Bacteria are the most ubiquitous life form on the earth. The biomass of bacteria exceeds that of plants or animals. Due to their abundance, most of the bacterial species have not been identified so far. The traditional identification of bacteria is based on the phenotypic characteristics, which are not accurate as genotypic methods. The comparison of the 16S rRNA sequence has emerged as a most preferred genotypic method for the identification of bacteria in their genus level. There are several reasons to use 16S rRNA as a housekeeping genetic maker, which will be explained further in detail.
Key Areas Covered
1. What is 16S rRNA
– Definition, Structure, Role
2. Why is 16S rRNA Used to Identify Bacteria
– Introduction, Reasons, Methods
3. What are the Applications of the 16S rRNA in Microbiology
Key Terms: Bacteria, Classification, Gene Sequence, Identification, Ribosome, 16S rRNA
What is 16S rRNA
The 16S rRNA is a component of the small subunit of the prokaryotic ribosome. The two subunits of the prokaryotic ribosome are 50S large subunit and the 30S small subunit. They form 70S ribosome. The small subunit is composed of 16S rRNA bound to 21 proteins. The 16S rRNA is made up of 1540 nucleotides. The secondary structure of 16S rRNA is shown in figure 1.
The 3’end of the 16S rRNA contains the anti-Shine-Dalgarno sequence that binds upstream to the start codon, AUG. The Shine-Dalgarno sequence is the ribosomal binding site of the bacterial mRNA. As 16S rRNA is essential for the functioning of the bacteria, the gene that encodes the 16S rRNA is highly conserved among bacterial species. The sequence of the 16S rRNA is widely used in the identification and classification of bacteria.
Why is 16S rRNA Used to Identify Bacteria
The traditional identification methods of bacteria are mainly based on the phenotypic characteristics of bacteria. However, the comparison of the 16S rRNA sequence has become a ‘gold standard’, replacing the traditional methods of bacterial identification. The analysis of the 16S rRNA sequence is better for the identification of phenotypically aberrant, poorly described or rarely isolated strains. It is also better for the identification of non-cultured bacteria and novel pathogens. The 16S rRNA gene occurs in the rRNA operon in the bacterial genome. The rRNA operon is shown in figure 2.
16S rRNA is suitable to be used as a housekeeping genetic marker due to several reasons. They are described below.
- 16S rRNA gene is a ubiquitous gene in the bacterial genome. Since the 16S rRNA function is essential for the bacterial cell during translation, almost all the bacterial genomes are composed of the 16S rRNA gene.
- The sequence of the 16S rRNA gene is highly conserved. As the function of the 16S rRNA is more general, the sequence of the 16S rRNA gene is highly conserved. The changes in the gene sequence can be considered as a measurement of time (evolution).
- The size of the 16S rRNA gene (1, 550 bp) is sufficient for bioinformatics purposes.
- 16S rRNA gene is a well-studied gene in the bacterial genome. Since the function of the 16S rRNA gene is vital for the cell, it is subjected to many studies.
Up to date, over 8, 168 bacterial species have been identified with the use of 16S rRNA gene sequence. The procedure of the identification process is described below.
- Extraction of genomic DNA
- PCR amplification of the 16S rRNA gene
- Obtain the nucleotide sequence of the amplified 16S rRNA gene
- Compare the sequence with the existing nucleotide sequences in the databases
The 16S rRNA sequence is about 1, 550 base pairs long and is composed of both variable and conserved regions. The universal primers, which are complementary to the conserved region of the gene, can be used for the amplification of the variable region of the gene by PCR. Generally, 540 base pairs region from the beginning of the gene or the whole gene is amplified by PCR. The PCR fragment is sequenced, and the sequence is compared with the existing nucleotide sequences of the 16S rRNA gene for the identification of the pre-isolated bacterial species. GenBank, the largest repository of nucleotide sequences, has over 20 million sequences of 90, 000 different 16S rRNA genes. If the bacterial species is novel, the sequence will not match with any 16S rRNA sequence in the databases.
Since the 16S rRNA gene sequence is found in almost all the bacterial species, the comparison of different 16S rRNA gene sequences can be used to differentiate bacteria up to species and subspecies levels. Similar bacterial species may have similar sequences of 16S rRNA gene. A phylogenetic tree of bacteria constructed by comparing the 16S rRNA gene sequence is shown in figure 3.
What are the Applications of 16S rRNA in Microbiology
The applications of the 16S rRNA in microbiology are listed below.
- 16S rRNA gene sequencing is used as the “gold standard” for the identification and taxonomic classification of bacterial species.
- Comparison of the 16S rRNA sequence can be used for the recognition of novel pathogens.
- The 16S rRNA sequencing can be used as a rapid and cheap alternative to the phenotypic methods of bacterial identification in medical microbiology.
The 16S rRNA is vital for the functioning of the bacteria as it provides a site for the binding of bacterial mRNA to the ribosome during translation. Since the function of the 16SrRNA is essential for the cell, its gene sequence is present in almost all bacterial cells. Moreover, its sequence is highly conserved. However, the 16S rRNA sequence is composed of variable regions as well, allowing the identification of bacterial species. In addition, bacterial species can be classified based on the gene sequence of 16S rRNA.
1. Janda, J. Michael, and Sharon L. Abbott. “16S rRNA Gene Sequencing for Bacterial Identification in the Diagnostic Laboratory: Pluses, Perils, and Pitfalls.” Journal of Clinical Microbiology, American Society for Microbiology, Sept. 2007, .
2. Clarridge, Jill E. “Impact of 16S rRNA Gene Sequence Analysis for Identification of Bacteria on Clinical Microbiology and Infectious Diseases.” Clinical Microbiology Reviews, American Society for Microbiology, Oct. 2004, .
1. “16S” By Squidonius – Own work (Public Domain) via
2. “Amit Yadav Phytoplasma rRNA operon” via
3. “Phylogenetic position of Mollicutes among bacteria” By Kenro Oshima, Kensaku Maejima and Shigetou Namba – Front. Microbiol., 14 August 2013 / doi: 10.3389/fmicb.2013.00230 via