streptovaricin-c and streptolydigin

Streptomyces lydicus NRRL2433 and S. spectabilis NRRL2494 produce two inhibitors of bacterial RNA polymerase: the 3-acyltetramic acid streptolydigin and the naphthalenic ansamycin streptovaricin, respectively. Both strains are highly resistant to their own antibiotics. Independent expression of the S. lydicus and S. spectabilis rpoB and rpoC genes, encoding the beta- and beta'-subunits of RNA polymerase, respectively, in S. albus showed that resistance is mediated by rpoB, with no effect of rpoC. Within the beta-subunit, resistance was confined to an amino acid region harboring the "rif region." Comparison of the beta-subunit amino acid sequences of this region from the producer strains and those of other streptomycetes and site-directed mutagenesis of specific differential residues located in it (L485 and D486 in S. lydicus and N474 and S475 in S. spectabilis) showed their involvement in streptolydigin and streptovaricin resistance. Other amino acids located close to the "Stl pocket" in the S. lydicus beta-subunit (L555, F593, and M594) were also found to exert influence on streptolydigin resistance.

Topics: Amino Acid Sequence; Aminoglycosides; Anti-Bacterial Agents; Binding Sites; DNA-Directed RNA Polymerases; Drug Resistance, Bacterial; Gene Expression Regulation, Bacterial; Microbial Sensitivity Tests; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Structure, Tertiary; Rifampin; RNA Polymerase III; Streptomyces; Streptovaricin

Transcription by RNA polymerase II occurs after formation of a transcription complex. This complex is assembled in stages by the interaction of transcription factors with the template and/or with each other. We report on the ability of six drugs to inhibit the assembly of the RNA polymerase II transcription complex. Assembly of the complex on the adenovirus major late promoter requires several transcription factors. The normal assembly process requires that the DNA first interact with TFIIA, then with TFIID, and finally with at least four additional transcription factors (one of which is RNA polymerase II). We observed that streptolydigin (10 micrograms/ml) inhibits association of ILA and IID, and at higher concentrations (100 micrograms/ml) inhibits that IIA/IID complex from binding to DNA. Streptovaricin (100 micrograms/ml) appears to inhibit the IIA/IID interaction with DNA and prevents reinitiation (at 500 micrograms/ml). Adriamycin (1 microgram/ml) inhibits the interaction of TFIID with the IIA/DNA complex and inhibits an additional event immediately prior to, or during, elongation. Daunorubicin may be an elongation inhibitor. Heparin at 10 micrograms/ml inhibits further assembly after the IIA/IID/DNA complex has formed, and at 100 micrograms/ml also inhibits a late event in the assembly process and blocks reinitiation. Rifamycin AF/013 (100 micrograms/ml) inhibits the early events necessary to form the IIA/IID/DNA complex and (at 10 micrograms/ml) an assembly event following formation of the IIA/IID/DNA complex. Therefore, these compounds should be useful as probes for further examination of the assembly process.

Topics: Aminoglycosides; Anti-Bacterial Agents; Daunorubicin; Doxorubicin; HeLa Cells; Heparin; Humans; Rifamycins; RNA Polymerase II; Streptovaricin; Transcription, Genetic

We investigated the effects of six drugs on an RNA polymerase III in vitro transcription system. Adriamycin, daunorubicin, heparin, rifamycin AF/013, streptolydigin, and streptovaricin all inhibit RNA synthesis from a tRNA gene or the adenovirus 2 (AD2) VA1 RNA gene. The completed RNA polymerase III transcription complex is formed by the sequential, ordered addition of protein factors. Although both genes reportedly use the same transcription fractions for in vitro RNA synthesis, some of these drugs interfere differentially with these genes. A drug concentration that inhibits transcription from one gene may not inhibit transcription from the other gene. Adriamycin seems to block transcription if added between the binding of the individual transcription fractions. Daunorubicin appears to inhibit VA transcription only if added prior to both transcription fractions, but inhibits tRNA synthesis before and during transcription factor binding. Heparin blocks both genes between factors binding to DNA and after factor binding. Rifamycin blocks VA synthesis more effectively than tRNA synthesis. Streptolydigin blocks transcription of both genes. Streptovaricin probably blocks transcription by inhibiting early transcription complex assembly events. These drugs appear useful as appropriate probes to investigate transcription complexes since several discriminate between complexes formed on different genes during the assembly process.

Topics: Adenoviruses, Human; Aminoglycosides; Anti-Bacterial Agents; Cytosol; Daunorubicin; DNA-Directed RNA Polymerases; Doxorubicin; Genes; Genes, Viral; HeLa Cells; Heparin; Humans; Rifamycins; RNA Polymerase III; Streptovaricin; Transcription, Genetic

Resistance to the endogenous antibiotic was studied in three actinomycetes that produce inhibitors of RNA polymerase. The three producers, Nocardia mediterranei (rifamycin producer), Streptomyces spectabilis (streptovaricin producer) and Streptomyces lydicus (streptolydigin producer), were each highly resistant to the antibiotic they produce (MIC greater than 200 micrograms ml-1) and in vivo RNA synthesis was also resistant. However, cross-resistance to the other RNA polymerase inhibitors was not found. Resistance to these antibiotics was due to target site modification, since the RNA polymerase enzymes of the three producing organisms were highly resistant in vitro to the corresponding antibiotic, and no antibiotic-inactivating enzymes were detected. A mutant was isolated from S. spectabilis which was sensitive to steptovaricin (its own product) and also showed an increased sensitivity to rifamycin and streptolydigin. This mutant had RNA polymerase which was extremely sensitive to the three antibiotics.

Topics: Actinomycetales; Aminoglycosides; Anti-Bacterial Agents; DNA-Directed RNA Polymerases; Drug Resistance, Microbial; Nocardia; Rifamycins; RNA, Bacterial; Streptomyces; Streptovaricin

Initiation of DNA replication has been observed in vitro with a DNA-membrane complex extracted from Bacillus subtilis. Antibiotics known to interfere with various aspects of initiation inhibited DNA synthesis significantly in vitro, whereas a mutant resistant to one inhibitor failed to respond to its presence. The inhibitory effects occurred primarily when the immediate RNA precursors (ribonucleoside triphosphates) were present in the assay solution but not significantly when the precursors were omitted. Complexes extracted from a temperature-sensitive initiation mutant were almost incapable of synthesizing DNA at the restrictive temperature but displayed extensive synthesis at the permissive temperature. A strong indication of semiconservative DNA synthesis was obtained in vitro after density-shift experiments involving incubation of the complex with a heavy-density DNA precursor, followed by neutral and alkaline CsCl density gradient centrifugation. A significant amount of chain elongation or repair (or both) was also observed.

Topics: Aminoglycosides; Anti-Bacterial Agents; Bacillus subtilis; Cell Membrane; DNA Replication; DNA, Bacterial; Nalidixic Acid; Streptovaricin

Bacteriophage PBS2 replication is unaffected by rifampicin and other rifamycin derivatives, which are potent inhibitors of Bacillus subtilis RNA synthesis. Extracts of gently-lysed infected cells contain a DNA-dependent RNA polymerase activity which is specific for uracil-containing PBS2 DNA. The PBS2-induced RNA polymerase is insensitive to rifamycin derivatives which inhibit the host's RNA polymerase.

Topics: Aminoglycosides; Anti-Bacterial Agents; Bacillus Phages; Bacillus subtilis; Chloramphenicol; Dactinomycin; DNA-Directed RNA Polymerases; Drug Resistance, Microbial; Lucanthone; Rifampin; Rifamycins; Streptovaricin; Viral Proteins; Virus Replication