Revisiting bacterial phylogeny: Natural and experimental evidence for horizontal gene transfer of 16S rRNA

Abstract

Current methods used for phylogenetic classification of prokaryotes largely rely on the sequences of 16S rRNA genes that are ubiquitously present in the cell. Theoretical basis of this methodology is based on the assumption that 16S rRNA genes are only vertically inherited and are thus indigenous to each species. However, microbial genomic analysis has revealed the existence of prokaryotic species containing two types of rRNA (rrn) operons of seemingly different origins. It has also been reported that some bacteria contain 16S rRNA that are mosaics of sequences from multiple species. This suggests that horizontal gene transfer (HGT) occurred for 16S rRNA genes. In addition, a recent HGT experiment mimicking the natural HGT process has shown that a wide range of foreign 16S rRNA genes can be transferred into Escherichia coli, including those from different phylogenetic classes (with a minimum sequence identity of 80.9% to the Escherichia coli 16S rRNA gene). Thus, in contrast to the complexity hypothesis that states informational genes are rarely horizontally transferred between species, 16S rRNA is occasionally amenable to HGT. Results of the current method for rapid identification and classification of prokaryotes based on the 16S rRNA gene should thus be carefully analyzed and interpreted.

Keywords: 16S ribosomal RNA; complexity hypothesis; horizontal gene transfer; phylogenetics.

Figure 1.(A) Genome-wide distribution of rrn operon and ribosomal protein-encoding genes. Location of ribosomal protein genes involved in 30S subunit (S1–S21) and 7 rrn operons on the genomic map of E. coli (0–100 min). (B) Assembly map of the 30S subunit. Assembly order of ribosomal protein to 16S rRNA is shown by arrows. Figure was constructed as descried by Culver et al.

Figure 2.(A) Schematic illustration of laboratory HGT experiment of 16S rRNA. Escherichia coli ribosomal 30S subunits (green, 16S rRNA; white, ribosomal proteins) were genetically modified to replace 16S rRNA with foreign 16S rRNA (orange, blue and cyan). Through functional screening, 16S rRNA genes that are active in E. coli can be selected (orange). (B)Escherichia coli genetic system for laboratory HGT experiment of 16S rRNA. Escherichia coli Δ7 strain KT101 was used to screen for foreign (metagenomically retrieved) 16S rRNA genes compatible with an E. coli genetic background. The rescue plasmid contained the rrnB operon of E. coli and the counterselectable marker sacB. Transformants containing pRB103 were referred to as KT103.

Figure 3. Neighbor-joining phylogenetic tree of 16S rRNA genes. Environmental 16S rRNA genes that were functional in E. coli are shown as the clone ID (A01–H03) with their closest relatives (nomenclature). Several other relevant strains are also shown.