It is a short presentation that I prepared about the 16s rRNA gene sequencing in bacterial identification as a part of my assignment in college.

1. 16S rRNA Sequencing for
Bacterial Identification
Sanam Parajuli
M.Tech. in Biotechnology
Kathmandu University

2. RNA
• Ribonucleic Acid
• Single stranded Nucleic Acid, except double stranded RNA (dsRNA) in some viruses (eg:
Rotaviruses)
• Base pairs: Guanine, Cytosine, Adenine, Uracil
• Types: Messenger RNA(mRNA), transfer RNA(tRNA) and, Ribosomal RNA(rRNA)

3. Different types
of RNA in
action
Image Source: Wikipedia

4. Lets Talk Ribosomes!
• Ribosomes: Complex
macromolecules; site for
biological protein synthesis in all
living cells
• Link amino acids together in the
order specified by mRNA
molecules
• Made of rRNA and dozens of
distinct proteins
• Two subunits: large and small
Image source: Wikipedia

5. Prokaryotic ribosomes!
• Made of 65% rRNA and 35% ribosomal proteins (Kurland, 1960)
• 70S with 50S + 30S subunits
• The two subunits fit together during translation

6. Ribosome Composition of E. coli (Garrett and Grisham, 2009)
Ribosome Subunit rRNAs r-proteins
70S
50S 23S (2904
nucleotides)
31
5S (120
nucleotides)
30S 16S (1542
nucleotides)
21

7. What is the deal with 16S rRNA?
• A component of the 30S subunit of the prokaryotic ribosome
• Binds to the Shine-Dalgarno sequence: ribosomal binding site on the mRNA generally
located around 8 bases upstream of the start codon AUG; consensus sequence of the SD
sequence : AGGAGG
• 3’ end of 16S rRNA binds to S1 and S21 genes needed for initiation of translation
• Genes coding for 16S rRNA known as 16S rRNA gene, used for constructing phylogenies
because rate of evolution in this gene really slow (Woese & Fox, 1977)
• Carl Woese and George E. Fox pioneered the use of 16S rRNA in phylogenetics in 1977

8. Image Source: Wikipedia

9. Significance of the 16S rRNA gene in phylogenetics
• Because of very slow evolution, the gene highly conserved between bacteria and archaea
(Coenye & Vandamme, 2003)
• 16S rRNA gene: a reliable molecular clock, 16S rRNA sequences from distantly related
bacterial lineages show similar functionality
• Multiple sequences of the 16S rRNA gene can exist within a single bacterium
• Since highly conserved, universal primers able to anneal to these sequences during
Polymerase Chain Reaction (PCR), facilitates sequencing a lot
• **In order Thermoproteales, multiple introns present in the 16S rRNA gene that can
interfere with annealing of universal primers

10. Commonly Used
Universal Primers
in 16S rRNA gene
sequencing

11. Hypervariable Regions
• Incidentally, both conserved and non-conserved sequences present in the 16S rRNA gene
• Hypervariable Regions (HVRs): the non-conserved portion, i.e., polymorphism among
organisms of various taxa
• Total of 9 such regions present in the 16S rRNA gene (V1 – V9)
• A HVR: 30-100 bp long
• Significance: involved in secondary structure of the small ribosomal unit

12. • HVRs contain species specific signature sequences
• Despite the presence of HVRs, 16S rRNA gene still has greater length
homogeneity than its eukaryotic counterpart, the 18S rRNA
• Not all HVR have same level of variability, different HVR provide resolution to
different taxonomic levels
• For eg: V4: semi conserved: mostly used for resolutions upto the phylum level
• Also, Chakravorty et al. in 2007: characterization of V1-V8 regions of
pathogens;
• Result: V3: genus level differentiation; V6: species level differentiation
• Universal Primers Anneal to the Conserved Regions
Image Source: researchgate.net

13. Image Source: researchgate.net

14. How is this all done?
• DNA Isolation from the bacteria/archaea
• PCR Amplification of the 16S rRNA gene or/and targeted HVR
with Universal Primers
• Gene Sequencing of PCR Products (Illumina, Sanger,
Pyrosequencing)
• Analysis and Identification of species using bioinformatic tools

15. 16S rRNA Gene and the Taxonomic Revolution
• Use of 16S rRNA genes for taxonomic studies: 1980s
• With advent of automatic sequencing, development of sequence databases like
GenBank, Greengenes, SILVA, EzTaxon, and comparison of sequence of isolate using
BLAST
• Published bacterial names, 1,800 in 1980 to 12,500 in 2013 (Parte, 2014)
• Many taxa reclassified and greater number created
• Cutoff % (sequence similarity): 95% for genus and 98.7% for species (Stackebrandt &
Ebers, 2006)
• However, like Edwardsiella (99.3% and 99.8%) (Janda and Abbott, 2007), and
Streptomyces and Chlorobium (78% and 86.1%) (Alexander et al., 2002), and many other
species may not follow the same cutoff %

16. Limitations
• Sequence similarity may result from horizontal gene transfer, may lead to identification
errors
• As mentioned earlier, not all bacteria and bacterial families follow similar cutoff % for
sequence similarity
Example: Enterobacteriaceae, Clostridiaceae, and Peptostreptococcaceae: species share
upto 99% 16S rRNA gene sequence similarity, only few nucleotides differ, hence, species-
level classification not precise only on the basis of select HVRs. Thus, care required while
adopting this method for nomenclature and classification

17. References
Garrett, R., & Grisham, C. (2009). Biochemistry (4th ed.). Cengage Learning Services.
Kurland, C. G. (1960). Molecular characterization of ribonucleic acid from Escherichia coli ribosomes: I. Isolation and molecular
weights. Journal of Molecular Biology, 2(2), 83-91.
Woese, C. R., & Fox, G. E. (1977). Phylogenetic structure of the prokaryotic domain: the primary kingdoms. Proceedings of the
National Academy of Sciences, 74(11), 5088-5090.
Coenye, T., & Vandamme, P. (2003). Intragenomic heterogeneity between multiple 16S ribosomal RNA operons in sequenced
bacterial genomes. FEMS Microbiology Letters, 228(1), 45-49.
Stackebrandt, E. (2006). Taxonomic parameters revisited: tarnished gold standards. Microbiol. Today, 33, 152-155.
Alexander, B., Andersen, J. H., Cox, R. P., & Imhoff, J. F. (2002). Phylogeny of green sulfur bacteria on the basis of gene
sequences of 16S rRNA and of the Fenna-Matthews-Olson protein. Archives of microbiology, 178(2), 131-140.
Chakravorty, S., Helb, D., Burday, M., Connell, N., & Alland, D. (2007). A detailed analysis of 16S ribosomal RNA gene segments
for the diagnosis of pathogenic bacteria. Journal of microbiological methods, 69(2), 330-339.