Blasticidin S is an

antibiotic An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting pathogenic bacteria, bacterial infections, and antibiotic medications are widely used in the therapy ...

that is used in biology research for selecting cells in

cell culture Cell culture or tissue culture is the process by which cell (biology), cells are grown under controlled conditions, generally outside of their natural environment. After cells of interest have been Cell isolation, isolated from living tissue, ...

. Cells of interest can express the blasticidin resistance genes BSD or bsr, and can then survive treatment with the antibiotic. Blasticidin S is a

nucleoside analogue Nucleoside analogues are structural analogues of a nucleoside, which normally contain a nucleobase and a sugar. Nucleotide analogues are analogues of a nucleotide, which normally has one to three phosphates linked to a nucleoside. Both types ...

antibiotic, resembling the nucleoside

cytidine Cytidine (symbol C or Cyd) is a nucleoside molecule that is formed when cytosine is attached to a ribose ring (also known as a ribofuranose) via a β-N1-glycosidic bond. Cytidine is a component of RNA. It is a white water-soluble solid that is ...

. Blasticidin works against human cells, fungi, and bacteria, all by disrupting

protein translation In biology, translation is the process in living cells in which proteins are produced using RNA molecules as templates. The generated protein is a sequence of amino acids. This sequence is determined by the sequence of nucleotides in the RNA. T ...

. It was originally described by Japanese researchers in the 1950s seeking antibiotics for rice blast fungus.

Chemistry

A nucleoside analog, blasticidin S resembles the nucleoside

. The chemical structure consists of a

cytosine Cytosine () (symbol C or Cyt) is one of the four nucleotide bases found in DNA and RNA, along with adenine, guanine, and thymine ( uracil in RNA). It is a pyrimidine derivative, with a heterocyclic aromatic ring and two substituents attac ...

molecule, linked to a

glucuronic acid Glucuronic acid (GCA, from ) is a uronic acid that was first isolated from urine (hence the name "uronic acid"). It is found in many natural gum, gums such as gum arabic ( 18%), xanthan, and kombucha tea and is important for the metabolism of ...

-derived ring, linked in turn to the

peptide Peptides are short chains of amino acids linked by peptide bonds. A polypeptide is a longer, continuous, unbranched peptide chain. Polypeptides that have a molecular mass of 10,000 Da or more are called proteins. Chains of fewer than twenty am ...

N-methyl β-arginine.

Uses

Blasticidin S is widely used in

for selecting and maintaining genetically manipulated cells. Cells of interest express the blasticidin S resistance genes BSD or bsr, and can then survive blasticidin S being added to the

culture media A growth medium or culture medium is a solid, liquid, or semi-solid designed to support the growth of a population of microorganisms or cells via the process of cell proliferation or small plants like the moss ''Physcomitrella patens''. Differen ...

. Blasticidin S is typically used at 2–300

microgram In the metric system, a microgram or microgramme is a unit of mass equal to one millionth () of a gram. The unit symbol is μg according to the International System of Units (SI); the recommended symbol in the United States and United Kingdom wh ...

s per milliliter of media, depending on the type of cell being grown.

Mechanism of action

Blasticidin prevents the growth of both

eukaryotic The eukaryotes ( ) constitute the Domain (biology), domain of Eukaryota or Eukarya, organisms whose Cell (biology), cells have a membrane-bound cell nucleus, nucleus. All animals, plants, Fungus, fungi, seaweeds, and many unicellular organisms ...

and

prokaryotic A prokaryote (; less commonly spelled procaryote) is a single-celled organism whose cell lacks a nucleus and other membrane-bound organelles. The word ''prokaryote'' comes from the Ancient Greek (), meaning 'before', and (), meaning 'nut' ...

cells. It works by inhibiting termination step of translation and peptide bond formation (to lesser extent) by the

ribosome Ribosomes () are molecular machine, macromolecular machines, found within all cell (biology), cells, that perform Translation (biology), biological protein synthesis (messenger RNA translation). Ribosomes link amino acids together in the order s ...

. This means that cells can no longer produce new proteins through

translation Translation is the communication of the semantics, meaning of a #Source and target languages, source-language text by means of an Dynamic and formal equivalence, equivalent #Source and target languages, target-language text. The English la ...

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 ...

. It is competitive with

puromycin Puromycin is an antibiotic protein synthesis inhibitor which causes premature chain termination during translation. Inhibition of translation Puromycin is an aminonucleoside antibiotic, derived from the '' Streptomyces alboniger'' bacterium, ...

suggesting a highly similar binding site.

Biosynthesis

The first step in blasticidin S biosynthesis is the combination of

UDP-glucuronic acid UDP-glucuronic acid is a sugar used in the creation of polysaccharides and is an intermediate in the biosynthesis of ascorbic acid (except in primates and guinea pigs). It also participates in the heme degradation process of human. It is made fro ...

with cytosine to form cytosylglucuronic acid (CGA). Given the product name, the enzyme that performs this combination is called CGA synthase. Cosmid cloning experiments from the Blasticidin S producer ''

Streptomyces griseochromogenes ''Streptomyces griseochromogenes'' is a bacterium species from the genus of ''Streptomyces'' which has been isolated from soil.Deutsche Sammlung von Mikroorganismen und Zellkulturen The Leibniz Institute DSMZ - German Collection of Microorga ...

'', followed by evaluation of the putative biosynthetic gene cluster via heterologous reconstitution of Blasticidin S production in '' Streptomyces lividans'', indicated that a 20 Kbp gene cluster with 19 genes, plus possibly a peptidase outside the gene cluster that acts on the final leucylblasticidin S (LBS) intermediate, was sufficient for reconstitution of Blasticidin S biosynthesis.

Resistance genes

Resistance to blasticidin S can be conferred by either of two deaminases: BSD, originally isolated from ''

Aspergillus terreus ''Aspergillus terreus'', also known as ''Aspergillus terrestris'', is a fungus (mold) found worldwide in soil. Although thought to be strictly asexual until recently, ''A. terreus'' is now known to be capable of sexual reproduction. This saprot ...

'' or bsr, isolated from ''

Bacillus cereus ''Bacillus cereus'' is a Gram-positive bacteria, Gram-positive Bacillus, rod-shaped bacterium commonly found in soil, food, and marine sponges. The specific name, ''cereus'', meaning "waxy" in Latin, refers to the appearance of colonies grown o ...

''. Both deaminases work by modifying blasticidin S directly, replacing the amine on the cytosine ring with a hydroxyl group, resulting in the inactive deaminohydroxy-blasticin S. bsr and BSD are the most commonly used resistance genes. The proteins produced from these genes enable the cells carrying them to produce proteins in the presence of blasticidin.

History

In the 1950s, a drug screening program was designed in

Japan Japan is an island country in East Asia. Located in the Pacific Ocean off the northeast coast of the Asia, Asian mainland, it is bordered on the west by the Sea of Japan and extends from the Sea of Okhotsk in the north to the East China Sea ...

to discover a new antibiotic that prevents

blast disease ''Magnaporthe grisea'', also known as rice blast fungus, rice rotten neck, rice seedling blight, blast of rice, oval leaf spot of graminea, pitting disease, ryegrass blast, Johnson spot, neck blast, wheat blast and , is a plant-pathogenic fungus ...

by the

fungus A fungus (: fungi , , , or ; or funguses) is any member of the group of eukaryotic organisms that includes microorganisms such as yeasts and mold (fungus), molds, as well as the more familiar mushrooms. These organisms are classified as one ...

Magnaporthe grisea ''Magnaporthe grisea'', also known as rice blast fungus, rice rotten neck, rice seedling blight, blast of rice, oval leaf spot of graminea, pitting disease, ryegrass blast, Johnson spot, neck blast, wheat blast and , is a plant-pathogenic fungus ...

''.Natural Products Isolation: Separation Methods for Antimicrobials, Antivirals, and Enzyme Inhibitors. Wagman G. H., Elsevier R. C.; p. 191 (1988).

References

{{Reflist

External links

Taxonomy of Streptomyces griseochromogenes
Protein synthesis inhibitor antibiotics Genetic engineering Pyrimidones Guanidines Eukaryotic selection compounds