Figure 1.
Outline of the pathways for 2-OG metabolism in cyanobacteria.
2-OG, produced from isocitrate in the TCA cycle, can be used by all cyanobacteria as backbone to incorporate ammonium, through the GS/GOGAT pathway. Alternatively, the majority of cyanobacteria can transform 2-OG to succinic semialdehyde, and later to succinate, through reactions catalyzed by 2-OG decarboxylase (2-OGDC) and succinic semialdehyde dehydrogenase (SSADH), respectively [28]. These reactions (shown in grey) are missing in marine Synechococcus and Prochlorococcus strains.
Figure 2.
ICDH Western blotting of cell extracts from Prochlorococcus sp. strain PCC 9511 cultures subjected to different conditions.
Cultures were subjected to the indicated conditions for 24(except in the case of iron starvation, which were starved for 8 h). C, control; -Fe, iron starvation; -N, nitrogen starvation; -P, phosphorus starvation; MSX, 100 µM methionine sulfoximine; AZA, 100 µM azaserine. The quantification of bands is shown below the picture, assigning an arbitrary value of 100 to the control conditions.
Figure 3.
Effect of nitrogen starvation.
A, Time course of ICDH activity in Prochlorococcus sp. strain PCC 9511 cultures (squares, control cells; circles, N-starved cells). B. Time course of 2-OG concentration (light grey, control cells; dark grey, N-starved cells). Values are the average of three independent biological replicates, each of them measured in triplicate. Error bars correspond to the standard deviation.
Figure 4.
Effect of a metal-catalyzed oxidative system (Fe3+/ascorbate) on ICDH activity and concentration.
A, Time course of ICDH activity in Prochlorococcus sp. PCC 9511 cell extracts with the following additions: ▪, control cells (no addition); •, 0.2 mM FeCl3; ▴, 0.2 mM FeCl3 +1 mM ascorbate; ⧫, 0.2 mM FeCl3 +5 mM ascorbate; ▾, 0.2 mM FeCl3 +10 mM ascorbate. Values are the average of three biological samples, each of them measured in triplicate. Error bars correspond to the standard deviation. B, Western blotting of ICDH in Prochlorococcus sp. PCC 9511 cell extracts after 60 min of incubation under the following additions: Control, no addition; 1, 0.2 mM FeCl3; 2, 0.2 mM FeCl3 +1 mM ascorbate; 3, 0.2 mM FeCl3 +5 mM ascorbate; 4, 0.2 mM FeCl3 +10 mM ascorbate. The quantification of bands is shown below the picture, assigning an arbitrary value of 100 to the control conditions. C, Coomasie-stained gel corresponding to a duplicate of that used for the Western blotting shown in B.
Figure 5.
Effects of azaserine addition on ICDH activity, icd expression, ICDH enzyme concentration and 2-OG concentration.
A, Effect on ICDH activity in Prochlorococcus sp. strain PCC 9511 cultures (▪, control cells; •, cells in the presence of 100 µM azaserine). B, Effect on icd expression in Prochlorococcus sp. PCC 9511 cultures. C, Western blotting from cultures under control conditions or subjected to 100 µM azaserine addition. Lanes are marked with C (control) or AZA (azaserine), followed by sampling time (in hours). Quantitation of bands is shown below the picture, assigning an arbitrary value of 100 to the time 0 of each series (control, azaserine). D, Time course of 2-OG concentration (light grey, control cells; dark grey, azaserine-treated cells). Values are the average of at least three independent biological samples. Error bars correspond to the standard deviation.
Figure 6.
Effect of darkness on the ICDH activity.
Time course of ICDH activity in Prochlorococcus sp. PCC 9511 cultures. ▪, control cells; •, cells under darkness. Each value shows relative values (cells in the dark vs control cells). Values are the average of three independent biological samples, each of them measured in triplicate. Error bars correspond to the standard deviation.
Figure 7.
Effect of DCMU and DBMIB on the ICDH activity.
A, Changes in ICDH activity in Prochlorococcus sp. PCC 9511 cultures after addition of 0.3 µM DCMU. Black, control cells; grey, cells subjected to the DCMU addition. B Changes in ICDH activity in Prochlorococcus sp. PCC 9511 cultures after addition of 0.06 µM DBMIB. Black, control cells; grey, cells subjected to the DBMIB addition. Values are the average of three independent biological samples, each of them measured in triplicate. Error bars correspond to the standard deviation.