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The Journal of Antibiotics Feb 1983
Topics: Capreomycin; Drug Resistance, Microbial; Mycobacterium; Viomycin
PubMed: 6187721
DOI: 10.7164/antibiotics.36.197 -
Microbiology and Immunology 1983The mechanism of resistance of Mycobacterium intracellulare strain 103 and other clinical isolates to a variety of drugs including aminoglycoside and peptide antibiotics...
The mechanism of resistance of Mycobacterium intracellulare strain 103 and other clinical isolates to a variety of drugs including aminoglycoside and peptide antibiotics was investigated. Enzymatic inactivation of aminoglycoside and peptide antibiotics could not be demonstrated. Ribosomes of the strain were found to be sensitive to the antibiotics. The levels of resistance of strain 103 and other clinical isolates decreased dramatically when the culture medium was changed from Dubos agar to Tween 80-containing agar. These results suggest that a permeability barrier is the reason for naturally occurring resistance in M. intracellulare.
Topics: Aminoglycosides; Anti-Bacterial Agents; Cell Membrane Permeability; Chloramphenicol; Culture Media; Drug Resistance, Microbial; Kanamycin; Mycobacterium; Nontuberculous Mycobacteria; Ribosomes; Streptomycin; Viomycin
PubMed: 6312275
DOI: 10.1111/j.1348-0421.1983.tb00601.x -
Journal of Bacteriology Aug 1982Determinants of antibiotic resistance have been cloned from four antibiotic-producing streptomycetes into Streptomyces lividans. Biochemical analyses of resistant clones...
Determinants of antibiotic resistance have been cloned from four antibiotic-producing streptomycetes into Streptomyces lividans. Biochemical analyses of resistant clones revealed the presence of enzymes that had previously been characterized as likely resistance determinants in the producing organisms. These included: 23S rRNA methylases from S. azureus and S. erythreus, which confer resistance to thiostrepton and erythromycin, respectively; viomycin phosphotransferase from S. vinaceus; and aminoglycoside phosphotransferase and acetyltransferase from the neomycin producer S. fradiae. In general, the levels of antibiotic resistance of the clones were similar to those of the producing organisms. Although the two aminoglycoside-modifying enzymes from S. fradiae could independently confer only low-level resistance to neomycin, the presence of both enzymes in the same strain resulted in a level of resistance comparable with that of the producing organism.
Topics: Acetyltransferases; Anti-Bacterial Agents; Cloning, Molecular; Erythromycin; Kanamycin Kinase; Methyltransferases; Phosphotransferases; R Factors; Streptomyces; Thiostrepton
PubMed: 6284707
DOI: 10.1128/jb.151.2.678-685.1982 -
Journal of Bacteriology Aug 1982Methodology which allows consistent shotgun cloning of streptomycete genes is presented. Parameters that increase transformation efficiency of Streptomyces lividans 66...
Methodology which allows consistent shotgun cloning of streptomycete genes is presented. Parameters that increase transformation efficiency of Streptomyces lividans 66 were adjusted to generate reproducibly a population of cloned genes likely to represent the entire genome. Factors which influence the recovery of viable transformants include: growth phase of the mycelium, ionic and osmotic characteristics of the medium during protoplast formation and transformation, and moisture content and protoplast density during regeneration. A modified transformation procedure was devised which increased transformation frequency more than 20-fold (allowing up to 10(7) primary transformants per microgram of SLP1.2 covalently closed circular DNA) and greatly facilitated the cloning of drug resistance genes and biosynthetic genes, using one of two plasmid vectors. Viomycin resistance genes on BamHI or PstI fragments were cloned from S. vinaceus genomic DNA into S. lividans, using the SLP1.2 vector. At least three different S. vinaceus BamHI fragments (1.9, 5.8, or 8.5 kilobases) confer viomycin resistance; only one PstI fragment (4.3 kilobases) was found. Recombinant plasmids were all able to produce lethal zygosis and to be transferred by conjugation within S. lividans. SCP2 was used to clone S. coelicolor A3(2) genes that "complemented" the auxotrophic mutation hisD3, argA1, or guaA1. Recombinant DNA technology can now be applied to economically and academically interesting problems unique to streptomycete molecular biology.
Topics: Cloning, Molecular; Genes, Bacterial; Plasmids; R Factors; Streptomyces; Transformation, Bacterial; Viomycin
PubMed: 6284706
DOI: 10.1128/jb.151.2.668-677.1982 -
Journal of Biochemistry May 1982Precise immunological recognition of anti-viomycin antiserum at detailed parts in the structure of viomycin was studied by cross reactivities of the antiserum to...
Precise immunological recognition of anti-viomycin antiserum at detailed parts in the structure of viomycin was studied by cross reactivities of the antiserum to viomycin and its ten analogs using an enzyme immunoassay of viomycin. The antiserum clearly recognized all minor modifications in the sixteen membered ring of viomycin, indicating that the antiserum clearly recognizes the whole structure of the sixteen membered ring. Recognition of the antiserum on the beta-lysine terminus was also examined showing that the antiserum was also recognized on this part. Thus, the anti-viomycin antiserum was deduced to recognize the whole structure of viomycin, from which the deduction was made that the anti-viomycin antibodies in the antiserum must possess cavities fitting the whole structure of viomycin. The crystal dimensions of viomycin are 13 A in length, 8 A in width, and 7 A in depth. Thus, the high dimensional structure of the binding sites of the anti-viomycin antibodies was deduced to possess cavities of a similar size to that of viomycin.
Topics: Animals; Antigens; Binding Sites, Antibody; Cross Reactions; Immunochemistry; Immunoenzyme Techniques; Immunoglobulins; Rabbits; Viomycin
PubMed: 6284729
DOI: 10.1093/oxfordjournals.jbchem.a133851 -
Antimicrobial Agents and Chemotherapy Dec 1981The effects of 61 synthetic tuberactinomycin derivatives on polypeptide synthesis were tested in bacterial cell-free systems. Di-beta-lys-capreomycin IIA was more...
The effects of 61 synthetic tuberactinomycin derivatives on polypeptide synthesis were tested in bacterial cell-free systems. Di-beta-lys-capreomycin IIA was more effective than the natural product. Palmitoyl tuberactinamine N inhibited the growth of tuberactinomycin-resistant mutants and was not a ribosome inhibitor.
Topics: Anti-Bacterial Agents; Bacteria; Capreomycin; Drug Resistance, Microbial; Enviomycin; Mutation; Ribosomes; Viomycin
PubMed: 6173016
DOI: 10.1128/AAC.20.6.834 -
Proceedings of the National Academy of... Sep 1981The binding of N-acetyl-Phe-tRNAPhe (an analogue of peptidyl-tRNA), Phe-tRNAPhe, and deacylated tRNAPhe to poly(U)-programmed tightly coupled 70S ribosomes was studied....
The binding of N-acetyl-Phe-tRNAPhe (an analogue of peptidyl-tRNA), Phe-tRNAPhe, and deacylated tRNAPhe to poly(U)-programmed tightly coupled 70S ribosomes was studied. The N-acetyl-Phe-tRNAPhe binding is governed by an exclusion principle: not more than one N-acetyl-Phe-tRNAPhe can be bound per ribosome, although this peptidyl-tRNA analogue can be present either at the aminoacyl-tRNA (A) site or the peptidyl-tRNA (P) site. Two Phe-tRNAPhe molecules are accepted by one ribosome in the presence of poly(U). This aminoacyl-tRNA binds enzymatically (in the presence of elongation factor Tu and GTP) and nonenzymatically to the A site and is then transferred to the P site, if that site is free. If this elongation factor G-independent movement is hampered, either by using an incubation temperature of 0 degrees C or by the addition of the translocation inhibitor viomycin, only one Phe-tRNAPhe per ribosome can be bound. The effect of the peptidyltransferase inhibitor chloramphenicol on the binding is similar to that of viomycin. In the absence of poly(U), Phe-tRNAPhe cannot bind to the ribosome. Deacylated [14C]tRNAPhe can bind in three copies to one ribosome. The new third tRNA binding site is called the "E" site. The sequence of filling the sites is P, E, and A. The apparent binding constants for the P and the E sites are both approximately 9 X 10(6) M-1 and that for the A site is 1.3 X 10(6) M-1. In the absence of poly(U), only one deacylated tRNAPhe can be bound per ribosome. This tRNAPhe most likely occupies the P site.
Topics: Binding Sites; Escherichia coli; Peptide Elongation Factors; RNA, Messenger; RNA, Transfer; RNA, Transfer, Amino Acyl; Ribosomes
PubMed: 7029532
DOI: 10.1073/pnas.78.9.5310 -
The Journal of Antibiotics Jun 1981The degrees of binding of [3H]dibekacin to LiCl-treated cores of E. coli ribosomes were reduced by increasing LiCl concentrations. The 1.15 M LiCl core lost 70...
The degrees of binding of [3H]dibekacin to LiCl-treated cores of E. coli ribosomes were reduced by increasing LiCl concentrations. The 1.15 M LiCl core lost 70 approximately 80% of the original binding capacity. The antibiotic attachment to the 1.15 M LiCl core was restored by reconstitution with the split proteins (SP), which were obtained by the treatment of 70S ribosomes with LiCl at concentrations of 0.8 approximately 1.15 M. The basic proteins, split off during the transition from 0.4 M LiCl core to 0.8 approximately 1.15 M LiCl core, seemed to be involved in the drug binding. SP0.4 approximately 1.15, which was obtained by the treatment of the 0.4 M LiCl core with 1.15 M LiCl, was fractionated by CM-Sephadex C-25 column chromatography, and each fraction was assayed for protein composition and the capability of restoring the ability of the 1.15 M LiCl core to bind the drug. Of ribosomal proteins eliminated with 1.15 M LiCl, the addition of either S9 or L6 alone to the 1.15 M LiCl core was observed to restore approximately 50% of the binding as compared to the 70S ribosome alone, and both proteins restored about 70% of the binding. The results suggested that ribosomal proteins S9 and L6 were involved in the attachment of [3H]dibekacin to the ribosome. The antibiotic binding to the 70S ribosome and 1.15 M LiCl core reconstituted with S9 and L6 was considerably inhibited by unlabelled dibekacin or kanamycin, and partially inhibited by gentamicin or neomycin, but was not significantly affected by streptomycin or viomycin.
Topics: Aminoglycosides; Anti-Bacterial Agents; Chlorides; Dibekacin; Escherichia coli; Kanamycin; Lithium; Lithium Chloride; Protein Binding; Ribosomal Protein S9; Ribosomal Proteins; Ribosomes
PubMed: 6268595
DOI: 10.7164/antibiotics.34.763 -
Journal of Bacteriology May 1981After treatment with mitomycin D and other antibacterial agents, a translucent, smooth-colony-forming mycobacterium, isolated from sputum and designated as Mycobacterium...
After treatment with mitomycin D and other antibacterial agents, a translucent, smooth-colony-forming mycobacterium, isolated from sputum and designated as Mycobacterium intracellulare strain 103, gave rise to variants forming opaque colonies. These opaque variants were more sensitive streptomycin, kanamycin, viomycin, and rifampin than were the wild-type translucent variants. Plasmid deoxyribonucleic acids taken from translucent strain cells and from cells of certain opaque variants were analyzed by agarose gel electrophoresis. Two plasmids of molecular weights of approximately 2 x 10(6) and 50 x 10(6), respectively, were found in the wild-type translucent cells; one of them, the 2 x 10(6)-molecular-weight plasmid, was always missing from deoxyribonucleic acids of the opaque variant cells. The results suggested that translucent colonial appearance and antibiotic resistance of the strain are plasmid-determined functions.
Topics: Anti-Bacterial Agents; DNA, Bacterial; DNA, Circular; Genetic Variation; Molecular Weight; Mycobacterium; Nontuberculous Mycobacteria; Plasmids
PubMed: 7217014
DOI: 10.1128/jb.146.2.656-659.1981 -
Nucleic Acids Research Dec 1980Binding studies were performed with a [14C]-labelled derivative of viomycin, tuberactinomycin 0 (TUM O). TUM O bound to 30S and 50S subunits. The binding component was...
Binding studies were performed with a [14C]-labelled derivative of viomycin, tuberactinomycin 0 (TUM O). TUM O bound to 30S and 50S subunits. The binding component was the RNA, since ribosomal proteins did not bind the drug. Other RNAs such as tRNA, phage RNA (MS2), and homopolynucleotides also bound the drug. Striking differences in the binding capacity of the various homopolynucleotides were found. Poly(U) bound strongly, poly(G) and poly(C) bound intermediately, whereas poly(A) showed a very low binding. DNA also bound TUM O, although with native DNA the binding was only weak. Finally the effects of viomycin on the assembly in vitro of the 50S subunit from E. coli were tested. A very strong inhibition was found: when the reconstitution was performed at 0.5 x 10(-6) M viomycin the particles formed sedimented at about 50S, but showed a residual activity of less than 10%. The inhibitory power of viomycin with respect to the in vitro assembly is more pronounced than that found in in vitro systems for protein synthesis.
Topics: Binding Sites; Enviomycin; Escherichia coli; Mycobacterium; Nucleic Acid Conformation; Polyribonucleotides; RNA, Ribosomal; Ribosomes; Viomycin
PubMed: 6258151
DOI: 10.1093/nar/8.23.5767