- Email: [email protected]
Antibiotic-Resistance Mutants
Antibiotic-Resistance Mutants J Parker, Southern Illinois University Carbondale, Carbondale, IL, USA
© 2013 Elsevier Inc. All rights reserved.
This article is reproduced from the previous edition, volume 1, pp 76–78, © 2001, Elsevier Inc., with revisions made by the Editor.
Glossary Antibiotic A substance that interferes with a particular step of cellular metabolism, thereby inhibiting growth or killing living organisms; sometimes restricted to those having a natural biological origin. Many derivatives have been obtained by chemical synthesis. Integrons DNA fragments that serve as sites for insertion of a variety of other DNA fragments (especially antibiotic resistance genes) and facilitate their transfer into other cells.
Antibiotic-resistant organisms can arise from antibiotic-sensi tive organisms in a number of ways. Genes, or sets of genes, conferring antibiotic resistance may be obtained via resistance plasmids (R plasmids), integrons, or transposable elements. These genes often encode proteins responsible for modifying or destroying the antibiotic, affecting the uptake or efflux of the antibiotic, or modifying the cellular target of the antibiotic. This mechanism is of great practical importance, as horizontal transfer of antibiotic resistance to pathogens is limiting the effectiveness of antibiotic therapy for treatment of infectious disease. Many of the resistance genes present on transposable ele ments, viruses, or specially constructed cassettes also have important uses as mutagenic agents in the laboratory, either in transposon mutagenesis or in various in vitro methods. In these cases, the antibiotic-resistance phenotype is of impor tance to the geneticist because it can be positively selected. Antibiotic resistance can also be the result of a mutation in one or more chromosomal genes in a sensitive strain. Sometimes these genes are also involved in uptake, destruction, or modification of the antibiotic. However, in some cases, the resistance arises because the mutation is in the gene encoding the cellular target of the antibiotic. These latter antibiotic-resis tance mutations have been invaluable for dissection of the complex reactions in macromolecular synthesis. In this article, we discuss a few examples of antibiotic resistance that result from such mutations in the bacterium Escherichia coli.
Coumarins The coumarins, such as coumermycin A and novobiocin, are antibiotics that inhibit certain DNA topoisomerases, enzymes that catalyze interconversions of different topological isomers of DNA. One of the topoisomerases that the coumarins inhibit is bacterial DNA gyrase. This enzyme introduces negative super coils into DNA and is an essential enzyme in DNA replication. Specifically, the coumarins inhibit the activity of the B subunit, which catalyzes the ATP hydrolysis involved in the enzyme
138
Plasmid A molecule of extrachromosomal DNA that replicates as an autonomous replicon in the cytoplasm, and is not required for growth of the host cell under certain conditions. Most plasmids are covalently closed circular (CCC) DNA, although examples of linear plasmids are known. Transposable elements A genetic element that, in addition to encoding the proteins required for its own transposition, confers one or more new observable phenotypes (often resistance to one or more specific drugs) on the host cell. Also known as transposons.
reaction. Some mutations of the gyrB gene, which encodes the B subunit of this enzyme, lead to resistance to these antibiotics.
Erythromycin Erythromycin is one of the macrolides, a large group of structu rally related antibiotics that inhibit protein synthesis. Erythromycin has been shown to bind to the large ribosomal subunit in the peptidyltransferase region of the 23S ribosomal RNA (rRNA). Resistance can arise from mutations in at least three different genes encoding large subunit ribosomal proteins. In E. coli, genetic elimination of ribosomal protein L11 makes the cells hypersensitive to erythromycin. Resistance can also arise from specific mutations in the gene encoding 23S rRNA, mutations which must be constructed in organisms such as E. coli that have multiple copies of this gene. These mutations are in a region of the 23S rRNA that is protected by specific methylation in the organ ism that produces erythromycin. Methylation at this site in E. coli leads to erythromycin resistance, but the gene that encodes the specific methylase must be acquired by horizontal gene transfer.
Fusidic Acid The antibiotic fusidic acid inhibits the translational elongation factor EF-G, which promotes translocation of the ribosome from one codon on the messenger RNA to the next. Mutants of EF-G are known that are resistant to fusidic acid, and they are responsible for the gene encoding this factor being termed fusA. Many such mutations inhibit the growth rate of the cell as well as the rate of translation elongation.
Kasugamycin The aminoglycoside kasugamycin acts as an inhibitor of transla tion initiation. Sensitivity to kasugamycin is dependent on the presence of two dimethyladenosine residues found near the 3′ end of 16S rRNA. Ribosomes whose 16S rRNA is missing these
Brenner’s Encyclopedia of Genetics, 2nd edition, Volume 1
doi:10.1016/B978-0-12-374984-0.00069-3
Antibiotic-Resistance Mutants methylated adenosine residues are resistant to the antibiotic and, therefore, mutants defective in the methylase (encoded by the gene ksgA) are kasugamycin resistant. Cells containing this undermethylated rRNA also have a reduced growth rate.
Kirromycin The polyenic antibiotic kirromycin inhibits the translational elongation factor, EF-Tu, blocking its exit from the ribosome. Kirromycin-resistant alleles of the tuf genes, which encode identical copies of EF-Tu, have been isolated. Individually, these mutations lead to amino acid substitutions at one of a small number of sites. Many of these alleles lead to an increase in various errors in translation. Many bacteria, including E. coli, have duplicate tuf genes (typically tufA and tufB) and the alleles conferring resistance are recessive to the wild-type alleles. Kirromycin-resistant cells grow more slowly than wild-type cells. Resistance to kirromycin-like antibiotics in some of the actinomyctes that produce them is also the result of these organisms having a resistant EF-Tu.
Quinolones The quinolones, such as nalidixic acid, inhibit bacterial DNA gyrase. Unlike coumarins (see above) these antibiotics specifi cally inhibit the A subunit, the nicking-ligating component of the enzyme. Certain mutants of the gene encoding this subunit, gyrA (formerly nalA), confer resistance to nalidixic acid and other quinolones.
Rifampicin The rifamycins are a group of antibiotics synthesized by certain Streptomyces species. One of these antibiotics, rifam picin, specifically inhibits the bacterial RNA polymerase by binding to the β subunit of this enzyme. Rifampicin blocks transcription at, or shortly after, the initiation of an RNA chain by the polymerase, but it does not block the elonga
139
tion of chains already initiated. Rifampicin-resistant mutants are readily isolated and are found to have mutations in rpoB (formerly rif), the gene encoding the β subunit. The muta tions are point mutations or small, in-frame insertions and deletions at a limited number of sites which result in amino acid substitutions, leading to the loss of the ability of the enzyme to bind rifampicin. Rifamycin-resistant mutations can have pleiotropic phenotypes, such as temperature sensi tivity or a change in the regulation of transcription of some genes.
Streptomycin The aminoglycoside antibiotic streptomycin inhibits protein synthesis in bacteria by binding to a specific site on 16S rRNA. Streptomycin-resistant mutants of E. coli were first reported in 1950. These mutants have amino acid substitutions in ribosomal protein S12, encoded by the rpsL gene (formerly strA). Streptomycin itself can increase many types of transla tional errors that occur on the ribosome. Many, but not all, streptomycin-resistant mutants are said to be restrictive in that they reduce the error frequency below that of wild-type ribo somes. All such restrictive mutations also result in a decreased growth rate and a decreased peptide chain elongation rate. In at least some cases, these effects can be compensated for by sec ond-site mutations outside rpsL without diminishing the level of streptomycin resistance. Streptomycin-resistant mutants are recessive in cells that also contain a wild-type allele. Streptomycin resistance can also arise from mutations in the genes encoding 16S rRNA. These mutations are also recessive to the wild-type allele and in organisms containing multiple rRNA genes these mutations are typically constructed by in vitro techniques.
See also: Antibiotic Resistance; DNA Replication; Elongation Factors: Translation; Escherichia coli ; Resistance to Antibiotics, Genetics of; Integrons; RNA Polymerase; Resistance Plasmids; Ribosomal RNA; Ribosomes; Streptomyces; Streptomycin; Topoisomerases; Transcription; Translation; Transposable Elements.
© 2013 Elsevier Inc. All rights reserved.
This article is reproduced from the previous edition, volume 1, pp 76–78, © 2001, Elsevier Inc., with revisions made by the Editor.
Glossary Antibiotic A substance that interferes with a particular step of cellular metabolism, thereby inhibiting growth or killing living organisms; sometimes restricted to those having a natural biological origin. Many derivatives have been obtained by chemical synthesis. Integrons DNA fragments that serve as sites for insertion of a variety of other DNA fragments (especially antibiotic resistance genes) and facilitate their transfer into other cells.
Antibiotic-resistant organisms can arise from antibiotic-sensi tive organisms in a number of ways. Genes, or sets of genes, conferring antibiotic resistance may be obtained via resistance plasmids (R plasmids), integrons, or transposable elements. These genes often encode proteins responsible for modifying or destroying the antibiotic, affecting the uptake or efflux of the antibiotic, or modifying the cellular target of the antibiotic. This mechanism is of great practical importance, as horizontal transfer of antibiotic resistance to pathogens is limiting the effectiveness of antibiotic therapy for treatment of infectious disease. Many of the resistance genes present on transposable ele ments, viruses, or specially constructed cassettes also have important uses as mutagenic agents in the laboratory, either in transposon mutagenesis or in various in vitro methods. In these cases, the antibiotic-resistance phenotype is of impor tance to the geneticist because it can be positively selected. Antibiotic resistance can also be the result of a mutation in one or more chromosomal genes in a sensitive strain. Sometimes these genes are also involved in uptake, destruction, or modification of the antibiotic. However, in some cases, the resistance arises because the mutation is in the gene encoding the cellular target of the antibiotic. These latter antibiotic-resis tance mutations have been invaluable for dissection of the complex reactions in macromolecular synthesis. In this article, we discuss a few examples of antibiotic resistance that result from such mutations in the bacterium Escherichia coli.
Coumarins The coumarins, such as coumermycin A and novobiocin, are antibiotics that inhibit certain DNA topoisomerases, enzymes that catalyze interconversions of different topological isomers of DNA. One of the topoisomerases that the coumarins inhibit is bacterial DNA gyrase. This enzyme introduces negative super coils into DNA and is an essential enzyme in DNA replication. Specifically, the coumarins inhibit the activity of the B subunit, which catalyzes the ATP hydrolysis involved in the enzyme
138
Plasmid A molecule of extrachromosomal DNA that replicates as an autonomous replicon in the cytoplasm, and is not required for growth of the host cell under certain conditions. Most plasmids are covalently closed circular (CCC) DNA, although examples of linear plasmids are known. Transposable elements A genetic element that, in addition to encoding the proteins required for its own transposition, confers one or more new observable phenotypes (often resistance to one or more specific drugs) on the host cell. Also known as transposons.
reaction. Some mutations of the gyrB gene, which encodes the B subunit of this enzyme, lead to resistance to these antibiotics.
Erythromycin Erythromycin is one of the macrolides, a large group of structu rally related antibiotics that inhibit protein synthesis. Erythromycin has been shown to bind to the large ribosomal subunit in the peptidyltransferase region of the 23S ribosomal RNA (rRNA). Resistance can arise from mutations in at least three different genes encoding large subunit ribosomal proteins. In E. coli, genetic elimination of ribosomal protein L11 makes the cells hypersensitive to erythromycin. Resistance can also arise from specific mutations in the gene encoding 23S rRNA, mutations which must be constructed in organisms such as E. coli that have multiple copies of this gene. These mutations are in a region of the 23S rRNA that is protected by specific methylation in the organ ism that produces erythromycin. Methylation at this site in E. coli leads to erythromycin resistance, but the gene that encodes the specific methylase must be acquired by horizontal gene transfer.
Fusidic Acid The antibiotic fusidic acid inhibits the translational elongation factor EF-G, which promotes translocation of the ribosome from one codon on the messenger RNA to the next. Mutants of EF-G are known that are resistant to fusidic acid, and they are responsible for the gene encoding this factor being termed fusA. Many such mutations inhibit the growth rate of the cell as well as the rate of translation elongation.
Kasugamycin The aminoglycoside kasugamycin acts as an inhibitor of transla tion initiation. Sensitivity to kasugamycin is dependent on the presence of two dimethyladenosine residues found near the 3′ end of 16S rRNA. Ribosomes whose 16S rRNA is missing these
Brenner’s Encyclopedia of Genetics, 2nd edition, Volume 1
doi:10.1016/B978-0-12-374984-0.00069-3
Antibiotic-Resistance Mutants methylated adenosine residues are resistant to the antibiotic and, therefore, mutants defective in the methylase (encoded by the gene ksgA) are kasugamycin resistant. Cells containing this undermethylated rRNA also have a reduced growth rate.
Kirromycin The polyenic antibiotic kirromycin inhibits the translational elongation factor, EF-Tu, blocking its exit from the ribosome. Kirromycin-resistant alleles of the tuf genes, which encode identical copies of EF-Tu, have been isolated. Individually, these mutations lead to amino acid substitutions at one of a small number of sites. Many of these alleles lead to an increase in various errors in translation. Many bacteria, including E. coli, have duplicate tuf genes (typically tufA and tufB) and the alleles conferring resistance are recessive to the wild-type alleles. Kirromycin-resistant cells grow more slowly than wild-type cells. Resistance to kirromycin-like antibiotics in some of the actinomyctes that produce them is also the result of these organisms having a resistant EF-Tu.
Quinolones The quinolones, such as nalidixic acid, inhibit bacterial DNA gyrase. Unlike coumarins (see above) these antibiotics specifi cally inhibit the A subunit, the nicking-ligating component of the enzyme. Certain mutants of the gene encoding this subunit, gyrA (formerly nalA), confer resistance to nalidixic acid and other quinolones.
Rifampicin The rifamycins are a group of antibiotics synthesized by certain Streptomyces species. One of these antibiotics, rifam picin, specifically inhibits the bacterial RNA polymerase by binding to the β subunit of this enzyme. Rifampicin blocks transcription at, or shortly after, the initiation of an RNA chain by the polymerase, but it does not block the elonga
139
tion of chains already initiated. Rifampicin-resistant mutants are readily isolated and are found to have mutations in rpoB (formerly rif), the gene encoding the β subunit. The muta tions are point mutations or small, in-frame insertions and deletions at a limited number of sites which result in amino acid substitutions, leading to the loss of the ability of the enzyme to bind rifampicin. Rifamycin-resistant mutations can have pleiotropic phenotypes, such as temperature sensi tivity or a change in the regulation of transcription of some genes.
Streptomycin The aminoglycoside antibiotic streptomycin inhibits protein synthesis in bacteria by binding to a specific site on 16S rRNA. Streptomycin-resistant mutants of E. coli were first reported in 1950. These mutants have amino acid substitutions in ribosomal protein S12, encoded by the rpsL gene (formerly strA). Streptomycin itself can increase many types of transla tional errors that occur on the ribosome. Many, but not all, streptomycin-resistant mutants are said to be restrictive in that they reduce the error frequency below that of wild-type ribo somes. All such restrictive mutations also result in a decreased growth rate and a decreased peptide chain elongation rate. In at least some cases, these effects can be compensated for by sec ond-site mutations outside rpsL without diminishing the level of streptomycin resistance. Streptomycin-resistant mutants are recessive in cells that also contain a wild-type allele. Streptomycin resistance can also arise from mutations in the genes encoding 16S rRNA. These mutations are also recessive to the wild-type allele and in organisms containing multiple rRNA genes these mutations are typically constructed by in vitro techniques.
See also: Antibiotic Resistance; DNA Replication; Elongation Factors: Translation; Escherichia coli ; Resistance to Antibiotics, Genetics of; Integrons; RNA Polymerase; Resistance Plasmids; Ribosomal RNA; Ribosomes; Streptomyces; Streptomycin; Topoisomerases; Transcription; Translation; Transposable Elements.