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The effects of disruptions in ribosomal active sites and in intersubunit contacts on ribosomal degradation in Escherichia coli.

ABSTRACT: Although ribosomes are very stable under most conditions, ribosomal degradation does occur in diverse groups of organisms in response to specific stresses or environmental conditions. While non-functional ribosome decay (NRD) in yeast is well characterized, very little is known of the mechanisms that initiate ribosomal degradation in bacteria. Here we test ribosome degradation in growing Escherichia coli expressing mutant ribosomes. We found that mutations in the 16S rRNA decoding centre (G530U and A1492C) and 23S rRNA active site (A2451G) do not lead to ribosomal degradation. In contrast, 23S rRNA mutation U2585A causes degradation of both the large and small ribosomal subunits in E. coli. We further tested mutations in 23S rRNA, which disrupt ribosomal intersubunit bridges B2a and B3. Deletion of helix 69 of 23S rRNA and the point mutation A1912G in the same helix did not destabilize ribosomes, while expression of mutations A1919G in H69 and A1960G in H71 led to degradation of both mutant and wild-type ribosomes. Our results suggest an actively induced mechanism requiring de novo protein synthesis for ribosomal degradation in E. coli, which degrades both structurally inactive and active ribosomes.

SUBMITTER: Paier A

PROVIDER: S-EPMC4289901 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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The effects of disruptions in ribosomal active sites and in intersubunit contacts on ribosomal degradation in Escherichia coli.

Paier Anton A Leppik Margus M Soosaar Aksel A Tenson Tanel T Maiväli Ülo Ü

Scientific reports 20150112

Although ribosomes are very stable under most conditions, ribosomal degradation does occur in diverse groups of organisms in response to specific stresses or environmental conditions. While non-functional ribosome decay (NRD) in yeast is well characterized, very little is known of the mechanisms that initiate ribosomal degradation in bacteria. Here we test ribosome degradation in growing Escherichia coli expressing mutant ribosomes. We found that mutations in the 16S rRNA decoding centre (G530U ...[more]

PMID: 25578614

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Project description:Tigecycline is a tetracycline derivative that is being used as an antibiotic of last resort. Both tigecycline and tetracycline bind to the small (30S) ribosomal subunit and inhibit translation. Target mutations leading to resistance to these antibiotics have been identified both in the 16S ribosomal RNA and in ribosomal proteins S3 and S10 (encoded by the rpsJ gene). Several different mutations in the S10 flexible loop tip residue valine 57 (V57) have been observed in tigecycline-resistant Escherichia coli isolates. However, the role of these mutations in E. coli has not yet been characterized in a defined genetic background. In this study, we chromosomally integrated 10 different rpsJ mutations into E. coli, resulting in different exchanges or a deletion of S10 V57, and investigated the effects of the mutations on growth and tigecycline/tetracycline resistance. While one exchange, V57K, decreased the minimal inhibitory concentration (MIC) (Etest) to tetracycline to 0.75 μg/ml (compared to 2 μg/ml in the parent strain) and hence resulted in hypersensitivity to tetracycline, most exchanges, including the ones reported previously in resistant isolates (V57L, V57D, and V57I) resulted in slightly increased MICs to tigecycline and tetracycline. The strongest increase was observed for the V57L mutant, with a MIC (Etest) to tigecycline of 0.5 μg/ml (compared to 0.125 μg/ml in the parent strain) and a MIC to tetracycline of 4.0 μg/ml. Nevertheless, none of these exchanges increased the MIC to the extent observed in previously described clinical tigecycline-resistant isolates. We conclude that, next to S10 mutations, additional mutations are necessary in order to reach high-level tigecycline resistance in E. coli. In addition, our data reveal that mutants carrying S10 V57 exchanges or deletion display growth defects and, in most cases, also thermosensitivity. The defects are particularly strong in the V57 deletion mutant, which is additionally cold-sensitive. We hypothesize that the S10 loop tip residue is critical for the correct functioning of S10. Both the S10 flexible loop and tigecycline are in contact with helix h31 of the 16S rRNA. We speculate that exchanges or deletion of V57 alter the positioning of h31, thereby influencing both tigecycline binding and S10 function.

Dataset Information

The effects of disruptions in ribosomal active sites and in intersubunit contacts on ribosomal degradation in Escherichia coli.

Publications

The effects of disruptions in ribosomal active sites and in intersubunit contacts on ribosomal degradation in Escherichia coli.

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