How conserved are the conserved 16S-rRNA regions?

Area of Science:

• Microbiology
• Molecular Biology
• Bioinformatics

Background:

• The 16S rRNA gene is widely used to study prokaryotic diversity across various environments.
• Existing 'universal' primers for the 16S rRNA gene have limitations, suggesting conserved regions may not be as conserved as assumed.

Purpose of the Study:

• To evaluate the actual conservation of regions flanking the hypervariable regions of the 16S rRNA gene.
• To assess the reliability of commonly used primer binding sites for prokaryotic diversity studies.

Main Methods:

• Utilized sequences from the SILVA database (release 123).
• Assembled primer matches into contigs and extracted 12-nucleotide fragments (12-mers).
• Performed frequency analysis of 12-mers across the entire SILVA database to identify conserved and variable segments.

Main Results:

• Identified significant variability within the so-called conserved regions, with some segments showing conservation below 90%.
• Extreme primer regions (1 and 10) exhibited the lowest frequencies, indicating poor conservation.
• Specific primer contigs (3, 4, 5b, 6a) were found in all analyzed sequences, but nucleotide frequency analysis revealed limited true conservation.

Conclusions:

• The conserved regions of the 16S rRNA gene display considerable variation.
• This variability must be accounted for when using the 16S rRNA gene as a biomarker for prokaryotic diversity.
• The findings challenge the universality of current 16S rRNA gene primers.