V. Kumar et al., EFFECT OF AMPLIFICATION OR TARGETED DISRUPTION OF THE BETA-LACTAMASE GENE OF NOCARDIA LACTAMDURANS ON CEPHAMYCIN BIOSYNTHESIS, Applied microbiology and biotechnology, 45(5), 1996, pp. 621-628
The bla gene of the cephamycin cluster of Nocardia lactamdurans has be
en subcloned in the shuttle plasmids pULVK2 and pULVK2A and amplified
in N. lactamdurans LC411. The transformants showed two- to threefold h
igher beta-lactamase activity. Formation of beta-lactamase preceded th
e onset of cephamycin biosynthesis. The beta-lactamase of N. lactamdur
ans inactivated penicillins and, to a lesser extent, cephalosporin C b
ut did not hydrolyse cephamycin C. This beta-lactamase was highly sens
itive to clavulanic acid (50% inhibition was observed at 0.48 mu g/ml
clavulanic acid). The N. lactamdurans bla gene was disrupted in vivo b
y insertion of the kanamycin-resistance gene. Three bin-disrupted muta
nts, BD4, BD8 and BD12, were selected that lacked beta-lactamase activ
ity. Overexpression of the bla gene resulted in N. lactamdurans transf
ormants that were resistant to penicillin whereas mutants in which the
bin gene was disrupted were supersensitive to this antibiotic. The th
ree N. lactamdurans mutants with the bla gene disrupted showed a signi
ficant increase of cephamycin biosynthesis in solid medium, whereas tr
ansformants with the amplified bla gene produced reduced levels of cep
hamycin. The cephamycin-overproducing Merck strain N. lactamdurans MA4
213 showed no detectable levels of beta-lactamase activity. The beta-l
actamase plays a negative role in cephamycin biosynthesis in solid med
ium, but not in liquid medium.