The current perception of evolutionary relationships and the natural diversity of ammonia-oxidizing bacteria (AOB) is mainly based on comparative sequence analyses of their genes encoding the 16S rRNA and the active site polypeptide of the ammonia monooxygenase (AmoA). However, only partial 16S rRNA sequences are available for many AOB species and most AOB have not yet been analyzed on the amoA level. In this study, the 16S rDNA sequence data of 10 Nitrosomonas species and Nitrosococcus mobilis were completed. Furthermore, previously unavailable 16S rRNA sequences were determined for three Nitrosomonas sp. isolates and for the gamma-subclass proteobacterium Nitrosococcus halophilus. These data were used to revaluate the specificities of published oligonucleotide primers and probes for AOB. In addition, partial amoA sequences of 17 AOB, including the above-mentioned 15 AOB, were obtained. Comparative phylogenetic analyses suggested similar but not identical evolutionary relationships of AOB by using 16S rRNA and AmoA as marker molecules, respectively. The presented 16S rRNA and amoA and AmoA sequence data from all recognized AOB species significantly extend the currently used molecular classification schemes for AOB and now provide a more robust phylogenetic framework for molecular diversity inventories of AOB. For 16S rRNA-independent evaluation of AOB species-level diversity in environmental samples, amoA and AmoA sequence similarity threshold values were determined which can be used to tentatively identify novel species based on cloned amoA sequences. Subsequently, 122 amoA sequences were obtained from 11 nitrifying wastewater treatment plants. Phylogenetic analyses of the molecular isolates showed that in all but two plants only nitrosomonads could be detected. Although several of the obtained amoA sequences were only relatively distantly related to known AOB, none of these sequences unequivocally suggested the existence of previously unrecognized species in the wastewater treatment environments examined.Chemolithoautotrophic ammonia-oxidizing bacteria (AOB) play a central role in the natural cycling of nitrogen by aerobically transforming ammonia to nitrite. From an anthropocentric point of view, the activity of AOB is considered to be both detrimental and beneficial. AOB oxidize urea and ammonia fertilizers to nitrite and, in conjunction with nitrite oxidizers which subsequently convert nitrite to nitrate, thus contribute to fertilizer loss from agricultural soils by producing compounds which are easily washed out or used as electron acceptors for denitrification (42). The former process is also responsible for significant pollution of water supplies with nitrite and nitrate. Furthermore, AOB can produce greenhouse gases (8, 74) and corrode, because of the produced acid, stonework and concrete (46). On the other hand, AOB activity is encouraged in wastewater treatment plants to reduce the ammonia content of sewage before discharge into the receiving waters (49). Reduction of ammonia releases into aquatic en...

The ammonia-oxidizing and nitrite-oxidizing bacterial populations occurring in the nitrifying activated sludge of an industrial wastewater treatment plant receiving sewage with high ammonia concentrations were studied by use of a polyphasic approach. In situ hybridization with a set of hierarchical 16S rRNA-targeted probes for ammonia-oxidizing bacteria revealed the dominance ofNitrosococcus mobilis-like bacteria. The phylogenetic affiliation suggested by fluorescent in situ hybridization (FISH) was confirmed by isolation of N. mobilis as the numerically dominant ammonia oxidizer and subsequent comparative 16S rRNA gene (rDNA) sequence and DNA-DNA hybridization analyses. For molecular fine-scale analysis of the ammonia-oxidizing population, a partial stretch of the gene encoding the active-site polypeptide of ammonia monooxygenase (amoA) was amplified from total DNA extracted from ammonia oxidizer isolates and from activated sludge. However, comparative sequence analysis of 13 amoA clone sequences from activated sludge demonstrated that these sequences were highly similar to each other and to the corresponding amoA gene fragments ofNitrosomonas europaea Nm50 and the N. mobilisisolate. The unexpected high sequence similarity between theamoA gene fragments of the N. mobilisisolate and N. europaea indicates a possible lateral gene transfer event. Although a Nitrobacter strain was isolated, members of the nitrite-oxidizing genus Nitrobacter were not detectable in the activated sludge by in situ hybridization. Therefore, we used the rRNA approach to investigate the abundance of other well-known nitrite-oxidizing bacterial genera. Three different methods were used for DNA extraction from the activated sludge. For each DNA preparation, almost full-length genes encoding small-subunit rRNA were separately amplified and used to generate three 16S rDNA libraries. By comparative sequence analysis, 2 of 60 randomly selected clones could be assigned to the nitrite-oxidizing bacteria of the genusNitrospira. Based on these clone sequences, a specific 16S rRNA-targeted probe was developed. FISH of the activated sludge with this probe demonstrated that Nitrospira-like bacteria were present in significant numbers (9% of the total bacterial counts) and frequently occurred in coaggregated microcolonies with N. mobilis.

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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...

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