16S‐23S rDNA intergenic spacer and 23S rDNA of anaerobic ammonium‐oxidizing bacteria: implications for phylogeny and <i>in situ</i> detection

<jats:p> Recently, anaerobic ammonium‐oxidizing bacteria (AAOB) were identified by comparative 16S rDNA sequence analysis as a novel, deep‐branching lineage within the <jats:italic>Planctomycetales</jats:italic> . This lineage consists currently of only two, not yet culturable bacteria which have been provisionally described as <jats:italic>Candidatus</jats:italic> ‘Brocadia anammoxidans’ and <jats:italic>Candidatus</jats:italic> ‘Kuenenia stuttgartiensis’. In this study, a large fragment of the rDNA operon, including the 16S rDNA, the intergenic spacer region (ISR) and approximately 2 000 bases of the 23S rDNA, was polymerase chain reaction (PCR) amplified, cloned and sequenced from both AAOB. The retrieved 16S rDNA sequences of both species contain an insertion at helix 9 with a previously overlooked pronounced secondary structure (new subhelices 9a and 9b). This insertion, which is absent in all other known prokaryotes, is detectable by fluorescence <jats:italic>in situ</jats:italic> hybridization (FISH) and thus present in the mature 16S rRNA. In contrast with the genera <jats:italic>Pirellula</jats:italic> , <jats:italic>Planctomyces</jats:italic> and <jats:italic>Gemmata</jats:italic> that possess unlinked 16S and 23S rRNA genes, both AAOB have the respective genes linked together by an ISR of approximately 450 bp in length. Phylogenetic analysis of the obtained 23S rRNA‐genes confirmed the deep branching of the AAOB within the <jats:italic>Planctomycetales</jats:italic> and allowed the design of additional specific FISH probes. Remarkably, the ISR of the AAOB also could be successfully detected by FISH via simultaneous application of four monolabelled oligonucleotide probes. Quantitative FISH experiments with cells of <jats:italic>Candidatus</jats:italic> ‘Brocadia anammoxidans’ that were inhibited by exposure to oxygen for different time periods demonstrated that the concentration of transcribed ISR reflected the activity of the cells more accurately than the 16S or 23S rRNA concentration. Thus the developed ISR probes might become useful tools for <jats:italic>in situ</jats:italic> monitoring of the activity of AAOB in their natural environment. </jats:p>