Fig 2.

Construction and confirmation of T. kodakarensis LC124 (ΔTK1413). (A) Plasmid pLC113 was generated from pTS535 (Table 1) by cloning amplicons from T. kodakarensis genomic DNA upstream and downstream of the TK0254-TK0664 cassette. Plasmid pLC124, generated by deletion of TK1413 from pLC113, was used as the donor DNA to transform T. kodakarensis TS517. The genome structure of the intermediate strain generated was confirmed, as illustrated, and recombination between the duplicated sequences deleted the cassette to yield T. kodakarensis LC124 with the genome as shown. The positions of primers (primers a to g [see Table S1 in the supplemental material for sequences]) used to generate diagnostic amplicons (C) and probes for Southern blotting (D to F) are indicated by arrows above the T. kodakarensis TS517 genome. The 2,905-bp PvuII restriction fragment to which the probes hybridized is indicated. (B) Expanded illustration of the primer locations (heavy arrows) and the amplicons generated. (C) Ethidium bromide-stained agarose gel for electrophoretic separation of the amplicons generated using the primer pairs a-b and c-d from T. kodakarensis LC124, LC125, and TS517 genomic DNAs. (D to F) Southern blots of PvuII-digested T. kodakarensis TS517, LC124, and LC125 DNAs probed with DIG-labeled amplicons (primer pairs c-d and f-g) or ³²P-labeled oligonucleotide primer e. In panel D, the 2,905-bp PvuII fragment that contains TK1413 in T. kodakarensis TS517 is indicated by an arrow. This fragment is not present in T. kodakarensis LC124. The PvuII fragment present in both genomic DNAs which contains TK2289 and thus cross-hybridizes with the probe is noted by an asterisk. In panel F, as the probe includes sequences immediately adjacent to TK1413, it hybridized to the PvuII fragment in T. kodakarensis LC124 DNA that contains the ΔTK1413 deletion (gray arrow). As the probe contains the entire TK1413 sequence, it cross-hybridized to the ∼1-kbp PvuII fragment present which contains TK2289 (asterisk) in both T. kodakarensis TS517 and LC124 DNAs. M, molecular size marker (in base pairs).