title: Effect of amplification or targeted disruption of the beta-lactamase gene of Nocardia lactamdurans on cephamycin biosynthesis.
abstract: The bla gene of the cephamycin cluster of Nocardia lactamdurans has been subeloned in the shuttle plasmids pULVK2 and pULVK2A and amplified in N. lactamdurans LC411. The transformants showed two- to threefold higher beta-lactamase activity. Formation of beta-lactamase preceded the onset of cephamycin biosynthesis. The beta-lactamase of N. lactamdurans inactivated penicillins and, to a lesser extent, cephalosporin C but did not hydrolyse cephamycin C. This beta-lactamase was highly sensitive to clavulanic acid (50% inhibition was observed at 0.48 microgram/ml clavulanic acid). The N. lactamdurans bla gene was disrupted in vivo by inertion of the kanamycin-resistance gene. Three bla-disrupted mutants, BD4, BD8 and BD12, were selected that lacked beta-lactamase activity. Overexpresion of the bla gene resulted in N. lactamdurans transformants that were resistant to penicillin whereas mutants in which the bla gene was disrupted were super-sensitive to this antibiotic. The three N. lactamdurans mutants with the bla gene disrupted showed a significant increase of cephamycin biosynthesis in solid medium, whereas transformants with the amplified bla gene produced reduced levels of cephamycin. The cephamycin-overproducing Merck strain N. lactamdurans MA4213 showed no detectable levels of beta-lactamase activity. The beta-lactamase plays a negative role in cephamycin biosynthesis in solid medium, but not in liquid medium.
authors.affiliation: Area of Microbiology, Faculty of Biology, University of León, Spain.
authors.forename: V
authors.initials: V
authors.lastname: Kumar
authors.forename: J L
authors.initials: JL
authors.lastname: de la Fuente
authors.forename: A L
authors.initials: AL
authors.lastname: Leitão
authors.forename: P
authors.initials: P
authors.lastname: Liras
authors.forename: J F
authors.initials: JF
authors.lastname: Martín
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism; D002513:Cephamycins / Q000096:biosynthesis*; D003001:Cloning, Molecular; D005784:Gene Amplification; D005798:Genes, Bacterial; D016296:Mutagenesis; D009615:Nocardia / Q000201:enzymology / Q000235:genetics*; D010403:Penicillin Resistance / Q000235:genetics*; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: D000900:Anti-Bacterial Agents; D002513:Cephamycins; D001618:beta-Lactamases