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 ϳ40% 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 ϳ2.5. 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.

The contribution of PCR artifacts to 16S rRNA gene sequence diversity from a complex bacterioplankton sample was estimated. Taq DNA polymerase errors were found to be the dominant sequence artifact but could be constrained by clustering the sequences into 99% sequence similarity groups. Other artifacts (chimeras and heteroduplex molecules) were significantly reduced by employing modified amplification protocols. Surprisingly, no skew in sequence types was detected in the two libraries constructed from PCR products amplified for different numbers of cycles. Recommendations for modification of amplification protocols and for reporting diversity estimates at 99% sequence similarity as a standard are given.Estimation of the extent of PCR-induced artifacts in microbial diversity studies remains an important task in the search for patterns and extent of microbial diversity. The basic types of PCR artifacts have been shown in controlled laboratory studies and can be divided into two categories: those resulting in sequence artifacts (PCR errors), and those skewing the distribution of PCR products due to unequal amplification (PCR bias) or cloning efficiency. Sequence artifacts may arise due to (i) the formation of chimerical molecules (3,10,14,15,25,26,37,38), (ii) the formation of heteroduplex molecules (25,27,29,32), and (iii) Taq DNA polymerase error (4, 25). PCR bias is thought to be due to intrinsic differences in the amplification efficiency of templates (23) or to the inhibition of amplification by the self-annealing of the most abundant templates in the late stages of amplification (31). However, it remains difficult to translate these results to environmental samples in which target genes are orders of magnitude more highly concentrated than in the simple mixtures of templates generally used in controlled laboratory studies.Here, we address the following questions. (i) To what extent do different PCR errors contribute to overestimation of microbial diversity? (ii) Do these PCR errors suggest differences in community structure? (iii) To what extent does PCR bias result in different template distributions after various cycle numbers? Finally, we derive and reiterate recommendations to minimize PCR artifacts.We have recently generated two large 16S rRNA gene libraries (ϳ1,000 sequences each) from a single bacterioplankton sample (1), providing an opportunity to evaluate PCR artifacts in a realistic setting. The first (standard) library was constructed using 35-cycle amplification to mimic commonly used protocols. The second (modified) library was based on the following amplification protocol to reduce the accumulation of PCR artifacts: limitation to 15 cycles of amplification to decrease PCR bias (23) and accumulation of Taq DNA polymerase errors and chimerical sequence formation (25), followed by 3 additional cycles in a fresh reaction mixture (reconditioning PCR step) to minimize the formation of heteroduplex and Taq DNA polymerase errors (32). In addition, we identified Taq DNA polymerase errors in sequences fr...

Although molecular data have revealed the vast scope of microbial diversity, two fundamental questions remain unanswered even for well-defined natural microbial communities: how many bacterial types co-exist, and are such types naturally organized into phylogenetically discrete units of potential ecological significance? It has been argued that without such information, the environmental function, population biology and biogeography of microorganisms cannot be rigorously explored. Here we address these questions by comprehensive sampling of two large 16S ribosomal RNA clone libraries from a coastal bacterioplankton community. We show that compensation for artefacts generated by common library construction techniques reveals fine-scale patterns of community composition. At least 516 ribotypes (unique rRNA sequences) were detected in the sample and, by statistical extrapolation, at least 1,633 co-existing ribotypes in the sampled population. More than 50% of the ribotypes fall into discrete clusters containing less than 1% sequence divergence. This pattern cannot be accounted for by interoperon variation, indicating a large predominance of closely related taxa in this community. We propose that such microdiverse clusters arise by selective sweeps and persist because competitive mechanisms are too weak to purge diversity from within them.

The genomic diversity and relative importance of distinct genotypes within natural bacterial populations have remained largely unknown. Here, we analyze the diversity and annual dynamics of a group of coastal bacterioplankton (greater than 99% 16 S ribosomal RNA identity to Vibrio splendidus ). We show that this group consists of at least a thousand distinct genotypes, each occurring at extremely low environmental concentrations (on average less than one cell per milliliter). Overall, the genomes show extensive allelic diversity and size variation. Individual genotypes rarely recurred in samples, and allelic distribution did not show spatial or temporal substructure. Ecological considerations suggest that much genotypic and possibly phenotypic variation within natural populations should be considered neutral.