Approaches to the Genetics of Escherichia coli Ribosomes

Apirion, David; Phillips, Stephen L.; Schlessinger, David

doi:10.1101/sqb.1969.034.01.018

Cited by 32 publications

(23 citation statements)

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“…2A, dashed lines), doubled the spiramycin MIC (Ϸ500 to Ϸ1000 g/ml; data not shown), and quadrupled the tylosin MIC (Ϸ500 M to Ϸ2 mM; data not shown). E. coli strains containing plasmidborne L22 variants lacking the titin-fusion domain behaved simi- larly (data not shown) as did strains with chromosomal alleles of wild-type L22 or ⌬MKR L22 (2,(11)(12)(13). Although sufficiently high concentrations of macrolides inhibited growth of both the L22-titin and ⌬MKR-L22-titin strains, as expected from our results in vitro, the differential macrolide sensitivity in vivo was not recapitulated in the in vitro assays.…”

Section: ⌬Mkr Ribosomes Are Inhibited By Erythromycin In Vitromentioning

confidence: 45%

“…The discovery that bacterial resistance to erythromycin can be caused by mutations in ribosomal proteins was first reported in 1967, and the ⌬MKR deletion in protein L22 was subsequently shown to cause this phenotype (8,11,12). Since these initial observations, consensus molecular mechanisms for macrolide inhibition of translation and for the effects of the ⌬MKR mutation have emerged from biochemical and structural studies (3,4,7,18,19).…”

Section: Discussionmentioning

confidence: 99%

“…Intriguingly, other mutations confer resistance despite the fact that macrolides still bind the mutant ribosome well (8)(9)(10). For example, deletion of the M 82 K 83 R 84 sequence in Escherichia coli ribosomal protein L22 (⌬MKR) allows growth in the presence of high levels of erythromycin and other macrolides (11)(12)(13). The same mutation makes Haemophilus influenzae resistant to numerous macrolides (14); different L22 mutations also confer macrolide resistance in other bacterial species (2).…”

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Revisiting the mechanism of macrolide-antibiotic resistance mediated by ribosomal protein L22

Moore

Sauer²

2008

Proc. Natl. Acad. Sci. U.S.A.

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Bacterial antibiotic resistance can occur by many mechanisms. An intriguing class of mutants is resistant to macrolide antibiotics even though these drugs still bind to their targets. For example, a 3-residue deletion (⌬MKR) in ribosomal protein L22 distorts a loop that forms a constriction in the ribosome exit tunnel, apparently allowing nascent-chain egress and translation in the presence of bound macrolides. Here, however, we demonstrate that ⌬MKR and wild-type ribosomes show comparable macrolide sensitivity in vitro. In Escherichia coli, we find that this mutation reduces antibiotic occupancy of the target site on ribosomes in a manner largely dependent on the AcrAB-TolC efflux system. We propose a model for antibiotic resistance in which ⌬MKR ribosomes alter the translation of specific proteins, possibly via changes in programmed stalling, and modify the cell envelope in a manner that lowers steady-state macrolide levels.ermC ͉ erythromycin ͉ ribosome ͉ TolC M any antibiotics inhibit bacterial protein synthesis. Understanding how microbes become antibiotic resistant is important both for developing effective treatment regimens and designing new therapeutics. Macrolides consist of a 14-to 16-member lactone ring with different appended sugars and comprise a key group of inhibitors of bacterial translation (1, 2). The inhibitory activity of macrolides, including erythromycin, depends on binding to a site near the polypeptide exit tunnel of the large ribosomal subunit (3, 4). Because macrolides do not bind to ribosomes with an occupied exit tunnel and cause the synthesis of 2-10 residue peptides in translation assays in vitro, it has been proposed that drug binding physically blocks elongation of nascent proteins beyond this size (5-7).Some macrolide-resistance mutations alter the ribosomal target site and prevent binding (4,8). Intriguingly, other mutations confer resistance despite the fact that macrolides still bind the mutant ribosome well (8-10). For example, deletion of the M 82 K 83 R 84 sequence in Escherichia coli ribosomal protein L22 (⌬MKR) allows growth in the presence of high levels of erythromycin and other macrolides (11-13). The same mutation makes Haemophilus influenzae resistant to numerous macrolides (14); different L22 mutations also confer macrolide resistance in other bacterial species (2). When binding has been measured, ribosomes with macrolideresistant alterations in L22 bind erythromycin with near wild-type affinity (8,10,12,15).In a crystal structure of the E. coli ribosome, the MKR sequence is part of an extended L22 loop, which together with a similar loop in protein L4 forms a narrow constriction in the exit tunnel (Fig. 1A) (16, 17). Cryo-EM studies initially revealed a widened exit tunnel in E. coli ⌬MKR ribosomes (18). This loop is also displaced to create an expanded tunnel in structures of ⌬MKR ribosomes from Thermus thermophilus and Haloarcula marismortui (4, 19). These results explain the altered chemical reactivity in E. coli ⌬MKR ribosomes of 23S-RNA bases (13). Together,...

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Section: ⌬Mkr Ribosomes Are Inhibited By Erythromycin In Vitromentioning

confidence: 45%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Revisiting the mechanism of macrolide-antibiotic resistance mediated by ribosomal protein L22

Moore

Sauer²

2008

Proc. Natl. Acad. Sci. U.S.A.

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“…This feature of drug efflux pump deficiency is intuitively obvious, but recent findings for the ribosome targeting macrolide antibiotic erythromycin suggest the existence of more subtle aspects of the interplay between drug efflux pump efficiency and drug susceptibility. An Escherichia coli mutant with an amino acid deletion in protein L22 of the large ribosomal subunit (10,11), causing reduced affinity of erythromycin to the ribosome (12), displayed reduced susceptibility to erythromycin in relation to a ribosome WT strain in a drug efflux pump proficient but the same susceptibility in a drug efflux pump deficient background (12,13). These experiments demonstrated how a mutation reducing drug affinity to a vital intracellular target may give a distinct growth advantage to drug exposed bacteria in a drug efflux pump proficient background but virtually no advantage in a drug efflux pump deficient background.…”

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confidence: 99%

Drug efflux pump deficiency and drug target resistance masking in growing bacteria

Fange

Nilsson²,

Tenson³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Recent experiments have shown that drug efflux pump deficiency not only increases the susceptibility of pathogens to antibiotics, but also seems to ''mask'' the effects of mutations, that decrease the affinities of drugs to their intracellular targets, on the growth rates of drug-exposed bacteria. That is, in the presence of drugs, the growth rates of drug-exposed WT and target mutated strains are the same in a drug efflux pump deficient background, but the mutants grow faster than WT in a drug efflux pump proficient background. Here, we explain the mechanism of target resistance masking and show that it occurs in response to drug efflux pump inhibition among pathogens with high-affinity drug binding targets, low cell-membrane drug-permeability and insignificant intracellular drug degradation. We demonstrate that target resistance masking is fundamentally linked to growth-bistability, i.e., the existence of 2 different steady state growth rates for one and the same drug concentration in the growth medium. We speculate that target resistance masking provides a hitherto unknown mechanism for slowing down the evolution of target resistance among pathogens.antibiotic resistance ͉ efflux pump inhibition ͉ macrolides

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“…Studies demonstrating assembly of ribosomes from a pool of subunits showed an excess of SpcS strains when isogenic merodiploids were constructed by introducing an SpcS allele episomally into a SpcR background [6]. Other merodiploids constructed between SpcR and SpcS strains were unstably SpcS, and segregated SpcR derivatives more frequently than expected from spontaneous mutation [7]. It is possible that SpcS alleles could be dominant in a heterozygote through incorporation of sensitive subunits into spcR ribosomes [6,8].…”

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confidence: 99%

Spectinomycin Resistance in rpsE Mutants is Recessive in Streptomyces roseosporus

Miao

Baltz

2005

J Antibiot

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Eight spontaneous mutants of Streptomyces roseosporus resistant to spectinomycin (SpcR) were mapped to distinct transversions or deletions in the rpsE gene. Most of the mutations were strongly recessive to the wild type SpcS allele. This suggests that some SpcR alleles of rpsE may be useful in a spectinomycin based counterselection system. Keywords spectinomycin, rpsE, ribosomal protein S5, Streptomyces reseosporus, resistance Spectinomycin is a bacteriostatic aminocyclitol antibiotic that inhibits protein synthesis. It affects ribosome function by binding to the 30S subunit and inhibiting translocation of peptidyl-tRNA from the A-site to the P-site [1,2]. Translocation involves local pivoting of the head of the ribosome relative to the body, and is sterically hindered by the presence of the antibiotic [3]. Amino acid substitutions in ribosomal protein S5 [4], the product of the rpsE gene, is associated with resistance to spectinomycin (SpcR). This protein is located on the 30S subunit and interacts with 16S rRNA and other proteins. Crystallographic studies of the Bacillus stearothermophilus S5 protein showed that it contains an N-terminal dsRNA binding region that interacts with 16S rRNA helix H3 [4,5].Early experiments on ribosome morphogenesis in E. coli hinted that sensitivity to spectinomycin might be a dominant phenotype. Studies demonstrating assembly of ribosomes from a pool of subunits showed an excess of SpcS strains when isogenic merodiploids were constructed by introducing an SpcS allele episomally into a SpcR background [6]. Other merodiploids constructed between SpcR and SpcS strains were unstably SpcS, and segregated SpcR derivatives more frequently than expected from spontaneous mutation [7]. It is possible that SpcS alleles could be dominant in a heterozygote through incorporation of sensitive subunits into spcR ribosomes [6,8]. In this study, we isolated a collection of spontaneous SpcR rpsE mutants of S. roseosporus, complemented them with a cloned rpsE gene and showed that SpcS is dominant over SpcR. Cloning of the S. roseosporus rpsE geneThe approach used for cloning the rpsE gene took advantage of the synteny observed in the chromosomes of model actinomycetes [9,10], in this case, the local order of genes around rpsE (Fig. 1A). Primers P949 and P950 (5ЈCGSGARGCSGGVCTSAAGTTC3Ј and 5ЈCTTSAG-CTTSGGVAGVCGCATGTG3Ј) targeting rplR and rplO were used to amplify (92°C 15 seconds, 52°C 15 seconds, 72°C 45 seconds, 30 cycles) an intervening region from S. roseosporus UA343 (NRRL 15998). The sequenced PCR product (deposited as Genbank AY772011) was found to include rpsE and rpmD, indicating that the rplR · rpsE · rpmD · rplO continguity was also conserved in S. roseosporus (Fig. 1B). The rpsE gene sequence was GC rich (69.3%) and the 200 residue deduced S5 peptide was

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Approaches to the Genetics of Escherichia coli Ribosomes

Cited by 32 publications

References 0 publications

Revisiting the mechanism of macrolide-antibiotic resistance mediated by ribosomal protein L22

Revisiting the mechanism of macrolide-antibiotic resistance mediated by ribosomal protein L22

Drug efflux pump deficiency and drug target resistance masking in growing bacteria

Spectinomycin Resistance in rpsE Mutants is Recessive in Streptomyces roseosporus

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