- Resistance genes (R genes)
- How are antibiotic resistance genes on some plasmids used in genetic engineering?
*Resistance genes (R genes):
Schematic representation of how antibiotic resistance evolves via natural selection. The top section represents a population of bacteria before exposure to an antibiotic. The middle section shows the population directly after exposure, the phase in which selection took place. The last section shows the distribution of resistance in a new generation of bacteria. The legend indicates the resistance levels of individuals.
- Diagram depicting antibiotic resistance through alteration of the antibiotic's target site, modeled after MRSA's resistance to penicillin. Beta-lactam antibiotics permanently inactivate PBP enzymes, which are essential for bacterial life, by permanently binding to their active sites. MRSA, however, expresses a PBP that will not allow the antibiotic into its active site.
Antibiotic resistance can be a result of horizontal gene transfer, and also of unlinked point mutations in the pathogen genome at a rate of about 1 in 108 per chromosomal replication. The antibiotic action against the pathogen can be seen as an environmental pressure; those bacteria which have a mutation allowing them to survive will live on to reproduce. They will then pass this trait to their offspring, which will result in the evolution of a fully resistant colony.
The four main mechanisms by which microorganisms exhibit resistance to antimicrobials are:
1.Drug inactivation or modification: for example, enzymatic deactivation of Penicillin G in some penicillin-resistant bacteria through the production of β-lactamases.
2.Alteration of target site: for example, alteration of PBP—the binding target site of penicillins—in MRSA and other penicillin-resistant bacteria.
3.Alteration of metabolic pathway: for example, some sulfonamide-resistant bacteria do not require para-aminobenzoic acid (PABA), an important precursor for the synthesis of folic acid and nucleic acids in bacteria inhibited by sulfonamides. Instead, like mammalian cells, they turn to utilizing preformed folic acid.
4.Reduced drug accumulation: by decreasing drug permeability and/or increasing active efflux (pumping out) of the drugs across the cell surface.
- There are three known mechanisms of fluoroquinolone resistance. Some types of efflux pumps can act to decrease intracellular quinolone concentration. In gram-negative bacteria, plasmid-mediated resistance genes produce proteins that can bind to DNA gyrase, protecting it from the action of quinolones. Finally, mutations at key sites in DNA gyrase or Topoisomerase IV can decrease their binding affinity to quinolones, decreasing the drug's effectiveness. Research has shown that the bacterial protein LexA may play a key role in the acquisition of bacterial mutations giving resistance to quinolones and rifampicin.
- Antibiotic resistance can also be introduced artificially into a microorganism through laboratory protocols, sometimes used as a selectable marker to examine the mechanisms of gene transfer or to identify individuals that absorbed a piece of DNA that included the resistance gene and another gene of interest. A recent study demonstrates the extent of horizontal gene transfer among Staphylococcus to be much greater than one previously expected, and encompasses genes with functions beyond antibiotic resistance and virulence, and beyond genes residing within the mobile genetic elements.
***Antibiotic resistance is an important tool for genetic engineering. By constructing a plasmid which contains an antibiotic resistance gene as well as the gene being engineered or expressed, a researcher can ensure that when bacteria replicate, only the copies which carry along the plasmid survive. This ensures that the gene being manipulated passes along when the bacteria replicates.
- The most commonly used antibiotics in genetic engineering are generally "older" antibiotics which have largely fallen out of use in clinical practice. These include:
- Industrially the use of antibiotic resistance is disfavored since maintaining bacterial cultures would require feeding them large quantities of antibiotics. Instead, the use of auxotrophic bacterial strains (and function-replacement plasmids) is preferred.