An improvement on labelled messenger RNA preparation for DNA-RNA hybridisation

An improved method for the removal of DNA and phenol residues after phenol extraction in m-RNA preparation has been developed. This involves repeated freeze-thaws of the aqueous layer under centrifugal force. As a model assay, five minutes pulse-labelled lambda cI47 late m-RNA is prepared and hybridised to the r-strain lambda DNA. After the centrifugation treatment, non-specific hybridisation is reduced to one-tenth of the untreated sample and the background with respect to input radioactivity for hybridisation is reduced to 0.3%. This low background m-RNA preparation is important especially in dealing with minor class m-RNA.     

Synthesis of nascent prothrombin and albumin in a heterologous system using rat liver messenger RNA purified in oligo (dT)-cellulose.

Highly active m-RNA was prepared by phenol extraction of rat liver polysomes followed by oligo (dT)-cellulose chromatography. This m-RNA preparation stimulated total protein synthesis in rabbit reticulocyte lysates and in wheat germ extracts. Nascent prothrombin and albumin synthesized in the reticulocyte system programmed with this m-RNA were precipitated with specific antibodies and identified by their electrophoretic mobilities on SDS-acrylamide gels.     

微生物燃料电池降解苯酚过程中微电场对阴极微生物多样性的影响

[背景]苯酚废水作为一种毒性强、难降解的废水而备受关注.目前,微生物燃料电池(microbial fuel cell,MFC)已经广泛用于苯酚废水的降解,MFC的产电效果和苯酚的降解效率与反应器内的微生物群落有着密切关系.[目的]为了提高MFC的产电效果及对有害物质的降解能力,需要对MFC中苯酚的降解和微生物群落结构进...     

Biological Activities of Oligonucleotides Linked to Poly(L-Lysine)

Abstract

We have developped a method to couple oligonucleotides to poly(L-lysine). This tool has been tested with anti m-RNA synthetic oligodeoxyribonucleotides and 2′-5′(A)_n and allows oligonucleotides to enter intact cells.     

新疆艾丁湖中度嗜盐苯酚降解菌多样性研究

高盐含酚废水属于极难处理的废水之一,筛选具有生物学降解能力的嗜盐菌有助于解决这一难题。从新疆艾丁湖盐湖中分离筛选能够降解苯酚的中度嗜盐菌,了解盐湖中度嗜盐苯酚降解菌的多样性组成和降解能力。研究结果表明,10%(质量分数)的盐浓度条件下,分离得到166株嗜盐菌,通过以苯酚为唯一碳源的培养基进行降解活性筛选后得到45株阳性菌,根据细菌16S rRNA基因序列系统进化分析,这45株菌分别归类到3个门,5个科,9个属。其中拟诺卡氏菌属(Nocardiopsis)是优势菌,占总量的68.8%,其余菌分布于Bacillus、Gracilibacillus、Pontibacillus、Halobacillus、Marinococcus和Halomonas属。在含100 mg/L苯酚的液体培养基,经过10 d培养后,这45株菌降解效率为1%~17%。本研究为工业应用提供了嗜盐微生物种质资源,极具进一步发掘和研究价值。     

Picomole Syntheses of High Quality Oligonucleotide Primers in Combination with the Preparation of Oligonucleotide Arrays

Abstract

The establishment of a new synthesis procedure for the preparation of oligonucleotide arrays is described. A modified phosphoramidite chemistry allowed the in situ synthesis of oligomer arrays on specially derivatized polypropylene membranes which can be used both for hybridisation experiments and for the isolation of the individual oligonucleotides.     

Potential of <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> as a bioremediation organism

Summary The capability of Rhodococcus erythropolis CCM 2595(ATCC 11048) to utilize phenol, pyrocatechol, resorcinol, p-nitrophenol, p-chlorophenol, hydroquinone and hydroxybenzoate, respectively, or as respective binary mixtures with phenol, was described. This capability was found to depend on the substrate and its initial concentration. Some monoaromatic compounds had a suppressive effect on the strain’s ability to utilize phenol in a binary mixture and easily utilizable monoaromatics were strong inducers of the phenol 2-monooxygenase (EC 1.14.13.7). The capacity of R. erythropolis to colonize a synthetic zeolite was demonstrated and the enhancement of phenol tolerance of biofilms utilizing phenol was observed. The effect of humic acids on phenol killing was described and discussed as well. To allow use of recombinant DNA technology for strain improvement, methods of genetic transfer (transformation and conjugation) in R. erythropolis were established.     

宁夏地区春季奶牛场牛舍土壤细菌群落特征

【背景】现代规模化生产模式下,牛舍环境管理是影响奶牛高效健康生产的重要因素。【目的】探讨牛场不同牛舍土壤细菌群落特征,为奶牛健康生产提供理论依据。【方法】采集宁夏某规模化奶牛场的哺乳犊牛岛、断奶犊牛舍、育成牛舍、低产泌乳牛舍、高产头胎泌乳牛舍、高产经产泌乳牛舍、干奶牛舍和病牛舍这8个不同牛舍的土样,每个牛舍6个重复,共48份土样。利用16S rRNA基因扩增子测序分析细菌群落结构与多样性,并对细菌群落的功能进行预测。【结果】不同牛舍土样细菌群落组成存在差异,并且8个牛舍中高产头胎泌乳牛舍土样的细菌群落多样性最高。哺乳犊牛岛土壤与其他牛舍土壤细菌群落在门水平上差异较大;泌乳期牛舍土样之间的细菌群落结构相似度较高。在门的水平上,拟杆菌门(Bacteroidetes)、变形菌门(Proteobacteria)、放线菌门(Actinobacteria)和厚壁菌门(Firmicutes)是这8个牛舍土样共有的优势菌门。在属的水平上,嗜盐碱的盐单胞菌属(Halomonas)、具有潜在降解特性的Fermentimonas和栖海面菌属(Aequorivita)及致病菌的鸟杆菌属(Ornithobacterium)是犊牛期牛舍土样的优势菌属;嗜盐碱的Truepera是育成牛舍土样的优势菌属;致病菌的不动杆菌属(Acinetobacter)和Parapedobacter、耐药菌的Pedobacter是泌乳期牛舍土样的优势菌属。【结论】致病菌和参与硝酸盐呼吸的细菌主要分布在哺乳犊牛岛,嗜盐碱菌主要分布在断奶犊牛舍和育成牛舍,产甲烷的细菌主要分布在高产头胎泌乳牛舍。本研究分析了不同牛舍土壤细菌群落多样性,为奶牛健康生产提供理论依据。     

基于16S rRNA基因序列分析法比较苏尼特双峰驼和阿拉善双峰驼自然发酵酸驼乳的微生物多样性

【目的】传统发酵乳制品是一类未经任何处理自然发酵而成的,其微生态环境未遭破坏,从而乳酸菌的生物学特性和基因多样性得到了很好的保留,具有开发和利用价值。自然发酵酸驼乳常用来治疗多种疾病且效果良好,与其中丰富的乳酸菌资源有着密不可分的联系。然而,目前有关自然发酵酸驼乳微生物菌群及多样性相关研究甚少。因此进一步挖掘内蒙古地区双峰驼自然发酵酸驼乳微生物群落结构和多样性是至关重要的。【方法】本研究采用IlluminaMiseq测序技术,测定了苏尼特和阿拉善双峰驼的自然发酵酸驼乳中微生物16S rRNA V3–V4区序列,并对群落结构和多样性进行了比较分析。【结果】多样性分析表明,苏尼特双峰驼酸驼乳中微生物群落丰富度和种群差异性比阿拉善双峰驼酸驼乳大,细菌多样性也高。在门水平上,苏尼特和阿拉善双峰驼酸驼乳中的菌群均以厚壁菌门(Firmicutes)和变形菌门(Proteobacteria)为主。在属水平上,苏尼特双峰驼酸驼乳主要以乳杆菌属(Lactobacillus)和乳球菌属(Lactococcus)为优势菌群,阿拉善双峰驼酸驼乳以乳杆菌属(Lactobacillus)和醋酸杆菌属(Acetobacter)为优势菌属。此外,肠杆菌属(Enterobacter)、拉乌尔菌属(Raoultella)和明串珠菌属(Leuconostoc)等的含有食源性致病菌和环境污染菌的菌属被检出。综上所述,不同地区不同品种酸驼乳的乳酸菌种类及优势菌群有较大差异,存在显著的地理差异。【结论】通过本研究,不仅对苏尼特和阿拉善双峰驼自然发酵酸驼乳乳酸菌的组成和种类有了明确的认知,为评估发酵酸驼乳微生物群落对消费者身体健康的影响提供了数据基础的同时为今后筛选优势菌群和挖掘新型益生菌奠定基础。     

Detection and Phylogenetic Relationships of Highly Diverse Uncultured Acidobacterial Communities in Altamira Cave Using 23S rRNA Sequence Analyses

Acidobacteria have been established as a novel and widespread bacterial phylum consistently detected during 16S rDNA-based molecular surveys. Recently, representatives of the phylum Acidobacteria were found to make up a large percentage of cloned sequences recovered from paleolithic paintings. The present study addresses the intraphylum diversity of Acidobacteria in Altamira Cave. In contrast to previous studies, we have focused on 23S rRNA-based phylogenetic analyses, exploiting the high information content of the 23S RNA gene. Comparison with existing studies was facilitated by the preparation of corresponding 16S rRNA clone libraries. Fourty-one distinct operational taxonomic units (OTU) could be identified. Of the eight described acidobacterial subgroups, five were represented in the cave; only subgroups 1, 2, and 8 were not found. Subgroup 4 was the group harbouring the highest number of OTUs. Various clusters that could not be assigned to any of the established subgroups, but were clearly grouped within the phylum and appeared to represent new taxa at elevated phylogenetic levels, were detected. Results from this study give a first insight into the enormous acidobacterial diversity existing in a single hypogean environment containing unique paleolithic paintings, the Altamira Cave. Novel taxa have been found within the phylum, suggesting a higher diversity of Acidobacteria than previously suspected even at a local scale.     

A comparison of ammonia-oxidiser populations in eutrophic and oligotrophic basins of a large freshwater lake

A combination of PCR amplification and oligonucleotide probing was used to investigate the populations of ammonia-oxidisers of the -Proteobacteria in the eutrophic and oligotrophic basins of Lake Windermere, a large temperate lake in the English Lake District. Numbers of ammonia-oxidisers (MPN) in the Windermere lakewater were low (< 100 cells ml^–1) throughout the year with the exception of peaks in August, which coincided with stratification, and November in the South Basin where overturn may have introduced ammonia-oxidising bacteria into the water column. Sediment samples contained larger populations of ammonia oxidisers, usually ca. 10⁴ per g. dry weight, which remained relatively constant throughout the seasonal cycle in both Basins. DNA was recovered from lakewater and sediment samples and Nitrosospiraand N. europaea-eutrophalineage16S rRNA genes amplified in a nested PCR reaction, with confirmation of identity by oligonucleotide hybridisation. Nitrosospira 16S rDNA was readily detected in all samples and therefore found to be ubiquitous. In contrast, nitrosomonad DNA of the N. europaea-eutropha lineage could only be detected in the oligotrophic North Basin. Enrichment cultures of lakewater samples only exhibited nitrification at low (0.67 mM) and medium (5 mM) ammonium concentrations, whilst sediment enrichments nitrified at all concentrations tested including high (12.5 mM) ammonium medium. These data suggest that ammonia-oxidiser populations may be physiologically distinguished between lakewater and sediment, and that species distribution in a single lake is non-uniform.     

In Situ Enrichment of a Diverse Community of Bacteria from a 4–5 km Deep Fault Zone in South Africa

The extreme environment of South Africa's ultra-deep gold mines offers an opportunity to discover novel microorganisms (e.g., Alkaliphilus transvaalensis), including extremophiles that may provide insight into the origins of life on earth and offer industrial applications because of their thermophilic enzymatic properties. This study employed culture-independent methods to examine the bacterial diversity in water (T = 55° C, pH = 9, Cl^? = 1000 ppm and age = 4–53 Ma) emanating from an exploration borehole in a South African Au mine that intersected a quartzite-hosted fault at 3.3 km below land surface. The more adhesive strains of sulfate reducing bacteria were effectively selected and enriched from the planktonic community by forcing water from a flowing borehole through a sand/agar cartridge that was installed within the borehole. The cartridge's agar contained sulfate and lactate that diffused from the agar into the cartridge. DNA was extracted from the sand, after which a bacterial 16S rDNA gene clone library was generated. Analysis of clone sequences indicated that the groundwater bacterial community was quite diverse, including members of the α-, β-, and γ-Proteobacteria (20%), Actinobacteria (6%), Firmicutes (57%), Chloroflexi (3%), and Deinococcus-Thermus (14%) phyla. One of the most frequently detected clone types was associated with Desulfotomaculum (a known SRB). Another predominant clone type was closely related to Meiothermus cerbereus. A proportion of Proteobacteria clones were closely related to Ralstonia, Alishewanella, Hydrogenophaga, or Methylobacterium species. Some of the Firmicutes clones were closely related to Alkaliphilus transvaalensis, which was isolated from a nearby South African Au mine, or to Clostridium thermocellum. Of the total 21 OTUs identified from the cartridge sand, 6 most likely represent novel species of Firmicutes given their dissimilarity to other 16S rDNA sequences in the GenBank database.     

TWO EFFICIENT POLYMERIC CHEMICAL PLATFORMS FOR OLIGONUCLEOTIDE MICROARRAY PREPARATION

ABSTRACT

In this report we describe two robust procedures for oligonucleotide microarray preparation based on polymeric coatings. The proposed chemical approaches include: 1) a glass functionalisation step with appropriate silanes (γ-aminopropyltriethoxysilane-APTES or 3-glycid-oxypropyltrimethoxysilane-GOPS), 2) a coating step using polymers (poly-L-Lysine or poly(acrylic acid-co-acrylamide) copolymer) covalently bound to the modified glass and 3) a surface activation step to allow for the attachment of amino-modified oligonucleotides. Results obtained using these chemistries in oligo microarray preparation show: 1) an overall high loading capacity and availability to hybridisation against targets, 2) a good uniformity, 3) resistance to consecutive probing/stripping cycles, 4) stability to thermal cycles, 5) effectiveness in hybridisation-mediated mutation detection procedures and 6) the possibility to perform enzymatic reactions, such as ligation.     

Characterization of Bacterial Flora from Afşin–Elbistan Lignite Mine for Potential Microbial Biotech Applications

Abstract

The energy demand is enhancing day by day all over the world with the increasing of huge population. Lignite, a fossil fuel with low energy efficiency, is used in many different fields. However, lignite is primarily an energy source and its use without treatment causes environmental pollution as well as low energy efficiency. Advantages are known for the use of microorganisms in lignite processing technologies, and research is underway to improve this process. In this research designed to meet this need, isolation and characterization of bacteria with potential for use in biotechnological applications resistant to extreme conditions were performed from lignite mine. Turkey’s largest lignite mine bacterial flora have been identified for the first time in this study. In addition to lignite processing technologies, a bacterial strain collection has been created to be used in other biotechnological studies. This collection of bacteria includes Methylorubrum zatmanii, Streptomyces argenteolus, Streptomyces galilaeus, Pseudomonas mandelii, Paenarthrobacter aurescens, Paeniglutamicibacter sulfureus, Arthrobacter humicola, Bacillus pumilus, Bacillus subtilis, Bacillus safensis, Kocuria polaris, Arthrobacter sp., Rhodococcus sp. This specific bacteria collection is open to sharing with other researchers who want to do field applications.     

南极乔治王岛冰锥洞微生物培养探索

【目的】南极洲具备独特的环境和相对的生物地理隔离,南极洲各类生境中蕴藏了大量尚未培养和难培养的微生物,也是新颖微生物物种的重要来源之一。本研究以南极冰锥洞这类特殊生境为研究对象,通过培养条件的多样化提升南极微生物的培养率和多样性,揭示南极冰锥洞可培养微生物类群多样性,为该环境可培养微生物功能研究奠定基础,也为南极极端环境未培养微生物的培养方法提供借鉴。【方法】通过采用不同培养基添加复苏促进因子(resuscitation promoting factor, Rpf)的方式,提高南极柯林斯冰盖冰锥洞生境中微生物的可培养率,探究该生境中微生物的多样性。采用4种不同营养水平的培养基,平行添加Rpf进行菌株培养,经分离纯化与16S rRNA基因鉴定,分析冰锥洞可培养微生物的多样性及培养条件对多样性的影响。【结果】本研究共分离培养细菌407株,涵盖5个门、18个科、29个属,其中：放线菌门(Actinomycetota)为优势门,占72.73%;微杆菌科(Microbacteriaceae)为优势科,占69.78%;Lacisediminihabitans属为优势属,占45.70%。从培养基效果...     

A rapid and cost-effective method of genomic DNA isolation from cyanobacterial culture,mat and soil suitable for genomic fingerprinting and community analysis

This study presents a phenol and lysozyme free protocol for genomic DNA isolation of cyanobacteria from culture, mats and soil. For an efficient and pure DNA isolation from cyanobacteria having tough cell wall, extra steps of glass beading and Sepharose 4B purification were added. The modified method gave a higher yield of DNA than the phenol: chloroform extraction method. Four parameters selected for purity testing of the isolated DNA were: (i) restriction digestion with Hind III, (ii) randomly amplified polymorphic DNA-PCR of axenic culture of cyanobacteria to assess phylogenetic relatedness, (iii) denaturing gradient gel electrophoretic (DGGE) analysis of cyanobacterial mat and soil to ascertain the applicability of the isolated DNA for community analysis, and (iv) sequencing of partial 16S rDNA of Hapalosiphon intricatus BHULCR1, Anabaena doliolum LCR1, Anabaena oryzae LCR2, Aulosira fertilissima LCR4, and Tolypothrix tenuis LCR7 and BLAST analysis to confirm their cyanobacterial identity. Data generated from above analyses lead us to conclude that the modified method in question is rapid, cost effective, health and time conscious and promising for genetic fingerprinting and community analysis of cyanobacteria from diverse habitats.     

高亚硝酸盐环境下不同生长速率凡纳滨对虾肠道微生物多样性研究北大核心

【目的】高亚硝酸盐环境中饲养的凡纳滨对虾(Litopenaeus vannamei),在养殖结束时其生长速率和体重往往差异较大。本研究旨在探讨在高亚硝酸盐环境下饲养的对虾生长速率与肠道菌群结构和功能的相关性。【方法】本研究通过收集高亚硝酸盐条件下快速生长对虾(rapidly growing,RG)、正常生长对虾(normally growing,NG)和缓慢生长对虾(slowly growing,SG)的肠道和海水样品,通过16S rRNA基因测序、线性判别分析[line discriminant analysis(LDA)effect size,LEfSe]等进行分析。【结果】发现SG的细菌群落多样性与RG和NG不同。主坐标分析(principal coordinate analysis,PCoA)分析表明,NG的群落组成与RG比SG更相似。通过LEfSe差异分析发现,RG中火色杆菌科(Flammeovirgaceae)、黄杆菌科(Flavobacteraceae)和浮酶菌科(Planctomycetaceae)的丰度较高,而SG中脱硫弧菌科(Desulfovibrionaceae)、希瓦氏菌科(Shewanellaceae)和弧菌科(Vibrionaceae)的丰度显著增加。【结论】本研究发现,在高亚硝酸盐环境下,肠道微生物群落的氮代谢能力是造成对虾不同生长速度的原因。该研究将为虾的工业化养殖提供指导。     

青藏高原阿里、那曲和海西地区土壤可培养放线菌的多样性

【目的】为了研究青藏高原北部地区土壤可培养放线菌的多样性,并比较不同选择性分离培养基对高原土壤放线菌的分离效果。【方法】使用9种分离培养基,并尝试添加藤黄微球菌发酵液,对采集自阿里、那曲和海西地区的14份土壤样品中的放线菌进行选择性分离。通过16S r RNA基因序列分析对分离菌株进行初步分类鉴定,并在不同分类水平上统计所分离得到的放线菌多样性。【结果】分离得到去重复后的放线菌255株,分布于放线菌门的8个目,14个科,23个属,包含94个可能的物种。其中至少25个物种可能为新种,分布于13个属。链霉菌属的菌株108株,可能的物种28个,是最主要的优势菌属。分离培养基中添加藤黄微球菌发酵液明显增加了放线菌分离菌株的数量和多样性,稀释的葡萄糖酵母麦芽汁培养基适合分离链霉菌,淀粉甘油脯氨酸培养基、丙酸钠酪蛋白培养基等则适合分离稀有放线菌。【结论】青藏高原北部土壤放线菌多样性非常丰富,并且存在较多的新颖放线菌类群;添加藤黄微球菌发酵液是提高放线菌分离效率的有效手段。     

Diversity of Degradation Pathways of Some Aromatic Compounds by Phenotype and Genotype Testing in Acinetobacter Strains

Phenotype and genotype testing were combined in this study to identify the diversity of five Acinetobacter strains to degrade some low-molecular-weight aromatic compounds. Three groups were identified: Group one, A. calcoaceticus PHEA-2 and A. baumanii UMI-95, which had the ability to degrade catechol and protocatchuate, and could catabolize vanillic acid and phenol; Group two, Acinetobacter sp. MM1, which had the ability to degrade protocatchuate, but not catechol, just could catabolize vanillic acid; and Group three, A. lwoffi UMI-8, A. junii 85, which degraded neither catechol nor protocatchuate, not could catabolize vanillic acid or phenol. This revised version was published online in July 2006 with corrections to the Cover Date.     

不同配方益生菌在高脂饮食诱导小鼠肥胖发生中的差异化作用

【目的】基于肠道微生物与宿主代谢的相互关系,研究不同配方的益生菌对小鼠肥胖的影响。【方法】50只C57BL/6J雄性小鼠随机平均分成10组,分别给予正常饲料、高脂饲料以及高脂饲料加8种不同配方的益生菌产品(50亿CFU/只),所有动物连续喂养9周,每周测量小鼠体重1次。最后一周测定空腹血糖、葡萄糖耐量试验(glucose tolerance test,GTT)、血脂相关指标,称取内脏重量,并留取小鼠盲肠内容物,提取小鼠肠道菌群总DNA,利用16S rDNA测序检测相关细菌含量。【结果】部分益生菌可引起小鼠体重增速加快,而部分益生菌可减缓小鼠肥胖和降低内脏脂肪重量,同时缓解高血脂症。丹尼斯克品牌益生菌配方组小鼠肠道中厚壁菌/拟杆菌比例(F/B)是正常饮食组的22.8倍,Akkermansia muciniphila(Akkermansia)细菌含量几乎为0;而菌拉丁品牌益生菌配方组小鼠F/B比例与正常饲料饮食组类似,Akkermansia含量为0.5%,为正常饮食对照组小鼠的一半左右。【结论】益生菌可影响小鼠体重和代谢,但不同配方的益生菌效果截然相反。特定的益生菌配方对肥胖和高血脂的改善可能是由于其选用的菌株本身的特性以及菌株之间的相互配比能够降低小鼠肠道中F/B比例以及升高Akkermansia的含量所带来的。此研究为进一步开发可改善代谢的益生菌产品提供了参考。