System. App!. Microbiol. 18,549-559 (1995) © Gu;;tav Fischer Verlag· Stuttgart· .lena· New York Phylogenetic Analysis of Seven New Isolates of Ammonia-Oxidizing Bacteria Based on 16S rRNA Gene Sequences JANNE B. UTAKER1, LARS BAKKEN 2 , QING QIAO JIANG 2 , and INGOLF F. NES 1 1 Laboratory of Microbial Gen'e Technology, Department of Biotechnology, Agricultural University of Norway, As, Norway Section of Microbiology, Department of Biotechnology, Agricultural University of Norway, As, Norway Received June 23, 1995 Summary In this report 165 rRNA gene sequences from seven new isolates of the genus Nitrosospira were deter- mined, and used in a phylogenetic analysis. The homology values for these 16S rDNA sequences ranged from 97.4% to complete homology. In general, the three closest related genera of ammonia-oxidizers - Nitrosospira, Nitrosoiobus and Nitrosovibrio - have 165 rDNA sequences with homology values from 96.9%, giving low resolution when the taxa are positioned in a phylogenetic tree. Thus, while these ammonia-oxidizers possess distinct morphological differences; these differences seem not to be reflected in the 165 ribosomal DNA sequences. This suggests that the morphological traits have evolved recently, and that 16S ribosomal RNA analysis alone is not sufficient to determine the evolutionary relationship be- tween them. The results of this phylogenetic analysis confirm the previously proposed emendation of the genus classification within these bacteria. Key words: 165 rRNA - Phylogeny - Ammonia-oxidizing bacteria - PCR Introduction The ammonia-oxidizing bacteria constitute a group of obligate chemoautotrophs which use carbon dioxide as the sole source of carbon, and obtain energy from oxida- tion of ammonia to nitrite (Watson et aI., 1989). Five genera of ammonia-oxidizers have been characterized; Nitrosomonas (Winogradsky, 1892), Nitrosococcus (Winogradsky, 1892). Nitrosospira (Winogradsky and Winogradsky, 1933), Nitrosolobus (Watson et aI., 1971) and Nitrosovibrio (Harms et aI., 1976), being classified as nitrifying bacteria and belonging to the Nitrobacteraceae family. The nitrifiers are found in most aerobic environ- ments where organic matter is mineralized, and have been isolated from soil, freshwater, brackish water, seawater, and sewage disposal system (Watson et aI., 1989). The earliest classification of ammonia-oxidizers was based on morphological characters, and the progress re- garding comparative studies on physiology, genetics and phylogeny of this group has been slow (MacDonald, 1986). One obvious reason for this is the fact that isolation and maintenance of ammonium-oxidizing bacteria is both difficult and time consuming. The difficulties are con- nected to elimination of heterotrophic organisms during isolation, since the ammonia-oxidizers have very low growth rates (8-24 hrs generation time) and Harms, 1985), and a low cell yield per mol substrate Uiang, 1995). Hence, a culture seldom grows to visible turbidity unless excessive amounts of substrate is added Uiang, 1995). High ammonium concentrations are not re- commended, however, due to the potential toxicity of am- monium and of the nitrite formed during growth (Schmidt and Belser, 1982). The ability to make stock cultures of ammonia-oxidizers is also limited, because of low survival both after preserveration by freeze drying and by freezing at -80°C Uiang, 1995). Because of the low availability of ammonium-oxidizers, molecular biology techniques are well suited for studies on these bacteria. The ammonia-oxidizers have been found to have very similar physiology (Koops and Moiler, 1992), and the DNA base composition (mol% G+C) among the genera of ammonia-oxidizers varies within the limited range of 45 to 56% GC (Koops and Harms, 1985). A study on lipid profiles (Blumer et aI., 1969), has also concluded that