16S Ribosomal RNA
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16 S ribosomal RNA (or 16 S
rRNA Ribosomal ribonucleic acid (rRNA) is a type of non-coding RNA which is the primary component of ribosomes, essential to all cells. rRNA is a ribozyme which carries out protein synthesis in ribosomes. Ribosomal RNA is transcribed from ribosoma ...
) is the RNA component of the
30S The prokaryotic small ribosomal subunit, or 30 S subunit, is the smaller subunit of the 70S ribosome found in prokaryotes. It is a complex of the 16S ribosomal RNA (rRNA) and 19 proteins. This complex is implicated in the binding of transfer ...
subunit of a
prokaryotic A prokaryote () is a single-celled organism that lacks a nucleus and other membrane-bound organelles. The word ''prokaryote'' comes from the Greek πρό (, 'before') and κάρυον (, 'nut' or 'kernel').Campbell, N. "Biology:Concepts & Connec ...
ribosome Ribosomes ( ) are macromolecular machines, found within all cells, that perform biological protein synthesis (mRNA translation). Ribosomes link amino acids together in the order specified by the codons of messenger RNA (mRNA) molecules to ...
(
SSU rRNA Small subunit ribosomal ribonucleic acid (SSU rRNA) is the smaller of the two major RNA components of the ribosome. Associated with a number of ribosomal proteins, the SSU rRNA forms the small subunit of the ribosome. It is encoded by SSU- rDNA. ...
). It binds to the Shine-Dalgarno sequence and provides most of the SSU structure. The genes coding for it are referred to as 16S rRNA gene and are used in reconstructing
phylogenies A phylogenetic tree (also phylogeny or evolutionary tree Felsenstein J. (2004). ''Inferring Phylogenies'' Sinauer Associates: Sunderland, MA.) is a branching diagram or a tree showing the evolutionary relationships among various biological spec ...
, due to the slow rates of
evolution Evolution is change in the heritable characteristics of biological populations over successive generations. These characteristics are the expressions of genes, which are passed on from parent to offspring during reproduction. Variation ...
of this region of the gene.
Carl Woese Carl Richard Woese (; July 15, 1928 – December 30, 2012) was an American microbiologist and biophysicist. Woese is famous for defining the Archaea (a new domain of life) in 1977 through a pioneering phylogenetic taxonomy of 16S ribosomal RNA, ...
and
George E. Fox George Edward Fox (born December 17, 1945) is an astrobiologist, a Professor Emeritus and researcher at the University of Houston. He is an elected fellow of the American Academy of Microbiology, the American Association for the Advancement of Sc ...
were two of the people who pioneered the use of 16S rRNA in phylogenetics in 1977. Multiple sequences of the 16S rRNA gene can exist within a single
bacterium Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were among ...
.


Functions

* Like the large (23S) ribosomal RNA, it has a structural role, acting as a scaffold defining the positions of the
ribosomal protein A ribosomal protein (r-protein or rProtein) is any of the proteins that, in conjunction with rRNA, make up the ribosomal subunits involved in the cellular process of translation. ''E. coli'', other bacteria and Archaea have a 30S small subunit an ...
s. * The 3-end contains the anti- Shine-Dalgarno sequence, which binds upstream to the AUG
start codon The start codon is the first codon of a messenger RNA (mRNA) transcript translated by a ribosome. The start codon always codes for methionine in eukaryotes and Archaea and a N-formylmethionine (fMet) in bacteria, mitochondria and plastids. The ...
on the
mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein. mRNA is ...
. The 3-end of 16S RNA binds to the proteins S1 and S21 which are known to be involved in initiation of
protein synthesis Protein biosynthesis (or protein synthesis) is a core biological process, occurring inside Cell (biology), cells, homeostasis, balancing the loss of cellular proteins (via Proteolysis, degradation or Protein targeting, export) through the product ...
* Interacts with 23S, aiding in the binding of the two ribosomal subunits ( 50S and
30S The prokaryotic small ribosomal subunit, or 30 S subunit, is the smaller subunit of the 70S ribosome found in prokaryotes. It is a complex of the 16S ribosomal RNA (rRNA) and 19 proteins. This complex is implicated in the binding of transfer ...
) * Stabilizes correct codon-anticodon pairing in the
A-site The A-site (A for aminoacyl) of a ribosome is a binding site for charged t-RNA molecules during protein synthesis. One of three such binding sites, the A-site is the first location the t-RNA binds during the protein synthesis process, the other ...
by forming a
hydrogen bond In chemistry, a hydrogen bond (or H-bond) is a primarily electrostatic force of attraction between a hydrogen (H) atom which is covalently bound to a more electronegative "donor" atom or group (Dn), and another electronegative atom bearing a ...
between the N1 atom of
adenine Adenine () ( symbol A or Ade) is a nucleobase (a purine derivative). It is one of the four nucleobases in the nucleic acid of DNA that are represented by the letters G–C–A–T. The three others are guanine, cytosine and thymine. Its derivati ...
residues 1492 and 1493 and the 2OH group of the mRNA backbone.


Structure


Universal primers

The 16S rRNA gene is used for
phylogenetic In biology, phylogenetics (; from Greek φυλή/ φῦλον [] "tribe, clan, race", and wikt:γενετικός, γενετικός [] "origin, source, birth") is the study of the evolutionary history and relationships among or within groups o ...
studies as it is highly conserved between different species of bacteria and archaea.
Carl Woese Carl Richard Woese (; July 15, 1928 – December 30, 2012) was an American microbiologist and biophysicist. Woese is famous for defining the Archaea (a new domain of life) in 1977 through a pioneering phylogenetic taxonomy of 16S ribosomal RNA, ...
pioneered this use of 16S rRNA in 1977. It is suggested that 16S rRNA gene can be used as a reliable
molecular clock The molecular clock is a figurative term for a technique that uses the mutation rate of biomolecules to deduce the time in prehistory when two or more life forms diverged. The biomolecular data used for such calculations are usually nucleoti ...
because 16S rRNA sequences from distantly related bacterial lineages are shown to have similar functionalities. Some
thermophilic A thermophile is an organism—a type of extremophile—that thrives at relatively high temperatures, between . Many thermophiles are archaea, though they can be bacteria or fungi. Thermophilic eubacteria are suggested to have been among the earl ...
archaea Archaea ( ; singular archaeon ) is a domain of single-celled organisms. These microorganisms lack cell nuclei and are therefore prokaryotes. Archaea were initially classified as bacteria, receiving the name archaebacteria (in the Archaebac ...
(e.g. order
Thermoproteales In taxonomy Taxonomy is the practice and science of categorization or classification. A taxonomy (or taxonomical classification) is a scheme of classification, especially a hierarchical classification, in which things are organized into g ...
) contain 16S rRNA gene
intron An intron is any nucleotide sequence within a gene that is not expressed or operative in the final RNA product. The word ''intron'' is derived from the term ''intragenic region'', i.e. a region inside a gene."The notion of the cistron .e., gene. ...
s that are located in highly conserved regions and can impact the annealing of "universal" primers. Mitochondrial and
chloroplast A chloroplast () is a type of membrane-bound organelle known as a plastid that conducts photosynthesis mostly in plant and algal cells. The photosynthetic pigment chlorophyll captures the energy from sunlight, converts it, and stores it in ...
ic rRNA are also amplified. The most common primer pair was devised by Weisburg ''et al.'' (1991) and is currently referred to as 27F and 1492R; however, for some applications shorter
amplicon In molecular biology, an amplicon is a piece of DNA or RNA that is the source and/or product of amplification (molecular biology), amplification or DNA replication, replication events. It can be formed artificially, using various methods including ...
s may be necessary, for example for 454 sequencing with titanium chemistry the primer pair 27F-534R covering V1 to V3. Often 8F is used rather than 27F. The two primers are almost identical, but 27F has an M instead of a C. AGAGTTTGATCMTGGCTCAG compared with 8F.


PCR and NGS applications

In addition to highly conserved primer binding sites, 16S rRNA gene sequences contain
hypervariable region A hypervariable region (HVR) is a location within nuclear DNA or the D-loop of mitochondrial DNA in which base pairs of nucleotides repeat (in the case of nuclear DNA) or have substitutions (in the case of mitochondrial DNA). Changes or repeats in ...
s that can provide species-specific signature sequences useful for identification of bacteria. As a result, 16S rRNA gene sequencing has become prevalent in
medical microbiology Medical microbiology, the large subset of microbiology that is applied to medicine, is a branch of medical science concerned with the prevention, diagnosis and treatment of infectious diseases. In addition, this field of science studies various ...
as a rapid and cheap alternative to
phenotypic In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology or physical form and structure, its developmental processes, its biochemical and physiological proper ...
methods of bacterial identification. Although it was originally used to identify bacteria, 16S sequencing was subsequently found to be capable of reclassifying bacteria into completely new
species In biology, a species is the basic unit of classification and a taxonomic rank of an organism, as well as a unit of biodiversity. A species is often defined as the largest group of organisms in which any two individuals of the appropriate s ...
, or even
genera Genus ( plural genera ) is a taxonomic rank used in the biological classification of living and fossil organisms as well as viruses. In the hierarchy of biological classification, genus comes above species and below family. In binomial nomenclat ...
. It has also been used to describe new species that have never been successfully cultured. With
third-generation sequencing Third-generation sequencing (also known as long-read sequencing) is a class of DNA sequencing methods currently under active development. Third generation sequencing technologies have the capability to produce substantially longer reads than secon ...
coming to many labs, simultaneous identification of thousands of 16S rRNA sequences is possible within hours, allowing
metagenomic Metagenomics is the study of genetic material recovered directly from environmental or clinical samples by a method called sequencing. The broad field may also be referred to as environmental genomics, ecogenomics, community genomics or microb ...
studies, for example of gut flora.


Hypervariable regions

The bacterial 16S gene contains nine hypervariable regions (V1–V9), ranging from about 30 to 100
base pair A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both DNA ...
s long, that are involved in the secondary structure of the small ribosomal subunit. The degree of conservation varies widely between hypervariable regions, with more conserved regions correlating to higher-level taxonomy and less conserved regions to lower levels, such as genus and species. While the entire 16S sequence allows for comparison of all hypervariable regions, at approximately 1,500 base pairs long it can be prohibitively expensive for studies seeking to identify or characterize diverse bacterial communities. These studies commonly utilize the Illumina platform, which produces reads at rates 50-fold and 12,000-fold less expensive than 454
pyrosequencing Pyrosequencing is a method of DNA sequencing (determining the order of nucleotides in DNA) based on the "sequencing by synthesis" principle, in which the sequencing is performed by detecting the nucleotide incorporated by a DNA polymerase. Pyrosequ ...
and
Sanger sequencing Sanger sequencing is a method of DNA sequencing that involves electrophoresis and is based on the random incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. After first being developed by Frederi ...
, respectively. While cheaper and allowing for deeper community coverage, Illumina sequencing only produces reads 75–250 base pairs long (up to 300 base pairs with Illumina MiSeq), and has no established protocol for reliably assembling the full gene in community samples. Full hypervariable regions can be assembled from a single Illumina run, however, making them ideal targets for the platform. While 16S hypervariable regions can vary dramatically between bacteria, the 16S gene as a whole maintains greater length homogeneity than its eukaryotic counterpart ( 18S ribosomal RNA), which can make alignments easier. Additionally, the 16S gene contains highly conserved sequences between hypervariable regions, enabling the design of universal primers that can reliably produce the same sections of the 16S sequence across different
taxa In biology, a taxon (back-formation from ''taxonomy''; plural taxa) is a group of one or more populations of an organism or organisms seen by taxonomists to form a unit. Although neither is required, a taxon is usually known by a particular nam ...
. Although no hypervariable region can accurately classify all bacteria from
domain Domain may refer to: Mathematics *Domain of a function, the set of input values for which the (total) function is defined **Domain of definition of a partial function **Natural domain of a partial function **Domain of holomorphy of a function * Do ...
to species, some can reliably predict specific taxonomic levels. Many community studies select semi-conserved hypervariable regions like the V4 for this reason, as it can provide resolution at the phylum level as accurately as the full 16S gene. While lesser-conserved regions struggle to classify new species when higher order taxonomy is unknown, they are often used to detect the presence of specific pathogens. In one study by Chakravorty et al. in 2007, the authors characterized the V1–V8 regions of a variety of pathogens in order to determine which hypervariable regions would be most useful to include for disease-specific and broad assays. Amongst other findings, they noted that the V3 region was best at identifying the genus for all pathogens tested, and that V6 was the most accurate at differentiating species between all CDC-watched pathogens tested, including anthrax. While 16S hypervariable region analysis is a powerful tool for bacterial taxonomic studies, it struggles to differentiate between closely related species. In the families '' Enterobacteriaceae'', ''
Clostridiaceae The Clostridiaceae are a family of the bacterial class Clostridia, and contain the genus '' Clostridium''. The family Clostridiaceae (scientific name) defined by the taxonomic outline of '' Bergey's Manual of Systematic Bacteriology '' contains ...
'', and ''
Peptostreptococcaceae The Peptostreptococcaceae are a family of Gram-positive bacteria in the class Clostridia. Several members of the Peptostreptococcaceae are well known inhabitants of the digestive tract. Microbiome studies of animal feces have corroborated this ...
'', species can share up to 99% sequence similarity across the full 16S gene. As a result, the V4 sequences can differ by only a few
nucleotide Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecules wi ...
s, leaving reference databases unable to reliably classify these bacteria at lower taxonomic levels. By limiting 16S analysis to select hypervariable regions, these studies can fail to observe differences in closely related taxa and group them into single taxonomic units, therefore underestimating the total diversity of the sample. Furthermore, bacterial genomes can house multiple 16S genes, with the V1, V2, and V6 regions containing the greatest intraspecies diversity. While not the most precise method of classifying bacterial species, analysis of the hypervariable regions remains one of the most useful tools available to bacterial community studies.


Promiscuity of 16S rRNA genes

Under the assumption that evolution is driven by vertical transmission, 16S rRNA genes have long been believed to be species-specific, and infallible as genetic markers inferring phylogenetic relationships among
prokaryotes A prokaryote () is a single-celled organism that lacks a nucleus and other membrane-bound organelles. The word ''prokaryote'' comes from the Greek πρό (, 'before') and κάρυον (, 'nut' or 'kernel').Campbell, N. "Biology:Concepts & Connec ...
. However, a growing number of observations suggest the occurrence of horizontal transfer of these genes. In addition to observations of natural occurrence, transferability of these genes is supported experimentally using a specialized ''
Escherichia coli ''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escher ...
'' genetic system. Using a null mutant of ''E. coli'' as host, growth of the mutant strain was shown to be complemented by foreign 16S rRNA genes that were phylogenetically distinct from ''E. coli'' at the phylum level. Such functional compatibility was also seen in ''
Thermus thermophilus ''Thermus thermophilus'' is a Gram-negative bacterium used in a range of biotechnological applications, including as a model organism for genetic manipulation, structural genomics, and systems biology. The bacterium is extremely thermophilic, w ...
''. Furthermore, in ''T. thermophilus'', both complete and partial gene transfer was observed. Partial transfer resulted in spontaneous generation of apparently random
chimera Chimera, Chimaera, or Chimaira (Greek for " she-goat") originally referred to: * Chimera (mythology), a fire-breathing monster of Ancient Lycia said to combine parts from multiple animals * Mount Chimaera, a fire-spewing region of Lycia or Cilici ...
between host and foreign bacterial genes. Thus, 16S rRNA genes may have evolved through multiple mechanisms, including vertical inheritance and
horizontal gene transfer Horizontal gene transfer (HGT) or lateral gene transfer (LGT) is the movement of genetic material between Unicellular organism, unicellular and/or multicellular organisms other than by the ("vertical") transmission of DNA from parent to offsprin ...
; the frequency of the latter may be much higher than previously thought.


16S ribosomal databases

The 16S rRNA gene is used as the standard for classification and identification of microbes, because it is present in most microbes and shows proper changes. Type strains of 16S rRNA gene sequences for most bacteria and archaea are available on public databases, such as
NCBI The National Center for Biotechnology Information (NCBI) is part of the United States National Library of Medicine (NLM), a branch of the National Institutes of Health (NIH). It is approved and funded by the government of the United States. The ...
. However, the quality of the sequences found on these databases is often not validated. Therefore, secondary databases that collect only 16S rRNA sequences are widely used. The most frequently used databases are listed below:


EzBioCloud

EzBioCloud database, formerly known as EzTaxon, consists of a complete hierarchical taxonomic system containing 62,988 bacteria and archaea species/phylotypes which includes 15,290 valid published names as of September 2018. Based on the phylogenetic relationship such as maximum-likelihood and OrthoANI, all species/subspecies are represented by at least one 16S rRNA gene sequence. The EzBioCloud database is systematically curated and updated regularly which also includes novel candidate species. Moreover, the website provides bioinformatics tools such as ANI calculator, ContEst16S and 16S rRNA DB for QIIME and Mothur pipeline.


Ribosomal Database Project

The Ribosomal Database Project (RDP) is a curated database that offers ribosome data along with related programs and services. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams and various software packages for handling, analyzing and displaying alignments and trees. The data are available via ftp and electronic mail. Certain analytic services are also provided by the electronic mail server. Due to its large size the RDP database is often used as the basis for bioinformatic tool development and creating manually curated databases.


SILVA

SILVA Silva is a surname in Portuguese language, Portuguese-speaking countries, such as Portugal and Brazil. It is derived from the Latin word , meaning "forest" or "woodland". It is the family name of the House of Silva. The name is also widespread i ...
provides comprehensive, quality checked and regularly updated datasets of aligned small (16S/18S, SSU) and large subunit (23S/28S, LSU) ribosomal RNA (rRNA) sequences for all three domains of life as well as a suite of search, primer-design and alignment tools (Bacteria, Archaea and Eukarya).


GreenGenes

GreenGenes is a quality controlled, comprehensive 16S rRNA gene reference database and taxonomy based on a ''de novo'' phylogeny that provides standard operational taxonomic unit sets. Beware that it utilizes taxonomic terms proposed from phylogenetic methods applied years ago between 2012 and 2013. Since then, a variety of novel phylogenetic methods have been proposed for Archaea and Bacteria.


References


External links


University of Washington Laboratory Medicine: Molecular Diagnosis , Bacterial Sequencing

The Ribosomal Database Project



SILVA rRNA database

Greengenes: 16S rDNA data and tools

EzBioCloud
{{DEFAULTSORT:16s Ribosomal Rna Ribosomal RNA Metagenomics