Figure 2.

Identification of genes required for B₁₂ transport and assimilation. (a) Schematic of the screening cascade. The ΔmetH Tn library was plated on selective medium containing 10 µg ml⁻¹ CNCbl. 612 ‘B₁₂-resistant’ clones were isolated and regrown in standard liquid medium, eliminating 35 mutants owing to poor (n = 14) or absent (n = 21) growth. A secondary screen tested the B₁₂ uptake ability of the remaining 577 insertion mutants in a four-well microtitre assay using 0.1% propionate (Prop) plus 20 µg ml⁻¹ kanamycin as base medium (well 1) supplemented with 10 µg ml⁻¹ CNCbl (well 2), 3NP (well 3) and 3NP plus 10 µg ml⁻¹ CNCbl (well 4). A total of 84 mutants failed to grow in well 4, suggesting impaired B₁₂ uptake. Each determination was performed in duplicate, and the results confirmed in batch culture. (b) Insertion mutants with disrupted B₁₂ uptake ability.