Effect of different iron supplements on Hyd-1 and Hyd-2 activities in PM06 ( feoB ::Tn 5 ) after growth in M9 minimal medium. (A) Aliquots of crude extracts (25 μg) derived from DHP-F2 (negative control) the wild type (MC4100) and PM06 grown anaerobically in M9 minimal medium with glucose and the iron sources indicated were separated by non-denaturing PAGE (7.5% w/v polyacrylamide) and subsequently stained for hydrogenase enzyme activity (see Methods). The iron sources were the following: 7.5 μM FeCl3; 15.3 μM hemin; 50 μM iron citrate (C6H5FeO7) (Fe3+); 10 μM potassium ferrocyanide (K4[Fe(CN)6]) (Fe2+); 10 μM potassium ferricyanide (K3[Fe(CN)6]) (Fe3+); 10 μM Fe(NH4)(SO4)2 (Fe3+). (B) Densitometric quantification of the activity bands corresponding to Hyd-1 (black bars) and Hyd-2 (white bars) from the activity gel. Values were calculated as relative values compared to the intensity of the activity bands in the wild type (MC4100) grown with iron-citrate.