FIG 1.

Cloning steps involved for construction of engineered Azotobacter. (A) Partial restriction map of the ∼7.5-kb BamHI genomic fragment from A. chroococcum CBD15, which contains regions homologous to nifL and nifA of A. vinelandii UW, cloned in the BamHI site of pUC7. The construct has been designated pCL6. The restriction subfragments that hybridize with nifL and nifA of A. vinelandii UW are shown by thick lines. (B) The ∼4.8-kb EcoRI fragment from pCL6 containing the regions homologous to nifL and nifA of A. vinelandii UW was cloned in pUC7, and this construct has been designated pCL6.2. (C) The ∼2.0-kb EcoRI fragment from pHP45ΩKm containing the interposon ΩKm was inserted into the SalI sites of pCL6.2, and this construct has been designated pCL6.3. (D) The construct pCL6.2 was digested with SalI, and deletion of 1,112 bp was achieved by subsequent Bal 31 treatment; the 374-bp EcoRI-BamHI fragment from pBR322 containing the Tet promoter was cloned there in the correct orientation. This construct has been designated pCL6.4. (E) The construct pCL6.3 was introduced into A. chroococcum CBD15 by electroporation. (F) Insertion of the kanamycin interposon into the nifL gene in the genome of A. chroococcum CBD15, as a result of homologous recombination between pCL6.3 and the A. chroococcum CBD15 genome. (G) The construct pCL6.4 was introduced by electroporation into A. chroococcum CBD15, which already had the kanamycin interposon inserted into the nifL gene in its genome. (H) Replacement of the region of the nifL gene comprising 1,112 bp around the two SalI sites and the kanamycin interposon in the genome of A. chroococcum CBD15, as a result of homologous recombination with pCL6.4. Abbreviations: E, EcoRI; B, BamHI; Nc, NcoI; S, SalI; Sa, SacII; Sm, SmaI; K, KpnI; Kn, kanamycin.