Confirmation of obtained mutants by Southern blotting.

<p>(A) Replacement of wild-type <i>recA</i> with its mutated copy, Δ<i>recA</i>. The chromosomal DNA was digested with <i>Kpn</i>I. The hybridization probe was amplified based on pMG 22 carrying Δ<i>recA</i> gene as a template, and contained 5′(377 bp) and 3′(75 bp) fragments of <i>recA</i>. The wild-type and mutant strains revealed a different hybridization patterns: I- 2530 bp band representing 3′ KpnI Δ<i>recA</i> (629 bp, carrying 75 bp of 3′<i>recA</i> end and 554 bp downstream region joined to <i>hyg</i>^R cassette fragment of 1901 bp); II- 2456 bp band representing 3′KpnI <i>recA</i> wild type carrying 1666 bp of 3′<i>recA</i> gene and 790 bp of downstream region; III- 1036 bp band representing 5′KpnI <i>recA</i> wild type carrying 707 bp of 5′ <i>recA</i> gene and 329 bp of upstream region; IV- 808 bp band representing 5′KpnI Δ<i>recA</i> (706 bp, carrying 377 bp of 5′ <i>recA</i> gene fragment and 329 bp of upstreamregion joined to <i>hyg</i>^R cassette fragment of 102 bp), Lanes: 1, Mtb wild-type (control); 2, DCO Δ<i>recA</i> (double-crossover homologous-recombination mutant); 3, SCO Δ<i>recA-recA</i> (single-crossover homologous-recombination mutant); 4, 1-kb marker. (B) Replacement of wild-type <i>ku</i> and <i>ligD</i> genes with their mutated copies, Δ<i>ku</i> and Δ<i>ligD</i>. The chromosomal DNA was digested with <i>Pvu</i>II. The 3′ end of the <i>ligD</i> gene was used as a hybridization probe. Lanes: 1, DCO Δ(<i>ku,ligD</i>); 2–5, DCO wild-type <i>ku,ligD</i>; 6, Mtb wild-type (control); 7, 1-kb marker. (C) Replacement of wild-type <i>recA</i> with its mutated copy (Δ<i>recA</i>) in a strain carrying inactivated <i>ku</i> and <i>ligD</i> genes. The chromosomal DNA was digested with <i>Kpn</i>I. The 3′ end of <i>recA</i> was used as a hybridization probe. The probe was amplified based on pMG 22 carrying Δ<i>recA</i> gene, and contained 5′ (377 bp) and 3′ (75 bp) fragments of <i>recA</i>. The wild-type and mutant strains revealed a different hybridization patterns: I- 2530 bp band representing 3′ KpnI Δ<i>recA</i> (629 bp, carrying 75 bp of 3′<i>recA</i> end and 554 bp downstream region joined to <i>hyg</i>^R cassette fragment of 1901 bp); II- 2456 bp band representing 3′KpnI <i>recA</i> wild type carrying 1666 bp of 3′<i>recA</i> gene and 790 bp of downstream region; III- 1036 bp band representing 5′KpnI <i>recA</i> wild type carrying 707 bp of 5′ <i>recA</i> gene and 329 bp of upstream region; IV- 808 bp band representing 5′KpnI Δ<i>recA</i> (706 bp, carrying 377 bp of 5′ <i>recA</i> gene fragment and 329 bp of upstream region joined to <i>hyg</i>^R cassette fragment of 102 bp). Lanes: 1 and 10, 1-kb marker; 2 and 3, Mtb wild-type (control); 4–7, DCO Δ(<i>ku,ligD,recA</i>); 8 and 9, SCO Δ(<i>ku,ligD,recA</i>)-<i>recA</i>.