16S rRNA analysis of isolates obtained from gram-negative, filamentous bacteria micromanipulated from activated sludge

Individual filaments of the Gram negative, bulking filamentous morphotypes Eikelboom Type 0092, Type 0411, TT;pe 0803 and Herpetosiphon sp. were identified in activated sludge mixed liquors and specifically isolated by micromanipulation. Although their isolation and partial phenotypic description have previously been reported, we sought them to determine their phylogenetic position and to compare our data with the previous descriptions. Direct cell lysis procedures and the polymerase chain reaction were used to obtain their 16S rRNA genes, These were sequenced and the data were analysed to phylogenetically place the filaments into their respective lines of descent in the domain Bacteria. Type 0803 is a member of the Rubrivivax subgroup of the beta proteobacterial subclass; Herpetosiphon sp. belongs in the Chloroflexus subdivision of the green non-sulfur lineage and its sequence is most similar to that of H. aurantiacus; while Type 0092 and Type 0411 belong in the Flexibacter-Cytophaga-Bacteroides phylum. The contemporary ''identification'' of these organisms is ambiguous and relies on subjective morphological criteria and staining reactions. However, the sequence data reported here are being examined for morphotype-specific regions to be exploited for RNA-directed, DNA probes for rapid, unequivocal, in situ identification of each filament type.