The phylogenetic relationship of 12 ammonia-oxidizing isolates (eight nitrosospiras and four nitrosomonads), for which no gene sequence information was available previously, was investigated based on their genes encoding 16S rRNA and the active site subunit of ammonia monooxygenase (AmoA). Almost full-length 16S rRNA gene sequences were determined for the 12 isolates. In addition, 16S rRNA gene sequences of 15 ammonia-oxidizing bacteria (AOB) published previously were completed to allow for a more reliable phylogeny inference of members of this guild. Moreover, sequences of 453 bp fragments of the amoA gene were determined from 15 AOB, including the 12 isolates, and completed for 10 additional AOB. 16S rRNA gene and amoA-based analyses, including all available sequences of AOB pure cultures, were performed to determine the position of the newly retrieved sequences within the established phylogenetic framework. The resulting 16S rRNA gene and amoA tree topologies were similar but not identical and demonstrated a superior resolution of 16S rRNA versus amoA analysis. While 11 of the 12 isolates could be assigned to different phylogenetic groups recognized within the betaproteobacterial AOB, the estuarine isolate Nitrosomonas sp. Nm143 formed a separate lineage together with three other marine isolates whose 16S rRNA sequences have not been published but have been deposited in public databases. In addition, 17 environmentally retrieved 16S rRNA gene sequences not assigned previously and all originating exclusively from marine or estuarine sites clearly belong to this lineage.
INTRODUCTIONChemolithoautotrophic ammonia-oxidizing bacteria (AOB) are capable of gaining energy via conversion of ammonia to nitrite and are thus of considerable importance in the global nitrogen cycle. Almost all aerobic environments in which organic matter is mineralized are possible habitats for AOB (Bock & Wagner, 2001). They have been detected in a variety of soil, marine, estuarine and freshwater systems and are crucial for the removal of nitrogen compounds in wastewater treatment plants (Painter, 1986), thus contributing to the impairment of anthropogenic damage to the environment. On the other hand, AOB activity causes deterioration of natural building stones (Bock & Sand, 1993) and enhances nitrogen fertilizer loss from arable soil (MacDonald, 1986). Due to their importance in natural and engineered systems, significant efforts have been made to characterize the diversity, distribution patterns and ecophysiology of AOB (for reviews see Koops & Pommerening-Röser, 2001; Koops et al., 2003).The first isolation of AOB was reported in 1890 (Frankland & Frankland, 1890;Winogradsky, 1890) and since then a considerable number of AOB isolates was obtained from various environments, leading to the description of 16 AOB species (reviewed by Koops et al., 2003). Comparative 16S rRNA gene sequence analyses of these species showed that 'Nitrosococcus halophilus' and Nitrosococcus oceani belong to the class 'Gammaproteobacteria', while the remaining...