Phylogeny and in situ identification of a morphologically conspicuous bacterium, Candidatus Magnospira bakii, present at very low frequency in activated sludge Jiri Snaidr, 1,2² Bernhard Fuchs, 1,2 Gu ¨ nter Wallner, 3 Michael Wagner, 1 Karl-Heinz Schleifer 1 and Rudolf Amann 1,2 * 1 Technische Universita ¨ t Mu ¨ nchen, Lehrstuhl fu ¨ r Mikrobiologie, Arcisstr. 16, D-80290 Munich, Germany. 2 Max-Planck-Institut fu ¨ r Marine Mikrobiologie, Celsiusstr. 1, D-28359 Bremen, Germany. 3 GSF-Forschungszentrum fu ¨ r Umwelt und Gesundheit, Durchflußzytometrie, D-85764 Neuherberg, Germany. Summary A morphologically conspicuous bacterium that consti- tuted a very small fraction (< 0.01%) of the total micro- bial community of activated sludge was enriched and analysed phylogenetically by a combination of culti- vation-independent molecular and physical methods. The large, corkscrew-shaped, filamentous bacteria were first detected in municipal activated sludge by light microscopy owing to their unusual rotating glid- ing motility. Various attempts at microbiological enrichment and pure culture isolation with traditional techniques failed, as did attempts to retrieve the mor- photype of interest by micromanipulation. In situ hybridization with the group-specific, rRNA-targeted oligonucleotide probe CF319a indicated a phyloge- netic affiliation to the Cytophaga–Flexibacter group of the Cytophaga –Flavobacterium–Bacteroides phy- lum. Based on strong morphological resemblance to members of the genus Saprospira, additional 16S rRNA-targeted oligonucleotides with more narrow spe- cificity were designed and evaluated for in situ hybridi- zation to the morphotype of interest. Flow cytometric cell sorting based on the fluorescence conferred by probe SGR1425 and forward scatter enabled a physi- cal enrichment of the helical coiled cells. Subsequent polymerase chain reaction (PCR) amplification of 16S rDNA fragments from whole fixed sorted cells with a primer pair based on probes CF319a and SGR1425 resulted in the retrieval of 12 almost identical partial 16S rDNA fragments with sequence similarities among each other of more than 99.2%. In situ hybridizations proved that the sequences that showed the highest similarity (88.4%) to the 16S rRNA of Saprospira grandis were indeed retrieved from the corkscrew- shaped filaments. The bacterium is likely to be a mem- ber of a genus of which no species has been cultured hitherto. It was consequently tentatively named ‘Mag- nospira bakii’ and has the taxonomic rank of Candida- tus Magnospira bakii, as the ultimate taxonomic placement has to await its cultivation. In this study, it was demonstrated that even bacteria occurring at very low frequencies in highly complex environmen- tal samples can be retrieved selectively without culti- vation for further molecular analysis. Introduction The real extent of bacterial diversity remained invisible for decades, because bacterial identification relied largely on cultivation-dependent methods. Pure cultures isolated from clinical and environmental samples were classified on the basis of their biochemical or physiological parameters. They formed the basis for the production of the only tools for in situ identification, antibodies that were applied, for example, in immunofluorescence assays (Bohlool and Schmidt, 1980). Consequently, bacterial population analy- sis targeted mainly microorganisms that grew readily on artificial nutrient media. However, it has long been recog- nized that only a minority of the microorganisms present in natural and engineered environmental samples can be cultivated by standard techniques (Wagner et al., 1993; Amann et al., 1995). Molecular studies indicate an aston- ishing microbial diversity (e.g. Torsvik et al ., 1990; Amann et al., 1996). It is the rRNA approach to microbial ecology and evolution (Olsen et al., 1986; Pace et al., 1986) that allows the analysis of this high natural microbial diversity in more detail today. Microorganisms can be analysed phylogenetically and identified in situ without cultivation by a combination of polymerase chain reaction (PCR)- assisted rDNA retrieval, comparative sequence analysis and fluorescence in situ hybridization (Amann et al., 1995). Yet, this molecular analysis remained restricted to the more dominant populations, as the screening and Environmental Microbiology (1999) 1(2), 125–135  1999 Blackwell Science Ltd Received 24 August, 1998; revised 11 November, 1998; accepted 16 November, 1998. ²Present address: Vermicon Engineering and Microbiology Aktiengesellschaft, Nymphenburger Strasse 81, D- 80636 Munich, Germany. *For correspondence at the Max-Planck- Institut, Bremen. E-mail ramann@mpi-bremen.de; Tel. (þ49) 421 2028 930; Fax (þ49) 421 2028 690.