The relative intensity of the activity-staining bands was quantified by densitometric analysis (Figure 1B) as described in the Methods section. The intensity of the

Hyd-1 and Hyd-2 activity-staining bands was similar when cells were grown fermentatively in the presence of iron citrate, ferric ammonium sulfate, ferricyanide or ferrocyanide. In cell-free extracts derived from PM06 grown with the three Fe3+ sources ferricyanide, ferric ammonium sulfate and ferric citrate the Hyd-1 activity-staining profile was similar to that of the wild type, however, the intensity was reduced by approximately 50% (Figure 1). On the other hand, Hyd-2 attained a level that was

only between 10 and 20% the intensity of the wild type grown with iron citrate, suggesting that the activity Pictilisib purchase of this enzyme is less readily complemented by addition of oxidized iron. Somewhat surprising, however, was the observation that although some activity of Hyd-2 could be observed after growth of the mutant in the presence of FeCl3, Hyd-1 activity was strongly reduced (Figure 1). Total hydrogenase enzyme activity measured in these extracts of PM06 was nevertheless near wild type (Table 1). MLN8237 cell line It must be noted, however, that under these growth conditions the contributions of Hyd-1 and Hyd-2 to the total activity are low (around 1% for Hyd-1 and 5-10% for Hyd-2), as can be deduced from a strain lacking Hyd-3 (CP971) that retained 4% of the wild type activity with iron chloride [3, 17]. This means that although

Hyd-1 or Hyd-2 activities could barely be observed by in-gel staining, the increase in total hydrogenase activity by addition of FeCl3 was due to Hyd-3 activity. Figure 1 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) Thymidylate synthase 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 YH25448 in vitro 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.