STRAIN CHARACTERIZATION AND CLASSIFICATION OF OXYPHOTOBACTERIA IN CLONE CULTURES ON THE BASIS OF 165 RIBOSOMAL-RNA SEQUENCES FROM THE VARIABLE REGIONS V6, V7, AND V8

A major problem in development of a polyphasic taxonomy is that the id entification of oxyphotobacterial strains (cyanobacteria and prochloro phytes) in culture collections may be incorrect, We have therefore dev eloped a diagnostic system using the DNA sequence polymorphism in the 16S rRNA regions V6 to V8 for individual strain characterization and i dentification, PCR primers amplifying V6 to V8 from oxyphotobacteria i n unialgal cultures were constructed. Direct solid-phase or cyclic seq uencing was used to determine the sequences from the amplified DNA, Th is survey includes 10 strains of Nostoc/Anabaena/Aphanizomenon (Nostoc category), 5 strains of Microcystis (Microcystis category), and 4 str ains of Planktothrix (Planktothrix category), Fifteen additional strai ns of cyanobacteria and two strains of prochlorophytes were included s uch that the major phyletic groups were represented, One of the strain s, Phormidium sp, NIVA-CYA 203, contained an Il-nucleotide insertion w ith 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 Pseudanaba ena 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 Plan ktothrix and Microcystis. Based on the sequence and phyletic informati on, group-specific PCR primers for the categories Nostoc, Planktothrix , and Microcystis were constructed, For the strains included in this w ork, the amplifications were specific for the relevant groups, By comb ination of magnetic solid-phase DNA isolation and group-specific PCR a mplifications, an accurate method for characterization, classification and identification of oxyphotobacterial clone cultures has been devel oped.