FIG. 3.
Generation of the mini-Tn5Tctmo/pcu transposon and its different locations in the chromosomes of DOT-T1EΔtodCpobA derivatives. (A) Construction. The 7.4-kb BamHI fragment of pMC4 containing the tmoXABCDEF genes was subcloned at the BamHI site in the polylinker of pUC19, generating plasmid pMIR32. The 7.6-kb MluI/NheI fragment of pPCU17 (Ben-Bassat et al., U.S. patent application 20020151003) containing the pcuRCAXB genes was subcloned at the HindII/XbaI sites of pUC18NotI to obtain pMIR40. The 7.4-kb BamHI fragment of pMIR32 was incorporated into pMIR40 at the unique BamHI site to obtain plasmid pMIR42. Finally, the 15-kb NotI fragment of pMIR42 containing the pcu and tmo gene clusters was subcloned at the unique NotI site of pUT/Tc, which was located within the transposable element of the mini-Tn5Tc transposon, yielding plasmid pMIR44. Restriction sites are indicated as follows: B, BamHI; N, NotI; H, HindIII. Insertion sequences delimiting transposable elements are indicated by solid boxes. (B) Southern blot of exconjugants. Portions (10 μg) of total DNA of five independent clones were digested with EcoRV, which did not cut in the tetracycline (Tc) resistance determinant gene, and the fragments were analyzed by Southern blotting with a 429-bp digoxigenin-labeled PCR probe. The probe was amplified with the oligonucleotides 5′-CAACCCAGTCAGCTCCTTCC-3′ and 5′-GGACAGCTTCAAGGATCGCT-3′ and annealed internal to the tetracycline resistance gene. λHindIII (λH)was used as molecular weight marker. Lane 1, DOT-T1E-21; lane 2, DOT-T1E-17; lane 3, DOT-T1E-22; lane 4, DOT-T1E-24; lane 5, DOT-T1E-1-10a.