THE PHYLOGENETIC POSITION AND ULTRASTRUCTURE OF THE UNCULTURED BACTERIUM ACHROMATIUM-OXALIFERUM

Achromatium oxaliferum is a large, morphologically conspicuous, sedime nt-dwelling bacterium. Nothing is known concerning its phylogeny and i t has eluded all attempts at laboratory cultivation. The limited physi ological Building, University of description of A. oxaliferum has been based on morphological features of the Newcastle upon Tyne, bacterium such as the presence of intracellular sulphur inclusions. A. oxalifer um cells were purified from a wetland region close to Rydal Water (Cum bria, UK). Scanning and transmission electron microscopy revealed that a number of morphologically distinct A. oxaliferum cell-types, based on cell surface features and the size and abundance of calcite and sul phur inclusions within the cells, were present in a single sample of p urified cells. PCR was used to amplify almost full-length 16S rRNA gen e sequences from DNA extracted from A. oxaliferum cells directly purif ied from sediments. The PCR products were cloned and partial sequences (approx. 400 bp) were determined for seven of the clones. Three diffe rent sequence clusters were recovered from the clone libraries. A near full-length (1489 bp) 16S rRNA gene sequence was determined for a rep resentative clone of the most dominant sequence-type (52 % of the sequ ences). Comparative sequence analysis showed A. oxaliferum to form a d eep branching lineage within the gamma-subdivision of the Proteobacter ia. A. oxaliferum was related most closely to the Chromatium assemblag e that includes sulphur-oxidizing symbiotic bacteria, purple sulphur b acteria, and sulpur- and iron-oxidizing thiobacilli. Phylogenetic infe rences made using distance, parsimony and maximum likelihood methods a ll placed A. oxaliferum with this group of bacteria. Bootstrap support for a relationship with any particular lineage within the assemblage was weak. The seven clone sequences recovered from the A. oxaliferum c ells however formed a monophyletic group well supported by bootstrap a nalysis (85-100% support depending on the analysis done). It was concl uded that A. oxaliferum was related to organisms of the Chromatium ass emblage but constituted a novel lineage within this group of bacteria. A. oxaliferum cells were confirmed as the source of the 16S rRNA sequ ence obtained, by the use of a fluorescently-labelled 16S rRNA-targete d oligonucleotide specific for the A. oxaliferum rRNA sequence.