极端嗜盐菌16SrDNA的PCR扩增

四株嗜盐菌Haloarcula vallismortis(EM201)、Haloferax denitrificans(EM303)、A_5和B_2已通过一对特定引物用PCR技术从总DNA中扩增出各自的16SrDNA片段,分子大小在1.47kd左右.DNA杂交也表明这些PCR产物具有嗜盐菌的同源性.     

利用定量PCR测定嗜盐古菌噬菌体SNJ1的滴度

[目的]利用定量PCR建立检测嗜盐古生菌噬菌体SNJ1滴度的新方法,为研究高盐环境中噬菌体的动态变化提供新的途径。[方法]采用定量PCR技术检测嗜盐古菌噬菌体的gene 23上的DNA片段,通过DNA片段丰度推断噬菌体的滴度。[结果]定量PCR方法绘制的噬菌体SNJ1的标准曲线与噬菌体的滴度间有非常高的相关性(R2=0.9956),且定量PCR扩增体系效率高(99.87%),样品间的变异系数小(0.257%~3.270%),扩增特异性强(融解曲线仅有1个峰)。[结论]利用定量PCR在检测嗜盐古生菌噬菌体SNJ1滴度时表现了较高的特异性与精确性,该方法为检测高盐环境中噬菌体变化提供了一种快速、简便的手段。     

一株嗜盐细菌的16SrRNA基因序列分析

目的:从舟山深海海泥中获得了底泥样品,并从中提取到了一株嗜盐细菌.方法:通过用不同盐浓度的培养基培养,挑取单菌落,反复划线纯化,得到了嗜盐菌的单菌落,通过菌株基因组DNA的提取、菌株的抗性实验、质粒的提取、16SrRNA的PCR扩增及克隆、16SrRNA的全序列分析等手段.结果:得到该菌株的16SrRNA的基因序列.结论:该株嗜盐菌是一株新色盐杆菌.     

岱山盐场可培养嗜盐菌的多样性及其产酶活性筛选

从岱山盐场采集样品,利用选择性培养基分离培养嗜盐菌,对盐田环境中可培养嗜盐菌的多样性及产酶活性进行研究.共分离得到181株嗜盐菌菌株,通过真细菌和古生菌两对通用引物扩增其16S rRNA 基因,并采用限制性内切酶Hinf I进行ARDRA(amplified rDNA restriction analysis)多态性分析,共分为21个不同的操作分类单元(operation taxonomy units, OTUs),其中嗜盐细菌有12个OTUs,嗜盐古菌有9个OTUs.选取具有不同酶切图谱的代表菌株进行克隆测序,BLAST 比对及系统发育分析将嗜盐细菌归于7个属,其中嗜盐单胞菌属(Halomonas)的菌株数占优势,是嗜盐细菌总数的46.8%;嗜盐古菌归于4个属,盐盒菌属(Haloarcula)的菌株数占优势,是嗜盐古菌总数的49.1%.对分离菌株的产酶活性进行检测表明,岱山盐田环境蕴含丰富的产淀粉酶、蛋白酶和脂肪酶等生物活性酶的嗜盐菌, 其中盐盒菌属产酶菌株数最丰富.研究结果表明,岱山盐田环境中具有较为丰富的嗜盐菌多样性,是筛选产酶菌株的重要资源库.     

科技信息

嗜盐古菌资源的研发极端嗜盐菌有嗜盐古菌(halophilicarchaea)之称,可分为6个属:(1)嗜盐碱杆菌(Na tronobacterium),(2)嗜盐碱球菌(Natronococcus),(3)嗜盐杆菌(Halobacterium),(4)嗜盐球菌(Halococcus),(5)嗜盐富盐杆菌(Haloferax),(6)盐盒菌(Haloarcula),后又增加了6属:即Hal     

基于高通量测序的陕西花马池和苟池微生物群落多样性分析

目的:研究陕西花马池和苟池盐湖嗜盐细菌和古菌的群落多样性特征。方法:从两地盐湖采集卤水样品,提取总DNA后对其16S rRNA基因进行PCR扩增,通过Miseq高通量测序和生物信息学分析得到花马池和苟池微生物群落结构。结果:细菌优化序列被分入222个操作分类单位(OTU),花马池细菌优化序列经筛选后得到26 698个序列,其中厚壁菌门(Firmicutes)占主要地位,占总序列数的24.13%,其次20.02%的序列归入变形菌门(Proteobacteria);苟池细菌优化序列经筛选得到31 329个序列,拟杆菌门(Bacteroidetes)是样品中最主要的门类,占总数的60.56%。古菌优化序列经筛选后有81 286条,被归入86个OTU中,绝大多数属于广古菌门(Euryarchaeota)。在属水平上,2个盐湖共有的优势古菌群落包括Halonotius、盐红菌属(Halorubrum)、盐杆状菌属(Halorhabdus)和Halobellus等。花马池特有Halolamina、盐杆菌属(Halobacterium)、Halobellus和Halovenus。结论:研究揭示了陕西花马池和苟池嗜盐细菌和古菌群落组成,表明陕西花马池和苟池盐湖微生物群落分布具有相似性和独特性,同时存在丰富的嗜盐菌资源和潜在的新类群,为新种的挖掘及嗜盐菌的开发提供了理论数据。     

极端嗜盐古菌蛋白类抗生素——嗜盐菌素

古菌(Archaea)是一类与细菌及真核生物显著不同的生命的第三种形式[1],大多生活在极端或特殊环境,主要包括产甲烷古菌(Methanogenic Achaea)、极端嗜盐古菌(Extremely Halophilic Archaea)和极端嗜热古菌(Extremely Thermophilic Archaea)等三大类.极端古菌是极端环境微生物的重要成员,也是极端环境微生物资源开发的重要领域.其中,嗜盐古菌可产生一类蛋白类抗生素,称为嗜盐菌素(halocin).     

嗜盐嗜碱杆菌属的一个新种

从内蒙古自治区哈马台碱湖分离到一株多形态嗜盐嗜碱菌(编号HAM—2),其生长的NaCI浓度范围为12％～30％,最适17.5％;生长的pH范围为7.8～10.4,最适pH9.0～9.5。革兰氏染色阴性。细胞为不规则杆状、椭圆形、三角形等多形态,细胞大小为1.0～2.0×2.0～5.Oμm。该菌株主要极性脂是磷脂酰甘油磷酯(PGP)和磷脂酰甘油(PG),还含有一种未知的次要磷脂成分(PL4)。DNA中G+C含量为59.5mol％。根据这些特征,菌株可归入嗜盐嗜碱杆菌属,又根据细胞形态和极性脂组份不同于该属正式承认的三个种,因此,鉴定此菌株为嗜盐嗜碱杆菌属(Natronobacterium)的—个新种,定名为内蒙古嗜盐嗜碱菌(Natronobacterium innermongoliae Sp.nov.)。     

西藏扎布耶茶卡盐碱湖古菌多样性的非培养技术分析

采用非培养技术,直接从西藏扎布耶茶卡盐碱湖样品中提取微生物总DNA。以样品总DNA为模板,PCR扩增湖中古菌的16S rDNA序列。扩增产物经过克隆并随机挑选60个克隆进行测序得到它们的16S rDNA部分序列,大部分序列与嗜盐碱古菌的16S rDNA相近。在系统发育树上,部分克隆与已知古菌属归于同一分支,主要分布在嗜盐菌科的Natronococcus、Natronorubrum、Natronobacterium、Natronomonas、Natrinema、Halorubrum、Haloterrigena和Halorhabdus等8个嗜盐古菌属中, 也有一些克隆形成了独立的分支。它们共同显示出扎布耶茶卡湖中的古菌具有丰富的多样性。     

嗜盐古菌资源的研发

极端嗜盐菌有嗜盐古菌(halophilic archaea)之称,可分为6个属：(1)嗜盐碱杆菌(Natronobacterium),(2)嗜盐碱球菌(Natronococcus),(3)嗜盐杆菌(Halobacterium),(4)嗜盐球菌(Halococcus),(5)嗜盐富盐杆菌(Haloferax),(6)盐盒菌(Haloarcula),后又增加了6属：即Halorubrum、Haloaculum、Natrialba、Haloterrigena、Natronorubrum、Natronomonas共12个。其实不止这些属。其中有一种来自大盐湖的嗜盐碱杆菌(Hatronobact sp．)不仅具嗜盐性,且有嗜碱性(pH10～12),在5．5mol／L的钠盐溶液中生长。     

Halobacteria: the evidence for longevity

Subterranean salt deposits are the remains of ancient hypersaline waters that presumably supported dense populations of halophilic microorganisms including representatives of the haloarchaea (halobacteria). Ancient subterranean salt deposits (evaporites) are common throughout the world, and the majority sampled to date appear to support diverse populations of halobacteria. The inaccessibility of deep subsurface deposits, and the special requirements of these organisms for survival, make contamination by halobacteria from surface sites unlikely. It is conceivable that these subterranean halobacteria are autochthonous, presumably relict populations derived from ancient hypersaline seas that have been revived from a state of dormancy. One would predict that halobacteria that have been insulated and isolated inside ancient evaporites would be different from comparable bacteria from surface environments, and that it might be possible to use a molecular chronometer to establish if the evolutionary position of the subsurface isolates correlated with the geological age of the evaporite. Extensive comparisons have been made between the 16S rRNA genes of surface and subsurface halobacteria without showing any conclusive differences between the two groups. A further phylogenetic comparison exploits an unusual feature of one particular group of halobacteria that possess at least two heterogeneous copies of the 16S rRNA gene, the sequences of which may have been converging or diverging over geological time. However, results to date have yet to show any gene sequence differences between surface and evaporite-derived halobacteria that might arguably be an indication of long-term dormancy. Received: January 22, 1998 / Accepted: February 16, 1998     

Multiple species of ectomycorrhizal fungi are frequently detected on individual oak root tips in a tropical cloud forest

The ecological importance of ectomycorrhizal (EM) fungi in tropical ecosystems is increasingly recognized, but few studies have used molecular methods to examine EM fungal communities in tropical forests. The diversity and composition of the EM community on Quercus crassifolia in a tropical montane cloud forest in southern Mexico were characterized using DNA sequencing of single root tips. Individual root tips commonly harbored multiple fungal species that resulted in mixed polymerase chain reaction (PCR) products. By cloning and performing gel extractions on mixed PCR samples, we identified two or more EM fungi on 26% of the root tips. When non-EM fungi were considered, this figure increased to 31% of root tips. A total of 44 EM taxa and nine non-EM taxa were detected on roots from 21 soil cores (104 root tips). Taxa in the families Russulaceae, Cortinariaceae, Inocybaceae, and Thelephoraceae were frequent. This is the first study to characterize the belowground EM community in a tropical montane cloud forest. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Orthopoxvirus Detection in Environmental Specimens during Suspected Bioterror Attacks: Inhibitory Influences of Common Household Products

After terrorists attacked the United States in 2001, the appearance of letters and other objects containing powdery substances with unknown potentials for biological threat focused attention on the speed, sensitivity, and reliability of diagnostic methods. This study summarizes the abilities and limitations of real-time PCR, electron microscopy (EM), and virus isolation when used to detect potential bioweapons. In particular, we investigated the inhibitory influences of different common household products present in environmental specimens on PCR yield, EM detection, and virus isolation. We used vaccinia virus as a model for orthopoxviruses by spiking it into specimens. In the second part of the study, we describe modifications of diagnostic methods to overcome inhibitory effects. A variety of PCR amplification enhancers, DNA extraction protocols, and applications of internal controls were evaluated to improve diagnostic simplicity, speed, and reliability. As a result, we strongly recommend using at least two different frontline techniques in parallel, e.g., EM and PCR. A positive result obtained by any one of these techniques should be followed by a biological method to confirm the putative diagnosis. Confirmatory methods include virus isolation followed by an agent-specific immunofluorescence assay to confirm the presence of replication-competent particles.     

Photophosphorylation elements in halobacteria: An A-type ATP synthase and bacterial rhodopsins

Photophosphorylation in halobacteria is carried out by two rather simple elements: an A-type ATP synthase and light-driven ion-pumping bacterial rhodopsins. The unique features of halobacterial ATP synthase, mostly common to archaebacteria (A-type), and of new members of the bacteriorhodopsin family are introduced along with studies performed in the authors' laboratory. This is the story of how we found that the A-type ATP synthase is close to V-type ATPase but far from F-type ATPase, although all three ATPases are believed to have the same ancestor. Archaerhodopsins, the new members of the proton-pumping retinal proteins, were found in Australian halobacteria and have been used in a comparative study of bacterial rhodopsins.     

Stringency and relaxation among the halobacteria.

Accumulation of stable RNA and production of guanosine polyphosphates (ppGpp and pppGpp) were studied during amino acid starvation in four species of halobacteria. In two of the four species, stable RNA was under stringent control, whereas one of the remaining two species was relaxed and the other gave an intermediate phenotype. The stringent reaction was reversed by anisomycin, an effect analogous to the chloroamphenicol-induced reversal of stringency in the eubacteria. During the stringent response, neither ppGpp nor pppGpp accumulation took place during starvation. In both growing and starved cells a very low basal level of the two polyphosphates appeared to be present. In the stringent species the intracellular concentration of GTP did not diminish but actually increased during the course of the stringent response. These data demonstrate that (i) wild-type halobacteria can have either the stringent or the relaxed phenotype (all wild-type eubacteria tested have been shown to be stringent); (ii) stringency in the halobacteria is dependent on the deaminoacylation of tRNA, as in the eubacteria; and (iii) in the halobacteria, ppGpp is not an effector of stringent control over stable-RNA synthesis.     

Thymidine incorporation in saltern ponds of different salinities: Estimation of in situ growth rates of halophilic archaeobacteria and eubacteria

Incorporation of [methyl-³H]thymidine was measured in solar saltern ponds of different salinities. Estimated doubling times of the bacterial communities were in the range of 1.1 to 22.6 days. Even at the highest salt concentrations (NaCl saturation), relatively rapid thymidine incorporation was observed. In an attempt to differentiate between activity of halophilic archaeobacteria (theHalobacterium group) and halophilic eubacteria, taurocholate, which causes lysis of the halobacteria without affecting eubacteria, was used. At salt concentrations exceeding 250 g/liter all thymidine incorporation activity could be attributed to halobacteria. Aphidicolin, a potent inhibitor of DNA synthesis in halobacteria, completely abolished thymidine incorporation at the highest salinities, but also caused significant inhibition at salinities at which halobacteria are expected to be absent. Attempts to use nalidixic acid to selectively inhibit DNA synthesis by the eubacterial communities were unsuccessful.     

Occurrence of coenzyme F420 and its gamma-monoglutamyl derivative in nonmethanogenic archaebacteria.

Analysis of the fluorescent compounds extracted from six different species of halobacteria and one species each of Sulfolobus and Thermoplasma revealed the universal occurrence of coenzyme F420, (N-[N-[O-[5-(8-hydroxy-5-deazaisoalloxazin-10-yl)-2,3,4-trihydroxy -4-pentoxyhydroxyphosphinyl]-L-lactyl]-L-gamma-glutamyl]-L -glutamic acid), or its gamma-monoglutamyl derivative or both. The total amount (approximately 100 pmol/mg [dry weight]) of these compounds found in the halobacteria studied was approximately 5% of the amount previously reported for methanogenic bacteria. The amount of F420 found in the Sulfolobus and Thermoplasma strains was approximately 1% of that found in the halobacteria. The major compound in all but one of the examined strains was the gamma-monoglutamyl derivative of F420; one strain of halobacteria contained only F420. For the halobacterium-derived samples, the additional glutamic acid was shown to be linked by a gamma-glutamyl peptide bond to the terminal glutamic acid of the F420 core structure by enzymatic hydrolysis of the samples with three different gamma-glutamyltranspeptidases. The product of this enzymatic hydrolysis was F420 with one less glutamic acid in the side chain.     

Diversity, abundance and novel 16S rRNA gene sequences of methanogens in rumen liquid, solid and epithelium fractions of Jinnan cattle

Three methanogen 16S rRNA gene clone libraries were constructed from liquid (LM), solid (SM) and epithelium (EM) fractions taken from the rumen of Jinnan cattle in China. After the amplification by PCR using methanogen-specific primers Met86F and Met1340R, equal quantities of PCR products from the same fractions from each of the four cattle were mixed together and used to construct the three libraries. Sequence analysis showed that the 268 LM clones were divided into 35 phylotypes with 18 sequences of phylotypes affiliated with the genus Methanobrevibacter (84.3% of clones). The 135 SM clones were divided into 19 phylotypes with 11 phylotypes affiliated with the genus Methanobrevibacter (77.8%). The 267 EM clones were divided into 33 phylotypes with 15 phylotypes affiliated with the genus Methanobrevibacter (77.2%). Clones closely related to Methanomicrobium mobile and Methanobrevibacter wolinii were only found in the LM library, and those to Methanobrevibacter ruminantium and Methanobrevibacter gottschalkii only in the SM library. LM library comprised 12.4% unidentified euryarchaeal clones, SM library 23.7% and EM library 25.5%, respectively. Five phylotypes (accession number: EF055528 and EF055531-EF055534) did not belong to the Euryarchaeota sequences we had known. One possible new genus (represented by phylotype E17, accession number EF055528) belonging to Methanobacteriaceae was identified from EM library. Quantitative real-time PCR for the first time revealed that epithelium fraction had significantly higher density of methanogens, with methanogenic mcrA gene copies (9.95 log 10 (copies per gram of wet weight)) than solid (9.26, P < 0.01) and the liquid (8.44, P < 0.001). The three clone libraries also appeared different in Shannon index (EM library 2.12, LM library 2.05 and SM library 1.73). Our results showed that there were apparent differences in the methanogenic diversity and abundance in the three different fractions within the rumen of Jinnan cattle, with Methanobrevibacter species predominant in all the three libraries and with epithelium fraction having more unknown species and higher density of methanogens.     

O‐8FALSE NEGATIVE BRONCHIAL CYTOLOGY

Introduction: BK virus nephropathy is an emerging cause of renal transplant failure accounting for allograft loss in 45–50% of recipients between 2–60 months post‐transplantation. The Renal Transplant Unit in Royal Free Hospital has devised a local surveillance programme for screening renal transplant patients at weekly intervals by urinary cytology (UC) and electron microscopy (EM), and confirmation of positive results by plasma PCR and allograft biopsy. Objective: (i) To monitor the implementation of local guidelines; (ii) To compare UC with EM and (iii) To identify areas for improvement. Methods: Decoy cell and EM positive cases were retrieved from the WinPath database for new renal transplant recipients (n = 55) during 1^st November 2004 to 31^st October 2005 in Royal Free Hospital. Plasma PCR was retrieved for positive cases. Results: Up to eight samples were sent for UC at random intervals from 47 patients, and up to 7 were sent for EM from 42 patients. Eleven were UC‐positive and one was EM‐positive. PCR was not requested in UC‐positive cases and 3 out of 10 were positive retrospectively. The EM‐positive case was PCR‐positive but UC‐negative. Discussion: Urine Cytology is more appropriate and cost effective for screening than Electron Microscopy. During the period covered by this audit, samples were sent in an inconsistent fashion after allograft dysfunction rather than for screening. A screening protocol has been agreed and a re‐audit is planned.     

Conserved and variable regions in the chromosomal and extrachromosomal DNA of halobacteria

Summary Total DNA from Halobacterium halobium and other halobacteria strains is separated into two fractions, FI and FII, which differ in their G+C content. FI DNA, which represents the major part of the genome is highly conserved in all purple-membrane-forming halobacteria. Fraction II (FII) consists in H. halobium of three DNA specimen: (a) the previously isolated plasmid pHH1, (b) a heterogeneous set of ccc-DNA molecules present in the cell in low copies, termed minor-circular DNA (MCD) and (c) a new type of more A-T rich DNA segments (chromosomal islands) which, as described here and by Pfeifer and Betlach (1985), are integrated in FI. Sequences homologous to pHH1 occur only in Halobacterium species closely related to H. halobium (like H. cutirubrum), whereas MCD sequences are present in all purple-membrane-forming halobacteria. The sequences of the newly identified chromosomal islands are only found like pHH1, in Halobacterium species, closely related to H. halobium. Total DNA from square halobacteria exhibits no extended homologies to FI or FII DNA from H. halobium. The only common DNA sequences found in all halobacteria are certain insertion elements (ISH), such as ISH26. Based on these data, halobacteria can be subdivided in at least three major groups.Dedicated to Prof. Dr. F. Lingens to his 60th anniversary