![Bildschirmfoto_2019-03-28_um_17](https://upload.wikimedia.org/wikipedia/commons/1/15/Bildschirmfoto_2019-03-28_um_17.23.07.png)
Microbial DNA barcoding is the use of DNA metabarcoding to characterize a mixture of
microorganism
A microorganism, or microbe,, ''mikros'', "small") and ''organism'' from the el, ὀργανισμός, ''organismós'', "organism"). It is usually written as a single word but is sometimes hyphenated (''micro-organism''), especially in olde ...
s.
DNA metabarcoding is a method of DNA barcoding that uses universal genetic markers to identify DNA of a mixture of organisms.
History
Using
metabarcoding
Metabarcoding is the barcoding of DNA/ RNA (or eDNA/ eRNA) in a manner that allows for the simultaneous identification of many taxa within the same sample. The main difference between barcoding and metabarcoding is that metabarcoding does ...
to assess
microbial communities Microbial population biology is the application of the principles of population biology to microorganisms.
Distinguishing from other biological disciplines
Microbial population biology, in practice, is the application of population ecology and popu ...
has a long history. Back in 1972,
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, ...
,
Mitchell Sogin Dr. Mitchell Sogin is a distinguished senior scientist at the Marine Biological Laboratory in Woods Hole, Massachusetts, whose research investigates the evolution and diversity of single-celled organisms.
Career
Dr. Sogin obtained a BS in Chemist ...
and Stephen Sogin first tried to detect several
families
Family (from la, familia) is a group of people related either by consanguinity (by recognized birth) or affinity (by marriage or other relationship). The purpose of the family is to maintain the well-being of its members and of society. Ideal ...
within
bacteria
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 ...
using the 5S
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 ...
gene. Only a few years later, a new
tree of life
The tree of life is a fundamental archetype in many of the world's mythological, religious, and philosophical traditions. It is closely related to the concept of the sacred tree.Giovino, Mariana (2007). ''The Assyrian Sacred Tree: A History ...
with three
domains was proposed by again Woese and colleagues, who were the first to use the
small subunit of the ribosomal RNA (SSU rRNA) gene to distinguish between bacteria,
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 ...
and
eukaryote
Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bacte ...
s. Out of this approach, the SSU rRNA gene made its way to be the most frequently used
genetic marker A genetic marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify individuals or species. It can be described as a variation (which may arise due to mutation or alteration in the genomic loci) that can be ...
for both
prokaryote
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 ...
s (16S rRNA) and eukaryotes (
18S rRNA). The tedious process of
cloning
Cloning is the process of producing individual organisms with identical or virtually identical DNA, either by natural or artificial means. In nature, some organisms produce clones through asexual reproduction. In the field of biotechnology, cl ...
those DNA fragments for
sequencing
In genetics and biochemistry, sequencing means to determine the primary structure (sometimes incorrectly called the primary sequence) of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succ ...
got fastened up by the steady improvement of sequencing technologies. With the development of
HTS (High-Throughput-Sequencing) in the early 2000s and the ability to deal with this massive data using modern bioinformatics and cluster algorithms, investigating microbial life got much easier.
Genetic markers
Genetic diversity is varying from species to species. Therefore, it is possible to identify distinct species by the recovery of a short DNA sequence from a standard part of the genome. This short sequence is defined as barcode sequence. Requirements for a specific part of the genome to serve as barcode should be a high variation between two different
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 ...
, but not much differences in the gene between two individuals of the same species to make differentiating individual species easier.
For both bacteria and archaea the 16S rRNA/rDNA gene is used. It is a common
housekeeping gene
In molecular biology, housekeeping genes are typically constitutive genes that are required for the maintenance of basic cellular function, and are expressed in all cells of an organism under normal and patho-physiological conditions. Although ...
in all prokaryotic organisms and therefore is used as a standard barcode to assess prokaryotic diversity. For protists, the corresponding 18S rRNA/rDNA gene is used. To distinguish different species of fungi, the ITS (
Internal Transcribed Spacer
Internal transcribed spacer (ITS) is the spacer DNA situated between the small-subunit ribosomal RNA (rRNA) and large-subunit rRNA genes in the chromosome or the corresponding transcribed region in the polycistronic rRNA precursor transcript.
I ...
) region of the ribosomal
cistron
A cistron is an alternative term for "gene". The word cistron is used to emphasize that genes exhibit a specific behavior in a cis-trans test; distinct positions (or loci) within a genome are cistronic.
History
The words ''cistron'' and ''gene ...
is used.
Advantages
The existing diversity of the microbial world is not unraveled completely yet, although we know that it is mainly composed by bacteria, fungi and unicellular eukaryotes.
Taxonomic identification of microbial eukaryotes requires exceedingly skillful expertise and is often difficult due to small sizes of the organisms, fragmented individuals, hidden
diversity
Diversity, diversify, or diverse may refer to:
Business
*Diversity (business), the inclusion of people of different identities (ethnicity, gender, age) in the workforce
*Diversity marketing, marketing communication targeting diverse customers
* ...
and
cryptic species
In biology, a species complex is a group of closely related organisms that are so similar in appearance and other features that the boundaries between them are often unclear. The taxa in the complex may be able to hybridize readily with each oth ...
. Further, prokaryotes can simply not be taxonomically assigned using traditional methods like
microscopy, because they are too small and
morphologically indistinguishable. Therefore, via the use of DNA metabarcoding, it is possible to identify organisms without taxonomic expertise by matching short High Throughput Sequences (HTS)-derived gene fragments to a reference sequence database, e.g.
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 ...
. These mentioned qualities make DNA barcoding a cost-effective, reliable and less time-consuming method, compared to the traditional ones, to meet the increasing need for large-scale environmental assessments.
Applications
A lot of studies followed the first usage of Woese et al., and are now covering a variety of applications. Not only in biological or ecological research
metabarcoding
Metabarcoding is the barcoding of DNA/ RNA (or eDNA/ eRNA) in a manner that allows for the simultaneous identification of many taxa within the same sample. The main difference between barcoding and metabarcoding is that metabarcoding does ...
is used. Also in medicine and human biology bacterial barcodes are used, e.g. to investigate the
microbiome
A microbiome () is the community of microorganisms that can usually be found living together in any given habitat. It was defined more precisely in 1988 by Whipps ''et al.'' as "a characteristic microbial community occupying a reasonably wel ...
and bacterial colonization of the human gut in normal and obese twins or comparison studies of newborn, child and adult gut bacteria composition. Additionally, barcoding plays a major role in biomonitoring of e.g. rivers and streams and grassland restoration. Conservation parasitology, environmental parasitology and paleoparasitology rely on barcoding as a useful tool in disease investigating and management, too.
Cyanobacteria
Cyanobacteria
Cyanobacteria (), also known as Cyanophyta, are a phylum of gram-negative bacteria that obtain energy via photosynthesis. The name ''cyanobacteria'' refers to their color (), which similarly forms the basis of cyanobacteria's common name, blu ...
are a group of
photosynthetic
Photosynthesis is a process used by plants and other organisms to convert light energy into chemical energy that, through cellular respiration, can later be released to fuel the organism's activities. Some of this chemical energy is stored in c ...
prokaryote
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 ...
s. Similar as in other prokaryotes,
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 groups or types. ...
of cyanobacteria using
DNA sequences
A nucleic acid sequence is a succession of bases signified by a series of a set of five different letters that indicate the order of nucleotides forming alleles within a DNA (using GACT) or RNA (GACU) molecule. By convention, sequences are us ...
is mostly based on similarity within the
16S ribosomal gene. Thus, the most common barcode used for identification of cyanobacteria is 16S
rDNA marker. While it is difficult to define
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 ...
within prokaryotic organisms, 16S marker can be used for determining individual operational taxonomic units (OTUs). In some cases, these OTUs can also be linked to traditionally defined species and can therefore be considered a reliable representation of the
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 ...
ary relationships.
![Dolichospermum sp](https://upload.wikimedia.org/wikipedia/commons/2/2e/Dolichospermum_sp..jpg)
However, when analyzing a taxonomic structure or
biodiversity
Biodiversity or biological diversity is the variety and variability of life on Earth. Biodiversity is a measure of variation at the genetic (''genetic variability''), species (''species diversity''), and ecosystem (''ecosystem diversity'') l ...
of a whole cyanobacterial community (see
DNA metabarcoding), it is more informative to use markers specific for cyanobacteria. Universal 16S bacterial primers have been used successfully to isolate cyanobacterial rDNA from
environmental samples, but they also recover many bacterial sequences. The use of cyanobacteria-specific
or phyto-specific 16S markers is commonly used for focusing on cyanobacteria only.
A few sets of such primers have been tested for barcoding or metabarcoding of environmental samples and gave good results, screening out majority of non-photosynthetic or non-cyanobacterial organisms.
Number of sequenced cyanobacterial genomes available in databases is increasing.
Besides 16S marker,
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 could therefore include also more variable sequences, such as sequences of
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
-coding genes (gyrB, rpoC, rpoD, rbcL, hetR, psbA, rnpB, nifH, nifD), internal transcribed spacer of ribosomal RNA genes (16S-23S rRNA-ITS)
or phycocyanin intergenic spacer (PC-IGS).
However, nifD and nifH can only be used for identification of nitrogen-fixing cyanobacterial strains.
DNA barcoding of cyanobacteria can be applied in various ecological, evolutionary and taxonomical studies. Some examples include assessment of cyanobacterial diversity and community structure, identification of harmful cyanobacteria in ecologically and economically important waterbodies and assessment of cyanobacterial
symbionts in
marine invertebrate
Invertebrates are a paraphyletic group of animals that neither possess nor develop a vertebral column (commonly known as a ''backbone'' or ''spine''), derived from the notochord. This is a grouping including all animals apart from the chordate ...
s.
It has a potential to serve as a part of routine
monitoring
Monitoring may refer to:
Science and technology Biology and healthcare
* Monitoring (medicine), the observation of a disease, condition or one or several medical parameters over time
* Baby monitoring
* Biomonitoring, of toxic chemical compounds, ...
programs for occurrence of cyanobacteria, as well as early detection of potentially
toxic
Toxicity is the degree to which a chemical substance or a particular mixture of substances can damage an organism. Toxicity can refer to the effect on a whole organism, such as an animal, bacterium, or plant, as well as the effect on a subst ...
species in waterbodies. This might help us detect harmful species before they start to form
blooms and thus improve our water management strategies. Species identification based on environmental DNA could be particularly useful for cyanobacteria, as traditional identification using microscopy is challenging. Their morphological characteristics which are the basis for species delimitation vary in different growth conditions.
[ Identification under microscope is also time-consuming and therefore relatively costly. Molecular methods can detect much lower concentration of cyanobacterial cells in the sample than traditional identification methods.
]
Reference databases
The reference database is a collection of DNA sequences, which are assigned to either a species or a function. It can be used to link molecular obtained sequences of an organism to pre-existing taxonomy. General databases like the 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 ...
platform include all kind of sequences, either whole genomes or specific marker genes of all organisms. There are also different platforms where only sequences from a distinct group of organisms are stored, e.g. UNITE database exclusively for fungi sequences or the PR2 database solely for protist ribosomal sequences. Some databases are curated, which allows a taxonomic assignment with higher accuracy than using uncurated databases as a reference.
See also
* Consortium for the Barcode of Life
The Consortium for the Barcode of Life (CBOL) was an international initiative dedicated to supporting the development of DNA barcoding as a global standard for species identification. CBOL's Secretariat Office is hosted by the National Museum of ...
*Algae DNA barcoding
DNA barcoding of algae is commonly used for species identification and phylogenetic studies. Algae form a phylogenetically heterogeneous group, meaning that the application of a single universal barcode/ marker for species delimitation is unfe ...
*DNA Barcoding
DNA barcoding is a method of species identification using a short section of DNA from a specific gene or genes. The premise of DNA barcoding is that by comparison with a reference library of such DNA sections (also called " sequences"), an indi ...
* DNA barcoding in diet assessment
* Fish DNA barcoding
References
{{Reflist
Authentication methods
Bioinformatics
Biometrics
Molecular genetics
Taxonomy (biology)
DNA barcoding