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

Abstract

A morphologically conspicuous bacterium that constituted a very small fraction (< 0.01%) of the total microbial community of activated sludge was enriched and analysed phylogenetically by a combination of cultivation-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 gliding motility. Various attempts at microbiological enrichment and pure culture isolation with traditional techniques failed, as did attempts to retrieve the morphotype of interest by micromanipulation. In situ hybridization with the group-specific, rRNA-targeted oligonucleotide probe CF319a indicated a phylogenetic affiliation to the Cytophaga-Flexibacter group of the Cytophaga-Flavobacterium-Bacteroides phylum. Based on strong morphological resemblance to members of the genus Saprospira, additional 16S rRNA-targeted oligonucleotides with more narrow specificity were designed and evaluated for in situ hybridization to the morphotype of interest. Flow cytometric cell sorting based on the fluorescence conferred by probe SGR1425 and forward scatter enabled a physical 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 member of a genus of which no species has been cultured hitherto. It was consequently tentatively named 'Magnospira bakii' and has the taxonomic rank of Candidatus 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 environmental samples can be retrieved selectively without cultivation for further molecular analysis.

Identification of Bacterial Populations in Dairy Wastewaters by Use of 16S rRNA Gene Sequences and Other Genetic Markers

“…For example, 16S rDNA sequences similar to those of Clostridium lituseburense (11), Clostridium celatum (17), Ruminococcus bromii (51), E. tenue (45), Turicibacter sanguinis (8), and AF371945 (26) have been isolated from the gastrointestinal tracts of humans and animals. In addition, 16S rDNA sequences similar to those of Acinetobacter johnsonii (32), Bacillus thermocloacae (25), Lactosphaera pasteurii (28), AF129864 (14), U81762 (14), U81676 (14), and AF087054 (46) have been isolated from anaerobic digestors and activated sludge, while others similar to those of Bacillus silvestris, Bacillus marinus (43), Salinococcus roseus (50), Thiocapsa rosea (48), and Alkalibacterium olivoapovliticus (38) appear to represent environmental organisms associated with plant rhizosphere, marine sediment, and both salt and fresh water.…”

Bacterial Community Structure and Physiological State within an Industrial Phenol Bioremediation System

“…Indeed, analyses of the system by microbiological agar isolation yielded few colonies typical of the CytophagaFlavobacterium group organisms (Ͻ0.04%), suggesting bias against these organisms during culture surveys with general agars (unpublished data). The exact functional nature and diversity of the members of this group within the processing system awaits resolution by the application of probes of higher resolution (32) and further characterization of their phylogeny and physiology through both culture and culture-independent analyses.…”

Fate of Heterotrophic Microbes in Pelagic Habitats: Focus on Populations

“…Direct sorting of microbial cells after FISH staining has first been shown in highly productive wastewater treatment systems (284). Recently this approach has also been adapted for bacterial cells from coastal marine bacterioplankton, taking advantage of the superior signal intensities of CARD-FISH staining (262).…”