Phylogeny and in situ identification of a morphologically conspicuous bacterium, Candidatus Magnospira bakii, present at very low frequency in activated sludge

A morphologically conspicuous bacterium that constituted a very small fract ion (< 0.01%) of the total microbial community of activated sludge was enri ched and analysed phylogenetically by a combination of cultivation-independ ent molecular and physical methods. The large, corkscrew-shaped, filamentou s bacteria were first detected in municipal activated sludge by light micro scopy owing to their unusual rotating gliding motility, Various attempts at microbiological enrichment and pure culture isolation with traditional tec hniques failed, as did attempts to retrieve the morphotype of interest by m icromanipulation. In situ hybridization with the group-specific, rRNA-targe ted oligonucleotide probe CF319a indicated a phylogenetic affiliation to th e Cytophaga-Flexibacter group of the Cytophaga-Flavobacterium-Bacteroides p hylum, Based on strong morphological resemblance to members of the genus Sa prospira, additional 16S rRNA-targeted oligonucleotides with more narrow sp ecificity were designed and evaluated for in situ hybridization to the morp hotype of interest. Flow cytometric cell sorting based on the fluorescence conferred by probe SGR1425 and forward scatter enabled a physical enrichmen t of the helical coiled cells. Subsequent polymerase chain reaction (PCR) a mplification of 16S rDNA fragments from whole fixed sorted cells with a pri mer pair based on probes CF319a and SGR1425 resulted in the retrieval of 12 almost identical partial 16S rDNA fragments with sequence similarities amo ng each other of more than 99.2%, In situ hybridizations proved that the se quences that showed the highest similarity (88.4%) to the 16S rRNA of Sapro spira grandis were indeed retrieved from the corkscrew-shaped filaments. Th e bacterium is likely to be a member of a genus of which no species has bee n cultured hitherto. It was consequently tentatively named 'Magnospira baki i' and has the taxonomic rank of Candidates Magnospira bakii, as the ultima te taxonomic placement has to await its cultivation. In this study, it was demonstrated that even bacteria occurring at very low frequencies in highly complex environmental samples can be retrieved selectively without cultiva tion for further molecular analysis.