RFLP of rRNA genes and sequencing of the 16S-23S rDNA intergenic spacer region of ammonia-oxidizing bacteria: a phylogenetic approach

It has been established that 16S rRNA gene-based phylogeny gives a low reso lution between members of the chemoautotrophic ammonia-oxidizing bacteria ( AOB) belonging to the beta-subclass of the Proteobacteria, In this study, 1 2 isolates of AOB were ribotyped, and the sequences of the 16S-23S rDNA int ergenic spacer region (ISR) were determined and used in a phylogenetic stud y. 16S and 23S rDNA ribotyping revealed that the AOB studied contain only o ne rm operon per genome, in contrast to most bacteria, which have 5-10 copi es of the rRNA genes per genome. It is likely that the presence of only one set of rRNA genes is related to the slow growth of the AOB, The 16S and 23 S rRNA genes of the AOB were shown to be arranged in the classical way: a 1 6S rRNA gene, an ISR and a 23S rRNA gene. Despite the close phylogenetic re lationship among the AOB, the relative location of the rRNA genes in the ge nome appears to vary considerably. The size of the ISR was approximately 40 0 bp in the Nitrosomonas isolates and 645-694 bp in the Nitrosospira isolat es, suggesting a species-specific size difference in the ISR, The ISR conta ined two potential tRNA genes in the 5' end in all isolates studied. The si milarity values between the ISR sequences of the AOB are low (42.9-96.2%) c ompared with the 16S rDNA sequence similarity values, and therefore the ISR sequences are valuable as a complementary phylogenetic tool in combination with 16S rRNA gene sequences. The phylogenetic analysis of the AOB based o n ISR sequences confirms the 16S rRNA gene-based phylogeny but has the bene fit of giving a higher resolution.