Divergence and redundancy of 16S rRNA sequences in genomes with multiple rrn operons.

The level of sequence heterogeneity among rrn operons within genomes determines the accuracy of diversity estimation by 16S rRNA-based methods. Furthermore, the occurrence of widespread horizontal gene transfer (HGT) between distantly related rrn operons casts doubt on reconstructions of phylogenetic relationships. For this study, patterns of distribution of rrn copy numbers, interoperonic divergence, and redundancy of 16S rRNA sequences were evaluated. Bacterial genomes display up to 15 operons and operon numbers up to 7 are commonly found, but 40% of the organisms analyzed have either one or two operons. Among the Archaea ,a single operon appears to dominate and the highest number of operons is five. About 40% of sequences among 380 operons in 76 bacterial genomes with multiple operons were identical to at least one other 16S rRNA sequence in the same genome, and in 38% of the genomes all 16S rRNAs were invariant. For Archaea, the number of identical operons was only 25%, but only five genomes with 21 operons are currently available. These considerations suggest an upper bound of roughly threefold overestimation of bacterial diversity resulting from cloning and sequencing of 16S rRNA genes from the environment; however, the inclusion of genomes with a single rrn operon may lower this correction factor to 2.5. Divergence among operons appears to be small overall for both Bacteria and Archaea, with the vast majority of 16S rRNA sequences showing <1% nucleotide differences. Only five genomes with operons with a higher level of nucleotide divergence were detected, and Thermoanaerobacter tengcongensis exhibited the highest level of divergence (11.6%) noted to date. Overall, four of the five extreme cases of operon differences occurred among thermophilic bacteria, suggesting a much higher incidence of HGT in these bacteria than in other groups. rRNA sequences play a central role in the study of microbial evolution and ecology. Particularly, the 16S rRNA genes have become the standard for the determination of phylogenetic relationships, the assessment of diversity in the environment, and the detection and quantification of specific populations (14, 16). Indeed, the rRNAs combine several properties which make them uniquely suited for such diverse applications. First, they are universally distributed, allowing the comparison of phylogenetic relationships among all extant organisms and thus the construction of a “tree of life.” Second, the rRNAs are generally thought to be part of a core of informational genes which are only weakly affected by horizontal gene transfer (HGT) (1, 8), so their relationships provide a solid framework for the assessment of evolutionary changes in lineages. Third, the rRNAs are functionally highly constrained mosaics of sequence stretches ranging from conserved to more variable. This enables the design of PCR primers and hybridization probes with various levels of taxonomic specificity and is exploited in microbial ecology when the number and distribution of different rRNA genes are taken as a measure of diversity (14). A testimony to the significance of these approaches is the vast and growing database of 16S rRNA genes, of which an increasing number are derived from the large majority of uncultured Bacteria and Archaea. Although many of these organisms appear to dominate in the environment, their distribution and relationships are only known from clone libraries derived from nucleic acids recovered from the en