-order decay of metarhodopsin is considerably faster than the kinetically more complicated recovery of rhodopsin.     

Function of rhodopsin in temperature discrimination in Drosophila

Many animals, including the fruit fly, are sensitive to small differences in ambient temperature. The ability of Drosophila larvae to choose their ideal temperature (18°C) over other comfortable temperatures (19° to 24°C) depends on a thermosensory signaling pathway that includes a heterotrimeric guanine nucleotide-binding protein (G protein), a phospholipase C, and the transient receptor potential TRPA1 channel. We report that mutation of the gene (ninaE) encoding a classical G protein-coupled receptor (GPCR), Drosophila rhodopsin, eliminates thermotactic discrimination in the comfortable temperature range. This role for rhodopsin in thermotaxis toward 18°C was light-independent. Introduction of mouse melanopsin restored normal thermotactic behavior in ninaE mutant larvae. We propose that rhodopsins represent a class of evolutionarily conserved GPCRs that are required for initiating thermosensory signaling cascades.     

藏系绵羊瘤胃古菌季节动态分析

在不同季节分别随机选取3只藏系绵羊,采用PCR-DGGE技术对12只藏系绵羊瘤胃内容物中的细菌区系进行分子生态学分析.对古菌16SrDNA的V2～V4可变区的扩增产物进行DGGE图谱分析、相似性分析和Shannon多样指数分析.结果表明:瘤胃内容物中古菌群落多样性指数冬季与春季间差异不显著(P>0.05),但冬季显著高于夏季(P<0.05),并极显著高于秋季(P<0.01);春季与夏季差异不显著(P>0.05),春季显著高于秋季(P<0.05);夏、秋季之间差异不显著(P>0.05).各季间瘤胃古菌区系组成结构相似性高于94%.冬、春季节,由于牧草中的纤维性物质含量增高,导致瘤胃古菌群落多样性增加,不同季节牧草中粗蛋白和酸性洗涤纤维含量的变化可以引起特异性的古菌群落发育,可能导致不同的优势种群.     

Identification of the Cdc48•20S proteasome as an ancient AAA+ proteolytic machine

Proteasomes are the major energy-dependent proteolytic machines in the eukaryotic and archaeal domains of life. To execute protein degradation, the 20S core peptidase combines with the AAA+ ring of the 19S regulatory particle in eukarya or with the AAA+ proteasome-activating nucleotidase ring in some archaea. Here, we find that Cdc48 and 20S from the archaeon Thermoplasma acidophilum interact to form a functional proteasome. Cdc48 is an abundant and essential double-ring AAA+ molecular machine ubiquitously present in archaea, where its function has been uncertain, and in eukarya where Cdc48 participates by largely unknown mechanisms in diverse cellular processes, including multiple proteolytic pathways. Thus, proteolysis in collaboration with the 20S peptidase may represent an ancestral function of the Cdc48 family.     

天山北坡垂直自然带土壤古菌多样性分析

[目的]了解天山北坡垂直自然带土壤古菌多样性、优势种群及影响其分布的主要因素.[方法]利用末端限制性片段长度多态性(Terminal Restriction Fragment Length Polymorphism,T- RFLP),对天山北坡垂直自然带(山地荒漠带(H1),山地草原带(H2),山地森林带(H3),亚高山草甸带(H4),高山草甸带(H5),冰雪带(H6))土壤古菌多样性及主要影响因素进行研究.[结果]古菌多样性指数在山地森林带最低,高山草甸带和冰雪带最高;聚类分析表明,山地荒漠带、亚高山草甸带、高山草甸带和冰雪带聚为一类,山地草原带和山地森林带聚为一类;典范对应分析(CCA)表明:香依指数(Shannon index,H)和均匀度指数(Evenness,E)均与C/N,土壤湿度及海拔成正相关,与pH,土壤温度成负相关;T- RFs与pH,全氮,有效磷成正相关.[结论]天山北坡垂直自然带土壤古菌多样性丰富度较低,土壤pH、湿度、全氮及植被类型可能是影响其多样性的关键因素.     

Transmembrane protein structure: spin labeling of bacteriorhodopsin mutants

Transmembrane proteins serve important biological functions, yet precise information on their secondary and tertiary structure is very limited. The boundaries and structures of membrane-embedded domains in integral membrane proteins can be determined by a method based on a combination of site-specific mutagenesis and nitroxide spin labeling. The application to one polypeptide segment in bacteriorhodopsin, a transmembrane chromoprotein that functions as a light-driven proton pump is described. Single cysteine residues were introduced at 18 consecutive positions (residues 125 to 142). Each mutant was reacted with a specific spin label and reconstituted into vesicles that were shown to be functional. The relative collision frequency of each spin label with freely diffusing oxygen and membrane-impermeant chromium oxalate was estimated with power saturation EPR (electron paramagnetic resonance) spectroscopy. The results indicate that residues 129 to 131 form a short water-exposed loop, while residues 132 to 142 are membrane-embedded. The oxygen accessibility for positions 131 to 138 varies with a periodicity of 3.6 residues, thereby providing a striking demonstration of an alpha helix. The orientation of this helical segment with respect to the remainder of the protein was determined.     

极端嗜盐古菌嗜盐特性研究及在废水处理中的应用

[目的]探讨极端嗜盐古菌的生理生化特性、最适生长条件以及对苯酚的降解效果。[方法]以一株盐生盐杆菌SYGJ-1为研究对象,分析了极端嗜盐古菌的生理生化特性以及不同生长条件(盐度、温度、pH)对其生长情况的影响,并探讨了该菌对苯酚的降解效果。[结果]极端嗜盐古菌SYGJ-1的最适盐度为19%,盐度17%或者25%时,生长量急剧下降;SEM图片表明,菌体在最适盐度时呈饱满短杆状,盐度小于15%时菌体呈球状,在盐度大于29%时会同时出现球状及长杆状菌体;最适温度为37℃,对数生长期为12~66 h;适宜pH范围为6.8~9.0;在苯酚浓度200 mg/L的培养基中生长良好,此时对苯酚的去除效率约为40%。[结论]极端嗜盐菌可有效降解有机污染物,并且菌体生长量与降解效率呈正相关。     

Isotopic signature of N(2)O produced by marine ammonia-oxidizing archaea

The ocean is an important global source of nitrous oxide (N(2)O), a greenhouse gas that contributes to stratospheric ozone destruction. Bacterial nitrification and denitrification are thought to be the primary sources of marine N(2)O, but the isotopic signatures of N(2)O produced by these processes are not consistent with the marine contribution to the global N(2)O budget. Based on enrichment cultures, we report that archaeal ammonia oxidation also produces N(2)O. Natural-abundance stable isotope measurements indicate that the produced N(2)O had bulk δ(15)N and δ(18)O values higher than observed for ammonia-oxidizing bacteria but similar to the δ(15)N and δ(18)O values attributed to the oceanic N(2)O source to the atmosphere. Our results suggest that ammonia-oxidizing archaea may be largely responsible for the oceanic N(2)O source.     

嗜盐古菌Haloferax sp. H4蛋白质表达差异研究

[目的]研究嗜盐古菌Haloferax sp.H4在不同环境中的蛋白表达差异。[方法]采用变性聚丙烯酰胺凝胶电泳（SDS-PAGE）检测不同NaCl浓度、pH、温度、有氧和无氧条件下的H4菌株蛋白表达差异。[结果]H4菌株在不同NaCl浓度、有氧和无氧条件下的蛋白表达存在明显特异性;而在不同pH和温度条件下,其蛋白表达差异性不明显。[结论]嗜盐古菌对不同环境因子的调控方式可能存在差异。     

转cry1Ab和epsps基因玉米C0030.3.5对土壤古菌丰度和多样性的影响

为探讨转基因玉米的生态安全问题,2015年分别于玉米拔节期、抽雄期、乳熟期和完熟期采集土壤样品,并采用荧光定量PCR(Quantitative real-time PCR,q PCR)和末端限制性片段长度多态性分析(Terminal restriction fragment length polymorphism,TRFLP)技术,研究了转cry1Ab和epsps基因玉米C0030.3.5种植对土壤古菌丰度和多样性的影响。结果表明:转cry1Ab和epsps基因玉米C0030.3.5(TM)和受体玉米DBN318(PM)的根际土和非根际土古菌数量为1.41×10~9~4.04×10~9copies·g~(-1)土,随生长时期的推进均呈现先升高后降低的变化趋势,同一生长时期和土壤样品采集区域,2种玉米古菌16S rRNA基因丰度间无显著差异。T-RFLP分析共获得15种不同长度的T-RFs,其中89 bp和184 bp片段为优势种群,同一生长时期和土壤样品采集区域每种优势种群在TM和PM间均无显著差异。土壤古菌Shannon指数除PM非根际土呈现先降低后升高的变化趋势外,其他均表现为先降低后升高再降低;TM和PM根际土古菌Evenness指数的变化趋势均为先降低后升高,而非根际土古菌Evenness指数表现为先降低后升高再降低。同一生长时期和土壤样品采集区域TM和PM的Shannon指数间及Evenness指数间差异均不显著。冗余分析(Redundancy analysis,RDA)显示土壤总氮和硝态氮含量对古菌群落结构有显著影响。主成分分析(Principal component analysis,PCA)表明,TM和PM土壤古菌群落在根际土和非根际土中均未发生明显分离,说明TM和PM土壤古菌群落组成无显著差异。上述结果表明:转cry1Ab和epsps基因玉米C0030.3.5土壤古菌丰度和群落结构组成与受体玉米DBN318无显著差异,古菌丰度和多样性主要受生长时期的影响,受土壤样品采集区域的影响不显著,土壤总氮和硝态氮是调控土壤古菌群落变化的关键因子。     

Photoelectric potential from photoreceptor cells in ventral eye of Limulus

Intense colored light from a gas laser evokes a photoelectric potential in the photoreceptor cells of the ventral eye of Limulus. This potential has two components, both of which have the action spectrum of a rhodopsin with an absorbancy maximum at 530 nm. The evidence is consistent with the hypothesis that the photoelectric potential arises directly from the orderly array of rhodopsin molecules which are an integral part of the photoreceptor cell membrane.     

Channelrhodopsin-1: a light-gated proton channel in green algae

Phototaxis and photophobic responses of green algae are mediated by rhodopsins with microbial-type chromophores. We report a complementary DNA sequence in the green alga Chlamydomonas reinhardtii that encodes a microbial opsin-related protein, which we term Channelopsin-1. The hydrophobic core region of the protein shows homology to the light-activated proton pump bacteriorhodopsin. Expression of Channelopsin-1, or only the hydrophobic core, in Xenopus laevis oocytes in the presence of all-trans retinal produces a light-gated conductance that shows characteristics of a channel selectively permeable for protons. We suggest that Channelrhodopsins are involved in phototaxis of green algae.     

One polypeptide with two aminoacyl-tRNA synthetase activities

The genome sequences of certain archaea do not contain recognizable cysteinyl-transfer RNA (tRNA) synthetases, which are essential for messenger RNA-encoded protein synthesis. However, a single cysteinyl-tRNA synthetase activity was detected and purified from one such organism, Methanococcus jannaschii. The amino-terminal sequence of this protein corresponded to the predicted sequence of prolyl-tRNA synthetase. Biochemical and genetic analyses indicated that this archaeal form of prolyl-tRNA synthetase can synthesize both cysteinyl-tRNA(Cys) and prolyl-tRNA(Pro). The ability of one enzyme to provide two aminoacyl-tRNAs for protein synthesis raises questions about concepts of substrate specificity in protein synthesis and may provide insights into the evolutionary origins of this process.     

Crystal structure of rhodopsin: A G protein-coupled receptor

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) respond to a variety of different external stimuli and activate G proteins. GPCRs share many structural features, including a bundle of seven transmembrane alpha helices connected by six loops of varying lengths. We determined the structure of rhodopsin from diffraction data extending to 2.8 angstroms resolution. The highly organized structure in the extracellular region, including a conserved disulfide bridge, forms a basis for the arrangement of the seven-helix transmembrane motif. The ground-state chromophore, 11-cis-retinal, holds the transmembrane region of the protein in the inactive conformation. Interactions of the chromophore with a cluster of key residues determine the wavelength of the maximum absorption. Changes in these interactions among rhodopsins facilitate color discrimination. Identification of a set of residues that mediate interactions between the transmembrane helices and the cytoplasmic surface, where G-protein activation occurs, also suggests a possible structural change upon photoactivation.     

Structural basis of DNA replication origin recognition by an ORC protein

DNA replication in archaea and in eukaryotes share many similarities. We report the structure of an archaeal origin recognition complex protein, ORC1, bound to an origin recognition box, a DNA sequence that is found in multiple copies at replication origins. DNA binding is mediated principally by a C-terminal winged helix domain that inserts deeply into the major and minor grooves, widening them both. However, additional DNA contacts are made with the N-terminal AAA+ domain, which inserts into the minor groove at a characteristic G-rich sequence, inducing a 35 degrees bend in the duplex and providing directionality to the binding site. Both contact regions also induce substantial unwinding of the DNA. The structure provides insight into the initial step in assembly of a replication origin and recruitment of minichromosome maintenance (MCM) helicase to that origin.     

冰岛硫化叶菌xpb基因缺失突变体的构建及遗传学分析

为在体内研究古菌核苷酸切除修复(nucleotide excision repair,NER)机制,利用基因敲除的方法,对泉古菌核苷酸切除修复机制进行研究,构建了冰岛硫化叶菌编码真核生物NER解旋酶XPB同源蛋白的基因xpb1和xpb2单缺失突变体,从而在体内分析xpb1和xpb2基因的功能。结果表明:基因xpb1或xpb2不是冰岛硫化叶菌存活的必需基因。表型分析发现,xpb1和xpb2基因单缺失突变体相较野生型菌株REY15A,对DNA损伤试剂4-NQO 4-硝基喹啉-1-氧化物(4-nitroquinoline 1-oxide,4-NQO)分别表现出轻微敏感性和不敏感性,暗示NER解旋酶功能在冰岛硫化叶菌体内存在多重冗余。