Introduction of the fdoG gene on a plasmid, however, restored the activity to the mutant (Figure 4A bottom panel). Notably, A 769662 replacing formate with hydrogen as electron donor revealed that
both enzymes also catalyzed the hydrogen-dependent reduction of PMS/NBT (Figure 4A, middle panel). A similar pattern for H2: PMS/NBT oxidoreductase activity was observed as was seen for formate: PMS/NBT oxidoreductase activity (compare the middle and bottom panels in Figure 4A). Taken together, these findings suggest that Fdh-N is the more effective enzyme at transferring the electrons from H2 to BV/TTC than to PMS/NBT. That Fdh-O is nevertheless effective at catalyzing H2-dependent BV reduction is shown in the lane containing an extract derived from CP1104
(labelled FTD147Δfnr in Figure 4) in which an fnr mutation was introduced into the hydrogenase-negative strain FTD147 (Figure 4, top panel). Synthesis of Fdh-N is absolutely dependent on the redox regulator FNR [1, 21] and thus is absent in an fnr mutant. In contrast, Fdh-O activity is apparently up-regulated in the fnr mutant (Figure 4A). Fdh-N/O show H2: BV and H2: PMS/NBT oxidoreductase activities in extracts after respiratory growth with nitrate Biosynthesis of Fdh-N is enhanced when E. coli is grown anaerobically in the presence of nitrate [1, 5, 21], while synthesis of Fdh-O is essentially constitutive [9]. The same strains selleckchem analyzed in Figure 4A were grown
anaerobically in the presence of nitrate and aliquots of crude extracts were separated by non-denaturing PAGE followed by staining for H2: BV oxidoreductase, Orotidine 5′-phosphate decarboxylase H2: PMS/NBT oxidoreductase and formate: PMS/NBT oxidoreductase activities. The gel presented in the top panel of Figure 4B shows clearly a H2: BV oxidoreductase activity in extracts of strains FTD147, CP1104 (FTD147Δfnr), as well as in the fdoG mutant. The activity in extracts of MC4100 shown in this experiment was only weakly discernable (Figure 4B, top panel, first lane). As anticipated [13], synthesis of Hyd-1 and Hyd-2 was strongly reduced in MC4100 after growth in the presence of nitrate (data not shown). The mutant with a deletion in the fdnG gene essentially lacked H2: BV oxidoreductase activity but this could be recovered by introduction of the fdnG gene on plasmid pCA24N-fdnG + (Figure 4B, top panel). Aliquots of the same extracts specifically stained to visualize H2: PMS/NBT oxidoreductase and formate: PMS/NBT oxidoreductase activities showed a strong Fdh-N-dependent H2: PMS/NBT oxidoreductase activity (Figure 4B, middle panel).