Analysis of the ribosomes of a micrococcin-resistant strain of Bacillus megaterium.
thesisposted on 19.11.2015, 09:07 by G. (Gary) Spedding
Micrococcin is a modified peptide antibiotic which contains sulphur. It is an inhibitor of bacterial protein synthesis and acts by binding directly to the 5OS ribosomal subunit. The drug also affects various partial reactions of protein synthesis. Of particular relevance here is the effect of micrococcin upon "uncoupled" hydrolysis of GTP catalysed jointly by the ribosome and the protein factor EF-G. Such GTPase which occurs in the absence of mRNA, tRNA and other factors normally required for protein synthesis is markedly stimulated by micrococcin. Moreover, a qualitative relationship exists between the level of stimulation of GTP hydrolysis and the level of inhibition of protein synthesis. In view of this, the ribosomal response to micrococcin in GTPase assays was used as an indicator of the levels of sensitivity. A mutant strain of Bacillus megaterium arising spontaneously and resistant to micrococcin possesses ribosomes which contain an altered form of protein BM-L11 (the homologue of Escherichia coli protein L11). The ribosomes from this mutant were highly resistant to micrococcin. Reconstitution analysis has revealed that the alteration to protein BM-L11 is the sole cause of resistance to micrococcin in this mutant. Ribosomes lacking protein BM-L11 were supplemented with the missing protein, purified from the wildtype or from the mutant. When the protein from the wildtype was employed, the reconstituted ribosomes exhibited wildtype characteristics, i.e. "uncoupled" GTP hydrolysis was stimulated by micrococcin. However, when protein from the resistant strain was employed GTP hydrolysis catalysed by the reconstituted ribosomes was not affected by the drug. A binding site for micrococcin can be constructed in vitro solely from 23S rRNA and protein BM-L11, although the fine details of the ribosomal target site are not known. However, methylation of the 23S rRNA at a single specific-residue confers total resistance to the drug. It is therefore probable that the antibiotic binds primarily to 23S rRNA and that protein (BM)-L11 promotes such binding. Consequently it is of interest to note that alterations in this protein can lead to resistance to micrococcin.