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Antibiotic-Resistance Mutants
76
Antibiotic-Resistance Mutants
infrequently may be integrated at other locations via site-specific recombination catalyzed by an integronencoded recombinase.
Russell AD and Chopra I (1996) Understanding Antibacterial Action and Resistance, 2nd edn. Hemel Hempstead, UK: Ellis Horwood.
Origins of Antibiotic Resistance Genes
See also: Bacterial Genetics; Transduction
When the Murray collection of bacteria made between 1914 and 1950 was examined for the presence of antibiotic resistance genes none were found; however a number of conjugative plasmids very similar to those carrying antibiotic resistance genes in ``modern bacteria'' were found. This implies that all of the mechanisms for antibiotic resistance gene dissemination existed prior to the use of antibiotics. Many antibiotic resistance genes have homologs in housekeeping genes found in bacteria, e.g., b-lactamases and PBPs suggesting they may have evolved by mutation. Antibiotic resistance genes are found in fungi and bacteria that produce antibiotics and it is probable that they have moved from that source. DNA sequencing studies of b-lactamases and aminoglycoside-inactivating enzymes show that despite similarities within the protein sequences, there are substantial DNA sequence differences. As the evolutionary time frame is less than 50 years it is not possible to derive a model in which evolution could have occurred by mutation alone. They must therefore be derived from a large and diverse gene pool occurring in environmental bacteria some of which produce antibotics. Mutation is an important process for the ``refinement'' of antibiotic resistance genes as has been seen in the last 10 years with the SHV and TEM plasmid encoded b-lactamases. The parental enzymes SHV-1 and TEM-1/2 are `pure' penicillinases but the substitution of Glu-237!Lys in SHV-5 and Glu102!Lys in TEM-9 extend activity to degrade cephalosporins like cefotaxime and ceftazidime. Mutations such as Arg244! Cys and Val69!Met in TEM b-lactamases confer resistance to inhibition by b-lactamase inhibitors like clavulanic acid. The selection pressure for the maintenance of antibiotic resistance genes is heavy and injudicious use of antibiotics, largely in medical practice (about 50% of production is used on humans, 20% in hospital, 80% in the community), is probably responsible. The addition of antibiotics to animal feed or water, either for growth promotion or, more significantly, for mass treatment or prophylaxis in factory-farmed animals is having an unquantified effect on resistance levels.
Antibiotic-Resistance Mutants
Further Reading
Lorian V (ed.) (1991) Antibiotics in Laboratory Medicine, 3rd edn. London: Williams & Wilkins. O'Grady LF, Lambert HP, Finch RG and Greenwood D (eds) (1997) Antibiotic and Chemotherapy, 7th edn. Edinburgh, UK: Churchill Livingstone.
J Parker Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.0058
Antibiotic-resistant organisms can arise from antibiotic-sensitive 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 putting severe constraints on antibiotic therapy for treatment of infectious disease. This topic is covered in the entries on Antibiotic Resistance, Drug Resistance, and Resistance Plasmids. Many of the resistance genes present on transposable elements, 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 (for example see Transposons as Tools). In these cases the antibiotic-resistance phenotype is of importance 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 antibioticresistance mutations have been invaluable for dissection of the complex reactions in macromolecular synthesis. In this entry we will discuss a few examples of antibiotic-resistance in the bacterium Escherichia coli which result from such mutations.
Coumarins The coumarins, such as coumermycin A and novobiocin, are antibiotics that inhibit certain DNA topoisomerases, enzymes that catalyze interconversions of
A n t i b i o t i c - R e s i s ta n c e M u t a n t s 77 different topological isomers of DNA. One of the topoisomerases that the coumarins inhibit is bacterial DNA gyrase. This enzyme introduces negative supercoils 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 reaction. Some mutationsofthegyrBgene,whichencodestheBsubunit of this enzyme, lead to resistance to these antibiotics.
Erythromycin Erythromycin is one of the macrolides, a large group of structurally 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. Curiously, 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 like E. coli that have multiple copies of this gene. These mutations are in a region of the 23S rRNA which is protected by specific methylation in the organism 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 fus. 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 translation initiation. Sensitivity to kasugamycin is dependent on the presence of two dimethyladenosine residues found near the 30 end of 16S rRNA. Ribosomes whose 16S rRNA is missing these 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 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 wildtype alleles. Kirromycin-resistant cells grow more slowly than wild-type cells. Resistance to kirromycinlike 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, also inhibit bacterial DNA gyrase. However, unlike coumarins (see above) these antibiotics specifically 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, rifampicin, specifically inhibits the bacterial RNA polymerase by binding to the b 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 elongation of chains already initiated. Rifampicin-resistant mutants are readily isolated and are found to have mutations in rpoB (formerly rif ), the gene encoding the b subunit. The mutations 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 sensitivity 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,
78
Antibody
encoded by the rpsL gene (formerly str). Streptomycin itself can increase many types of translational 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 ribosomes. 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 second-site mutations outside rpsL without diminishing the level of streptomycin resistance. Streptomycinresistant 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; Drug Resistance; Elongation Factors; Escherichia coli; Integrons; Resistance Plasmids; Resistance to Antibiotics, Genetics of; Ribosomal RNA (rRNA); Ribosomes; RNA Polymerase; Streptomyces; Streptomycin; Topoisomerases; Transcription; Translation; Transposable Elements; Transposons as Tools
Antibody Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.1759
An antibody is a protein (immunoglobulin) produced by B lymphocytes that recognizes and binds to a particular foreign `antigen.' See also: Antigen; Immunity
Anticodons A Liljas Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.0059
The anticodon is the part of the tRNA that decodes the genetic message contained in the mRNA. This leads to the incorporation of amino acids into the growing polypeptide. The anticodon is composed of three nucleotides, located approximately in the middle of the tRNA sequences and at one end of these elongated molecules.
tRNA Crick, in his adaptor hypothesis, proposed that small RNA molecules would be the adaptors that could be charged with amino acids by specific enzymes and that could identify the codons (triplets of nucleotides) of the mRNA by base-pairing. These adaptors could thus participate in incorporating the amino acids into a growing polypeptide. Subsequently these adaptors were identified and are now known as the tRNA molecules. From the nucleotide sequences of numerous tRNA molecules, the secondary structure of the tRNA, the classic cloverleaf, has been identified. Of the three loops, the middle one contains the anticodon of the tRNA. The three-dimensional structure of tRNA has the shape of an ``L.'' Here the anticodon is located at one end and the 30 acceptor for amino acids Ê away. This is at the opposite end, approximately 80 A means that the anticodon has no possibility of interacting with the amino acid. This also means that, when the tRNA assists in the incorporation of the amino acid into the growing polypeptide on the ribosome, the interaction of the anticodon with the mRNA is far from the site of peptidyl transfer. The anticodons are frequently posttranscriptionally modified. This concerns the bases as well as the riboses.
Code, Codons, and Codon Usage With a universal triplet genetic code and four different nucleotides in the mRNA, there are 64 words or codons in the genetic code. Even though the genetic code is universal, there are variations in the meaning of some code words. In bacteria three codons designate stop and are normally not read by tRNAs. Since there are 20 different amino acids in the regular protein the code is degenerate. Thus there are between one and six codons that correspond to the different amino acids. The tRNAs that bind the same amino acid are called isoacceptor tRNAs. The number of tRNAs that decode the message is variable for different organisms. The codons used in the tRNAs must be read by some tRNA expressed by the organism. In some organisms, the codon usage is limited to a small set of tRNAs (minimally 20), while in others there are different tRNAs for almost all codons. Thus the codon usage is different for different organisms.
Anticodons The anticodon is composed of three nucleotides, normally positions 34±36 of the tRNA, that read the codons of the mRNA, primarily by Watson±Crick base-pairing. However, the same tRNA can base-pair with different nucleotides in the third position of the
Antibiotic-Resistance Mutants
infrequently may be integrated at other locations via site-specific recombination catalyzed by an integronencoded recombinase.
Russell AD and Chopra I (1996) Understanding Antibacterial Action and Resistance, 2nd edn. Hemel Hempstead, UK: Ellis Horwood.
Origins of Antibiotic Resistance Genes
See also: Bacterial Genetics; Transduction
When the Murray collection of bacteria made between 1914 and 1950 was examined for the presence of antibiotic resistance genes none were found; however a number of conjugative plasmids very similar to those carrying antibiotic resistance genes in ``modern bacteria'' were found. This implies that all of the mechanisms for antibiotic resistance gene dissemination existed prior to the use of antibiotics. Many antibiotic resistance genes have homologs in housekeeping genes found in bacteria, e.g., b-lactamases and PBPs suggesting they may have evolved by mutation. Antibiotic resistance genes are found in fungi and bacteria that produce antibiotics and it is probable that they have moved from that source. DNA sequencing studies of b-lactamases and aminoglycoside-inactivating enzymes show that despite similarities within the protein sequences, there are substantial DNA sequence differences. As the evolutionary time frame is less than 50 years it is not possible to derive a model in which evolution could have occurred by mutation alone. They must therefore be derived from a large and diverse gene pool occurring in environmental bacteria some of which produce antibotics. Mutation is an important process for the ``refinement'' of antibiotic resistance genes as has been seen in the last 10 years with the SHV and TEM plasmid encoded b-lactamases. The parental enzymes SHV-1 and TEM-1/2 are `pure' penicillinases but the substitution of Glu-237!Lys in SHV-5 and Glu102!Lys in TEM-9 extend activity to degrade cephalosporins like cefotaxime and ceftazidime. Mutations such as Arg244! Cys and Val69!Met in TEM b-lactamases confer resistance to inhibition by b-lactamase inhibitors like clavulanic acid. The selection pressure for the maintenance of antibiotic resistance genes is heavy and injudicious use of antibiotics, largely in medical practice (about 50% of production is used on humans, 20% in hospital, 80% in the community), is probably responsible. The addition of antibiotics to animal feed or water, either for growth promotion or, more significantly, for mass treatment or prophylaxis in factory-farmed animals is having an unquantified effect on resistance levels.
Antibiotic-Resistance Mutants
Further Reading
Lorian V (ed.) (1991) Antibiotics in Laboratory Medicine, 3rd edn. London: Williams & Wilkins. O'Grady LF, Lambert HP, Finch RG and Greenwood D (eds) (1997) Antibiotic and Chemotherapy, 7th edn. Edinburgh, UK: Churchill Livingstone.
J Parker Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.0058
Antibiotic-resistant organisms can arise from antibiotic-sensitive 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 putting severe constraints on antibiotic therapy for treatment of infectious disease. This topic is covered in the entries on Antibiotic Resistance, Drug Resistance, and Resistance Plasmids. Many of the resistance genes present on transposable elements, 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 (for example see Transposons as Tools). In these cases the antibiotic-resistance phenotype is of importance 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 antibioticresistance mutations have been invaluable for dissection of the complex reactions in macromolecular synthesis. In this entry we will discuss a few examples of antibiotic-resistance in the bacterium Escherichia coli which result from such mutations.
Coumarins The coumarins, such as coumermycin A and novobiocin, are antibiotics that inhibit certain DNA topoisomerases, enzymes that catalyze interconversions of
A n t i b i o t i c - R e s i s ta n c e M u t a n t s 77 different topological isomers of DNA. One of the topoisomerases that the coumarins inhibit is bacterial DNA gyrase. This enzyme introduces negative supercoils 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 reaction. Some mutationsofthegyrBgene,whichencodestheBsubunit of this enzyme, lead to resistance to these antibiotics.
Erythromycin Erythromycin is one of the macrolides, a large group of structurally 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. Curiously, 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 like E. coli that have multiple copies of this gene. These mutations are in a region of the 23S rRNA which is protected by specific methylation in the organism 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 fus. 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 translation initiation. Sensitivity to kasugamycin is dependent on the presence of two dimethyladenosine residues found near the 30 end of 16S rRNA. Ribosomes whose 16S rRNA is missing these 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 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 wildtype alleles. Kirromycin-resistant cells grow more slowly than wild-type cells. Resistance to kirromycinlike 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, also inhibit bacterial DNA gyrase. However, unlike coumarins (see above) these antibiotics specifically 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, rifampicin, specifically inhibits the bacterial RNA polymerase by binding to the b 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 elongation of chains already initiated. Rifampicin-resistant mutants are readily isolated and are found to have mutations in rpoB (formerly rif ), the gene encoding the b subunit. The mutations 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 sensitivity 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,
78
Antibody
encoded by the rpsL gene (formerly str). Streptomycin itself can increase many types of translational 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 ribosomes. 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 second-site mutations outside rpsL without diminishing the level of streptomycin resistance. Streptomycinresistant 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; Drug Resistance; Elongation Factors; Escherichia coli; Integrons; Resistance Plasmids; Resistance to Antibiotics, Genetics of; Ribosomal RNA (rRNA); Ribosomes; RNA Polymerase; Streptomyces; Streptomycin; Topoisomerases; Transcription; Translation; Transposable Elements; Transposons as Tools
Antibody Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.1759
An antibody is a protein (immunoglobulin) produced by B lymphocytes that recognizes and binds to a particular foreign `antigen.' See also: Antigen; Immunity
Anticodons A Liljas Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.0059
The anticodon is the part of the tRNA that decodes the genetic message contained in the mRNA. This leads to the incorporation of amino acids into the growing polypeptide. The anticodon is composed of three nucleotides, located approximately in the middle of the tRNA sequences and at one end of these elongated molecules.
tRNA Crick, in his adaptor hypothesis, proposed that small RNA molecules would be the adaptors that could be charged with amino acids by specific enzymes and that could identify the codons (triplets of nucleotides) of the mRNA by base-pairing. These adaptors could thus participate in incorporating the amino acids into a growing polypeptide. Subsequently these adaptors were identified and are now known as the tRNA molecules. From the nucleotide sequences of numerous tRNA molecules, the secondary structure of the tRNA, the classic cloverleaf, has been identified. Of the three loops, the middle one contains the anticodon of the tRNA. The three-dimensional structure of tRNA has the shape of an ``L.'' Here the anticodon is located at one end and the 30 acceptor for amino acids Ê away. This is at the opposite end, approximately 80 A means that the anticodon has no possibility of interacting with the amino acid. This also means that, when the tRNA assists in the incorporation of the amino acid into the growing polypeptide on the ribosome, the interaction of the anticodon with the mRNA is far from the site of peptidyl transfer. The anticodons are frequently posttranscriptionally modified. This concerns the bases as well as the riboses.
Code, Codons, and Codon Usage With a universal triplet genetic code and four different nucleotides in the mRNA, there are 64 words or codons in the genetic code. Even though the genetic code is universal, there are variations in the meaning of some code words. In bacteria three codons designate stop and are normally not read by tRNAs. Since there are 20 different amino acids in the regular protein the code is degenerate. Thus there are between one and six codons that correspond to the different amino acids. The tRNAs that bind the same amino acid are called isoacceptor tRNAs. The number of tRNAs that decode the message is variable for different organisms. The codons used in the tRNAs must be read by some tRNA expressed by the organism. In some organisms, the codon usage is limited to a small set of tRNAs (minimally 20), while in others there are different tRNAs for almost all codons. Thus the codon usage is different for different organisms.
Anticodons The anticodon is composed of three nucleotides, normally positions 34±36 of the tRNA, that read the codons of the mRNA, primarily by Watson±Crick base-pairing. However, the same tRNA can base-pair with different nucleotides in the third position of the