Alteration of ribosomal protein L6 in mutants of Escherichia coli resistant to gentamicin

Buckel, Peter; Buchberger, Alexander; Böck, August; Wittmann, H. G.

doi:10.1007/bf00455118

Cited by 64 publications

(59 citation statements)

References 15 publications

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“…Recently, we reported the substitution of CtrL6e into E. coli ribosomes without any deleterious effect despite its overproduction (13). To (3). These studies prompted us to look for such a situation with CtrL6e, the chlamydial EcoL6 homolog.…”

Section: Discussionmentioning

confidence: 99%

Cloning and sequence analysis of the Chlamydia trachomatis spc ribosomal protein gene cluster

et al. 1992

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We identified and sequenced a segment of Chlamydia trachomatis chromosomal DNA that shows homology to the Escherichia coli spc and distal region of the S10 ribosomal protein (r-protein) operons. Its sequence revealed a high degree of nucleotide and operon context conservation with the E. coli r-protein genes. The C. trachomatis spc operon contains the r-protein genes for L14, L24, L5, S8, L6, L18, S5, L15, and Sec Y along with the genes for r-proteins L16, L29, and S17 of the S10 operon. The two operons are separated by a 16-bp intragenic region which contains no transcription signals. However, a putative promoter for the transcription of the spc operon was found 162 nucleotides upstream of the CtrL14e start site; it revealed significant homology to the E. coli consensus promoter sequences. Interestingly, our results indicate the absence of any structure resembling an EcoS8 regulatory target site on C. trachomatis spc mRNA in spite of significant amino acid identity between E. coli and C. trachomatis r-proteins. Also, the intrinsic aminoglycoside resistance in C. trachomatis is unlikely to be mediated by CtrL6e since E. coli expressing CtrL6e remained susceptible to gentamicin (MIC < 0.5 ,ug/ml).Chlamydia trachomatis infections represent major public health problems in both developing and industrialized countries (30). Chlamydia species have evolved a complex and unique developmental cycle which involves two distinct forms: the small (0.2 to 0.3 ,um) extracellular, rigid elementary bodies and the large (1 ,um) intracellular, fragile reticulate bodies (44). The genetics of chlamydial regulation is largely undefined, mainly because of the lack of any convenient system for gene transfer and also because of the paucity of information about the signals and machinery that govern gene expression.Ribosomes constitute the protein-synthesizing machinery of both prokaryotes and eukaryotes. The entire prokaryotic ribosome comprises three rRNAs with sedimentation coefficients of 23S, 16S, and 5S and approximately 52 ribosomal proteins (r-proteins) that are organized into 19 different operons (26,33). Earlier attempts at characterizing the rRNA from Chlamydia species have met with some success. Tamura and Iwanaga (42) identified 21S, 16S, and 4S rRNA fractions in Chlamydia psittaci; of these, 21S and 16S were more predominant forms in reticulate bodies, whereas 4S predominated in the elementary bodies. Sarov and Becker found similar rRNA species in C. trachomatis (39). On the basis of their 16S rRNA gene sequence, chlamydiae have been identified as eubacterial in origin, related peripherally to planctomyces (45). Although a few other ribosomal determinants have been identified, r-protein gene organization and expression have not been well characterized (7-10, 20, 21). Interestingly, the organization and transcriptional regulation of r-protein operons appear to be well conserved among eubacteria; however, their regulation among evolutionary distant species remains unelucidated (15).Recently, we reported the cloning and sequen...

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Section: Discussionmentioning

confidence: 99%

Cloning and sequence analysis of the Chlamydia trachomatis spc ribosomal protein gene cluster

et al. 1992

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“…Escherichia coli strain GE20-8/AB2834-5 (a gift from Dr Bock) was obtained by transferring the rplF gene from the original isolate GE20-8 (resistant to gentamicin and other 2-deoxystreptamine aminoglycosides) to strain AB2834 [14]. It possesses a mutated L6 protein.…”

Section: Bacterial Strainsmentioning

confidence: 99%

Mechanism of action of gentamicin components

Tangy¹,

Moukkadem²,

Vindimian³

et al. 1985

European Journal of Biochemistry

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The binding of gentamicin (Gm) to Escherichia coli ribosomes and ribosomal subunits has been studied. By means of equilibrium dialysis and of statistical interpretation of the data it was found that [3H]gentamicin Cz and 6'-N-[3H]methylgentamicin C1, interact with three classes of sites on tight-coupled 70-S species: (a) a first class concerning the tight and non-cooperative interaction with one drug molecule (Kd = 0.6 pM), (b) a second class in which about five Gm molecules bind cooperatively (mean Kd = 10 pM), and (c) a third class of very high capacity in which up to 70 drug molecules may interact. The extreme cooperativity of the third class of sites induces such an increase in the affinity for Gm that it may allow the shift of molecules already bound from highaffinity sites towards lower-affinity sites. The alteration of a ribosomal protein, L6, in a gentamicin-resistant mutant of E. coli abolished the multiclass and the cooperative aspects of ribosomes -gentamicin interaction. The large ribosomal subunits from E. coli MRE 600 strain interact cooperatively with Gm, whereas 50-S particles from the resistant mutant bind the drug in a diffuse way with high capacity and low affinity. The small subunits from both strains behave identically towards Gm. A good correlation is observed in comparing the gentamicin concentrations capable of saturating the different ribosomal classes of sites with concentrations inducing its multiphasic effects on protein synthesis.

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“…which confers resistance to thiostrepton [lo]. Otherwise, transduction analysis had rendered it highly likely that rcsistance to erythromycin can result from changes affecting protein L4 [27], that low-level resistance to gentamicin can be caused by alterations to protein L6 [28] and that the state of protein L3 or L4 governs the ribosomal response to tiamulin [29].…”

Section: Discussionmentioning

confidence: 99%

Identification of the altered ribosomal component responsible for resistance to micrococcin in mutants of Bacillus megaterium

Spedding

Cundliffe

1984

Eur J Biochem

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A mutant strain of Bacillus megaterium, arising spontaneously and resistant to micrococcin, possesses ribosomes which contain an altered form of protein BM-L1 1 (the homologue of Escherichia coli protein L11). Reconstitution analysis has revealed that the alteration to protein BM-L11 is the sole cause of resistance in this strain.Micrococcin, a modified peptide antibiotic, is related in structure to the better known compound thiostrepton (for revised structures, see [l, 21. The relationship between the two drugs also extends to their modes of action. Both inhibit bacterial protein synthesis by binding directly to the 50s ribosomal subunit and both affect various partial reactions of the peptide chain elongation cycle, probably all involving the ribosomal A site [3 -51. Of particular relevance in the present context are the effects of micrococcin and thiostrepton upon 'uncoupled' hydrolysis of GTP catalysed jointly by the ribosome and the protein factor, EF-G. Such GTPase activity, which occurs in the absence of other components (e. g. mRNA, tRNA etc.) normally required for protein synthesis, is powerfully inhibited by thiostrepton [6] but is markedly stimulated by micrococcin [5]. Quite how this novel effect of micrococcin is related to the drug's inhibitory action upon protein synthesis is unclear (for speculation, see [5]) but, since it occurs exclusively upon ribosomes from micrococcin-sensitive organisms, its significance cannot be doubted. This effect also represents the only recorded disparity between the actions of micrococcin and thiostrepton and we have exploited it here in some of our crucial experiments.Recently the ribosomal binding sites for micrococcin and thiostrepton were shown to be closely related. Both antibiotics bind to complexes formed between ribosomal protein L11 and 23s rRNA and methylation of the latter at a single specific site (residue A-1067) renders ribosomes totally resistant to the action of either drug [5,7]. Given such an intimate relationship between the target sites for micrococcin and thiostrepton, it might have been expected that genetic lesions resulting in resistance to either agent would confer cross-resistance to the other. However: although thiostrepton-resistant mutants of Bacillus subtilis were cross-resistant to micrococcin, resistant strains selected on the latter drug remained sensitive to thiostrepton [S]. (Since micrococcin, like thiostrepton, does not penetrate into gram-negative organisms, Escherichia coli could not be used in such experiments, although ribosomes from that organism are fully sensitive to the two drugs in vitro.) On detailed examination, 50s ribosomal subunits from thiostrepton-resistant mutants of B. subtilis [9] or Bacillus megaAbbreviations. Me,SO, dimethylsulphoxide; SDS, sodium do-terium [lo] proved to be devoid of a single protein, homologous with ribosomal protein L11 of E. coli, and this deficiency evidently accounted for the phenotype of those strains. In contrast, the ribosomes from micrococcin-resistant mutants possessed a full com...

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Alteration of ribosomal protein L6 in mutants of Escherichia coli resistant to gentamicin

Cited by 64 publications

References 15 publications

Cloning and sequence analysis of the Chlamydia trachomatis spc ribosomal protein gene cluster

Cloning and sequence analysis of the Chlamydia trachomatis spc ribosomal protein gene cluster

Mechanism of action of gentamicin components

Identification of the altered ribosomal component responsible for resistance to micrococcin in mutants of Bacillus megaterium

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