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E Nyssen

Publications and source records attributed to E Nyssen.

10 recordsLinked to original sources
PubMed1998

pGR71 plasmid promotor sequence temporally regulated in Bacillus subtilis.

pGR71, a composite of plasmids pUB110 and pBR322, replicates in Escherichia coli and in Bacillus subtilis. It carries the chloramphenicol resistance gene (cat) from Tn9, which is not transcribed in either host by lack of a promoter. The cat gene is preceded by a Shine-Dalgarno sequence functional in E. coli but not in B. subtilis. Deleted pGR71 plasmids were obtained in B. subtilis when cloning foreign viral DNA upstream of this cat sequence, as well as by BAL31 exonuclease deletions extending upstream from the cat into the pUB110 moiety. These mutant plasmids expressed chloramphenicol acetyltransferase (CAT), conferring on B. subtilis resistance to high chloramphenicol concentrations. CAT expression peaked at the early postexponential phas of B. subtilis growth. The transcription initiation site of cat, determined by primer extension, was located downstream of a putative promoter sequence within the pUB110 moiety. N-terminal amino acid sequencing showed that native CAT was produced by these mutant plasmids. The cat ribosome-binding site, functional in E. coli, was repositioned within the pUB110 moiety and had consequently an extended homology with B. subtilis 16S rRNA, explaining the production of native enzyme.

Bacillus subtilis↗
PubMed1997

Inhibition of protein synthesis by streptogramins and related antibiotics.

The streptogramins and related antibiotics (the lincosamides and macrolides) (MLS) are important inhibitors of bacterial protein synthesis. The key reaction in this process is the formation of a peptide bond between the growing peptide chain (peptidyl-tRNA) linked to the P-site of the 50S ribosome and aminoacyl-tRNA linked to the A site. This reaction is catalysed by the peptidyl transferase catalytic centre of the 50S ribosome. Type A and B streptogramins in particular have been shown to block this reaction through the inhibition of substrate attachment to the A and P sites and inhibition of peptide chain elongation. Synergy between type A and B components results from conformational changes imposed upon the peptidyl transferase centre by type A compounds and by inhibition of both early and late stages of protein synthesis. The conformational change increases ribosomal affinity for type B streptogramins. Microbial resistance to the MLSB antibiotics is largely attributable to mutations of rRNA bases, producing conformational changes in the peptidyl transferase centre. This can result in resistance to a single inhibitor or to a group of antibiotics (MLSB). The activity of type A streptogramin is retained thus explaining the improved inhibitory action of the combined streptogramins against macrolide and lincosamide-resistant strains. However, the development of resistance to the streptogramins may be less of a problem because of the synergic effect of type A and B compounds which has also been demonstrated in strains resistant to MLSB i.e., high level resistance to the combined streptogramins is only likely when type A streptogramin resistance determinants are present along with type B streptogramin resistance determinants.

Anti-Bacterial Agents↗
PubMed1991

Interaction between virginiamycin S and ribosomes is partly provided by a salt bridge with a Mg2+ ion.

Type B streptogramins, such as virginiamycin S (VS), are cyclic hexadepsipeptides, inhibiting protein synthesis in prokaryotes. L-Thr connects a 3-hydroxypicolinyl residue (3-OH-Pic) to the peptide lactone ring. The fluorescence intensity of 3-OH-Pic is strongly increased by chelation to alkaline earth cations or binding to ribosomes. Similar behavior of the ribosome-VS complex and the VS-Mg chelate provides strong evidence for the presence of a VS-Mg chelate within the ribosomal binding site. Different models involving the ribosome binding of either members of the VS-Mg2+ chelate or both have been tested by fluorescence lifetime measurements, equilibrium titrations, and stopped-flow spectrofluorometry. Our data strongly suggest that (a) the interaction between VS and the ribosome is partly provided by a salt bridge between suitable acceptor atoms of the ribosome and the 3-OH-Pic residue, (b) Mg2+ can be exchanged by Mn2+ without dissociation of the ribosome-VS complex, (c) Mg2+ coordinates to the negative form of the 3-OH-Pic residue, probably via an interaction with the phenolate oxygen and the amide carboxyl group, and (d) the picolinyl residue is essential for the biological activity, as indicated by the lack of activity when the latter is replaced by a serine derivative.

Binding Sites↗
PubMed1991

Do you also have problems with the file format syndrome?

In a biomedical data processing environment, an essential requirement is the ability to integrate a large class of standard modules for the acquisition, processing and display of the (image) data. Our approach to the management and manipulation of the different data formats is based on the specification of a common standard for the representation of data formats, called 'data nature descriptions' to emphasise that this representation not only specifies the structure but also the contents of data objects (files). The idea behind this concept is to associate each hardware and software component that produces or uses medical data with a description of the data objects manipulated by that component. In our approach a special software module (a format convertor generator) takes care of the appropriate data format conversions, required when two or more components of the system exchange data.

Electronic Data Processing↗
PubMed1990

Affinity labeling of the virginiamycin S binding site on bacterial ribosome.

Virginiamycin S (VS, a type B synergimycin) inhibits peptide bond synthesis in vitro and in vivo. The attachment of virginiamycin S to the large ribosomal subunit (50S) is competitively inhibited by erythromycin (Ery, a macrolide) and enhanced by virginiamycin M (VM, a type A synergimycin). We have previously shown, by fluorescence energy transfer measurements, that virginiamycin S binds at the base of the central protuberance of 50S, the putative location of peptidyltransferase domain [Di Giambattista et al. (1986) Biochemistry 25, 3540-3547]. In the present work, the ribosomal protein components at the virginiamycin S binding site were affinity labeled by the N-hydroxysuccinimide ester derivative (HSE) of this antibiotic. Evidence has been provided for (a) the association constant of HSE-ribosome complex formation being similar to that of native virginiamycin S, (b) HSE binding to ribosomes being antagonized by erythromycin and enhanced by virginiamycin M, and (c) a specific linkage of HSE with a single region of 50S, with virtually no fixation to 30S. After dissociation of covalent ribosome-HSE complexes, the resulting ribosomal proteins have been fractionated by electrophoresis and blotted to nitrocellulose, and the HSE-binding proteins have been detected by an immunoenzymometric procedure. More than 80% of label was present within a double spot corresponding to proteins L18 and L22, whose Rfs were modified by the affinity-labeling reagent. It is concluded that these proteins are components of the peptidyltransferase domain of bacterial ribosomes, for which a topographical model, including the available literature data, is proposed.

Affinity Labels↗
PubMed1989

Analysis of the reversible binding of virginiamycin M to ribosome and particle functions after removal of the antibiotic.

Type A synergimycins (VM) were shown to act catalytically and to induce two ribosomal alterations: (a) inability to promote polypeptide synthesis; (b) high-affinity binding of type B synergimycins (VS). A claim for irreversible binding of type A synergimycins to ribosomes has promoted the present reinvestigation. Submission of ribosomes from VM-treated bacteria to a purification procedure (supposed to remove the drug, according to a low association constant previously reported) yielded particles still holding residual VM. The formation of VM.ribosome complexes, more stable than previously inferred but without covalent linkage, was deduced from the extractability of complexed VM by organic solvents. Moreover, incubation of these complexes with increasing amounts of anti-VM immunoglobulins progressively restored ribosome activity in protein synthesis. Binding of VS to ribosomes, by fluorimetric titrations in the presence of substoichiometric concentrations of VM, was incompatible with catalytic action of type A synergimycins. Ribosomes from VM-treated bacteria displayed also a higher affinity for VS than did control ribosomes. This property did not disappear when ribosome.VM complexes were incubated with anti-VM IgG, nor when VM-IgG complexes were withdrawn from the reaction mixture by protein A-agarose binding. We can conclude that VM binding produces: (1) an inhibition of ribosome-promoted peptide bond formation, which occurs only in the presence of the drug; and (2) an increase of ribosome affinity for VS, which lasts after VM removal. The linkage of this drug with ribosomes is tight but reversible and its action is stoichiometric.

Anti-Bacterial Agents↗
PubMed1988

Action of erythromycin and virginiamycin S on polypeptide synthesis in cell-free systems.

Erythromycin (a 14-membered macrolide) and virginiamycin S (a type B synergimycin) block protein biosynthesis in bacteria, but are virtually inactive on poly(U)-directed poly(Phe) synthesis. We have recently shown, however, that these antibiotics inhibit the in vitro polypeptide synthesis directed by synthetic copolymers: this effect is analyzed further in the present work. We were unable to find any consistent alteration produced by these antibiotics on coupled and uncoupled EF-G- and EF-Tu-dependent GTPases, on the EF-Tu-directed binding of aminoacyl-tRNA to ribosomes, and on the EF-G- and GTP-mediated translocation of peptidyl-tRNA bound to poly(U,C).ribosome complexes. With these complexes, the peptidyl transfer reaction, as measured by peptidylpuromycin synthesis, was 10-30% inhibited by virginiamycin S and erythromycin. A direct relationship between the virginiamycin S- and erythromycin-promoted inhibition of poly(A,C)-directed polypeptide synthesis, on the one hand, and the EF-G concentration and the rate of the polymerization reaction, on the other hand, was observed, in agreement with a postulated reversible inhibitor action of these antibiotics. The increased inhibitory activity, which was observed during the first 4-6 rounds of elongation, in the presence of virginiamycin S or erythromycin, was suggestive of a specific action of these antibiotics on the correct positioning of peptidyl-tRNA at the P site. The marked stimulation of premature release of peptidyl-tRNA from poly(A,C).ribosome complexes can be referred to an altered interaction of the C-terminal aminoacyl residue of the growing peptidyl chain with the ribosome. We conclude that the action of virginiamycin S and erythromycin entails a template-dependent alteration of the interaction of peptidyl-tRNA with the donor site of peptidyltransferase, which may lead to a transient functional block of the ribosome and in some instances to a premature release of peptidyl-tRNA and termination of the elongation process.

Erythromycin↗
PubMed1988

Inhibition of polypeptide synthesis in cell-free systems by virginiamycin S and erythromycin. Evidence for a common mode of action of type B synergimycins and 14-membered macrolides.

Macrolides, lincosamides and type B synergimycins are powerful inhibitors of protein synthesis in vivo, but many of them were found to be inactive in vitro. In the present work, we confirm that virginiamycin S (a type B synergimycin) and erythromycin (a 14-membered macrolide) have no effect on poly(U)-directed poly(Phe) synthesis. However, the amino-acid polymerization reactions directed by poly(U,G), poly(U,C), poly(A,G) and poly(A,C) were increasingly inhibited (20-50%) by both antibiotics. The action of these inhibitors proved to be template-dependent and favored by the incorporation of proline and of basic amino acids into peptides. Under these conditions, virginiamycin S and erythromycin markedly stimulated a release of peptidyl-tRNA from the ribosomes. In the poly(A,C) model system, these antibiotics produced a 50% inhibition of amino-acid incorporation into total peptides, a 70% release of ribosome-bound peptidyl-tRNA, and a 95% repression of the synthesis of long peptide chains. The production of equivalent effects at saturating concentrations of these antibiotics in the four model systems examined is suggestive of a similarity in their mode of action. Our results indicate that 14-membered macrolides and type B synergimycins can act on ribosomes during the whole elongation process. The functional block produced by both antibiotics is usually reversible, but may result in a premature release of peptidyl-tRNA when the stability of ribosomal complexes is lowered by the incorporation of basic amino acids.

Anti-Bacterial Agents↗
PubMed1987

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.

Erythromycin↗