bEver since Carl Woese introduced the use of 16S rRNA genes for determining the phylogenetic relationships of prokaryotes, this method has been regarded as the "gold standard" in both microbial phylogeny and ecology studies. However, intragenomic heterogeneity within 16S rRNA genes has been reported in many investigations and is believed to bias the estimation of prokaryotic diversity. In the current study, 2,013 completely sequenced genomes of bacteria and archaea were analyzed and intragenomic heterogeneity was found in 952 genomes (585 species), with 87.5% of the divergence detected being below the 1% level. In particular, some extremophiles (thermophiles and halophiles) were found to harbor highly divergent 16S rRNA genes. Overestimation caused by 16S rRNA gene intragenomic heterogeneity was evaluated at different levels using the full-length and partial 16S rRNA genes usually chosen as targets for pyrosequencing. The result indicates that, at the unique level, full-length 16S rRNA genes can produce an overestimation of as much as 123.7%, while at the 3% level, an overestimation of 12.9% for the V6 region may be introduced. Further analysis showed that intragenomic heterogeneity tends to concentrate in specific positions, with the V1 and V6 regions suffering the most intragenomic heterogeneity and the V4 and V5 regions suffering the least intragenomic heterogeneity in bacteria. This is the most up-to-date overview of the diversity of 16S rRNA genes within prokaryotic genomes. It not only provides general guidance on how much overestimation can be introduced when applying 16S rRNA gene-based methods, due to its intragenomic heterogeneity, but also recommends that, for bacteria, this overestimation be minimized using primers targeting the V4 and V5 regions. F or decades, 16S rRNA genes, which encode the small subunit of rRNA in prokaryotes, have been widely used in taxonomic assignment and phylogenetic relationship determination (1, 2). The specific properties of the 16S rRNA gene, including its ubiquitous distribution, mosaic structure (3), and relative stability (4), qualify it as an optimal choice to fulfill these applications. Although some argue that 16S rRNA genes alone may not be sufficient to identify closely related species (5, 6) and the use of monocopy genes like rpoB to perform similar studies has been proposed (7), 16S rRNA genes are undoubtedly the most widely used molecular markers in microbial ecological studies due to well-maintained databases (8) and their easy accessibility.For many years, researchers have been trying to estimate the microbial diversity of complex environments, such as soil (9), marine systems (10), and animal gut systems (11, 12). Various techniques have been developed, from culture-dependent methods to 16S rRNA genes-based methods of clone library (13, 14), denaturing gradient gel electrophoresis (DGGE) (15), terminal restriction fragment length polymorphism (T-RFLP) (16), and the recently developed next-generation sequencing (17). However, the question of how diverse an e...

Bats (order Chiroptera) are one of the most diverse and widely distributed group of mammals with a close relationship to humans. Over the past few decades, a number of studies have been performed on bat viruses; in contrast, bacterial pathogens carried by bats were largely neglected. As more bacterial pathogens are being identified from bats, the need to study their natural microbiota is becoming urgent. In the current study, fecal samples of four bat species from different locations of China were analyzed for their microbiota composition. Together with the results of others, we concluded that bat microbiota is most commonly dominated by Firmicutes and Proteobacteria; the strict anaerobic phylum Bacteroidetes, which is dominant in other terrestrial mammals, especially humans and mice, is relatively rare in bats. This phenomenon was interpreted as a result of a highly specified gastrointestinal tract in adaptation to the flying lifestyle of bats. Further comparative study implied that bat microbiota resemble those of the order Carnivora. To discover potential bacterial pathogens, a database was generated containing the 16S rRNA gene sequences of known bacterial pathogens. Potential bacterial pathogens belonging to 12 genera were detected such as Salmonella, Shigella, and Yersinia, among which some have been previously reported in bats. This study demonstrated high resolution and repeatability in detecting organisms of rare existence, and the results could be used as guidance for future bacterial pathogen isolation.

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