Figure 3.

Bioassay for the detection of cobamides synthesized in vitro. (A and B) Detection of cobamides synthesized from AdoCbi and DMB (i.e., NLA reactions). (C) Detection of cobamides synthesized from AdoCbi-GDP and α-ribazole-5′-P (i.e., cobalamin synthase reactions). A 5-μl sample of each reaction mixture was applied to an overlay of strain JE212 on minimal glucose medium supplemented with 0.1 mM histidine. (A) Results from reaction mixtures containing cell-free extract (from a strain carrying pNLA1) as the source of CobU, CobS, and CobT. Shown are the complete reaction mixture (1); the vector-only control reaction (2); a reaction mixture lacking DMB (3); 70 pmol (CN)₂Cbi standard (4); and 14 pmol CNCbl standard (5). (B) Results from reactions performed by using homogeneous CobU and CobT and partially purified (His)₆CobS. Shown are the complete reaction (1); reactions in which CobU (2), CobS (3), and CobT (4) were individually omitted; and 14 pmol CNCbl standard (5). (C) Shown are the complete reaction with a cell-free extract of a strain carrying pCOBS4 as a source of CobS (1); the vector-only control reaction (2); a reaction mixture lacking α-ribazole-5′-P (3); the complete reaction with (His)₆CobS (4); and 14 pmol CNCbl standard (5).