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Flux Balance Analysis of Cyanobacterial Metabolism: The Metabolic Network of Synechocystis sp. PCC 6803

Figure 4

Experimental validation of the glyoxylate shunt.

(A) Enzymatic test to determine isocitrate lyase activity in cell-free extract of Synechocystis PCC 6803. Phenylhydrazon formation were measured as increase of A324 nm with time. Trace A - crude cell extract of Synechocystis cells, Trace B - crude cell extract passed over a PD-10 gel filtration column both corresponding to a protein concentration of reaction volume. Arrows mark the stepwise addition of phenylhydrazin (Phe, 5 mM), isocitrate (IC, 1 mM) and NADP ( mM), the latter as a control for isocitrate dehydrogenase activity. Increase in A324 nm in Trace A after adding of phenylhydrazin results from phenylhydrazine reactive metabolites, further increase after addition of IC results from internal NADP as a co-substrate of isocitrate dehydrogenase within the crude extract. Both traces show the same trend after addition of NADP, corresponding to the same isocitrate dehydrogenase activity. (B) The capability for utilization of acetate as sole carbon source was tested in spot assays in the presence of the photosystem II inhibitor DCMU (photoheterotrophic growth). Respective controls were conducted without the addition of DCMU (putative photomixotrophic growth) and without both sodium acetate and DCMU, respectively (photoautotrophic growth) to BG11 agar. 1∶1, 1.10 and 1∶100 represent dilution factors of the stock cell suspension of Synechocystis sp. PCC 6803 that contains chlorophyll a per ml cell suspension.

doi: https://doi.org/10.1371/journal.pcbi.1003081.g004