Rudi K, Skulberg O M, Larsen F, Jakobsen K S
Department of Biology, University of Oslo, Norway.
Appl Environ Microbiol. 1997 Jul;63(7):2593-9. doi: 10.1128/aem.63.7.2593-2599.1997.
A major problem in development of a polyphasic taxonomy is that the identification of oxyphotobacterial strains (cyanobacteria and prochlorophytes) in culture collections may be incorrect. We have therefore developed a diagnostic system using the DNA sequence polymorphism in the 16S rRNA regions V6 to V8 for individual strain characterization and identification. PCR primers amplifying V6 to V8 from oxyphotobacteria in unialgal cultures were constructed. Direct solid-phase or cyclic sequencing was used to determine the sequences from the amplified DNA. This survey includes 10 strains of Nostoc/Anabaena/Aphanizomenon (Nostoc category), 5 strains of Microcystis (Microcystis category), and 4 strains of Planktothrix (Planktothrix category). Fifteen additional strains of cyanobacteria and two strains of prochlorophytes were included such that the major phyletic groups were represented. One of the strains, Phormidium sp. NIVA-CYA 203, contained an 11-nucleotide insertion with no homology to other known 16S rRNA sequences. Based on parsimony and neighbor-joining trees, the phyletic relationships of the strains were investigated. Thirteen major branches were found, with Pseudanabaena limnetica NIVA-CYA 276/6 as the most divergent strain. The strain categories Nostoc, Planktothrix, and Microcystis were all monophyletic. The sequence polymorphism within Nostoc was higher than that in Planktothrix and Microcystis. Based on the sequence and phyletic information, group-specific PCR primers for the categories Nostoc, Planktothrix, and Microcystis were constructed. For the strains included in this work, the amplifications were specific for the relevant groups. By combination of magnetic solid-phase DNA isolation and group-specific PCR amplifications, an accurate method for characterization, classification and identification of oxyphotobacterial clone cultures has been developed.
多相分类学发展中的一个主要问题是，培养物保藏中心中氧光细菌菌株（蓝细菌和原绿藻）的鉴定可能有误。因此，我们开发了一种诊断系统，利用16S rRNA区域V6至V8中的DNA序列多态性对单个菌株进行特征分析和鉴定。构建了从单藻培养物中的氧光细菌扩增V6至V8的PCR引物。使用直接固相测序或循环测序来确定扩增DNA的序列。这项调查包括10株念珠藻属/鱼腥藻属/束丝藻属（念珠藻类别）、5株微囊藻属（微囊藻类别）和4株席藻属（席藻类别）。还包括另外15株蓝细菌菌株和2株原绿藻菌株，以便涵盖主要的系统发育类群。其中一个菌株，席藻属NIVA-CYA 203，含有一个11个核苷酸的插入片段，与其他已知的16S rRNA序列无同源性。基于简约树和邻接树，研究了这些菌株的系统发育关系。发现了13个主要分支，其中湖沼假鱼腥藻NIVA-CYA 276/6是最具分歧性的菌株。念珠藻、席藻和微囊藻类别均为单系类群。念珠藻内的序列多态性高于席藻和微囊藻。根据序列和系统发育信息，构建了念珠藻、席藻和微囊藻类别的组特异性PCR引物。对于本研究中包含的菌株，扩增对相关类群具有特异性。通过磁固相DNA分离和组特异性PCR扩增相结合，开发了一种准确的方法用于氧光细菌克隆培养物的特征分析、分类和鉴定。
