Biomedical subjects

C Cocito

Publications and source records attributed to C Cocito.

At least 73 records · Page 4Linked to original sources

PubMed1987

Ribosome protection by tRNA derivatives against inactivation by virginiamycin M: evidence for two types of interaction of tRNA with the donor site of peptidyl transferase.

Virginiamycin M (VM) was previously shown to interfere with the function of both the A and P sites of ribosomes and to inactivate tRNA-free ribosomes but not particles bearing peptidyl-tRNA. To explain these findings, the shielding ability afforded by tRNA derivatives positioned at the A and P sites against VM-produced inactivation was explored. Unacylated tRNA(Phe) was ineffective, irrespective of its position on the ribosome. Phe-tRNA and Ac-Phe-tRNA provided little protection when bound directly to the P site but were active when present at the A site. Protection by these tRNA derivatives was markedly enhanced by the formation of the first peptide bond and increased further upon elongation of peptide chains. Most of the shielding ability of Ac-Phe-tRNA and Phe-tRNA positioned at the A site was conserved when these tRNAs were translocated to the P site by the action of elongation factor G and GTP. Thus, a 5-10-fold difference in the protection afforded by these tRNAs was observed, depending on their mode of entry to the P site. This indicates the occurrence of two types of interaction of tRNA derivatives with the donor site of peptidyl transferase: one shared by acylated tRNAs directly bound to the ribosomal P site (no protection against VM) and the other characteristic of aminoacyl- or peptidyl-tRNA translocated from the A site (protection of peptidyl transferase against VM). To explain these data and previous observations with other protein synthesis inhibitors, a new model of peptidyl transferase is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-Bacterial Agents↗

PubMed1987

Immunological properties of antigen 60 of BCG. Induction of humoral and cellular immune reactions.

Antigen 60 (A60), a member of the thermostable macromolecular antigen family (TMA) and main component of old tuberculin and purified protein derivative (PPD), has been purified from the cytoplasm of Mycobacterium bovis BCG; its structure and metabolism have already been described. In the present paper, the action of A60 on humoral immunity has been analysed by an ELISA type immunoassay, and that on cellular immunity by the mouse footpad swelling test. Injection of very low A60 doses into unprimed mice produced an undetectable level of anti-A60 antibodies; the effect of a booster inoculation was not appreciable in the absence of incomplete Freund's adjuvant, but was evident when the latter was added. Higher doses of the antigen produced an appreciable primary response, and a sharp and long-lasting secondary response, which had a 10-fold higher intensity in the presence of incomplete adjuvant. No detectable delayed hypersensitivity reactions were observed in unprimed mice after footpad injection of A60, whereas clear responses were elicited in primed mice. This effect was more pronounced when the footpad was injected after a secondary response than after a primary response, and it was invariably magnified by incomplete adjuvant. It is concluded that A60 is a powerful immunogen, which is able to induce primary and secondary responses and delayed hypersensitivity reactions, effects that are adjuvant-modulated and develop concurrently.

Animals↗

PubMed1986

Localization of virginiamycin S binding site on bacterial ribosome by fluorescence energy transfer.

Virginiamycin S, a type B synergimycin inhibiting protein synthesis in bacteria, competes with erythromycin for binding to the 50S ribosomal subunits; the mechanism of action of the two antibiotics is unclear. Energy-transfer experiments between virginiamycin S (which is endowed with inherent fluorescence due to its hydroxypicolinyl moiety) and fluorescent coumarinyl derivatives of ribosomal proteins L7 and L10 have been carried out to locate the binding site of this antibiotic on the ribosome. Previous studies have indicated that two L7/L12 dimers can attach respectively to a strong binding site located on the central protuberance and to a weak binding site located on the stalk of the 50S subunits and that protein L10 is located at the base of the stalk. The distance between ribosome-bound virginiamycin S and a fluorophore located at the strong binding site of proteins L7/L12 (Lys-51 of L7) was found to be 56 (+/- 15) A. Virginiamycin S, on the other hand, was located at a distance exceeding 67 A from the weak binding site of L7/L12 dimers. A fluorophore positioned on the unique cysteine (Cys-70) of protein L10 and ribosome-bound virginiamycin S proved to be more than 60 A apart. From data available on the location of proteins L7/L12 and L10, a model is proposed, whereby the virginiamycin S binding site is placed at the base of the central protuberance of the 50S subunits, in proximity of the presumptive peptidyl transferase center. The binding sites of macrolides and lincosamides (related antibiotics of the MLS group) are expected to be very close to that of virginiamycin S.

Binding Sites↗

PubMed1986

Antagonistic interactions of macrolides and synergimycins on bacterial ribosomes.

The affinity of ribosomes for VS (virginiamycin S, a type B synergimycin) is known to be increased by VM (virginiamycin M, a type A synergimycin). Erythromycin, a macrolide, displaces ribosome-bound VS in the absence of VM, but is ineffective in its presence. In the present work, the ability of spiramycin and tylosin (macrolide subgroups) derivatives to displace ribosome-bound VS, in the presence and in the absence of VM, has been explored. All macrolides with in-vitro activity displaced ribosome-bound VS: the displacement curves produced by tylosin and spiramycin derivatives virtually overlapped. When VM was added to these systems, displaced VS became readily attached to ribosomes in the case of erythromycin, did not bind appreciably within 20 min incubation in the presence of tylosin, and underwent a slow binding in the case of an N-substituted tylosin. The 16-membered macrolides (leucomycin, spiramycin and tylosin subgroups) can therefore be distinguished from the 14-membered macrolides (erythromycin subgroup) by the antagonistic effect displayed toward VM.

Bacteria↗

PubMed1986

Chemical composition of antigen 60 from Mycobacterium bovis BCG.

Antigen 60 (A60), the main thermostable immunogen of tuberculin and PPD, has been purified from Mycobacterium bovis BCG cytoplasm, and identified by crossed immunoelectrophoresis with anti-BCG polyclonal antiserum. Two A60 fractions, free lipids and lipid-conjugated compounds, have been recognized. The free lipids represented about 30% (dry weight), and consisted essentially of C16-C18 fatty acids, and of phosphatidyl-inositol-mannosides. Lipoconjugates, upon DEAE-cellulose chromatography and gel filtration, yielded two main fractions of neutral and polar components. Chromatography of delipidated and deproteinized A60 on Sephadex G-100 yielded: a high molecular weight fraction (Al, 18%, a lipoglucan of congruent to 10(6)), and a low molecular weight fraction (B, 10%, a lipopeptidoglycan of congruent to 10(4)) containing mannose, glucose, and small amounts of arabinose. The polysaccharide moieties of fractions Al and B were submitted to acetylation, methylation, and acid hydrolysis, and the structure of the hydrolysed polymer was deduced by combined gas chromatography/mass spectrometry analysis. The results indicated a branched structure involving 1,4-, 1,6-, and 1,4,6-linked D-gluco- or D-manno-pyranosyl residues. Glucan- and peptidoglycan-bound fatty acids were identified as saturated (C16-C18) and monounsaturated linear acids (C12-C18). Immunodiffusion on agarose gel indicated that delipidation and proteolysis did not suppress the ability of A60 to yield immunoprecipitates with anti-A60 antiserum. The high polymer fractions obtained by chromatography on DEAE cellulose and Sephadex G-100 were also reactive. It is concluded that A60 is made of free lipids and of lipopeptidoglycans of high molecular weights (10(6)-10(7)) endowed with immunogenic properties.

Antigens, Bacterial↗

PubMed1986

Preparation and properties of antigen 60 from Mycobacterium bovis BCG.

Antigen 60 (A60) is the main thermostable immunogen of both 'old tuberculin' (OT) and 'purified protein derivative' (PPD), known reagents for cutaneous tests in tuberculosis. It is recognized by bidimensional immunoelectrophoresis with anti-BCG antiserum, where it appears as the less mobile component. A60 was prepared from the cytoplasm of Mycobacterium bovis BCG, and purified by exclusion gel chromatography and lectin affinity chromatography. Labelled A60 was obtained by radioiodination and used for a radioimmunoassay. Composition of A60 was explored by use of organic solvents, chemicals and enzymes. It contained two fractions of free and bound lipids, as well as protein and polysaccharide moieties. After removal of both free and bound lipid fractions, the core still retained the ability to form immunoprecipitinogen lines with anti-BCG antiserum. The lipopolysaccharide and lipo-protein moieties of A60, as well as the free lipid fraction, were also complexed by antibodies. It is concluded that A60 is a lipopolysaccharide-protein complex of 10(6) to 10(7) daltons, which is a major immunogenic component of mycobacterial cytoplasm. The detailed structure of this antigen, its immunological properties, and its use for an ELISA type immunoassay for tuberculosis are described in two other publications.

Antigens, Bacterial↗

PubMed1985

A microanalytical procedure for determination of the base composition of DNA.

A new procedure for the determination of the percentage guanine plus cytosine (% G+C; mol/100 mol) values of microquantities of DNA is described. Its principle is a DNA-polymerase-I-directed nick translation of DNA in the presence of dGTP, dTTP, [3H]dCTP, and [alpha-32P]dATP. Kinetics experiments indicate that the plateau value is reached in about 20 min of incubation under our experimental conditions. Percentage G+C is obtained from the linear relation 1/(% G+C) = 0.01 K [32P]/[3H] + 0.01, where the ratio of trichloroacetic-acid-precipitable radioactivity is taken into account, the K value being determined for each experiment by using a few reference DNAs of known composition. This procedure has proven suitable for analysis of plasmidic, viral and cellular DNAs of different base composition (25-75% G+C), shape (linear and circular double-stranded DNA) and size (100-150 000 base pairs). Usual methods for % G+C analysis (buoyant density and melting temperature determinations) yield unreliable results in the presence of either modified or unusual bases: the double-labeling procedure is still valid under these conditions. The latter is, therefore, the method of choice for analysis or rare DNA species which are available in very small quantities (it requires amounts of DNA as low as 1 ng, i.e. several order of magnitude lower than those used for chromatographic analysis of DNA hydrolysates). Since the obtention of highly purified DNA is an essential prerequisite for the double-labeling procedure, a method for purification of bacterial DNA is detailed in the present work.

Base Composition↗

PubMed1985

Biological, chemical, immunological and staining properties of bacteria isolated from tissues of leprosy patients.

Two kinds of microorganisms are found in tissue of leprosy patients: Mycobacterium leprae (ML) and leprosy derived corynebacteria (LDC). ML from untreated patients has an alcohol-acid-fastness, which is lost upon treatment with antibiotics and immune response (tuberculoid leprosy). Vulnerable ML thus produced can be reversibly de-stained by organic solvent: in tissue sections from tuberculoid and treated patients, more bacteria are, thus, revealed by the Wade-Fite than by the Ziehl-Neelsen procedure. Organisms of genera Corynebacterium, Mycobacterium and Nocardia (CMN group), have DNA with %GC contents of 50-70, 69-72, and 68-70 respectively. GC values of DNA from ML and LDC are close to 56%. DNA from different LDC strains display high homology among them and low homology with reference corynebacteria. CMN cell wall consists of interconnected peptidoglycan and polysaccharide-mycolate complex. Peptidoglycan of LDC (and known CMN) has the polysaccharide backbone linked to a tetrapeptide of L-Ala, D-Glu, m-DAP (meso-diaminopimelate), D-Ala. In ML, L-Ala is replaced by glycine. Mycobacterial wall polysaccharides (that of ML is unknown) are branched arabinogalactans with end arabinoses linked to C70 to C90 mycolates. LDC peripheral polysaccharides are arabinogalactomannans with arabinose and mannose lateral strands. Mycolic acids of LDC are of corynomycolic type (C32, C34 and C36 with 1-4 double bonds) and those of ML are of mycobacterial type. Components of CMN wall and cytoplasm are immunologically active as antigens (polysaccharides, proteins), haptens (lipids) and adjuvants (peptidoglycans). Strong intrageneric and weak intergenera crossreactions are observed among CMN bacteria: LDC preparations, however, crossreact strongly with ML and mycobacteria, and weakly with reference corynebacteria. LDC in leprosy tissues can, thus, be revealed as well by fluorescent anti-LDC antisera as by anti-ML antisera. The main crossreacting component is antigen M1 of LDC, which corresponds to antigens Ag 7 of ML and Ag60 of BCG, the active components of lepromin and tuberculin (known reagents for cutaneous tests). Antigen M1 has a polysaccharide moiety crossreacting with the wall polysaccharide of LDC.(ABSTRACT TRUNCATED AT 400 WORDS)

Corynebacterium↗

PubMed1985

Cross-reactivity of antigens from the cytoplasm and cell walls of some corynebacteria and mycobacteria.

Leprosy-derived corynebacteria (LDC) are non-acid-fast organisms isolated from leprosy lesions in humans. In this study 20 antigens of native LDC cytoplasm were identified by immunoelectrophoresis, and autoclaving yielded the M1 component, which strongly cross-reacted with antigen 60 of Mycobacterium bovis BCG (bacille Calmette-Guérin) and antigen 7 of Mycobacterium leprae. The polysaccharide moiety of M1 was immunologically related to the LDC cell wall polysaccharide previously characterized as arabinogalactomannan. The latter polysaccharide competitively inhibited the formation of immune complexes by labeled M1 and antisera to the LDC cell wall; cytoplasm and wall polysaccharides from other bacteria produced lower-level inhibition. In a radioimmunoassay with 125I-labeled antigen 7 of M. leprae, sera from patients with leprosy and antisera to the LDC cell wall yielded overlapping curves. Sera from patients with tuberculoid leprosy and those from patients with lepromatous leprosy afforded different levels of inhibition in this radioimmunoassay; this result indicated a difference in antibody specificity in the two forms of leprosy. In conclusion, the cell wall polysaccharide of LDC corresponds to the main thermostable cytoplasmic antigen M1, which strongly crossreacts with sera from patients with leprosy and, more specifically, with antigen 7 of M. leprae.

Antigens, Bacterial↗

PubMed1985

Molecular mechanism of action of virginiamycin-like antibiotics (synergimycins) on protein synthesis in bacterial cell-free systems.

Synergimycins contain two types of components, A and B, which synergistically inhibit in-vivo protein synthesis. In-vitro, both components interact with 50 s ribosomal subunits. B components bind in stoichiometric amounts (KA = 2.5 X 10(6) M-1): the association constant undergoes a ten-fold increase in the presence of A components. The latter inhibitors act in substoichiometric concentration (KA = 0.32 X 10(6) M-1) and produce lasting ribosome damage due to a conformational alteration requiring proteins L7/L12, L8 and L16. Such alteration entails a permanent block of the peptidyltransferase substrate acceptor site, whereby aminoacyl tRNA enzymically bound to ribosomes is released. Macrolides (KA = 7.2 X 10(7) M-1) displace ribosome-bound B components, but they are unable to compete in the presence of A components, which reduce both the affinity of ribosomes for macrolides, and the dissociation rate constant of B components. These findings provide a molecular explanation to the synergistic action of A and B components in-vivo.

Bacteria↗

PubMed1984

Analysis of fluorescence quenching of ribosome-bound virginiamycin S.

The two virginiamycin components VM and VS interact synergistically with bacterial ribosomes in vitro and in vivo. Ribosome affinity for virginiamycin S increases about 10-fold upon incubation with virginiamycin M. This effect has been previously traced by spectrofluorimetric measurement based on the enhancement of virginiamycin S fluorescence upon its binding to the 50 S ribosomal subunit. In the present work the action of two virginiamycin S fluorescence quenchers, acrylamide and iodide, has been explored to gather information about the accessibility of ribosome-bound virginiamycin S and the variation of the accessibility level in the presence of virginiamycin M. Both acrylamide (non-ionized quencher) and iodide (ionized quencher) proved powerful quenchers of free virginiamycin S solutions. Since a comparable effect was obtained on 3- hydroxypicolinamide , the latter was indicated as the part of the molecule involved in the fluorescence effect. Fluorescence quenching by either agent was of the dynamic, i.e. collisional, type. Such an inference was based on the fact that these quenchers merely modified the emission spectrum (not the absorption spectrum), the bimolecular rate constant for the quenching process decreased linearly with the viscosity of the medium (static-type quenching is viscosity-independent), and that linear Stern-Volmer plots were obtained. The quenching ability of both agents underwent a sharp decrease in the presence of ribosomes; however, the Stern-Volmer equation was followed only in the case of acrylamide, whereas Lehrer 's relationship had to be applied in the case of iodide. When ribosomes were incubated with virginiamycin M, the fluorescence quenching ability of acrylamide and iodide was significantly reduced. Conclusions are as follows: a) the 3- hydroxypicolinyl residue of virginiamycin S is buried within an open well on the ribosome surface and is likely to be involved in the interaction with the binding site; b) the accessibility to the well is partly controlled by electrostatic forces; c) interaction of ribosomes with virginiamycin M entails a conformational change whereby the access to the well is reduced. These findings provide a molecular explanation for the previously observed increase of the association constant of virginiamycin S to ribosomes incubated with virginiamycin M which was found to be due to the decrease of the dissociation rate constant (the association rate constant remains practically the same).

Anti-Bacterial Agents↗

PubMed1983

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.

Erythromycin↗

PubMed1983

Action of ions and pH on the binding of virginiamycin S to ribosomes.

Virginiamycin S (VS) binds to the 50 S ribosomal subunits (KaVS = 2.5 X 10(6) M-1); the affinity of ribosomes for VS undergoes a 6-fold increase (KaVS = 15 X 10(6) M-1) in the presence of virginiamycin M (VM). In the present work, the action of inorganic ions and pH on the binding reaction of VS to ribosomes was analyzed by a spectrofluorimetric technique, in the presence and in the absence of VM. Preliminary to this study, the interaction of ions with free VS was also explored. In aqueous solution and in the absence of VM, chelation by VS of K+, NH4+, Na+, Mg2+ and Ca2+ was observed. The binding of Mg2+ was strongly influenced by monovalent ions and pH between 7 and 8: the association constants for the VS:Mg2+ complex were 12.5 M-1 (+NH4+) and 45 M-1 (-NH4+). Binding of VS to ribosomes occurred as well in the presence of NH4+ as with K+, but was suppressed upon replacement of these ions by Na+. The association constant of the VS binding reaction to ribosomes was strongly enhanced by VM in the presence of either NH4+ or K+ but, while a plateau value was observed over the entire range of NH4+ concentrations, 100-fold higher values were obtained at low (25 mM) K+. This VM-induced increase of ribosome affinity for VS (KaVS) was dependent also on Mg2+, which displayed a higher synergistic action with K+ than with NH4+ (the highest KaVS values were recorded for 5 mM Mg2+ and 100 mM K+). In this reaction, Mg2+ could be replaced by Ca2+, whereas spermidine was ineffective. Also, the VM-promoted enhancement of the KaVS value increased with the pH. In conclusion, the two ribosomal functions analyzed in the present work, VS-binding to ribosome and VM-promoted enhancement of ribosome affinity for VS, rely on the concentration of monovalent and bivalent ions and on the pH. Both functions require either NH4+ or K+, and either Mg2+ or Ca2+ (the elements of each couple are not truly equivalent), from which ribosome conformation depends.

Binding Sites↗

PubMed1983

Comparison of the physical maps and redundant ends of the chromosomes of phages 2C, SP01, SP82 and phi e.

The physical map of 2C DNA (cf. following paper in this journal) was compared to the maps of SP01, SP82 and phi e (three other Bacillus subtilis phages containing hydroxymethyluracil in place of thymine in their DNA). The overall organization of the four genomes was remarkably similar, as indicated by the topology of HaeIII and SalI cleavage segments. The proof was gathered for the presence in the four phage DNAs of large redundant ends carrying a single HaeIII recognition site. The location of the latter proved identical for 2C and SP01, but was shifted in the DNAs of SP82 and phi e. Since the redundant end components of these hydroxymethyluracil genomes are colinear, as shown by cross-hybridization studies, the shifting of the HaeIII cleavage site is presumably due to two base substitutions, suppressing an endonuclease recognition site and establishing a new site elsewhere. Relatedness between the genomes of this family of viruses was evaluated from the fraction of conserved restriction fragments. According to these calculations, 6% base substitutions have occurred within the four viral DNAs, in the course of evolution. However, specific segments of 2C DNA were not present in SP01 and phi e DNA, as shown by cross-hybridization with restriction fragments. These data indicate the occurrence of deletions, in addition to base substitutions, as evolutionary mechanisms prevailing in the genomes of this family of phages.

Bacillus subtilis↗

PubMed1983

Physical map of phage 2 C DNA: evidence for the existence of large redundant ends.

The chromosome of the Bacillus subtilis phage 2C, a linear molecule of double-stranded DNA of about 10(8) Da, in which thymine is completely replaced by hydroxymethyluracil, was cleaved by different endonucleases. In some cases restriction segments were much fewer than expected, suggesting a possible interference of the unusual base with the recognition mechanism of endonucleases. The physical map of 2C DNA was established by use of SalI and HaeIII restriction endonucleases, which yielded a limited number of fragments. The expected number of fragments was 240 for HaeIII and 23 for SalI; in reality, five segments were observed upon cleavage with HaeIII and four with SalI. The terminal fragments of the genome were first identified; the other fragments were ordered by hybridization and molecular weight determination of restriction fragments obtained by cleavage with the two endonucleases. In addition, hybridization of restriction fragments showed the presence of homologous regions at the ends of the 2C genome. The structure of these direct repetitive sequences was analyzed by cleavage with HaeIII and hybridization with EcoRI restriction fragments. Their size (9.2 MDa) was found to be about 1/11 of that of the whole chromosome.

Bacillus subtilis↗

PubMed1983

The in vitro and in vivo inactivation of ribosomes by virginiamycin M does not entail an alteration of 5, 16 and 23 S ribosomal RNA.

The M component of virginiamycin blocks protein synthesis by inactivating catalytically the 50 S ribosomal subunits: the in vitro interaction of 50 S with virginiamycin M, followed by removal of the antibiotic, results in a lasting damage of the particle. This enzyme-like inactivation of 50 S subunits resembles that of 30 S subunits by colicin E3, which entails the cleavage of 16 S rRNA. To explore this possibility, rRNA obtained from particles incubated in vivo and in vitro with virginiamycin M were analyzed. Electrophoretic analysis of 5, 16 and 23 S rRNA did not reveal major changes, nor did it show the appearance of additional fragments. To exclude the possibility of terminal alterations, the 5'- and 3'-extremities of these RNA were also sequenced and found unchanged. Conclusions drawn in the present work parallel those of an accompanying paper (Moureau, P., Di Giambattista, M. and Cocito, C. (1983) Biochim. Biophys. Acta 739, 164-172) describing the dissociation and reassociation of ribosomes incubated with virginiamycin M: the lasting ribosome damage by this antibiotic appears to be due to a conformational rather than to a structural alteration.

Bacillus subtilis↗

PubMed1983

The lasting ribosome alteration produced by virginiamycin M disappears upon removal of certain ribosomal proteins.

Transient incubation of bacterial ribosomes with virginiamycin M produces a lasting damage of 50 S ribosomal subunits, whereby the elongation of peptide chains is still blocked after removal of the antibiotic. To elucidate the mechanism of this inactivation, ribosomal proteins were stepwise removed from 50 S subunits previously incubated with virginiamycin M, and cores were submitted to three functional tests. Total removal of proteins L7, L8, L12 and L16, and partial removal of L6, L9, L10 and L11, resulted in a loss of the virginiamycin M-induced alteration. When the split protein fractions were added back to these cores, unaltered functional particles were obtained. The reconstituted subunits, on the other hand, proved fully sensitive to virginiamycin M in vitro as they underwent, upon transient contact with the antibiotic, an alteration comparable to that of native particles. It is concluded that the virginiamycin M-induced ribosome damage is due to the production of a stable conformational change of the 50 S subunit. These data parallel those of an accompanying paper (Cocito, C., Vanlinden, F. and Branlant, C. (1983) Biochim. Biophys. Acta 739, 158-163) showing the intactness of all rRNA species from ribosomes treated in vivo and in vitro with virginiamycin M.

Cell-Free System↗

PubMed1983

Inhibitory action of virginiamycin components on cell-free systems for polypeptide formation from Bacillus subtilis.

Although virginiamycin components VM and VS are known to exert in vivo a synergistic inhibition of bacterial growth and viability, in cell-free systems only VM has proven active. In the present work, the in vivo and in vitro activities of VM and VS on Bacillus subtilis have been compared. Peptide formation in homogenates of bacteria previously incubated with either VM or VS was found strongly repressed; the 2 components acted synergistically. Ribosomes were fully responsible for this effect, as shown by mixed reconstitution experiments. On the other hand, cytoplasm from control bacteria disrupted in 10 mM Mg2+ buffer was refractory to in vitro inhibition by virginiamycin, whereas ribosomes prepared in 1 mM Mg2+ were sensitive to VM. VS was inactive on poly(U)-directed poly(phenylalanine) formation, and displayed some activity on the poly(A)-poly(lysine) system. In a cell-free system from Bacillus subtilis infected with phage 2C, both VM and VS were active and blocked synergistically protein synthesis in vitro. When the host cells were incubated with VS and the corresponding homogenate was then treated with VM, a complete inhibition of protein synthesis was observed. The present work, thus, describes the techniques for investigating the in vivo and in vitro action of synergimycins on the same organism, and for reproducing in vitro the synergistic interaction of type A and B components previously observed only in vivo.

Bacillus subtilis↗