Summary auto-generated
This study applied ribotyping and 16S-23S rDNA intergenic spacer (ISR) sequencing to 12 ammonia-oxidizing bacteria (AOB) isolates to improve phylogenetic resolution. Ribotyping revealed that AOB contain only one copy of the rrn operon per genome, unlike most bacteria which have 5-10 copies, likely related to the slow growth of AOB. The 16S and 23S rRNA genes are organized in the classical tandem arrangement with an intervening spacer. ISR size differed markedly between genera: approximately 400 bp in Nitrosomonas isolates versus 645-694 bp in Nitrosospira isolates, suggesting species-specific characteristics. All isolates contained two potential tRNA genes (Ala-tRNA and Ile-tRNA) near the 5' end of the ISR. ISR sequences showed low overall similarity (42.9-96.2%), compared to high 16S rDNA similarity, making ISR sequencing valuable as a complementary phylogenetic tool. Phylogenetic trees based on ISR sequences were topologically similar to 16S rDNA-based trees but provided significantly improved resolution for distinguishing closely related AOB, particularly at the strain and species levels.
Key findings
- Ammonia-oxidizing bacteria contain only one rrn operon (one copy each of 16S and 23S rRNA genes) per genome, contrasting with most bacteria that have multiple copies
- ISR size is species-characteristic, with Nitrosomonas isolates having ~400 bp and Nitrosospira isolates having 645-694 bp intergenic spacers
- ISR sequence similarity (42.9-96.2%) is substantially lower than 16S rDNA similarity, making ISR sequencing a more discriminatory phylogenetic tool for closely related bacteria
- All AOB isolates contain conserved tRNA genes (Ala-tRNA and Ile-tRNA) at the 5' end of the ISR
- ISR-based phylogenetic analysis provides superior resolution and higher bootstrap support than 16S rDNA-based phylogeny for distinguishing AOB strains and species
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Abstract
It has been established that 16S rRNA gene-based phylogeny gives a low resolution between members of the chemoautotrophic ammonia-oxidizing bacteria (AOB) belonging to the β-subclass of the Proteobacteria. In this study, 12 isolates of AOB were ribotyped, and the sequences of the 16S-23S rDNA intergenic spacer region (ISR) were determined and used in a phylogenetic study. 16S and 23S rDNA ribotyping revealed that the AOB studied contain only one rrn operon per genome, in contrast to most bacteria, which have 5--1 (copies 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 23S rRNA genes of the AOB were shown to be arranged in the classical way: a 16S rRNA gene, an ISR and a 23S rRNA gene. Despite the close phylogenetic relationship among the AOB, the relative location of the rRNA genes in the genome appears to vary considerably. The size of the ISR was approximately 400 bp in the Nitrosomonas isolates and 645--694 bp in the Nitrosospira isolates, suggesting a species-specific size difference in the ISR. The ISR contained two potential tRNA genes in the 5' end in all isolates studied. The similarity values between the ISR sequences of the AOB are low (42·9–96·2%) compared 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 on ISR sequences confirms the 16S rRNA gene-based phylogeny but has the benefit of giving a higher resolution.