FIG. 3.

Replacement of a 30-kb region containing the acbC homologue by aac(3)IV in strain 5008. (A) Schematic representation of the replacement of the 30 kb of DNA mediated by 2.1-kb genomic fragments flanking both sides of the 1.4-kb aac(3)IV apramycin resistance determinant in pHZ2236. The mutants generated by double crossover (YU-1-1 to YU-1-4) had a fusion of the 10-kb leftward BamHI fragment with the BglII end of the 1.4-kb aac(3)IV to form an 11.4-kb new BamHI fragment, but keep the rightward 7-kb fragment unchanged. (B) Southern hybridization using the α-[³²P]dCTP-labeled 5.6 (2.1 + 1.4 + 2.1)-kb insert from pHZ2236 as the probe after genomic DNAs of the wild-type 5008 and its derivatives (YU-1-1 to YU-1-4) were digested with BamHI. (C) HPLC analysis demonstrating that validamycin A is produced by wild-type strain 5008, but not by YU-1.