Construction of <i>Cba. limnaeum bciD</i> gene inactivated mutant.

<p>(A) Schematic map of genes arrangement around <i>bciD</i> gene in the genome of <i>Cba. limnaeum</i> RK-j-1, and its insertional inactivation. The <i>aadA1</i> gene, conferring resistance to streptomycin and spectinomycin, was inserted in <i>bciD</i>. Arrows represent the primers bciD-F (i), bciD-R (ii), bciD-inf-F (iii), bciD-inf-R (iv), bciD-comf-F (v), and bciD-comf-R (vi). (B) PCR confirmation of gene interruption. The <i>bciD</i> gene was amplified from genomic DNA extracted from the wild type (lanes 1 and 2) and a mutant (lanes 3 and 4) of <i>Cba. limnaeum</i>, using above bciD-comf-F and -R primers. The products in lanes 2 and 4 were then digested by restriction enzyme <i>Eco</i>RV, and the fragments yielded from wild type and the mutant were 1.36 and 0.78, and 2.22 kbp, respectively. Lane M, molecular size marker (the sizes of bands are indicated at left).