Use of 16S rRNA and rpoB genes as molecular markers for microbial ecology studies

Abstract

Several characteristics of the 16S rRNA gene, such as its essential function, ubiquity, and evolutionary properties, have allowed it to become the most commonly used molecular marker in microbial ecology. However, one fact that has been overlooked is that multiple copies of this gene are often present in a given bacterium. These intragenomic copies can differ in sequence, leading to identification of multiple ribotypes for a single organism. To evaluate the impact of such intragenomic heterogeneity on the performance of the 16S rRNA gene as a molecular marker, we compared its phylogenetic and evolutionary characteristics to those of the single-copy gene rpoB. Full-length gene sequences and gene fragments commonly used for denaturing gradient gel electrophoresis were compared at various taxonomic levels. Heterogeneity found between intragenomic 16S rRNA gene copies was concentrated in specific regions of rRNA secondary structure. Such "heterogeneity hot spots" occurred within all gene fragments commonly used in molecular microbial ecology. This intragenomic heterogeneity influenced 16S rRNA gene tree topology, phylogenetic resolution, and operational taxonomic unit estimates at the species level or below. rpoB provided comparable phylogenetic resolution to that of the 16S rRNA gene at all taxonomic levels, except between closely related organisms (species and subspecies levels), for which it provided better resolution. This is particularly relevant in the context of a growing number of studies focusing on subspecies diversity, in which single-copy protein-encoding genes such as rpoB could complement the information provided by the 16S rRNA gene.

FIG. 1.

Mapping of the 16S rRNA positions where intragenomic heterogeneity was observed among the 111 bacterial species analyzed. Intragenomic heterogeneity is represented by nucleotide substitutions between multiple copies of the 16S rRNA gene present in a single organism. Heterogeneous positions were mapped on the secondary structure of one of the E. coli K-12 16S rRNAs. Helices (H) are numbered as described by Cannone et al. (8). Positions indicated in blue are heterogeneous in one species, and those in red are heterogeneous in two or more species.

FIG. 2.

Evolutionary rates and intragenomic heterogeneity at specific sites across the lengths of the bacterial RpoB protein and the 16S rRNA gene. (A) Across-site evolutionary rate variation for the amino acid translation of the rpoB gene (gray line). Sites that are slowly evolving or invariable have low rate categories, and quickly evolving sites have high rate categories. The black bar above the graph illustrates the fragment targeted in DGGE analysis (11). (B) Across-site evolutionary rate variation for the 16S rRNA gene (gray line) and mapping of the number of species displaying intragenomic heterogeneity at all sites across the length of this gene (black line). Bars above the graph illustrate fragments of the 16S rRNA gene targeted for clone libraries (gray bar), T-RFLP analysis (dashed bar indicating variable length), and DGGE (black bar) (34).

FIG. 3.

Comparison of the best maximum likelihood trees for the 16S rRNA gene and the RpoB protein for the domain Bacteria.

FIG. 4.

Evaluation of 16S rRNA and rpoB genes as phylogenetic markers at the subspecies level. (A) Best maximum likelihood tree for the 16S rRNA genes of Escherichia coli and Shigella flexneri strains (all seven copies from each strain are included). (B) Random sampling analysis of 16S rRNA paralogous gene copies from multiple E. coli and S. flexneri strains. One thousand trees were constructed by randomly choosing one 16S rRNA gene copy from each strain for every tree. Values above the nodes represent the numbers of trees in which the given nodes were recovered. (C) Best maximum likelihood tree for the 16S rRNA genes of E. coli and S. flexneri strains for which positions displaying intragenomic heterogeneity were recoded as ambiguous. (D) Best maximum likelihood tree for the rpoB genes of E. coli and S. flexneri strains.