rifampin and streptolydigin

Topics: Amidines; Aminoglycosides; Anti-Bacterial Agents; Bacteria; Bacteriocins; DNA-Directed RNA Polymerases; Enzyme Inhibitors; Hydroxylamines; Models, Chemical; Molecular Conformation; Molecular Structure; Rifampin

Topics: Aminoglycosides; Anti-Bacterial Agents; Bacterial Proteins; Binding Sites; Catalysis; DNA-Directed RNA Polymerases; Escherichia coli; Genes, Bacterial; Mutagenesis, Site-Directed; Nucleic Acid Synthesis Inhibitors; Promoter Regions, Genetic; Protein Conformation; Rifampin; Structure-Activity Relationship; Terminator Regions, Genetic; Transcription, Genetic

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

A new method for determination of RNA polymerase (RNAP) activity is presented. The method uses nucleoside tri- and tetraphosphate derivatives carrying 4-methylumbelliferone residue at the terminal phosphate. Incorporation of such compounds in RNA by RNA polymerase is accompanied by release of di- and triphosphate derivatives of 4-methylumbelliferone. Subsequent treatment by alkaline phosphatase produces free 4-methylumbelliferone that is highly fluorescent and can be easily detected. The sensitivity of the method is higher than that reported in previous studies. The validity of the assay has been demonstrated by retrieving the RNAP inhibitors from a collection of 16,000 compounds.

Topics: Adenosine; Aminoglycosides; Combinatorial Chemistry Techniques; Coumarins; DNA-Directed RNA Polymerases; Enzyme Inhibitors; Heparin; Hymecromone; Rifampin; Sensitivity and Specificity; Spectrophotometry, Ultraviolet

Rifampicin and streptolydigin are antibiotics which inhibit prokaryotic RNA polymerase at the initiation and elongation steps, respectively. In Escherichia coli, resistance to each antibiotic results from alterations in the beta subunit of the core enzyme. However, in Bacillus subtilis, reconstitution studies found rifampicin resistance (RifR) associated with the beta subunit and streptolydigin resistance (StlR) with beta'. To understand the basis of bacterial StlR, we isolated the B. subtilis rpoC gene, which encodes a 1,199-residue product that is 53% identical to E. coli beta'. Two spontaneous StlR mutants carried the same D796G substitution in rpoC, and this substitution alone was sufficient to confer StlR in vivo. D796 falls within Region F, which is conserved among the largest subunits of prokaryotic and eukaryotic RNA polymerases. Among eukaryotes, alterations in Region F promote resistance to alpha-amanitin, a toxin which inhibits transcription elongation; among prokaryotes, alterations in Region F cause aberrant termination. To determine whether alterations in the beta subunit of B. subtilis could also confer StlR, we made three StlR substitutions (A499V, G500R, and E502V) in the rif region of rpoB. Together these results suggest that beta and beta' interact to form an Stl binding site, and that this site is important for transcription elongation.

Topics: Amino Acid Sequence; Aminoglycosides; Anti-Bacterial Agents; Bacillus subtilis; Base Sequence; DNA Primers; DNA-Directed RNA Polymerases; Drug Resistance, Microbial; Genes, Bacterial; Molecular Sequence Data; Polymerase Chain Reaction; Restriction Mapping; Rifampin; Sequence Homology, Amino Acid

Mutations to rifampicin resistance (RifR) in Escherichia coli alter the beta subunit of RNA polymerase (RNAP). A series of new point RifR mutations was isolated with the aid of random polymerase chain reaction-mediated mutagenesis followed by selection on rifampicin (Rif). All of the new RifR mutants, including changes in two novel positions, fell into the two principal clusters previously identified in the middle section of beta. Disruption of the spacer between the two clusters with deletions and insertions led to RNAP that was functional and sensitive to Rif in vitro, indicating that the spacer is not directly involved in Rif binding. However, most of the spacer mutants were strongly resistant to streptolydigin, suggesting that this area is involved in the binding of streptolydigin. An insertion of six consecutive histidines into the spacer was constructed that could be used to anchor RNAP on a Nichelate matrix without the loss of enzymatic activity, indicating that this region is looped out of the RNAP molecule.

Topics: Amino Acid Sequence; Aminoglycosides; Anti-Bacterial Agents; Base Sequence; DNA-Directed RNA Polymerases; DNA, Single-Stranded; Drug Resistance, Microbial; Escherichia coli; Molecular Sequence Data; Mutagenesis, Insertional; Mutagenesis, Site-Directed; Mutation; Rifampin; Sequence Alignment; Sequence Deletion; Transcription, Genetic

Cellular RNA polymerase in association with plasmid DNA segregates into the minicells of minicell-producing strains. In general, one "plasmid equivalent" of RNA polymerase, reflecting the size of the segregating plasmid DNA and its efficiency of segregation, entered the minicell with the plasmid. The amount of RNA polymerase (measured as the amount of enzyme activity purified from minicells and the rate of RNA synthesis in plasmid-containing minicells), and not the DNA content, appeared to be rate-limiting in plasmid-mediated transcription in minicells. The purified minicell and cellular RNA polymerases showed the same sensitivity to rifampin and streptolydigin; both were associated with sigma factor, although the minicell enzyme appeared to have slightly less than the cellular enzyme. These studies demonstrate that transcription of plasmid DNA in minicells is a function of the efficiency of segregation and the amount of RNA polymerase which enters with the plasmid DNA. Because RNA polymerase is limiting, plasmids with relatively weak promoters for the vector genes should be used when attempting to identify products from inserted foreign DNA.

Topics: Aminoglycosides; Anti-Bacterial Agents; DNA-Directed RNA Polymerases; DNA, Bacterial; Escherichia coli; Gene Expression Regulation; Plasmids; Rifampin; RNA, Bacterial; Templates, Genetic; Transcription, Genetic

We describe crude in vitro systems from T4-infected cells which reflect in vivo T4 regulation. Lysates from cells which had been infected in the presence of chloramphenicol manifest the same polarity of RNA synthesis as did the infected cells. Next, we describe a complementation system between lysates which have no RNA synthetic capacity and purified RNA polymerase; in this system, delayed early RNA synthesis in vitro depends on the presence of an active mot gene product. Mot activity controls middle mode gene expression in vivo. In vitro, not activity in the lysate directs RNA polymerase to initiate on regions of DNA that are otherwise inaccessible. This mot-dependent delayed early RNA synthesis in vitro is seen at 0.1 and 0.2 M KCl, but not at 0.05 M KCl. We present a model in which mot is a DNA melting protein necessary for recognition of a middle promoters by either Escherichia coli or T4-modified RNA polymerase which contains E. coli sigma subunit.

Topics: Aminoglycosides; Anti-Bacterial Agents; Chloramphenicol; DNA-Directed RNA Polymerases; Escherichia coli; Kinetics; Lysogeny; Rifampin; RNA, Viral; T-Phages; Transcription, Genetic

The incorporation of [3H]thymidine into deoxyribonucleic acid by exponentially growing cells of Bacillus brevis was inhibited by streptolydigin and rifampin in the same concentration range in which these drugs inhibit ribonucleic acid synthesis. Complete inhibition occurred within one-third generation time after drug addition, suggesting an effect on deoxyribonucleic acid chain elongation.

Topics: Aminoglycosides; Anti-Bacterial Agents; Bacillus; DNA Replication; DNA-Directed RNA Polymerases; DNA, Bacterial; Rifampin; RNA, Bacterial

When exponentially growing cells of Bacillus subtilis were treated with rifampin or lipiarmycin, both inhibitors of the initiation of ribonucleic acid synthesis, large amounts of (p)ppGpp accumulated. This accumulation appears to be independent of the ribosome-dependent stringent factor reaction because both relA and relC mutants responded in a manner similar to that of the wild type. The possibility that ribonucleic acid polymerase is directly involved in (p)ppGpp metabolism is discussed.

Topics: Aminoglycosides; Anti-Bacterial Agents; Bacillus subtilis; DNA-Directed RNA Polymerases; Fidaxomicin; Guanine Nucleotides; Guanosine Pentaphosphate; Guanosine Tetraphosphate; Rifampin; Transcription, Genetic

The mechanism of streptolydigin inhibition of RNA synthesis has been investigated with a combination of steady state kinetics and product analysis by employing the abortive initiation reaction of Escherichia coli RNA polymerase. The pattern of inhibition by streptolydigin on the poly[d(A-T)] . poly[d(A-T)]template (non-competitive versus AMP; competitive versus UTP) was consistent with one inhibitor binding site and with an ordered addition of AMP followed by UTP. The more complicated patterns observed on the poly[d(I-C)] . poly[d(I-C)] template and the bacteriophage T7 A2 promotor (noncompetitive versus CTP) were explained by assuming that streptolydigin could stabilize the translocated ternary complex containing the product dinucleotide. Product analysis of two other abortive initiation reactions showed that the product did not dissociate from the inhibitor-bound translocated ternary complex. Finally, rifampicin and streptolydigin were shown to be functionally independent during initiation on several templates.

Topics: Aminoglycosides; Anti-Bacterial Agents; DNA-Directed RNA Polymerases; Escherichia coli; Kinetics; Rifampin; T-Phages; Templates, Genetic; Transcription, Genetic

Topics: Aldehyde-Lyases; Aminoglycosides; Anti-Bacterial Agents; Chromosome Mapping; Deoxyribonucleosides; Deoxyribose; Enzyme Induction; Escherichia coli; Genes; Glucosephosphates; Kinetics; Lac Operon; Operon; Phosphotransferases; Purine-Nucleoside Phosphorylase; Ribosemonophosphates; Rifampin; RNA, Messenger; Templates, Genetic; Transcription, Genetic

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