Alteration of ribosomal protein L6 in gentamicin-resistant strains of Escherichia coli. Effects on fidelity of protein synthesis

Kühberger, R; Piepersberg, Wolfgang; A, Petzet; Buckel, Peter; Böck, August

doi:10.1021/bi00568a028

Cited by 84 publications

(40 citation statements)

References 32 publications

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“…Neomycin, gentamicin, and streptomycin all impair the proofreading control (reviewed in references 5 and 9), and our results lead us to suggest that the 2660 loop belongs to the ribosomal site which controls the proofreading step. The situation encountered with mutations in the 2660 loop of 23S rRNA is reminiscent of what was previously observed with mutations in the 50S protein L6 (21), which were also found to counteract the misreading effect of either streptomycin or gentamicin. L6, a protein located at the subunit interface, has been mapped in the proximity of the 2660 loop (27) and probably modulates the conformation of functional sites in 23S rRNA.…”

Section: Discussionmentioning

confidence: 54%

Single-base mutations at position 2661 of Escherichia coli 23S rRNA increase efficiency of translational proofreading

1992

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Two single-base substitutions were constructed in the 2660 loop of Escherichia coli 23S rRNA (G2661-C or U) and were introduced into the rrnB operon cloned in plasmid pKK3535. Ribosomes were isolated from bacteria transformed with the mutated plasmids and assayed in vitro in a poly(U)-directed system for their response to the misreading effect of streptomycin, neomycin, and gentamicin, three aminoglycoside antibiotics known to impair the proofreading control of translational accuracy. Both mutations decreased the stimulation of misreading by these drugs, but neither interfered with their binding to the ribosome. The response of the mutant ribosomes to these drugs suggests that the 2660 loop, which belongs to the elongation factor Tu binding site, is involved in the proofreading step of the accuracy control. In vivo, both mutations reduced read-through of nonsense codons and frameshifting, which can also be related to the increased efficiency in proofreading control which they confer to ribosomes.Streptomycin, an error-enhancing aminoglycoside antibiotic, perturbs translational fidelity primarily by interfering with the proofreading control (39,42,48). The proofreading control of translational accuracy triggers the rejection of near-cognate tRNAs which escaped the initial discrimination step of tRNA decoding. According to the two-step model for tRNA decoding, the initial discrimination step is mediated by the reversible binding of the ternary complex of aminoacyl-tRNA.EF-Tu.GTP to the ribosome, and the proofreading control takes place following hydrolysis of the GTP complexed to elongation factor Tu (EF-Tu). Thompson and coworkers have shown that proofreading efficiency depends upon the rate of dissociation of EF-Tu.GDP from the ribosome (reviewed in reference 47); the lower this rate, the higher the probability of rejecting a near-cognate tRNA before allowing the incorporation of the aminoacyl residue into the peptide bond. This view has been challenged by Ehrenberg et al. (13), who think that EF-Tu.GDP has left the ribosome before proofreading of tRNA. Although EF-Tu interacts with the 30S subunit, the 50S subunit provides the major determinants for EF-Tu binding (reviewed in references 28 and 34). One can thus assume that the 50S subunit should play an important part in proofreading control.Attempts to understand the interference of streptomycin with proofreading control have focused on 30S mutants which fail to bind the drug and consequently abolish its misreading effect. This was the case for the classical streptomycin-resistant mutants with altered ribosomal protein S12 (35) and for mutations in the 915 region or the stem-loop 530 region of 16S rRNA (25,26,38). We have hypothesized that it should be possible to obtain mutants which, while still binding the drug, would decrease its effect. In this study, we have investigated whether mutations in the 2660 loop of 23S rRNA interfere with the action of streptomycin. This loop is known to be located in the binding site of 27,33), which makes it a likely candidat...

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Section: Discussionmentioning

confidence: 54%

Single-base mutations at position 2661 of Escherichia coli 23S rRNA increase efficiency of translational proofreading

1992

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“…Bacteria carrying these extrachromosomal elements inactivate antibiotics by means of enzymatic reactions. Gentamicin resistance obtained in vitro is frequently due to failure of the antibiotic to reach the ribosomal target or to structural alteration of the ribosomal binding site for gentamicin (1,9). Mutations induced by the light used in phototherapy most probably involve one of the in vitro mechanisms.…”

Section: Discussionmentioning

confidence: 99%

Induction of Gentamicin Resistance by Visible Light

et al. 1981

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Pediatr. Res. 15: 952-955 (1981) SummaryRecent studies have demonstrated the ability of visible light or phototherapy to modify the intracellular DNA of prokaryotic and eukaryotic cells. The present study was undertaken to determine the effect of light used in phototherapy on antibiotic resistance in prokaryotic cells using tester strains of gentamicin-sensitive Escherichia coli and Staphylococcus aureus. A growing population of the tester microorganisms was inoculated on plates containing nutrient medium and gentamicin. Experiments were performed to determine the effect of blue light on the induction of gentamicinresistant mutants. The plates were divided into two populations, one of which was illuminated, while the other was kept in the dark to serve as a control. During photoirradiation, the plates were protected from direct sunlight and air cooled to maintain a temperature of 27°C. The sample distance from the light source was adjusted to maintain a fluence rate (450 nm) of 141 uW/cm2.Control experiments were performed to investigate the effect of photoirradiation on the media and gentamicin. An increased frequency of mutation to gentamicin resistance was seen in the irradiated population of bacteria. The mutagenic effect was observed over a wide range of gentamicin concentrations and correlated in a linear fashion with increasing duration of photoirradiation. There was an inverse correlation between the size of the bacterial inoculum and the recovery of mutant bacteria. SpeculationIn view of the demonstrated ability of high-intensity visible light to modify intracellular DNA and induce mutations in bacterial populations, the relationship between the widespread use of phototherapy and the emergence of multiple antibiotic-resistant bacteria in nursery populations needs to be determined. ducing large numbers of resistant organisms. The purpose of the present study is to investigate the role of phototherapy in the induction of gentamicin resistant bacteria. MATERIALS AND METHODS BACTERIAL STRAINSTwo strains of Escherichia coli (A and B) and a single Staphylococcus aureus were isolated from blood culture specimens by the Children's Hospital of Philadelphia Microbiology Laboratory. A third strain of E. coli (C) was provided by the Babies Hospital Microbiology Laboratory, Columbia Presbyterian Medical Center, New York. Organisms were maintained on nutrient agar slants and inoculated weekly on MacConkey plates (E. colq or sheep blood agar ( S . aureus). The minimum inhibitory concentrations to gentamicin for E. coli A and B were 5 1 pg/ml and for S. aureus, 5 2 pg/ml. The minimum bactericidal concentration for E. coli C was 5 pg/ml. A minimum inhibitory concentration was not determined for this organism.MEDIA AND ANTIBIOTIC PREPARATION Nutrient agar, containing beef extract and peptone (Difco Laboratories) was used for all experiments. Media were prepared following manufacturer's instructions, and the gentamicin was added to the media just before pouring the plates.Gentamicin (Clinical Laboratory Standard; Sch...

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“…Protein L6 can affect the accuracy of translation [4]: mutants containing altered L6 were resistant to gentamycin, which induces misreading, and failed to translate a 'leaky' amber codon. On the other hand, L6 has no significant effect on the peptidyl transferase activity of the 50-S subunit [5].…”

Section: -S Rnamentioning

confidence: 99%

“…Results were analysed using the classical Stern-Volmer approach : (4) where F, T and [Q] are fluorescence intensity, lifetime and quencher concentration respectively : the subscript o implies the absence of quencher, K,, is the Stern-Volmer constant and k, the collisional rate constant. This treatment ignores the so-called static quenching, which reduces F but not T , and the possibility of inefficient quenching (in which the probability of quenching, given collision, is less than unity).…”

Section: Quenching Titrationsmentioning

confidence: 99%

Structure of Ribosomal Protein L6 from Escherichia coli

Steinhäuser

Woolley

Epe

et al. 1982

European Journal of Biochemistry

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Protein L6 from the 50-S ribosomal subunit has been investigated using fluorimetric techniques. The intrinsic fluorophore Trp-61 and fluorescent labels (acetylaminoethyl-dansyl and acetylaminofluorescein) attached to the residue Cys-124 were used. It proved possible to incorporate fluorescence-labelled L6 into the 50-S ribosome.Trp-61 is exposed to solvent, as shown by its emission wavelength and by quenching experiments; the latter also show that it lies in a pocket with a high positive charge due to the basic residues in the N-terminal fragment. Cys-124 lies in a less strongly positive region. Upon incorporation into the 50-S subunit, the label on Cys-124 becomes less accessible for quenching but its positive potential rises, showing the absence of direct contact with Protein L6 can affect the accuracy of translation [4]: mutants containing altered L6 were resistant to gentamycin, which induces misreading, and failed to translate a 'leaky' amber codon. On the other hand, L6 has no significant effect on the peptidyl transferase activity of the 50-S subunit [5]. Available structural data on L6 are at present restricted to the sequence [6] and the hydrodynamic axial ratio [7]. The aim of this investigation was to find out as much as possible about the structure of L6 in the regions surrounding its intrinsic fluorophore Trp-61 and, using a fluorescent label, its single cysteine at position 124. MATERIALS A N D METHODS Prcpurution of' Biological MutcriulBu!f&s were as follows. I : NaCl350 mM, PO:-20 mM, dithioerythritol 0.5 mM, benzamidine 0.1 mM, phenylmethanesulphonyl fluoride 0.1 mM, pH 7.0. Ia: as I but acetate replaces chloride and 5 mM 2-mercaptoethanol replaces dithioAhhrevicitions. TP50, total protein from the 50-S subunit of the E,vc./zerichiu coli ribosome; IAEDANS, 5-[2-(iodoacetyl)aminoethyl]-aminonaphthalene-1-sulphonic acid; IAFI, 5-iodoacetamidofluorescein; AEDANSL6. FILL6, protein L6 labelled with IAEDANS or IAFI, respectively; AcTrpNHl, N-acetyl-tryptophanamide; AcTyrNHl. Nacetyl-tyrosinamide; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid; AEDANS-PME and FI-PME, conjugates of 2-mercaptoethanol with IAEDANS and IAFI respectively; SDS, sodium dodecyl sulphate; Bicine, N,N-bis(2-hydroxyethyl)glycine. ~~~ erythritol. 11: KCI 3.50 mM, MgC12 20 mM, Hepes 15 mM, dithioerythritol 0.5 mM, pH 7.5. 111: NaCl 100 mM, EDTA 10 mM, Hepes 10 mM, 2-mercaptoethanol 6 mM, pH 7.9. IV: ('polymix', cf. Jelenc [S]) KCI 95 mM, NH4Cl 5 mM. MgCl2 5 mM, CaCI2 0.5 mM, K2HP04 5 mM, putrescine 8 mM, spermidine 1 mM, dithioerythritol 1 mM, pH 7.5. V: NH4CI 150 m M , magnesium acetate 5 mM, Tris 50 mM, dithioerythritol 1 mM, pH 7.5.Ribosomes from Escherichiu coli (MRE 600) were prepared by the method of Hapke and Noll [9].Ribosomal Subunits and Protein L6 were purified by the method of Dijk and Littlechild [lo] avoiding denaturing reagents. 0.8 M LiCl cores were prepared by the method given by Homann and Nierhaus [l I]. The cores were pelleted, dissolved in buffer I1 and dialysed against buffer I1 for 5 h (Spectrapor...

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Alteration of ribosomal protein L6 in gentamicin-resistant strains of Escherichia coli. Effects on fidelity of protein synthesis

Cited by 84 publications

References 32 publications

Single-base mutations at position 2661 of Escherichia coli 23S rRNA increase efficiency of translational proofreading

Single-base mutations at position 2661 of Escherichia coli 23S rRNA increase efficiency of translational proofreading

Induction of Gentamicin Resistance by Visible Light

Structure of Ribosomal Protein L6 from Escherichia coli

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