Molecular evidence for genus level diversity of bacteria capable of catalyzing anaerobic ammonium oxidation

Recently, a bacterium capable to oxidize ammonium anaerobically at a high r ate was identified as novel member of the Planctomycetales (STROUS, M., FUE RST, J. A., KRAMER, E. H. M., LOGEMANN, S., MUYZER, G., VAN DE PAS-SCHOONEN , It. T., WEBB, R. I., KUENEN, J. G., and JETTEN, M. S. M.: Nature 400, 446 -449, 1999). Here we investigated the microbial community structure of a tr ickling filter biofilm with a high anaerobic ammonium oxidation activity. F luorescence in situ hybridization (FISH) with a set of nine probes designed for specific identification of the recently described anaerobic ammonium o xidizer demonstrated that only one probe hybridized to bacteria within the biofilm. For phylogenetic characterization of putative biofilm anaerobic am monium oxidizers a full-cycle 16S rDNA approach was performed by using a Pl anctomycetales-specific forward primer for PCR amplification. Of the twenty -five 16S rDNA fragments (1364 bp in length) amplified from the biofilm, ni ne were affiliated to the Planctomycetales. Comparative analysis showed tha t these sequences were more than 98.9% similar to each other but only dista ntly related to the previously recognized anaerobic ammonium oxidizer (belo w 91% similarity) and all other organisms represented in public 16S rRNA da tabases (similarities of below 79%). The retrieved sequences and the previo usly recognized anaerobic ammonium oxidizer represent two well-separated gr oups of a deep-branching lineage within the Planctomycetales. Quantitative FISH analysis with a newly designed specific probe showed that the novel ba cterium, provisionally classified as "Candidatus Kuenenia stuttgartiensis" constituted the dominant fraction of the biofilm bacteria. in situ probing revealed that ammonia-oxidizing bacteria of the beta-subclass of Proteobact eria were also present, albeit in significant smaller amounts, within the a noxic biofilm. Comparative sequence analysis of a stretch of the gene encod ing ammonia monooxygenase (amoA) demonstrated the occurrence of the DNA of at least three different populations of beta-subclass ammonia oxidizers wit hin the biofilm.