嗜盐古生菌AJ4中BR蛋白基因部分片段和16S rRNA基因序列研究

为了研究分析新疆阿尔金山国家自然保护区阿牙克库木湖嗜盐古生菌物种与细菌视紫红质(bacteriorhodopsin ,BR)蛋白资源 ,对分离纯化到的极端嗜盐古生菌AJ4 ,采用PCR方法扩增出其 16SrRNA基因 (16SrDNA)和编码螺旋C至螺旋G的BR蛋白基因片断 ,并测定了基因的核苷酸序列 .通过BR蛋白部分片段序列分析表明 ,BR蛋白中对于完成质子泵功能以及与视黄醛结合的关键性氨基酸残基均为保守序列 ,位于膜内侧的序列比位于膜外侧的序列更保守 ;基于BR蛋白基因和16SrDNA序列的同源性比较以及 16SrDNA序列的系统发育学研究表明 ,AJ4是Haloarcula属中新成员 .由此建立了一种快速筛选具有新BR蛋白的新嗜盐古生菌的方法 .     

一个新的Br蛋白基因部分序列测定及分析

从新疆北部艾比湖分离纯化到极端嗜盐古生菌AB1,采用PCR方法扩增了其165 rRNA基因(16S rDNA)和编码螺旋C至螺旋G的细菌视紫红质(bacteriorhodopsin,Br)蛋白基因片段,并测定了基因的核苷酸序列。基于16S rDNA序列的同源性比较及系统发育学研究表明,AB1是Natronococcus属中新成员。通过对菌株AB1的Br蛋白亲水性分析表明,AB1的Br蛋白与已报道Br蛋白有类似的超二级结构,进一步的蛋白质序列聚合比对结果表明,AB1中Br蛋白螺旋C至螺旋G的氨基酸序列与其他菌株差异明显。研究结果表明菌株AB1的Br蛋白是一种新的Br蛋白。     

新疆艾比湖嗜盐菌的细菌视紫红质和16S rRNA基因序列的研究

为了研究分析嗜盐古生菌物种与细菌视紫红质(BR)蛋白基因资源,从40份土壤、湖水及淤泥样品中分离出148株嗜盐菌,对其中6株菌采用聚合酶链式反应(PCR)方法对其编码螺旋C至螺旋G的蛋白基因片段和16SrRNA基因进行了扩增,并测定了基因的核苷酸序列。与已报道的相应片段进行对比,ABDH10,ABDH1I和ABDH40中的螺旋C至螺旋G的蛋白与其他菌株差异显著。基于16SrRNA序列的同源性比较以及系统发育学研究表明,ABDH10和ABDH40是Natronorubrum属下的新成员和Natrinema属下的新成员,ABDH40的16SrRNA序列已登录到GenBank,其序列号为AY989910。ABDH11中的螺旋C至螺旋G的蛋白与其他菌株差异显著。     

一个新的Br蛋白基因部分序列测定及分析

从新疆北部艾比湖分离纯化到极端嗜盐古生菌AB1,采用PCR方法扩增了其16S rRNA基因（16S rDNA）和编码螺旋C至螺旋G的细菌视紫红质（bacteriorhodopsin,Br）蛋白基因片段,并测定了基因的核苷酸序列。基于16S rDNA序列的同源性比较及系统发育学研究表明,AB1是Natronococcus属中新成员。通过对菌株AB1的Br蛋白亲水性分析表明,AB1的Br蛋白与已报道Br蛋白有类似的超二级结构,进一步的蛋白质序列聚合比对结果表明,AB1中Br蛋白螺旋C至螺旋G的氨基酸序列与其他菌株差异明显。研究结果表明菌株AB1的Br蛋白是一种新的Br蛋白。Abstract: A strain of halophilic archaeum AB1 was isolated and purified from Aibi Lake located in the north of Xinjiang Uygur Autonomous Region. Partial DNA fragment encoding a bateriorhodopsin (Br) protein as well as 16S rRNA of AB1 was amplified by PCR, and their nucleotide sequences were determined subsequently. On the basis of homology and phylognetic analysis about 16S rRNA gene (16S rDNA), it could be speculated that the strain AB1 is a novel member of the genus Natronococcus. The hydrophathy analysis of Br fragment revealed that the AB1 Br had a transmembrane heptahelical structure similar to that of other Brs. On the other hand, homology alignment using the deduced partial amino acid sequence of Br protein of AB1 with other Br proteins showed that AB1 Br protein is obviously different to others. These facts indicated that the Br in halophilic archaeum AB1 is a new Br protein.     

瘤胃甲硫氨酸降解菌的分离及其16 S rDNA全序列分析

为分析研究山羊瘤胃液中甲硫氨酸降解菌群的物种资源,对经分离纯化获得的一株甲硫氨酸降解菌MB6-1,采用PCR方法扩增其16 S rDNA基因,并测定其基因的核苷酸全序列。基于16 S rDNA序列的同源性比较和系统发育学分析(ribosomal database projectⅡ;简称RDPⅡ数据库),发现MB6-1可能是普罗威登斯菌属(Providencia)中的一个新种。菌株MB6-1的16 S rDNA序列已经被GenBank数据库收录,其序列号为DQ436917。     

云南省德宏州含羞草β-根瘤菌多样性及系统发育研究

【目的】通过对分离自云南德宏州的含羞草β-根瘤菌进行遗传与表型多样性研究,揭示我国含羞草β-根瘤菌的物种多样性。【方法】应用16S rDNA PCR-RFLP、全细胞蛋白SDS-PAGE电泳及16S rDNA全序列分析对分离得到的60株含羞草根瘤菌进行多样性研究。【结果】16S rDNA PCR-RFLP及全细胞蛋白SDS-PAGE图谱分析将供试菌株分为2个遗传型群和2个表型群,分别与贪铜菌属(Cupriavidus)和伯克霍尔德菌属(Burkholderia)参比菌株聚群。经16S rDNA全序列分析,供试菌株被归到台湾贪铜菌(Cupriavidus taiwanensis)、含羞草伯克霍尔德菌(Burkholderia mimosarum)及结瘤伯克霍尔德菌(Burkholderia phymatum)等3个种群。【结论】云南德宏州的含羞草β-根瘤菌主要为贪铜菌及伯克霍尔德菌类群,其中贪铜菌占绝对优势,且存在遗传和表型的丰富多样性,该研究揭示了含羞草β-根瘤菌的物种多样性并丰富了我国β-根瘤菌菌种资源。     

苏云金芽孢杆菌S1478-1分离株的特性鉴定及cry1Ac同源基因克隆

本研究对从海南岛尖峰岭热带雨林自然保护区的土壤样品中分离出的Bt菌株S1478-1进行了特性鉴定,研究表明S1478-1分离株菌落形态和生长特征和Bt参照菌株HD73极其相似.16S rDNA序列分析表明,S1478-1分离株与其它B.thuringiensis、B.cereus和B.anthracis的16S rDNA序列相似性达到99%.分离株能产菱形伴胞晶体,SDS-PAGE蛋白电泳分析表明,菌株在生长后期,形成芽孢同时分泌130 kD大小的晶体蛋白.生物测定表明S1478-1分离株对小菜蛾具有很高的毒杀活性,LC50卯值高达5.159 ×108cfu/mL.初步显示S1478-1分离株可作为防治鳞翅目害虫的生物农药菌株.利用PCR-RFLP方法鉴定S1478-1分离株含有cry1Ac同源基因,以PCR粘性端克隆方法扩增全长基因,序列测定表明该基因ORF为3 537bp,编码1178个氨基酸,推定的编码蛋白分子量为133.3 kD,与其它cry1Ac基因序列最高达到99%同源,因此,该基因可作为杀虫工程菌及培育转基因抗虫作物的候选基因.     

纤溶酶产生菌—藤黄微球菌的筛选、鉴定及纤溶酶基因的克隆

通过血粉培养基富集、纤维蛋白培养基筛选,从自然环境中分离到一株纤溶酶产生菌,命名为ML909.利用传统细菌分类鉴定方法对其形态和生理生化特征进行了研究,发现其与藤黄微球菌的特征相符,16S rDNA序列分析的结果也表明它与藤黄微球菌的16S rDNA序列的同源性高达99%,因此该茵在系统分类学上属于微球茵属(Micrococcus Cohn)藤黄微球菌(Micrococcus luteus).通过PCR方法对其纤溶酶的编码区序列进行了克隆和序列分析,基因登录GenBank,登录号为EU232121.     

新疆罗布泊周边地区极端环境嗜盐菌的研究

为了研究分析新疆罗布泊周边地区pH值5-6的盐湖嗜盐古菌资源。从湖中分离筛选出一批嗜盐古菌,对其进行了生理生化特性研究,发现其中6株菌的生理特性和产酶特性比较特殊,并采用PCR方法扩增出其16SrRNA基因（16S rDNA）,并测定了基因的核苷酸序列。基于16S rDNA序列的同源性比较以及16S rDNA序列的系统发育学研究表明,菌株B20-RDX是盐盒菌属Haloarchaeon属中新种成员,GenBank登录号为FJ561285,该菌株为革兰氏阴性菌,最适盐浓度25%,最适pH 8.0,能产过氧化氢酶、淀粉酶,对四环素有抗性,能利用精氨酸和丁二酸盐。迄今为止,国内极少有关罗布泊周边地区极端环境微生物研究的报道,该研究可为今后研究同类极端环境中新的物种资源开发应用以及微生物多样性研究提供素材和参考。     

1株虎源致病性肠球菌的分离鉴定及序列分析

从病死老虎肺脏中分离到1株肠球菌,并对该菌做了生理生化鉴定、药敏试验,致病性试验。本菌对多种抗生素高度耐药,对小白鼠有强致病性,其LD50为2.7×109.2cfu。并用PCR方法扩增分离菌株16S rDNA基因,获得1 415 bp片段,该片段核苷酸序列提交GenBank,登陆号为HM346186,将分离株的16S rDNA核苷酸序列与GenBank上其他肠球菌进行同源性分析。结果表明,分离株的16S rDNA核苷酸序列与肠球菌(EU285587)的同源性为100%,因此该分离菌株被鉴定为致病性肠球菌,命名为YN-1株(云南-1株)。     

Genetic analysis of the br gene in halophilic archaea isolated from Xinjiang region, China

Some novel members of extremely halophilic archaea, strains AJ 11, AJ 12 and AJ 13, were isolated from the Aularz Lake located in the Altun Mountain National Nature Reserve of Xinjiang, Uygur Autonomous Region in China. Partial DNA fragments encoding a bacteriorho-dopsin (BR), as well as for 16S rRNA of isolated strains, were amplified by PCR and their DNA sequences were determined subsequently. On the basis of homology and phylogenetic analysis of the 16S rDNA, we thought that the isolated strains forming a microbiological population are the members of the genus Natrinema. The results of genetic analysis, such as GC content, transition/transver-sion (Ti/Tv) rate ratios and synonymous substitution rates (Ks) indicate that the br fragments, with a high level of genetic divergence, are faced with both purifying selection and bias mutation pressure. The study provides the basis for use of species and BR proteins resources.     

Genetic analysis of the br gene in halophilic archaea isolated from Xinjiang region, China

Some novel members of extremely halophilic archaea, strains AJ11, AJ12 and AJ13, were isolated from the Aularz Lake located in the Altun Mountain National Nature Reserve of Xinjiang, Uygur Autonomous Region in China. Partial DNA fragments encoding a bacteriorhodopsin (BR), as well as for 16S rRNA of isolated strains, were amplified by PCR and their DNA sequences were determined subsequently. On the basis of homology and phylogenetic analysis of the 16S rDNA, we thought that the isolated strains forming a microbiological population are the members of the genus Natrinema. The results of genetic analysis, such as GC content, transition/transversion (Ti/Tv) rate ratios and synonymous substitution rates (Ks) indicate that the br fragments, with a high level of genetic divergence, are faced with both purifying selection and bias mutation pressure. The study provides the basis for use of species and BR proteins resources. __________ Translated from Hereditas (Beijing), 2007, 29(3): 376–380 [译自: 遗传]     

Bacillus beringensis sp. nov., a psychrotolerant bacterium isolated from the Bering Sea

Psychrotolerant Bacillus-like strains BR035(T) and BR011 were isolated from seawater of the Bering Sea and were characterized by means of a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that these strains were related to the members of the genus Bacillus and had the highest 16S rRNA gene sequence similarity with Bacillus korlensis ZLC-26(T). DNA-DNA hybridization experiments confirmed that strains BR035(T) and BR011 belonged to the same species and were distinct from their closest relatives. The cells were Gram-positive, rods, motile, spore-forming and psychrotolerant. The temperature range for growth was 4-42°C. The main respiratory quinone was MK-7. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unknown aminolipid and two unknown phospholipids. The major cellular fatty acids were iso-C15:0, anteiso-C15:0, iso-C14:0 and C16:1ω7c alcohol. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The genomic DNA G + C content was 37.6-37.8 mol%. On the basis of the phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness data, a novel species Bacillus beringensis is proposed and the type strain is BR035(T) (=CGMCC 1.9126(T)=DSM 22571(T)).     

ADSRRS-fingerprinting and PCR MP techniques for studies of intraspecies genetic relatedness in Staphylococcus aureus

The present study was designed to evaluate the usefulness of two novel molecular typing methods, amplification of DNA fragments surrounding rare restriction sites (ADSRRS-fingerprinting) and the PCR melting profile (PCR MP), for Staphylococcus aureus strain differentiation. Thirty-seven S. aureus strains isolated from patients with a history of furunculosis were studied. The strains were identified by determining several phenotypic properties and were genotyped using three differentiation methods: macrorestriction analysis of the chromosomal DNA by pulsed-field gel electrophoresis (REA-PFGE), ADSRRS-fingerprinting, and PCR MP technique. In some cases the results obtained showed that the S. aureus isolated from the nose was identical to the one from the furuncle of the same patient. The same genotype was also identified for S. aureus strains isolated from two different members of a family with a history of recurrent furunculosis, although the active lesions were present in only one of them when the investigation was done. Results from strain genotyping illustrated that the recently developed ADSRRS-fingerprinting and PCR MP techniques are useful for studies of intraspecies genetic relatedness of S. aureus strains. They are as effective in discriminating closely related strains as the PFGE method, which is currently considered to be "the gold standard" for epidemiological studies.     

长春花黄化植原体（PY）株系的检测与鉴定

植原体 (Ｐｈｙｔｏｐｌａｓｍａ) (原称类菌原体Ｍｙｃｏｐｌａｓｍａ ｌｉｋｅＯｒｇａｎｉｓｍ ,简称ＭＬＯ)是一类无细胞壁、存在于植物筛管细胞内的原核生物。植原体自 1 967年被日本学者土居养二首次发现后 ,迄今为止 ,世界上报道的植物植原体病害多达 30 0余种 ,早期对植原体的鉴定主要是通过生物学特性 ,如症状特征、与昆虫介体的相互关系等进行的。这些方法费时费力 ,结果往往也不是很可靠。 80年代 ,随着血清学、分子探针以及ＰＣＲ技术的发展应用 ,为植原体的检测提供了一种相对简单、灵敏、可靠的方法。通过对 1 6ＳｒＲＮＡ基…     

DNA-based classification and sequence heterogeneities in the 16S rRNA genes of Lactobacillus casei/paracasei and related species

The sequence differences within the 16S rRNA genes of Lactobacillus casei/paracasei and related species, Lactobacillus zeae and Lactobacillus rhamnosus, were investigated. Thirty-seven strains of mostly human or cheese origin were grouped by restriction endonuclease analysis (REA) of the total chromosomal DNA and by temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rRNA gene fragments. REA verified that all strains were genomically unique and singled out three major clusters, one L. rhamnosus-cluster and two clusters containing L. paracasei strains. The groups obtained by TTGE corresponded with one exception to the REA-clusters. In the TTGE clustering all L. paracasei strains formed one general group with one TTGE-band in common, and this group was sub-divided into five subgroups due to the presence of more than one TTGE-band in four of the subgroups. The occurrence of multiple TTGE-bands was investigated by amplifying and cloning of the 16S rRNA genes from the strains showing this phenomenon, thereby 12 clones from each strain were sequenced, demonstrating polymorphisms in almost all the cases. Subjecting the clones displaying sequence variations to TTGE as well as sequencing of 16S rDNA revealed by ribotyping of the strains, verified the presence of polymorphisms within the 16S rRNA genes. The migration characteristic of amplified DNA from a single clone corresponded to a specific band in the TTGE-pattern of the strain from which the clone originated. Southern blot hybridisation with a 16S rDNA probe demonstrated the presence of at least five 16S rRNA genes in L. casei/paracasei. A higher degree of variable positions than previously reported was observed in the 16S rRNA gene fragments of the members in the complex. Sequence comparison between the 16S rRNA gene copies of L. casei (CCUG 21451T) and L. zeae (CCUG 35515T) demonstrated that the two species shared almost the same sequence in some copies while the others were more different. Our results provide one explanation for the difficulties in reaching clear-cut taxa within the L. casei/paracasei complex.     

胡枝了属根瘤菌的多相分类研究

采用数值分类,全细胞可溶性蛋白电泳分析,ＤＮＡ,Ｇ＋Ｃｍｏｌ％和ＤＮＡ相关性的测定以及１６ＳｒＤＮＡＰＣＲ－ＲＦＬ分析等多相分类技术对来源于不同地区的１６种寄主的胡枝子根瘤菌进行了系统的分类研究,数值分类的结果表明,在６７％的相似性水平上,全部供试菌可以为快生型根瘤菌和慢性型根瘤菌两大群,在８０％的相似性水平上又可分为两个亚群。在此基础上,对各亚群的胡枝子根瘤菌进行了ＤＮＡ相关性的测定,以进一步证     

Metalloid Reducing Bacteria Isolated from Deep Ocean Hydrothermal Vents of the Juan de Fuca Ridge, Pseudoalteromonas telluritireducens sp. nov. and Pseudoalteromonas spiralis sp. nov

Five strains of Gram-negative, rod, curved rod and spiral-shaped bacteria were isolated from the vicinity of deep ocean hydrothermal vents along the Main Endeavour Segment of the Juan de Fuca Ridge in the Pacific Ocean. All strains showed remarkable resistance to high levels of toxic metalloid oxyanions, and were capable of reducing the oxyanions tellurite and selenite to their less toxic elemental forms. Phylogenetic analysis of four strains identified these isolates as close relatives of the genus Pseudoalteromonas within the class Gammaproteobacteria. Pseudoalteromonas agarivorans was the closest relative of strains Te-1-1 and Se-1-2-red^T, with, respectively, 99.5 and 99.8% 16S rDNA sequence similarity. Strain Te-2-2^T was most closely related to Pseudoalteromonas paragorgicola, with 99.8% 16S rDNA sequence similarity. The DNA G+C base composition was 39.6 to 41.8 mol%, in agreement with other members of the genus Pseudoalteromonas. However, the isolates showed important morphological and physiological differences from previously described species of this genus, with one group forming rod-shaped bacteria typical of Pseudoalteromonas and the other forming vibrioid- to spiral-shaped cells. Based on these differences, and on phylogenetic data, we propose the creation of the new species Pseudoalteromonas telluritireducens sp. nov., with strain Se-1-2-red^T (DSMZ=16098^T=VKM B-2382^T) as the type strain, and Pseudoalteromonas spiralis sp. nov., with strain Te-2-2^T (DSMZ=16099^T=VKM B-2383^T) as the type strain. The EMBL accession numbers for the 16S rDNA sequences are: Te-1-1, AJ314843; Te-2-2^T, AJ314842; Se-1-2-or, AJ314844; Se-1-2-red^T, AJ314845.