The peptide antibiotic microcin B17 induces double-strand cleavage of DNA mediated by E. coli DNA gyrase.
Microcin B17 (MccB17) is a bactericidal peptide antibiotic which inhibits DNA replication. Two Escherichia coli MccB17 resistant mutants were isolated and the mutations were shown to map to 83 min of the genetic map. Cloning of the mutations and Tn5 insertional analysis demonstrated that they were located inside gyrB. The approximate location of the mutations within gyrB was determined by constructing hybrid genes, as a previous step to sequencing. Both mutations were shown to consist of a single AT----GC transition at position 2251 of the gene, which produces a Trp751----Arg substitution in the amino acid sequence of the GyrB polypeptide. The inhibitory effect of MccB17 on replicative cell-free extracts was assayed. In this in vitro system, interaction of MccB17 with a component of the extracts induced double-strand cleavage of plasmid DNA. In vivo treatment with MccB17 also induced a well-defined cleavage pattern on chromosomal DNA. These effects were not observed with a MccB17-resistant, gyrB mutant. Altogether, our results indicate that MccB17 blocks DNA gyrase by trapping an enzyme-DNA cleavable complex. Thus, the mode of action of this peptide antibiotic resembles that of quinolones and a variety of antitumour drugs currently used in cancer chemotherapy. MccB17 is the first peptide shown to inhibit a type II DNA topoisomerase.
Vizán
J L
JL
Unidad de Genética Molecular, Hospital Ramón y Cajal, Madrid, Spain.
Hernández-Chico
C
C
del Castillo
I
I
Moreno
F
F
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001430:Bacteriocins / Q000494:pharmacology*; D003001:Cloning, Molecular; D004261:DNA Replication / Q000187:drug effects*; D004250:DNA Topoisomerases, Type II / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000187:drug effects / Q000378:metabolism*; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010641:Phenotype; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D059005:Topoisomerase II Inhibitors; D014161:Transduction, Genetic
D000900:Anti-Bacterial Agents; D001430:Bacteriocins; D004269:DNA, Bacterial; D059005:Topoisomerase II Inhibitors; C020411:microcin; D004250:DNA Topoisomerases, Type II