A comparison of the phylogeny of 38 isolates of chemolithoautotrophic ammonia-oxidizing bacteria (AOB) based on 16S rRNA gene sequences, 16S-235 rDNA intergenic spacer region (ISR) sequences and species affiliations based on DNA homology values was performed. The organisms studied all belong to the beta-subclass of the Proteobacteria and included representatives of Nitrosomonas, Nitrosococcus and Nitrosospira. The similarity values of the 16S rDNA sequences were high, particularly within the Nitrosospira genus, and based on these sequences it is difficult to determine the phylogenetic position of some AOB. As an alternative and supplement to 16S rRNA gene sequencing, the ISR was sequenced and analysed phylogenetically. Due to considerably lower similarity values, the ISR-based phylogeny gives a better resolution than the phylogeny based on the functional 16S rRNA gene. Since the ISR-based phylogeny of AOB is highly consistent with the 16S rDNA based phylogeny, ISR sequencing appears as a suitable tool for resolving the detailed phylogeny of AOB. The phylogenetic position of two isolates of the former genus 'Nitrosolobus' (now included in the Nitrosospira genus) is not clear. These organisms are close relatives of the former Nitrosospira spp. and 'Nitrosovibrio' spp. (now Nitrosospira), but based on their marginal positions in the phylogenetic trees, DNA-DNA hybridization data and phenotypic characteristics, it is suggested that 'Nitrosolobus' should be a separate genus. DNA homology determination of 11 Nitrosospira isolates revealed two new species of Nitrosospira. The phylogeny of AOB reflected in the trees based on the rDNA sequences is consistent with the species affiliations of AOB by DNA homology values. This observation will probably be important for the interpretation of results from studies of natural diversity of AOB.


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