16S-23S rDNA intergenic spacer and 23S rDNA of anaerobic ammonium-oxidizing bacteria: implications for phylogeny and in situ detection

Recently, anaerobic ammonium-oxidizing bacteria (AAOB) were identified by c omparative 16S rDNA sequence analysis as a novel, deep-branching lineage wi thin the Planctomycetales. This lineage consists currently of only two, not yet culturable bacteria which have been provisionally described as Candida tus 'Brocadia anammoxidans' and Candidatus 'Kuenenia stuttgartiensis'. In t his study, a large fragment of the rDNA operon, including the 16S rDNA, the intergenic spacer region (ISR) and approximately 2000 bases of the 23S rDN A, was polymerase chain reaction (PCR) amplified, cloned and sequenced from both AAOB. The retrieved 16S rDNA sequences of both species contain an ins ertion at helix 9 with a previously overlooked pronounced secondary structu re (new subhelices 9a and 9b). This insertion, which is absent in all other known prokaryotes, is detectable by fluorescence in situ hybridization (FI SH) and thus present in the mature 16S rRNA. In contrast with the genera Pi rellula, Planctomyces and Gemmata that possess unlinked 16S and 23S rRNA ge nes, both AAOB have the respective genes linked together by an ISR of appro ximately 450 bp in length. Phylogenetic analysis of the obtained 23S rRNA-g enes confirmed the deep branching of the AAOB within the Planctomycetales a nd allowed the design of additional specific FISH probes. Remarkably, the I SR of the AAOB also could be successfully detected by FISH via simultaneous application of four monolabelled oligonucleotide probes. Quantitative FISH experiments with cells of Candidatus 'Brocadia anammoxidans' that were inh ibited by exposure to oxygen for different time periods demonstrated that t he concentration of transcribed ISR reflected the activity of the cells mor e accurately than the 16S or 23S rRNA concentration. Thus the developed ISR probes might become useful tools for in situ monitoring of the activity of AAOB in their natural environment.