Microbial Community Analysis Using rRNA Techniques

Ribosomal RNA (rRNA) techniques underpin current investigations into the composition, function and dynamics of microbial communities across diverse ecosystems. By targeting conserved regions of the small subunit (SSU) rRNA genes or the rRNA molecules themselves, these approaches circumvent the limitations of culture-based methods and enable high-resolution taxonomic profiling from environmental or clinical samples. Polymerase chain reaction (PCR) amplification of 16S (bacterial and archaeal) and 18S (eukaryotic) rRNA genes, followed by separation techniques such as denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP) or single-strand conformation polymorphism (SSCP), reveals operational taxonomic units (OTUs) that serve as proxies for diversity. Next-generation sequencing of PCR amplicons or direct sequencing of reverse-transcribed SSU rRNA captures broader diversity, including rare or primer-mismatched taxa. Complementary methods such as fluorescence in situ hybridisation (FISH) provide spatial resolution and validate molecular inferences by visualising specific taxa within their native context. Together, these molecular rRNA-based techniques afford a comprehensive view of microbial biodiversity, ecological interactions and functional potential, with applications ranging from bioremediation and agricultural soil management to human and animal health.

Research from Nature Portfolio

A cultivation-independent study of polychlorinated biphenyls (PCBs)-contaminated soils employed SSCP analysis of 16S rRNA genes to monitor bacterial community succession during bioremediation. The single-strand conformation profiles revealed increasing community complexity, from initial low-diversity assemblages to richer phylum-level representation over time. PCR re-amplification and cloning of 51 distinct SSCP products identified members of Acidobacteria, Actinobacteria and Proteobacteria, while pure culture isolation yielded novel PCB-degrading strains bearing 2,3-dihydroxybiphenyl dioxygenase as a molecular marker. These isolates demonstrated the capacity to utilise PCBs as sole carbon sources, illustrating how rRNA-guided community profiling can guide the discovery and monitoring of functional degrader taxa and enhance bioremediation strategies.

Research from all publishers

A theoretical-to-actual delta approach to 16S rDNA PCR compared in silico mock communities with laboratory mock extractions, uncovering severe losses (2.3 to 8.2-fold) in detectable OTUs due to template type, primer bias and environmental DNA interference. This work emphasised the need for careful primer selection and validated fingerprinting protocols to preserve true community diversity in molecular surveys.

Direct sequencing of reverse-transcribed SSU rRNA molecules from the canine oral microbiome bypassed PCR amplification biases and detected one additional phylum, multiple novel genera and significant archaeal and eukaryotic RNA pools compared to conventional 16S amplicons. Implementation of an open-source pipeline for taxonomic classification strengthened the case for direct rRNA sequencing as a higher-resolution tool in microbial ecology.

Hybridisation chain reaction FISH protocols have been refined to visualise fungal endobacteria within hyphae, overcoming permeability and probe abundance challenges. By optimising permeabilisation steps, culture media selection and probe design targeting 16S rRNA sequences, researchers achieved high-resolution imaging of bacterial-fungal interactions, complementing amplicon-based surveys with spatial context and confirming specific symbiotic associations.

Microbial Community Analysis Using rRNA Techniques publication trend

The graph below shows the total number of articles in microbial community analysis using rrna techniques across all publications each year (not limited to Nature Index journals).

Technical terms

SSU rRNA gene: Gene encoding the small subunit ribosomal RNA, widely used as a phylogenetic marker for prokaryotes or eukaryotes.

Operational taxonomic unit (OTU): A cluster of similar rRNA sequences used as a proxy for microbial species or taxa in community analyses.

Single-strand conformation polymorphism (SSCP): A gel-based method that separates single-stranded PCR amplicons by conformational differences to profile sequence variants.

Reverse-transcribed SSU rRNA (RT-SSU): A method in which rRNA molecules are converted into complementary DNA for direct sequencing, reducing primer bias.

Fluorescence in situ hybridisation (FISH): A microscopy technique that employs fluorescently labelled probes to visualise specific rRNA sequences within intact cells or tissues.

References

1. A 16S rDNA PCR-based theoretical to actual delta approach on culturable mock communities revealed severe losses of diversity information. BMC Microbiology (2019).
2. Characterising the Canine Oral Microbiome by Direct Sequencing of Reverse-Transcribed rRNA Molecules. PLOS ONE (2016).
3. Biotechnology methods for succession of bacterial communities in polychlorinated biphenyls (PCBs) contaminated soils and isolation novel PCBs-degrading bacteria. Scientific Reports (2022).
4. Advances and Challenges in Fluorescence in situ Hybridization for Visualizing Fungal Endobacteria. Frontiers in Microbiology (2022).