Fluorescence stopped flow analysis of the interaction of virginiamycin components and erythromycin with bacterial ribosomes

The kinetics of the interaction between the 50 S subunits (R) of bacterial ribosomes and the antibiotics virginiamycin S (VS), virginiamycin M (VM), and erythromycin have been studied by stopped flow fluorimetric analysis, based on the enhancement of VS fluorescence upon its binding to the 50 S ribosomal subunit. Virginiamycin components M and S exhibit a synergistic effect in vivo, which is characterized in vitro by a 5- to 10-fold increase of the affinity of ribosomes for VS, and by the loss of the ability of erythromycin to displace VS subsequent to the conformational change (from R to R*) produced by transient contact of ribosomes with VM. Our kinetic studies show that the VM-induced increase of the ribosomal affinity for VS (K*VS = 25 X 10(6) M-1 instead of KVS = 5.5 X 10(6) M-1) is due to a decrease of the dissociation rate constant (k*-VS = 0.008 s-1 instead of 0.04 s-1). The association rate constant remains practically the same (k+VS approximately k*+VS = 2.8 X 10(5) M-1 s-1), irrespective of the presence of VM. VS and erythromycin bind competitively to ribosomes. This effect has been exploited to determine the dissociation rate constant of VS directly by displacement experiments from VS . 50 S complexes, and the association rate constant of erythromycin (k+Ery = 3.2 X 10(5) M-1 S-1) on the basis of competition experiments for binding of free erythromycin and VS to ribosomes. By making use of the change in competition behavior of erythromycin and VS, after interaction of ribosomes with VM, the conformational change induced by VM has been explored. Within the experimentally available concentration region, the catalytic effect of VM has been shown to be coupled to its binding kinetics, and the association rate constant of VM has been determined (k+VM = 1.4 X 10(4) M-1 S-1). Evidence is presented for a low affinity binding of erythromycin (K*Ery approximately 3.3 X 10(4) M-1) to ribosomes altered by contact with VM. A model involving a sequence of 5 reactions has been proposed to explain the replacement of ribosome-bound erythromycin by VS upon contact of 50 S subunits with VM.     

Binding of erythromycin to the 50S ribosomal subunit is affected by alterations in the 30S ribosomal subunit

Summary Expression of resistance to erythromycin in Escherichia coli, caused by an altered L4 protein in the 50S ribosomal subunit, can be masked when two additional ribosomal mutations affecting the 30S proteins S5 and S12 are introduced into the strain (Saltzman, Brown, and Apirion, 1974). Ribosomes from such strains bind erythromycin to the same extent as ribosomes from erythromycin sensitive parental strains (Apirion and Saltzman, 1974).Among mutants isolated for the reappearance of erythromycin resistance, kasugamycin resistant mutants were found. One such mutant was analysed and found to be due to undermethylation of the rRNA. The ribosomes of this strain do not bind erythromycin, thus there is a complete correlation between phenotype of cells with respect to erythromycin resistance and binding of erythromycin to ribosomes.Furthermore, by separating the ribosomal subunits we showed that 50S ribosomes bind or do not bind erythromycin according to their L4 protein; 50S with normal L4 bind and 50S with altered L4 do not bind erythromycin. However, the 30s ribosomes with altered S5 and S12 can restore binding in resistant 50S ribosomes while the 30S ribosomes in which the rRNA also became undermethylated did not allow erythromycin binding to occur.Thus, evidence for an intimate functional relationship between 30S and 50S ribosomal elements in the function of the ribosome could be demonstrated. These functional interrelationships concerns four ribosomal components, two proteins from the 30S ribosomal subunit, S5, and S12, one protein from the 50S subunit L4, and 16S rRNA.     

Erythromycin resistance by L4/L22 mutations and resistance masking by drug efflux pump deficiency

We characterized the effects of classical erythromycin resistance mutations in ribosomal proteins L4 and L22 of the large ribosomal subunit on the kinetics of erythromycin binding. Our data are consistent with a mechanism in which the macrolide erythromycin enters and exits the ribosome through the nascent peptide exit tunnel, and suggest that these mutations both impair passive transport through the tunnel and distort the erythromycin‐binding site. The growth‐inhibitory action of erythromycin was characterized for bacterial populations with wild‐type and L22‐mutated ribosomes in drug efflux pump deficient and proficient backgrounds. The L22 mutation conferred reduced erythromycin susceptibility in the drug efflux pump proficient, but not deficient, background. This ‘masking’ of drug resistance by pump deficiency was reproduced by modelling with input data from our biochemical experiments. We discuss the general principles behind the phenomenon of drug resistance ‘masking’, and highlight its potential importance for slowing down the evolution of drug resistance among pathogens.     

Mutation from guanine to adenine in 25S rRNA at the position equivalent to E. coli A2058 does not confer erythromycin sensitivity in Sacchromyces cerevisae

The macrolide erythromycin binds to the large subunit of the prokaryotic ribosome near the peptidyltransferase center (PTC) and inhibits elongation of new peptide chains beyond a few amino acids. Nucleotides A2058 and A2059 (E. coli numbering) in 23S rRNA play a crucial role in the binding of erythromycin, and mutation of nucleotide A2058 confers erythromycin resistance in both gram-positive and gram-negative bacteria. There are high levels of sequence and structural similarity in the PTC of prokaryotic and eukaryotic ribosomes. However, eukaryotic ribosomes are resistant to erythromycin and the presence of a G at the position equivalent to E. coli nucleotide A2058 is believed to be the reason. To test this hypothesis, we introduced a G to A mutation at this position of the yeast Saccharomyces cerevisiae 25S rRNA and analyzed sensitivity toward erythromycin. Neither growth studies nor erythromycin binding assays on mutated yeast ribosomes indicated any erythromycin sensitivity in mutated yeast strains. These results suggest that the identity of nucleotide 2058 is not the only determinant responsible for the difference in erythromycin sensitivity between yeast and prokaryotes.     

Direct Measurements of Erythromycin’s Effect on Protein Synthesis Kinetics in Living Bacterial Cells

Macrolide antibiotics, such as erythromycin, bind to the nascent peptide exit tunnel (NPET) of the bacterial ribosome and modulate protein synthesis depending on the nascent peptide sequence. Whereas in vitro biochemical and structural methods have been instrumental in dissecting and explaining the molecular details of macrolide-induced peptidyl-tRNA drop-off and ribosome stalling, the dynamic effects of the drugs on ongoing protein synthesis inside live bacterial cells are far less explored. In the present study, we used single-particle tracking of dye-labeled tRNAs to study the kinetics of mRNA translation in the presence of erythromycin, directly inside live Escherichia coli cells. In erythromycin-treated cells, we find that the dwells of elongator tRNA^Phe on ribosomes extend significantly, but they occur much more seldom. In contrast, the drug barely affects the ribosome binding events of the initiator tRNA^fMet. By overexpressing specific short peptides, we further find context-specific ribosome binding dynamics of tRNA^Phe, underscoring the complexity of erythromycin’s effect on protein synthesis in bacterial cells.     

Properties of human mitochondrial ribosomes

Mammalian mitochondrial ribosomes (55S) differ unexpectedly from bacterial (70S) and cytoplasmic ribosomes (80S), as well as other kinds of mitochondrial ribosomes. Typical of mammalian mitochondrial ribosomes, the bovine mitochondrial ribosome has been developed as a model system for the study of human mitochondrial ribosomes, to address several questions related to the structure, function, biosynthesis and evolution of these interesting ribosomes. Bovine mitochondrial ribosomal proteins (MRPs) from each subunit have been identified and characterized with respect to individuality and electrophoretic properties, amino acid sequence, topographic disposition, RNA binding properties, evolutionary relationships and reaction with affinity probes of ribosomal functional domains. Several distinctive properties of these ribosomes are being elucidated, including their antibiotic susceptibility and composition. Human mitochondrial ribosomes lack several of the major RNA stem structures of bacterial ribosomes but they contain a correspondingly higher protein content (as many as 80 proteins), suggesting a model where proteins have replaced RNA structural elements during the evolution of these ribosomes. Despite their lower RNA content they are physically larger than bacterial ribosomes, because of the 'extra' proteins they contain. The extra proteins in mitochondrial ribosomes are 'new' in the sense that they are not homologous to proteins in bacterial or cytoplasmic ribosomes. Some of the new proteins appear to be bifunctional. All of the mammalian MRPs are encoded in nuclear genes (a separate set from those encoding cytoplasmic ribosomal proteins) which are evolving more rapidly than those encoding cytoplasmic ribosomal proteins. The MRPs are imported into mitochondria where they assemble coordinately with mitochondrially transcribed rRNAs into ribosomes that are responsible for translating the 13 mRNAs for essential proteins of the oxidative phosphorylation system.     

A gratuitous inducer of cat-86, amicetin, inhibits bacterial peptidyl transferase.

Expression of the chloramphenicol resistance gene cat-86 is regulated by translation attenuation. Among the three ribosomally targeted antibiotics that can induce the gene, only amicetin has an unknown mode of action. Here we demonstrate that the nucleoside antibiotic amicetin is an inhibitor of bacterial peptidyl transferase. Thus, the three inducers of cat-86, chloramphenicol, erythromycin, and amicetin, interact with the peptidyl transferase region of bacterial ribosomes.     

Macrolide-ketolide inhibition of MLS-resistant ribosomes is improved by alternative drug interaction with domain II of 23S rRNA

The macrolide antibiotic erythromycin and its 6-O-methyl derivative (clarithromycin) bind to bacterial ribosomes primarily through interactions with nucleotides in domains II and V of 23S rRNA. The domain II interaction occurs between nucleotide A752 and the macrolide 3-cladinose moiety. Removal of the cladinose, and substitution of a 3-keto group (forming the ketolide RU 56006), results in loss of the A752 interaction and an approximately 100-fold drop in drug binding affinity. Within domain V, the key determinant of drug binding is nucleotide A2058 and substitution of G at this position is the major cause of drug resistance in some clinical pathogens. The 2058G mutation disrupts the drug-domain V contact and leads to a further > 25 000-fold decrease in the binding of RU 56006. Drug binding to resistant ribosomes can be improved over 3000-fold by forming an alternative and more effective contact to A752 via alkyl-aryl groups linked to a carbamate at the drug 11/12 position (in the ketolide antibiotics HMR 3647 and HMR 3004). The data indicate that simultaneous drug interactions with domains II and V strengthen binding and that the domain II contact is of particular importance to achieve binding to the ribosomes of resistant pathogens in which the domain V interaction is perturbed.     

Accumulation in gram-postive and gram-negative bacteria as a mechanism of resistance to erythromycin

Erythromycin was recovered in high yield after incubation with gram-negative bacteria. The cell-free protein-synthesizing preparation from gram-negative bacteria is equally as susceptible to the antibiotic as is that from gram-positive bacteria. Thus, neither destruction of erythromycin nor the absence of the step susceptible to the antibiotic plays an important role in the resistance mechanism of gram-negative bacteria. A 100-fold difference in accumulation of erythromycin between gram-positive and gram-negative bacteria was observed. This alone explains the resistance of gram-negative bacteria to erythromycin. Furthermore, data showed that the inhibition of growth is closely related to the accumulation of erythromycin. The concentration of intracellular erythromycin in gram-positive bacteria was found to be 44- to 90-fold greater than that of the extracellular medium. However, the antibiotic did not accumulate on the cell walls, nor was the accumulation energy-dependent. It is proposed that it takes place by the binding of erythromycin to the bacterial ribosomes, forming a very stable complex. The dissociation constants of erythromycin-Staphylococcus aureus complex and erythromycin-Bacillus subtilis complex were determined to be 1.1 x 10(-7) and 3.4 x 11(-7)m, respectively.     

Interaction of bovine mitochondrial ribosomes with Escherichia coli initiation factor 3 (IF3)

Mammalian mitochondrial ribosomes are distinguished from their bacterial and eukaryotic-cytoplasmic counterparts, as well as from mitochondrial ribosomes of lower eukaryotes, by their physical and chemical properties and their high protein content. However, they do share more functional homologies with bacterial ribosomes than with cytoplasmic ribosomes. To search for possible homologies between mammalian mitochondrial ribosomes and bacterial ribosomes at the level of initiation factor binding sites, we studied the interaction of Escherichia coli initiation factor 3 (IF3) with bovine mitochondrial ribosomes. Bacterial IF3 was found to bind to the small subunit of bovine mitochondrial ribosomes with an affinity of the same order of magnitude as that for bacterial ribosomes, suggesting that most of the functional groups contributing to the IF3 binding site in bacterial ribosomes are conserved in mitochondrial ribosomes. Increasing ionic strength affects binding to both ribosomes similarly and suggests a large electrostatic contribution to the reaction. Furthermore, bacterial IF3 inhibits the Mg2+-dependent association of mitochondrial ribosomal subunits, suggesting that the bacterial IF3 binds to mitochondrial small subunits in a functional way.     

Use of 50 S-binding antibiotics to characterize the ribosomal site to which peptidyl-tRNA is bound.

Five antibiotics (puromycin, erythromycin, lincomycin, sparsomycin, and virginiamycin M1) that bind specifically to the 50 S ribosomal subunit near the peptidyl transferase center were used to compare and characterize the positions of bound AcylPhe-tRNA in the puromycin-reactive and -unreactive states. Binding of the antibiotics was quantitatively measured by their perturbation of fluorescence from probes attached to the alpha-amino group of Phe-tRNA. Derivatives of three probes with differing chemical characteristics and environmental sensitivities were used: a coumarin, an aminonaphthalenesulfonate, and a pyrene. The effects of the antibiotics on the fluorescence of labeled AcylPhe-tRNAs in the two states, while generally qualitatively similar, are nonetheless quantitatively distinct, as are the calculated binding constants for the antibiotics. Puromycin, as reported earlier, binds to both the puromycin-reactive and -unreactive states, but its dissociation constant is higher for the latter state. Erythromycin binds tightly to ribosomes bearing labeled AcylPhe-tRNA in either the puromycin-reactive or -unreactive state. Its effect on the fluorescence of the labeled tRNA is very similar in the two states, except with the pyrene probe, where it has a larger effect in the puromycin-reactive state. Lincomycin and sparsomycin bind to both ribosomal states, but both bind more tightly to the puromycin-reactive state, the extent of the difference varying with the identity of the fluorescent probe. Virginiamycin M1 binds to ribosomes with AcylPhe-tRNA in the puromycin-reactive site, but its binding could not be detected to ribosomes with AcylPhe-tRNA in the puromycin-unreactive site.     

Interaction between the erythromycin and chloramphenicol binding sites on the Escherichica coli ribosome.

The effects of chloramphenical on the binding kinetics of a fluorescein isothiocyanate derivative of 9(S)-erythromycylamine with 70S and 50S ribosomes have been studied by direct fluorimetric measurements. While chloramphenicol had little effect on the second-order 70S binding rate of the erythromycin analogue, it substantially reduced the dissociation rate of the fluorescent antibiotic-70S ribosome complex. This could be explained by simultaneous binding of both antibiotics to the 70S ribosome. The kinetic results suggest that chloramphenicol-saturated 70S particles bind the erythromycin analogue four times stronger and this was confirmed by direct binding studies. In additon, chloramphenicol causes a twofold increase in the intrinsic fluorescence of the 70S-bound analogue. This increase in fluorescence was used to study the kinetics of chloramphenicol binding to 70S ribosomes containing the fluorescent derivative. The fluorescence change followed first-order kinetics, suggesting that chloramphenicol induces a conformational change in the 70S particle. This could explain both its effect on erythromycin binding and on the fluorescence of bound analogue. Less detailed results with the 50S particle indicate a qualitively similar picture of erythromycin-chloramphenicol interactions.     

Ribosomes from spiramycin resistant mutants of Escherichia coli Q13

Summary Mutants from Escherichia coli Q13 were selected for resistance to leucomycin, tylosin or spiramycin. Most of the mutants so selected exhibited cross resistance to all the macrolide antibiotics tested including erythromycin. A few mutants however seem to be less resistant to erythromycin. One mutant, QSP008, was highly resistant to tylosin, leucomycin and spiramycin but relatively sensitive to erythromycin. Another mutant, QSP006, was highly resistant to spiramycin but less resistant to erythromycin, tylosin and leucomycin. This selective resistance of cells to specific antibiotics could be due to the extent of conformational alteration of their ribosomes, which may be demonstrated by the extent of ¹⁴C-erythromycin binding to these ribosomes. The ribosomes from QSP008 cells were found to contain an altered 50-8 protein of the 50s ribosomal subunit, while in the ribosomes from QSP006 no such protein change could be detected by the methods used.A preliminary data of part of this work has been published (Tanaka, Teraoka, Tamaki, Watanabe, Osawa, Otaka, and Takata, 1971).     

Partition of ribosomes in two-polymer aqueous phase systems

Two-polymer aqueous phase systems are described in which ribosomes selectively partition into one of the phases. One of the phase systems is used to determine rapidly and conveniently binding of thiostrepton and erythromycin to Escherichia coli ribosomes under equilibrium conditions.     

Further similarities between chloroplast and bacterial ribosomes

Summary Protein synthesis by chloroplasts isolated under aseptic conditions from Phaseolus vulgaris leaves is inhibited by the bacterial antibiotics spectinomycin, lincomycin, and erythromycin; that by chloroplasts from Nicotiana tabacum leaves is inhibited by spectinomycin and lincomycin but not by erythromycin. Protein synthesis by cytoplasmic ribosomes from plants and animals is not inhibited by these compounds, nor is amino acid activation by the soluble fraction from bean chloroplasts. These results suggest that chloroplast ribosomes possess sites which bind several unrelated bacterial antibiotics and support the idea that chloroplasts originated from prokaryotic cells. These antibiotics may be useful in studying the process of chloroplast formation in intact cells.     

A plasmid-coded and site-directed mutation in Escherichia coli 23S RNA that confers resistance to erythromycin: implications for the mechanism of action of erythromycin

Primer-directed mutagenesis was employed to introduce an A2058----G transition in plasmid-encoded Escherichia coli 23S RNA at a site that has been implicated, indirectly, in erythromycin binding. The mutation raises the growth tolerance of cells from 30 to 300 micrograms/ml of erythromycin, and cells grown in the presence of erythromycin contain ribosomes with high levels of mutated 23S RNA. In these cells, wild type 50S subunits 'fall off' the message and are selectively degraded, possibly as a result of an erythromycin-induced conformational change. A fast in vitro poly(U) assay revealed minimal effects of erythromycin on elongation beyond tetrapeptides. We correlated these results with the literature data and concluded that erythromycin acts immediately post-initiation and directly, or indirectly, destabilizes mRNA-bound 70S ribosomes, and prevents their recycling by causing 50S subunit degradation.     

Antibacterial Activity of 2′-Esters of Erythromycin

The effect of esterification at the 2'-position of desosamine on the antibacterial activity of erythromycin was investigated by determining the bacteriostatic and bactericidal activities of erythromycin and a number of its 2'-esters on S. aureus and relating these activities to the hydrolysis rates of the esters. These studies, together with comparison of the inhibition of protein synthesis in a cell-free system isolated from S. aureus, lead to the conclusion that 2'-esters of erythromycin are inactive until hydrolyzed. Loss of activity appears to result from inability of erythromycin esters to bind to bacterial ribosomes and thus inhibit synthesis of protein.     

Evolution of a protein-rich mitochondrial ribosome: implications for human genetic disease

Mitochondrial ribosomes comprise the most diverse group of ribosomes known. The mammalian mitochondrial ribosomes (55S) differ unexpectedly from bacterial (70S) and cytoplasmic ribosomes (80S), as well as other kinds of mitochondrial ribosomes. The bovine mitochondrial ribosome has been developed as a model system for the study of human mitochondrial ribosomes to address several questions related to the structure, function, biosynthesis and evolution of these interesting ribosomes. Bovine mitochondrial ribosomal proteins (MRPs) from each subunit have been identified and characterized with respect to individuality and electrophoretic properties, amino acid sequence, topographic disposition, RNA binding properties, evolutionary relationships and reaction with affinity probes of ribosomal functional domains. Several distinctive properties of these ribosomes are being elucidated, including their antibiotic susceptibility and composition. Mammalian mitochondrial ribosomes lack several of the major RNA stem structures of bacterial ribosomes but they contain a correspondingly higher protein content (as many as 80 proteins), suggesting a model where proteins have replaced RNA structural elements during the evolution of these ribosomes. Despite their lower RNA content they are physically larger than bacterial ribosomes, because of the 'extra' proteins they contain. The extra proteins in mitochondrial ribosomes are 'new' in the sense that they are not homologous to proteins in bacterial or cytoplasmic ribosomes. Some of the new proteins appear to be bifunctional. All of the mammalian MRPs are encoded in nuclear genes (a separate set from those encoding cytoplasmic ribosomal proteins) which are evolving more rapidly than those encoding cytoplasmic ribosomal proteins. The MRPs are imported into mitochondria where they assemble coordinately with mitochondrially transcribed rRNAs into ribosomes that are responsible for translating the 13 mRNAs for essential proteins of the oxidative phosphorylation system. Interest is growing in the structure, organization, chromosomal location and expression of genes for human MRPs. Proteins which are essential for mitoribosome function are candidates for involvement in human genetic disease.     

Effect of viomycin on dihydrostreptomycin binding to bacterial ribosomes.

Viomycin, a peptide antibiotic, reduced the amounts of dihydrostreptomycin bound to ribosomes of Myobacterium smegmatis and Escherichia coli, although they have different modes of action. The [3H]dihydrostreptomycin binding to ribosomes could not exchanged with streptomycin or dihydrostreptomycin, but not with unrelated antibiotics, namely, kanamycin, neomycin, spectinomycin, capreomycin, tuberactinomycin-N, chloramphenicol and erythromycin. We suggest that there is a significant interaction between the binding sites of viomycin and streptomycin on ribosomes.     

Kinetics of binding of macrolides, lincosamides, and synergimycins to ribosomes

The synergistic effect of type A (virginiamycin M (VM)) and type B (virginiamycin S (VS)) synergimycins and their antagonistic effect against erythromycin (a 14-membered macrolide) for binding to the large ribosomal subunit (50 S) have been related. This investigation has now been extended to 16-membered macrolides (leucomycin A3 and spiramycin) and to lincosamides (lincomycin). A dissociation of VS-ribosome complexes was induced as well by 16-membered macrolides as by lincosamides. The observed dissociation rate constant of VS-ribosome complexes was identified with the kappa-vs in the case of 16-membered macrolides, but linearly related to lincomycin concentration, suggesting a direct binding of the latter antibiotic to VS-ribosome complexes and the triggering of a conformational change of particles entailing VS release. Two different mechanisms were also involved in the VM-promoted reassociation to ribosomes of VS previously displaced by either macrolides or lincosamides. By binding to lincosamide-ribosome complexes, VM induced a conformational change of ribosomes resulting in higher affinity for VS and lower affinity for lincosamides. On the contrary, an incompatibility for a simultaneous binding of VM and 16-membered macrolides to ribosomes was observed. These results have been interpreted by postulating specific (nonoverlapping) and aspecific (overlapping) antibiotic binding sites at the peptidyltransferase domain. All the kinetic constants of five antibiotic families (type A and B synergimycins, 14- and 16-membered macrolides, and lincosamides) and a topological model of peptidyltransferase are presently available.