PCR and Southern hybridization of wild type 920A6 and ΔrtfA mutant, 213R.4. (A) PCR of wild type M. avium 920A-6 (lane 3) yielded a single 1.9 kb band corresponding rtfA whereas the rtfA mutant 213R.4 yielded a 3.2 kb band corresponding to rtfA with an inserted 1.3 kb hygromycin resistance gene cassette (rtfA::hyg, lane 2). Vector pVAP39 served as a positive control (lane 4). Lane 1 represents a molecular weight marker. (B) Southern blot analysis of genomic DNA from wt M. avium 920A6 and clone 213R.4 was digested with HindIII and probed for rtfA. M. avium 920A6 yielded a 11.13 kb band (lane 2) whereas clone 213R.4 (lane 1) yielded a 12.49 kb band, corresponding to the incorporation of the 1.3 kb hyg gene.