FIG. 3.

Gene disruption of 71ORF2 (traA). (a and b) Diagrams showing gene disruption of 71ORF2 (traA). The box labeled 71ORF2(traA)∗ represents the internal fragment of 71ORF2 (traA) which was amplified by PCR with primers having EcoRI recognition sequences at the 5′ ends and cloned into the suicide vector pMG226, creating pMG227. The approximate locations of the primers are indicated by horizontal arrows below the 71ORF2(traA)∗ box. The vertical arrows labeled ClaI and HindIII indicate the recognition sites of ClaI and HindIII, respectively. The dotted line between the two HindIII sites indicates the HindIII fragment contained in clone #71. ClaI fragments A and B are cointegrate plasmid specific and are expected to hybridize with the HindIII fragment contained in clone #71. (c) Southern hybridization of cointegrate plasmid pMG229. For pMG1 (lane 1) and pMG229 (lane 2), DNA was digested with ClaI. DNA fragments were separated on an agarose gel, and the gel was treated with alkali transfer buffer (0.4 N NaOH, 1.5 M NaCl) for 20 min; then the DNA fragments were transferred to a nylon membrane by capillary action with alkali transfer buffer (2, 22). After transfer, the membrane was neutralized with 0.5 M Tris-HCl (pH 7.0) for 5 min and rinsed with 2× SSC for 2 min (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The membrane was air dried and baked at 80°C for 2 h. Hybridization and signal detection were performed with a DIG DNA labeling and detection kit (Roche Diagnostics GmbH). The insert of clone #71 was used as a probe. The arrows on the left indicate the positions of the fragments which hybridized with the probe in each lane. The numbers indicate the sizes of the fragments (in kilobase pairs) estimated on the basis of mobility. The arrows on the right indicate the positions of the molecular size markers, a λ HindIII digest; the numbers indicate their sizes (in kilobase pairs). Arrow A indicates the position of the cointegrate plasmid-specific ClaI fragment shown in panel b.