Southern blot analysis of the recA locus. (A) BCG Frappier. Lane 1, parental M. bovis BCG Frappier; lane 2, single-crossover transformant obtained with plasmid pGEM7-recA::3xstop-hsp60-sacB-hyg-aph; lanes 3-4, recA (unmarked) knock-out mutants obtained after counterselection of a single-crossover transformant. (B) BCG Russia. Lanes 1–9 transformants obtained with plasmid pGEM7-recA::3xstop-hsp60-sacB-hyg-aph resulting from illegitimate recombination; lane 10, parental strain. (C) Schematic drawing of the BCG recA locus: the wild-type locus is shown along with the vector used for inactivation, 5' and 3' single-crossover transformant, knock-out mutant.