中国和美国原始土壤中非高温泉古菌的发现和鉴定

近年来在非极端环境中已经发现有古菌(Archaea)的存在, 但在中国原始土壤中还未见报道。本研究的目的是调查古菌是否存在于两个分别取自中国新疆和广西的土壤及两个美国亚利桑那州南部地区的土壤中。我们分别构建了这四个原始土壤的古菌16S rDNA文库并对28个克隆的16S rDNA进行了鉴定。所有这些16S rDNA的序列都归类于古菌的泉古菌门(Crenarchaeota)。进化树分析表明, 这些泉古菌的16S rDNA属于非高温陆地环境中的泉古菌种群, 明显区别于海洋和淡水地带的泉古菌种群。这个泉古菌种群又有两个分支, 这两个分支在16S rDNA序列上和G C含量上有明显的区别。本研究在两个中国和两个美国原始土壤中鉴定了非高温泉古菌的存在, 由此证明泉古菌的存在范围不只局限于高温等极端环境。另外, 美国原始土壤中的泉古菌只属于一个进化分支, 这说明非高温泉古菌种群的类型和土壤的地理位置及土壤特性有关。     

一个新的古菌类群———奇古菌门(Thaumarchaeota)

基于16S rRNA基因的系统发育关系,古菌域(Archaea)被分为两个主要类群:广古菌门(Euryarchaeota)和泉古菌门(Crenarchaeota)。近20年来,微生物分子生态学技术的快速发展和应用显示,在中温环境中广泛存在着大量的未培养古菌,而且它们可能在自然界重要元素(N、C)的生物地球化学循环中发挥着重要作用。最初,这些未培养古菌因在16S rRNA基因系统发育上与泉古菌关系较密切而被称作中温泉古菌(non-thermophilic Crenarchaeota)。而近年来,对更多新发现的中温古菌核糖体RNA基因序列和其它分子标记物进行的分析均不支持中温泉古菌由嗜热泉古菌进化而来的假设,而揭示其可能代表古菌域中一个独立的系统发育分支。基因组学、生理生态特征等分析也显示中温泉古菌与泉古菌具有明显不同的特征。因而专家建议将这些古菌(中温泉古菌)划分为一个新的门,成为古菌域的第三个主要类群—Thaumarchaeota(意译为奇古菌门)。这一新古菌门提出后得到其他研究证据的支持和认可。本文对目前已知的奇古菌门的分类地位演化、基因组学、多样性和生理代谢特征等作一简要综述。     

海洋古菌多样性研究进展

海洋古菌是海洋微生物中的一个大的类群,然而绝大多数的古菌不能分离培养.近年来分子生物学的方法广泛地应用于微生物多样性的研究中,研究发现,海洋古菌广泛地生活在各类海域环境中,而不仅仅是生活在极端的环境中.海洋古菌为海洋生态系统中主要的原核细胞成分,在海洋生态系统中的物质与能量循环中扮演着重要角色.主要阐述了生活在海洋不同环境中海洋古菌的多样性,有海洋浮游古菌的多样性、海底环境及海洋沉积物中古菌的多样性、附着或寄共生古菌多样性等的研究状况,以及研究海洋古菌多样性的分子生物学的主要方法.     

嗜盐古菌噬菌体研究进展

嗜盐古菌噬菌体是噬菌体的一个重要分支,在群体生态学以及生命的起源与进化历程中扮演重要的角色.综述了嗜盐古菌噬菌体的形态多样性、研究方法、起源与进化几方面的研究报道,指出目前研究中存在的问题,对未来研究进行了展望.     

若尔盖高原湿地土壤氨氧化古菌的多样性

【目的】研究自然界中氨氧化古菌(ammonia-oxidizing archaea,AOA)对于理解全球氮循环起着至关重要的作用,但人们对高原湿地AOA种群生态还知之甚少。本研究旨在了解若尔盖高原湿地土壤AOA群落组成及多样性。【方法】从若尔盖高原阿西(A'xi)、麦西(Maixi)和分区(Fenqu)3个典型牧区采集土壤样品,提取土壤总DNA,利用AOA氨单加氧酶(ammonia monooxygenase,amoA)基因通用引物扩增amoA基因,构建amoA基因克隆文库。从每个克隆文库中随机挑选80个阳性克隆子用于后续限制性酶切片段长度多态性(restriction fragment length polymorphism,RFLP)分析,挑选不同酶切类型的克隆子进行测序、比对,利用MEGA 5.0软件构建amoA基因系统发育树。【结果】从3个克隆文库共240个AOA amoA基因阳性克隆中得到15条代表序列,通过Mothur软件进行OTUs(operational taxonomic units)分类得到7个不同的分类单元。其中OTU 6为优势类群,在3个克隆文库均有发现,约占所有特异性克隆子的27%。15条amoA基因序列分属于Zoige Wetland Clade 1(4 OTUs)、Zoige Wetland Clade 2(2 OTUs)和Zoige Wetland Clade 3(1OTU)3个系统发育分支。BLAST分析显示所有OTUs均归于泉古菌门(Crenarchaeota)。相关性分析表明,若尔盖高原湿地AOA多样性指数与土壤铵态氮和硝态氮含量存在显著的相关性(P0.05)。【结论】若尔盖高原湿地中AOA多样性较低,均属于泉古菌,且与土壤中氨态氮和硝态氮密切相关。     

施氮肥对华北平原土壤氨氧化细菌和古菌数量及群落结构的影响

利用荧光定量PCR、末端限制性片段长度多样性(T-RFLP)和基因克隆文库技术,比较了4种施氮水平(不施氮肥,0 kg N/hm~2,CK;施低水平氮肥,75 kg N/hm~2,N1;施中水平氮肥,150 kg N/hm~2,N2;施高水平氮肥,225 kg N/hm~2,N3)下华北平原地区小麦季表层(0—20 cm)土壤总细菌、氨氧化细菌(AOB)和氨氧化古菌(AOA)的丰度和群落结构。结果表明,土壤总细菌、AOB和AOA数量分别在每克干土5.74×10~9—7.50×10~9、8.89×10~6—2.66×10~7和3.83×10~8—7.78×10~8之间。不同施氮量土壤AOA数量均高于AOB数量,AOA/AOB值在81.72—14.38之间。增施氮肥显著显著提高AOB数量(P0.05),对总细菌和AOA数量的影响不显著(P0.05)。与CK相比,处理N1、N2和N3中AOB数量分别提高了0.64、1.50和1.99倍。增施氮肥显著改变了AOB和AOA的群落结构,且不同施氮量处理中AOB群落结构差异更大。系统进化分析显示,施氮肥小麦土壤AOB主要为Nitrosospira属类群,分布在Cluster 3的两个分支中;AOA分布在Cluster S的4个分支中。相关性分析显示,AOB数量与全氮和铵态氮含量呈显著正相关关系,与土壤pH和碳氮比呈显著负相关关系(P0.05);AOA数量与硝态氮含量和土壤pH呈显著正相关关系,与铵态氮含量呈显著负相关关系(P0.05)。研究结果表明:增施氮肥可显著改变华北平原地区碱性土壤AOB数量与群落结构,该地区小麦土壤中AOB比AOA对氮肥响应更敏感。     

长期施肥对稻田土壤细菌、古菌多样性和群落结构的影响

稻田土壤是“迷失碳”的重要吸纳场所之一,也是温室气体(CH4和N2O等)的重要排放源.大气温室气体的动态变化与土壤碳氮转化的微生物过程紧密相关.以湖南桃江国家级稻田肥力变化长期定位试验点为平台,采用PCR-克隆测序和实时荧光定量PCR技术,研究不施肥(CK)、施氮磷钾肥(NPK)和氮磷钾肥+秸秆还田(NPKS)3种长期施肥制度(＞25 a)对稻田土壤细菌和古菌群落结构及数量的影响.细菌和古菌16S rRNA基因文库分析结果表明:稻田土壤细菌主要类群为变形菌、酸杆菌、绿弯菌,而古菌主要为泉古菌和广古菌.长期施肥导致土壤细菌和古菌种群结构产生明显差异,与CK相比,NPK和NPKS处理稻田土壤的变形菌、酸杆菌和泉古菌相对丰度增加.LIBSHUFF软件分析结果也表明,16S rRNA基因文库在CK、NPK及NPKS处理间存在显著差异.3种施肥处理的稻田土壤细菌16S rRNA基因拷贝数为每克干土0.58× 1010～1.06×1010个,古菌为每克干土1.16×106 ～ 1.72×106个.施肥(NPK和NPKS)后,细菌和古菌的多样性和数量增加,且NPKS＞NPK.说明长期施肥显著影响土壤细菌和古菌群落结构、多样性及数量.     

古菌蛋白激酶的研究进展

磷酸化是蛋白质翻译后修饰(post-translational modification)的主要方式,可由蛋白激酶、磷酸转移酶、磷酸化酶等多种方式催化进行。其中,由蛋白激酶(protein kinases)/磷酸酶(protein phosphatases)介导的可逆的蛋白磷酸化是细胞中信号转导的重要机制,在DNA复制、转录、蛋白质翻译、DNA损伤修复等生命过程中起广泛的调节作用。目前,古菌中蛋白激酶的研究尚属于初期阶段。虽然磷酸化蛋白质组学研究表明,古菌中存在大量的磷酸化蛋白质,但是我们对其具体催化作用的酶及调控机制尚不清楚。本文总结了古菌中已报道的蛋白激酶所参与的生命过程,包括古菌的DNA代谢、细胞代谢、细胞周期和运动机制等四个方面,并对今后的研究提出展望。     

古菌中蛋白甲基化修饰的研究进展

蛋白甲基化修饰是翻译后修饰的主要方式之一,越来越多的报道证实古菌中存在这类蛋白修饰。目前古菌中一些甲基转移酶已经鉴定出来,但对其作用机制还不太清楚。本文对目前古菌中已经发现的蛋白质甲基转移酶和甲基化修饰可能的作用进行了总结。古菌中蛋白的甲基化修饰能够提高蛋白稳定性、影响侧链构象变化及与其他分子的相互作用,涉及DNA损伤修复和应激反应等途径。最后,本文对今后古菌中蛋白甲基化修饰的研究方向进行了展望。     

甲烷代谢古菌分离培养研究进展

全球变暖是全人类面临的一个巨大挑战,而温室气体排放持续上升是全球变暖的关键因素,并引发一系列生态环境问题。甲烷是第二温室气体,对全球变暖的贡献达20%。然而,在甲烷代谢中发挥重要作用的产甲烷古菌和厌氧甲烷氧化古菌(anaerobic methanotroph,ANME)较难培养,极大地限制了人们对甲烷代谢及其影响碳源-汇关系与机制的研究。本文综述了最新产甲烷古菌和ANME富集、分离和培养方法,包括富集培养、原位培养、共培养、微流控技术、稀释分离和固体分离技术、ANME反应器和培养瓶富集培养,以及宏基因组预测和反向基因组学,并对这些方法的优缺点进行了评估,对未来甲烷代谢古菌的富集、分离和培养提出新的建议。     

免耕与留茬对土壤微生物量C、N及酶活性的影响

2005～2008年在内蒙古呼和浩特市清水河县进行定位试验,设免耕留低茬(NL)、免耕留高茬覆盖(NHS)和传统耕翻(T)3种耕作处理方式.结果表明:(1)免耕留高茬覆盖及免耕留低茬长期实施,能显著提高表层土壤有机质、全氮、全钾、碱解氮、速效磷和速效钾含量,且免耕留高茬覆盖处理比传统耕翻分别提高了11%、41%、22%、15%、29%、27%、13%;在测定各个时期内,土壤各营养指标含量整体趋势为NHS＞NL＞T.(2)免耕留高茬覆盖及免耕留低茬耕作方式有利于提高土壤微生物量C、N含量,在各测定时期均以免耕留高茬覆盖处理的土壤微生物量C、N含量最高,传统耕翻最低.与传统耕翻相比,免耕留高茬覆盖处理土壤微生物量C、N含量分别平均提高了69%、43%;测定各个时期,不同处理土壤生物量C、N含量均以7月份含量最高、5月份次之、10月份最低.(3)免耕留高茬覆盖及免耕留低茬处理土壤碱性磷酸酶、蔗糖酶、过氧化氢酶活性和脲酶活性高于传统耕翻,整个测定期内免耕留高茬覆盖处理4种酶平均活性,分别较传统耕翻增加了57%、82%、93%和25%;春季土壤酶活性开始增强,在7月份蔗糖酶、过氧化氢酶和脲酶活性达到最大值,而碱性磷酸酶的峰值出现在6月份.土壤微生物量C、N及土壤酶活性是评价土壤质量的重要因子.     

Viruses, plasmids and other genetic elements of thermophilic and hyperthermophilic Archaea

We review and update the work on genetic elements, e.g., viruses and plasmids (excluding IS elements and transposons) in the kingdom Crenarchaeota (Thermoproteales and Sulfolobales) and the orders Thermococcales and Thermoplasmales in the kingdom Euryachaeota of the archael domain, including unpublished data from our laboratory. The viruses of Crenarchaeota represent four novel virus families. The Fuselloviridae represented by SSV1 of S. shibatae and relatives in other Sulfolobus strains have the form of a failed spindle. The envelope is highly hydrophobic. The DNA is double-stranded and circular. Members of this group have also been found in Methanococcus and Haloarcula. The Lipothrixviridae (e.g., T TV1 to 3) have the form of flexible filaments. They have a core containing linear double-stranded DNA and DNA-binding proteins which is wrapped into a lipid membrane. The ‘Bacilloviridae’ (e.g., TTV4 and SIRV) are stiff rods lacking this membrane, but also featuring linear double-stranded DNA and DNA-binding proteins. Both virus type carry on both ends structures involved in the attachment to receptors. Both types are represented in Thermoproteus and Sulfolobus. The droplet-formed novel Sulfolobus virus SNDV represents the ‘Guttaviridae’ containing circular double-stranded DNA. Though head and tail viruses distantly resembling T phages or lambdoid phages were seen electronmicroscopically in solfataric water samples, no such virus has so far been isolated. SSV1 is temperate, TTV1 causes lysis after induction, the other viruses found so far exist in carrier states. The hosts of all but TTV1 survive virus production. We discuss the implications of the nature of these viruses for understanding virus evolution. The plasmids found so far range in size from 4.5 kb to about 40 kb. Most of them occur in high copy number, probably due to the way of their detection. Most are cryptic, pNOB8 is conjugative, the widespread pDL10 alleviates in an unknown way autotrophic growth of its host Desulfurolobus by sulfur reduction. The plasmid pTIK4 appears to encode a killer function. pNOB8 has been used as a vector for the transfer of the lac S (β-galactosidase) gene into a mutant of S. solfataricus.     

High abundance of Crenarchaeota in a temperate acidic forest soil

The objective of the study was to elucidate the depth distribution and community composition of Archaea in a temperate acidic forest soil. Numbers of Archaea and Bacteria were measured in the upper 18 cm of the soil, and soil cores were sampled on two separate occasions using quantitative PCR targeting 16S rRNA genes. Maximum numbers of Archaea were 0.6-3.8 x 10(8) 16S rRNA genes per gram of dry soil. Numbers of Bacteria were generally higher, but Archaea always accounted for a high percentage of the total gene numbers (12-38%). The archaeal community structure was analysed by the construction of clone libraries and by terminal restriction length polymorphism (T-RFLP) using the same Archaea-specific primers. With the reverse primer labelled, T-RFLP analysis led to the detection of four T-RFs. Three had lengths of 83, 185 and 218 bp and corresponded to uncultured Crenarchaeota. One (447 bp) was assigned to Thermoplasmales. Labelling of the forward primer allowed further separation of the T-RF into Crenarchaeota Group I.1c and Group I.1b, and indicated that Crenarchaeota of the Group I.1c were the predominant 16S rRNA genotype (相似文献   

Soil pH regulates the abundance and diversity of Group 1.1c Crenarchaeota

Archaeal communities in many acidic forest soil systems are dominated by a distinct crenarchaeal lineage Group 1.1c. In addition, they are found consistently in other acidic soils including grassland pasture, moorland and alpine soils. To determine whether soil pH is a major factor in determining their presence and abundance, Group 1.1c community size and composition were investigated across a pH gradient from 4.5 to 7.5 that has been maintained for > 40 years. The abundances of Group 1.1c Crenarchaeota, total Crenarchaeota and total bacteria were assessed by quantitative PCR (qPCR) targeting 16S rRNA genes and the diversity of Group 1.1c crenarchaeal community was investigated by denaturing gradient gel electrophoresis (DGGE) and phylogenetic analysis. The abundance of Group 1.1c Crenarchaeota declined as the pH increased, whereas total Crenarchaeota and Bacteria showed no clear trend. Community diversity of Group 1.1c Crenarchaeota was also influenced with different DGGE bands dominating at different pH. Group 1.1c Crenarchaeota were also quantified in 13 other soils representing a range of habitats, soil types and pH. These results exhibited the same trend as that shown across the pH gradient with Group 1.1c Crenarchaeota representing a greater proportion of total Crenarchaeota in the most acidic soils.     

Analysis of the non-thermophilic Crenarchaeota phylogeny in the swamp soil of Zoige plateau wetland

Zoige wetland of Tibetan plateau is a model low temperature ecosystem in a low latitude (33°56′N, 102°52′E) and high altitude region. Its organism has a unique phylogeny. To better evaluate the resource of the non-thermophilic Crenarchaeota in such an ecosystem, both restriction fragment length polymorphism (RFLP) and clone techniques were employed to study the diversity and phylogenetics of the non-thermophilic Crenarchaeota in the wetland soil. Archaeal 16S rRNA genes were amplified with the archaea-specific primers, and a library consisting of 240 clones was established. The non-thermophilic Crenarchaeota phylogenetic tree was constructed using the ARB phylogenetic analysis software. Based on the results of the RFLP experiments, the clones of all three Zoige wetland swamp soil samples were grouped into 16 different restriction cleavage patterns, all the clone coverage indices were above 91%, showing high library coverage. The correlations analysis indicated that the biodiversity of the non-thermophilic Crenarchaeota be positively correlated with soil moisture. The phylogenetic analysis revealed that all of the 16 Crenarchaeota sequences were clustered into two groups: 13 sequences in the Group 1.1b and 3 in the Group 1.3, similar to those archaeal sequences obtained from grassland soil, freshwater reservoirs, and seawater above boreholes, and radioactive groundwater and hot springs.     

Genetic elements in the extremely thermophilic archaeon Sulfolobus

This minireview summarizes what is known about genetic elements in the archaeal crenarchaeotal genus Sulfolobus, including recent work on viruses, cryptic plasmids, a novel type of virus satellite plasmids or satellite viruses, and conjugative plasmids (CPs), mostly from our laboratory. It does not discuss IS elements and transposons. Received: January 22, 1998 / Accepted: February 16, 1998     

水稻土中硫酸盐还原微生物研究进展

硫是水稻必需的营养元素之一.硫酸盐还原是硫元素生物地球化学循环中的关键步骤,在稻田土壤表层和水稻根际都十分活跃.介导硫酸盐还原过程的硫酸盐还原菌(sulfate- reducing bacteria, SRB)是稻田土壤中重要的功能菌群.它们不仅是硫元素生物地球化学循环的重要参与者,也是土壤中有机污染物降解的主要力量之一,发挥着重要的生态和环境功能.综述了稻田土壤中微生物参与的硫酸盐还原过程、SRB的生物多样性以及目前研究稻田土壤SRB主要采用的分子生态学方法,如末端限制性片段长度多样性(T-RFLP)、变性梯度凝胶电泳(DGGE)、实时荧光定量PCR(real-time PCR)、荧光原位杂交(FISH),并对水稻土壤中SRB的分子生态学研究方向进行了展望.     

黄土高原半干旱区苜蓿草地土壤干燥化特征与粮草轮作土壤水分恢复效应

实地测定了黄土高原半干旱区固原不同生长年限苜蓿草地和连作8a苜蓿草地翻耕轮作不同年限粮食作物后深层土壤水分特征,分析了苜蓿草地土壤干燥化特征和粮草轮作对土壤水分的恢复效应.结果表明:(1)苜蓿连作1a、5a、8a和12a等4类苜蓿草地0～1000cm土层平均土壤湿度值为6.6%,平均土壤水分过耗量702.8mm,平均土壤干燥化速率147.1mm/a,达到强烈干燥化程度,苜蓿连作5a土壤干层深度超过1000cm,苜蓿连作8a土壤干层深度超过1360cm,苜蓿草地合理利用年限为7a.(2)连作8a苜蓿草地翻耕并轮作4～7a和25a粮食作物等5类粮田0～1000cm土层土壤湿度介于6.74%～11.95%,土壤贮水量恢复值介于210.6～887.3mm,平均土壤水分恢复速率为80.8mm/a.轮作6a后粮田土壤干层轻度恢复程度以上深度达到1000cm.通过粮草轮作使苜蓿草地土壤湿度恢复到当地土壤稳定湿度需要13a以上.黄土高原半干旱区适宜的粮草轮作模式为:7a苜蓿→13a粮食作物.