Microscopic life in the soil, water, and the human body cannot be distinguished by sight alone. To identify these microbes, scientists use molecular tools to differentiate species and understand their roles in health, disease, and the environment. Genetic analysis methods have been developed to provide a clear identity for these organisms.
Ribosomal RNA as a Genetic Fingerprint
Ribosomes are complex structures in every living cell responsible for synthesizing proteins. Because these structures are fundamental to all life, the genes that code for ribosomal RNA (rRNA), a main component of ribosomes, provide a unique opportunity for universal identification.
rRNA genes are effective identification markers because they contain both “conserved” and “hypervariable” regions. The conserved sections change very slowly over evolutionary time and are similar across broad categories of life, such as all bacteria. These stable sequences act as anchor points for scientific analysis.
In contrast, the hypervariable regions evolve much more rapidly, accumulating mutations that lead to significant sequence differences even between closely related species. This dual nature of conserved and variable regions allows the rRNA gene to function as a genetic fingerprint.
Researchers design tools called primers that bind to the conserved regions, which allows them to isolate and copy the rRNA gene from any organism in a sample. Sequencing the hypervariable regions then provides the specific signature needed to distinguish between different microbial species.
16S rRNA for Prokaryotic Identification
The 16S rRNA gene is the marker for identifying prokaryotes, a group that includes bacteria and archaea. This gene is a component of the small subunit (30S) of the prokaryotic ribosome. Its sequence is about 1,500 base pairs long and contains nine hypervariable regions (V1-V9) that serve as the primary targets for distinguishing between species.
Sequencing the entire 16S gene is not always necessary or cost-effective. Researchers target specific hypervariable regions that offer sufficient information for their research question. For example, the V3-V4 region is amplified in studies of the human gut microbiome, as it provides good resolution for identifying the bacteria present.
In human health, 16S rRNA sequencing is used to catalog bacteria in the gut, linking shifts in this microbial community to conditions like inflammatory bowel disease and obesity. In environmental studies, scientists use it to assess bacterial diversity in soil to improve agriculture or to monitor water quality by detecting harmful bacteria.
This method allows for a comprehensive census of the prokaryotic world without the need to culture each organism in a lab, a process that is impossible for a majority of bacterial species. Analyzing 16S rRNA sequences helps researchers gain an understanding of the composition and potential function of entire microbial ecosystems.
18S rRNA for Eukaryotic Identification
For identifying eukaryotes—organisms with a cell nucleus like fungi, protists, and microscopic animals—scientists use the 18S rRNA gene. This gene is the counterpart to 16S rRNA and is located in the small subunit of eukaryotic ribosomes. It serves as a genetic barcode for classifying organisms that are not bacteria or archaea.
The 18S rRNA gene is larger and less variable than the 16S gene. This lower rate of evolution can make it more challenging to differentiate between very closely related species. Its stability makes it a good tool for identifying organisms at broader taxonomic levels, such as the family or genus.
The use of 18S rRNA sequencing is prominent in mycology and parasitology. Agricultural scientists sequence the 18S gene from soil to understand fungal diversity and its impact on crop health. Public health researchers analyze water for the 18S rRNA of parasitic protists, like Giardia, to monitor for waterborne disease.
18S rRNA sequencing provides a view into the diversity of microscopic eukaryotes. It helps complete the picture of microbial communities by accounting for the non-prokaryotic members, offering insights into their ecological roles and interactions with other forms of life.
A Comparative Guide for Selection
Choosing between 16S and 18S rRNA sequencing depends on the organism of interest and the study’s goals. The primary distinction lies in their targets: 16S rRNA is the standard for identifying prokaryotes (bacteria and archaea), while 18S rRNA is used for eukaryotes (fungi, protists, and small animals). This targeting difference is the main guide for selection.
The gene’s structure also informs the choice. The 16S rRNA gene has a higher degree of variability, making it effective for distinguishing between different species of bacteria. In contrast, the more conserved nature of the 18S rRNA gene is better suited for classifying eukaryotes at the genus or family level.
The history of research has influenced the resources available for each marker. Due to the focus on bacteria in medical research, databases for 16S rRNA sequences, such as Greengenes and SILVA, are more extensive than those for 18S rRNA. In many ecosystem-level studies, researchers may use both markers to capture a complete picture of the microbial diversity present.