Hygromycin B is an antibiotic that is used in recombinant cloning to select cells that have been transformed with a desired DNA sequence. It acts to kill the untransformed, susceptible cells by inhibiting protein synthesis. This compound can be used in bacterial, fungal, plant, and mammalian cell cultures. Produced by the soil-dwelling actinomycete Streptomyces hygroscopicus, hygromycin B is also added to feedstocks for chicken and swine to keep them from being infested by parasites such as worms.
Actinomycetes are a type of bacteria that are common soil inhabitants. They produce a variety of natural products that are toxic to other organisms to help them compete and survive in the soil. Many different types of antibiotics have been isolated from these organisms, including streptomycin and actinomycin. Hygromycin B was discovered in 1953. Although toxic to humans, its first uses were to augment the food of poultry and pigs to prevent worm infestation.
Hygromycin B is an example of the class of aminoglycoside antibiotics. It is comprised of sugars that have amino groups on them. Several different kinds of bacteria have been found to have resistance to this antibiotic. This resistance is activated by the transfer of a phosphate group to a hydroxyl—OH—group on the antibiotic molecule. The protein that carries out this reaction is known as hygromycin B phosphotransferase (Hph).
The mechanism of action of hygromycin B is to inhibit protein synthesis by affecting the translation of messenger RNA (mRNA). Proteins are made up of long chains of amino acids. Normally, ribosomes move along a strand of mRNA and make an amino acid according to a group of three bases of mRNA known as a codon. When the antibiotic is present, the ribosomes do not read the codon properly and mis-translate. Generally, this results in the termination of protein synthesis.
With the advent of recombinant DNA technology, scientists were able to clone the Hph genes from several different types of bacteria. One type of gene was found in the actinomycete that makes the antibiotic, and another was found in the gram negative bacteria Escherichia coli (E. coli) and Klebsiella pneumoniae. Since E. coli is so commonly manipulated in genetic engineering, its gene is the one most commonly used in experiments. The mode of action of this antibiotic is different than that of many others, allowing it to often be used in combination with an additional type of antibiotic.
This hygromycin B resistance gene is widely used as a selectable marker during cloning experiments. Thus, it is used on a recombinant plasmid that also contains a gene that scientists wish to introduce into an organism such as a fungus or a plant cell line. Only a few cells in a large mixture of cells generally will have the desired plasmid. When an antibiotic is added to the cellular mixture, it will kill the susceptible cells that did not take up the recombinant plasmid. This hygromycin B selection ensures that the remaining cells will continue to contain the gene of interest.