Evaluation of four molecular typing methodologies as tools for determining taxonomy relations and for identifying species among Yersinia isolates

In the last few decades, molecular typing has become an important tool in taxonomic, phylogenetic and identification studies of numerous groups of bacteria, including the yersiniae. In this study, Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR), Pulsed-Field Gel Electrophoresis (PFGE), 16S rRNA gene sequencing and Multilocus Sequence Analysis (MLSA) were performed to determine the ability of these techniques to be used in taxonomy and identification of Yersinia strains. A total of 60 Yersinia strains were genotyped by ERIC-PCR and PFGE. Moreover, an in silico analysis was carried out for 16S rRNA gene sequencing and MLSA, using 68 and 49 Yersinia strains, respectively. A phylogenetic tree constructed from the ERIC-PCR, 16S rRNA gene sequencing and MLSA data grouped most of the Yersinia species into distinct species-specific clusters. In the PFGE assay these clusters were not observed. On this basis, ERIC-PCR, 16S rRNA gene sequencing and MLSA seem to be valuable techniques for use in taxonomic and identification studies of the genus Yersinia, whereas PFGE does not. Furthermore, ERIC-PCR has the advantage of being a cheaper, easier and faster assay than 16S rRNA gene sequencing or MLSA, and for these reasons can be considerate an alternative tool in taxonomic studies of yersiniae.     

Enterococcus columbae, a species from pigeon intestines

Gram-positive cocci which dominate in the intestinal flora of domestic pigeons were found to constitute a new species of the genus Enterococcus. The strains were most closely related to Enterococcus cecorum, originally described as Streptococcus cecorum, a carboxyphilic species from chicken intestines, and to Enterococcus avium. The pigeon strains resemble E. cecorum and also many E. avium strains in their lack of group D antigen and in being more sensitive to NaCl than other enterococci. The type strain is strain STR 345 (= NCIMB 13013).     

Phylogenetic relationships among Rhacophorinae (Rhacophoridae, Anura, Amphibia), with an emphasis on the Chinese species

The partial nucleotide sequence of mitochondrial 12S and 16S rRNA genes was determined for 23 Chinese species of Rhacophoridae (Amphibia: Anura), representing four of the eight recognized genera. Using Buergeriinae as the outgroup, phylogenetic analyses (maximum parsimony, maximum likelihood, and Bayesian inference) were performed in combination with already published mitochondrial 12S and 16S sequences of Rhacophorinae frogs. In all cases, Philautus romeri Smith, 1953 is recovered as the sister taxon to all other Rhacophorinae, although the support values are weak. Chirixalus doriae Boulenger, 1893 is closer to Chiromantis [ Chiromantis rufescens (Günther, 1868) and Chiromantis xerampelina Peters, 1854] than to Chirixalus vittatus (Boulenger, 1887). The clade { Philautus odontotarsus Ye & Fei, 1993, [ Philautus idiootocus (Kuramoto & Wang, 1987), Kurixalus eiffingeri (Boettger, 1895)]} is recovered with strong support. The monophyly of Theloderma and Rhacophorus rhodopus Liu & Hu, 1959 is not supported. It is suggested that Philautus albopunctatus Liu & Hu, 1962 should be placed into the synonymy of Theloderma asperum (Boulenger, 1886), and that Philautus rhododiscus Liu & Hu, 1962 should be assigned to Theloderma , so as to correct the paraphyly. Additionally, the monophyly of ' Aquixalus ' is not supported, and this requires further examination. Results also indicate that the Rhacophorus leucomystax (Gravenhorst, 1829)/ Rhacophorus megacephalus (Hallowell, 1861) complex needs further revision. Studies employing broader sampling and more molecular markers will be needed to resolve the deep relationships within the subfamily Rhacophorinae.  © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society, 2008, 153 , 733–749.     

Genetic relatedness among fish species of Genus Channa using mitochondrial DNA genes

The family Channidae is represented by 26 species, out of which 23 species are found in Asia. However, the taxonomy and phylogeny of the Channid fishes found in India are poorly understood. In the present study, eight species of Channa (Channa striata, Channa punctatus, Channa marulius, Channa gachua, Channa stewartii, Channa aurantimaculata, Channa barca and Channa bleheri) were investigated using partial sequences of 16S rRNA and Cytochrome c Oxidase subunit I (COI) of mitochondrial genes to differentiate among the eight species and study their relationships. The sequence analysis of the genes revealed two distinct groups, which are genetically distant from each other and exhibit identical phylogenetic resolution. The partial sequences of both the genes provided sufficient phylogenetic information to distinguish all the eight species of Channa.     

Molecular typing of isolates of the fish pathogen, Flavobacterium columnare, by single-strand conformation polymorphism analysis

Flavobacterium columnare intraspecies diversity was revealed by analyzing the 16S rRNA gene and the 16S-23S internal spacer region (ISR). Standard restriction fragment length polymorphism (RFLP) of these sequences was compared with single-strand conformation polymorphism (SSCP). Diversity indexes showed that both 16S-SSCP and ISR-SSCP improved resolution (D>or=0.9) when compared with standard RFLP. ISR-SSCP offered a simpler banding pattern than 16S-SSCP while providing high discrimination between isolates. SSCP analysis of rRNA genes proved to be a simple, rapid, and cost-effective method for routine fingerprinting of F. columnare.     

迟缓爱德华菌溶血相关基因的测序和初步的功能分析

溶血素是迟缓爱德华菌（Edwardsiella tarda简称ET）的重要致病因子。用鸟枪法从ET－12菌的染色体中克隆到1株含有溶血活性的克隆子,经测定其序列的大小为4264bp,和已报道的ET两种溶血素基因无同源性,其中开放阅读框3（ORF3）424bp序列和伤寒沙门氏菌溶血调控基因（slyA0序列有68％同源性。含有完整的ORF3的亚克隆子有溶血性,而卡那霉素基因插入ORF3内的酶切位点,其转化子无溶血性,斑点杂交和Southern blot证实,该基因片段来源于供体菌ET－12,而且也存在于其他ET菌染色体上,但这些ET菌表型不一定溶血。含有溶血相关基因重组质粒的大肠杆菌（Escherichia coli简称E.coli）JM109、E.coli LE392有溶血现象。含有溶血相关基因重组质粒的ET菌,不一定有溶血现象。该基因不是溶血结构基因,而是溶血相关基因。     

Genetic differences among three species of the genus Trichiurus (Perciformes: Trichiuridae) based on mitochondrial DNA analysis

The mitochondrial DNA segment encoding the 16S ribosomal RNA (16S rRNA) gene sequence (ca. 600 bp) was compared among Trichiurus sp. 2 (sensu Nakabo, 2002) (obtained from various areas of Japan), T. japonicus Temminck and Schlegel (collected from various localities within Japan), and true T. lepturus Linnaeus (caught off the Atlantic coast of the United States and Brazil) of the family Trichiuridae using 10, 10, and 15 specimens, respectively. Based on phylogenetic analysis using a neighbor-joining (NJ) algorithm, the haplotypes of Trichiurus sp. 2, T. japonicus, and T. lepturus indicated three distinct monophyletic lineages, being supported by 100% bootstrap values with no haplotypes overlapping or sharing among the lineages. Trichiurus sp. 2, T. japonicus, and T. lepturus are genetically different from each other, suggesting that they are three distinct species.     

大黄鱼16SrRNA和18SrRNA基因的测定与序列分析

利用多对引物,扩增并测定出大黄鱼16SrRNA基因和18SrRNA基因的部分序列,其长度分别为1202bp和1275bp,16SrRNA基因序列的GC含量为46．12％,18SrRNA基因的Gc含量为53．oo％。将大黄鱼16SrRNA基因序列与GenBank中15种硬骨鱼类的同源序列结合,同时将其18SrRNA基因序列与GenBank中9种脊索动物的同源序列相结合,运用软件获得各自序列间差异百分比,转换和颠换数值等信息。基于这两种基因序列,利用NJ法和BI法,分别构建16种硬骨鱼类和10种脊索动物的分子系统树。18SrRNA构建的系统树包括三大支,一支为哺乳类、鸟类和爬行类共6个物种,一支为两栖类的1个物种,另一支为2种硬骨鱼类。16SrRNA构建的系统树显示大黄鱼所在的石首鱼科与鲈科和盖刺鱼科亲缘关系较近。此外还讨论了这两个基因的序列特征。     

Identification and characterization of bacterial isolates from the Mir space station

Twenty bacterial isolates (supplied by NASA) from the Mir space station water system were identified using Vitek GNI+ test card, API 20NE, and 16S rRNA gene sequencing. The identification of only one isolate agreed among the three techniques. The utility of the API 20NE and Vitek GNI+ test card approaches for identifying these isolates was Limited. Although 16S rRNA gene sequencing effectively identified many of the bacteria to the genus level, 74% of the isolates could not be identified to the species level. Isolates were also characterized based on motility and hydrophobicity. About 40% of the isolates were motile and four isolates were hydrophobic, suggesting that many of the bacteria have the potential to colonize surfaces and form biofilms. These findings demonstrate the difficulties in identifying bacteria from some environments to the species level and have implications for determining the risks of contamination in water systems of space shuttles and stations.     

Using in situ hybridization to expand the daily egg production method to new fish species

The capacity to reliably identify fish eggs is critical in the application of the daily egg production method (DEPM) to estimate biomass of commercially important species. This application has largely been confined to species that have easily identifiable eggs. Various molecular strategies have been used to extend the DEPM to a broader range of species, with recent approaches like in situ hybridization (ISH) that preserves the integrity of whole eggs, embryos or larvae recommended as a suitable alternative over destructive procedures like PCR. Here, we designed and validated an ISH approach for the identification of whole eggs and larvae from Snapper (Chrysophrys auratus) from environmental samples using the mitochondrial 16S rRNA gene as a target for specific horseradish peroxidase (HRP)‐conjugated oligonucleotide probes. This colorimetric assay allowed the highly specific detection of positive hybridization signals from intact C. auratus larvae and eggs from mixed‐species samples comprising closely related taxa. Furthermore, evaluation of whole eggs across a range of developmental stages revealed the sensitivity of the approach for discerning early stages, thereby guiding staging and the identification of otherwise indistinguishable eggs from environmental samples. This approach represents a major advance from current molecular‐based strategies as it is nondestructive and allows for the simultaneous identification and staging of fish eggs (and larvae). The resultant 100% egg identification certainty we have achieved allows the DEPM to be applied to a wider array of fish species and is particularly applicable to species in areas where morphologically similar eggs are being spawned at the same time.     

16S rRNA gene sequence analyses and inter- and intrageneric relationships of Xanthomonas species and Stenotrophomonas maltophilia

The nearly complete, PCR-amplified, 16S rRNA gene sequences have been determined from the representative type strains of eight xanthomonad phena, including six validly described species of the genus Xanthomonas and Stenotrophomonas maltophilia. Pairwise sequence comparisons and phylogenetic analysis demonstrated that the xanthomonads comprise a monophyletic lineage within the γ-subclass of the Proteobacteria. Although the genus Xanthomonas was observed to comprise a cluster of very closely related species, the observed species-specific primary sequence differences were confirmed through sequencing additional strains belonging to the respective species.     

耐热嗜盐菌的分离及16S rRNA基因序列分析

从位于西藏自治区澜沧江边一个47℃的盐井中分离筛选到一株耐热嗜盐菌菌株YJ0238, 对其进行了生理生化特性研究, 采用PCR方法扩增其16S rRNA基因序列, 并进行了测定。基于生理生化特性和16S rRNA基因序列的同源性比较, 以及系统发育分析, 发现菌株YJ0238是Idiomarina属中成员zobellii的一个亚种, 其16S rRNA基因序列已被GenBank数据库收录, 序列号为EF693953。迄今为止, 国内极少有关高温、高盐环境中微生物研究的报道, 本研究可为今后研究同类极端环境中新的物种资源以及微生物多样性提供素材和参考。     

Thekopsora ostryae (Pucciniastraceae,Pucciniales), a new species from Gansu,northwestern China

Thekopsora ostryae, a new rust fungus on leaves of Ostrya japonica collected from Gansu Province was described. Morphological examination using light and scanning electron microscopy showed that this new species is distinct from other species of Thekopsora in the characteristics of ostiolar peridial cells of uredinia and the spinules on the surface of urediniospores. Analyses of the ITS1-5.8S-ITS2 and 28S rRNA gene partial sequences showed that T. ostryae could be a distinct lineage in the genus Thekopsora.     

一株耐热嗜盐菌的分离及16SrRNA基因序列分析

目的利用盐固体分离培养基,从西藏自治区澜沧江边康宁镇一个47℃的盐井样品中分离纯化到一株耐热嗜盐菌菌株YJ0232。方法通过形态观察、生理生化特性和16srRNA基因序列分析,鉴定嗜盐菌菌株YJ0232分类学地位。结果菌株YJ0232初步鉴定为中度嗜盐菌,属于盐单胞菌属（Halomonassp．）菌株。其16SrRNA基因序列已被GenBank数据库收录,序列号为EU029645。结论本研究对澜沧江高盐环境微生物资源进行了初步探索研究。可为今后研究同类极端环境中新的物种资源以及微生物多样性提供参考。     

耐热嗜盐菌的分离及16S rRNA基因序列分析

从位于西藏自治区澜沧江边一个47℃的盐井中分离筛选到一株耐热嗜盐菌菌株 YJ0238.对其进行了生理生化特性研究,采用PCR方法扩增其16S rRNA基因序列,并进行了测定.基于生理生化特性和16S rRNA基因序列的同源性比较,以及系统发育分析,发现菌株YJ0238是Idiomarina属中成员zobellii的一个亚种,其16S rRNA基因序列已被GenBank数据库收录,序列号为EF693953.迄今为止,国内极少有关高温、高盐环境中微生物研究的报道,本研究可为今后研究同类极端环境中新的物种资源以及微生物多样性提供素材和参考.     

我国南北大豆产区慢生大豆根瘤菌的遗传多样性和系统发育研究

利用16S rRNA基因RFLP、16S rRNA基因序列分析以及16S-23S rRNA IGS PCR RFLP技术对分离自我国南北大豆产区的慢生大豆根瘤菌进行了群体遗传多样性和系统发育研究。16S rRNA基因PCR RFLP分析以及16S rRNA基因序列分析结果表明:所有供试慢生大豆根瘤菌可分为B.japonicum和B.elkanii两个类群,其中属于B.japonicum的为优势种群,占供试菌株的91%,属于B.elkanii的仅占9%,多样性水平较低。16S-23S rRNA IGS PCRRFLP研究结果表明:属于B.japonicum的慢生根瘤菌具有较丰富的遗传多样性,在69%的相似性水平上可分为群Ⅰ和群Ⅱ两大类群。群I的菌株以分离自黑龙江和河北等北部区域的菌株为代表,群Ⅱ的菌株以分离自广西和江苏等南部地域的菌株为代表,反映出明显的地域特征。两群菌株在系统发育上均与USDA6、USDA110和USDA122等B.japonicum的模式或代表菌株有差异。     

Characterization and identification of Borrelia isolates as Borrelia valaisiana in Taiwan and Kinmen Islands

Eleven pure cultures of Borrelia from 3 species of wild rodents (Apodemus agrarius, Mus formosanus, Rattus losea) captured in Taichung, located in the center of Taiwan island, and on Kinmen Island were characterized. Five isolates showed restriction fragment length polymorphism (RFLP) patterns of 5S-23S rRNA gene intergenic spacer sequences identical to those of strains 5MT and 10MT, identified as Borrelia valaisiana, which were isolated in the southern tip of South Korea. Although the remaining six isolates showed novel RFLP patterns, these isolates showed more similarity to members of B. valaisiana from Korea, Japan and Europe based on 16S rRNA gene and flagellin gene sequences. This led us to speculate that transmission and proliferation of this type of borrelia occurred between Taiwan and the southern part of South Korea.