FIG. 4.

Disruption of the vbs gene. To disrupt the vbs gene, a double-crossover disruption vector, pVBS-DD1, was constructed (A). The vector was linearized and then transformed into A. parasiticus SYS-4 (= NRRL 2999). Double-crossover recombination led to replacement of most of the vbs gene containing the start codon with the selectable marker ptrA. The long arrows show gene directions. The short arrows indicate the positions of primers used for confirmation of gene disruption. The transformants accumulating yellow pigment on a GY agar plate were obtained as the vbs disruptants. The vbs gene disruptions were confirmed by PCR analyses (B) (lanes a, recipient train SYS-4; lanes b, c, and d, vbs disruptants VBS-DD1-16, VBS-DD1-23, and VBS-DD1-27, respectively; lane M, 1-kb molecular marker) with primer pairs P5-P6 (lanes 1), P1-P2 (lanes 2), P7-P10 (lanes 3), P8- P9 (lanes 4), P3-P10 (lanes 5), and P4-P9 (lanes 6).