Second, as shown in Figure 5, PEPCK is required to convert PEP into OAA in the partial reductive TCA (rTCA) cycle. Without assimilating CO2 by PEPCK, carbon flux through the partial rTCA cycle cannot take place. Possible functions of PFOR and FNR during chemotrophic growth To evaluate the mTOR kinase assay function of PFOR and FNR in pyruvate metabolism in darkness, we examine the culture growth in acetate-supported medium with and without the addition of HCO3 – and acetate excretion from pyruvate-grown cultures. No CO2-enhanced growth in acetate-supported

medium can be detected, and cell growth in acetate medium is extremely slow in darkness (data not shown). Also, approximately 44% of the pyruvate in pyruvate-grown cultures is converted into acetate during chemotrophic growth (Table 3). Madigan and coworkers reported a large amount of CO2 by analyzing the gas phase of chemotrophic-grown HMPL-504 concentration heliobacterial cultures [21]. Together, the following roles of PFOR and FNR during chemotrophic growth can be proposed (Figure 8): (1) PFOR provides energy and reducing power for cellular functions. PFOR catalyzes pyruvate fermentation to acetyl-CoA, CO2, 2 Fdred and 2 H+ (equation 1). Fdred is used for carbon and nitrogen metabolism

in PLX3397 cell line darkness (Figure 7), and 2 Fdred and 2 H+ from the oxidation of pyruvate can generate H2 by [FeFe]-hydrogenase (2 Fdred + 2 H+ → 2 Fdox + H2) (Figure 8). 2 Fdox can be then used for pyruvate fermentation. Further, acetyl-CoA can be utilized to generate acetate and produce ATP through substrate-level phosphorylation catalyzed by ACK (Table 3 and Figure 5). This

ATP production process may partially explain pyruvate being the most favorable nutrition source; and (2) FNR produces NADPH during chemotrophic growth. As mentioned above, essential genes in the oxidative pentose phosphate and ED pathways, two potential sources producing NADPH, are missing in the H. modesticaldum genome. While NADPH is generated by FNR via the light-induced Molecular motor electron transfer during phototrophic growth, NADPH production is also required during chemotrophic growth. It is likely that some Fdred molecules produced by pyruvate fermentation in H. modesticaldum are used to produce NADPH by FNR during chemotrophic growth (equation 2). When this occurs, Fdox is regenerated for pyruvate fermentation (Figure 8). In summary, since [FeFe]-hydrogenase and FNR compete for using 2 Fdred and 2 H+ produced from pyruvate fermentation, intracellular NAD(P)H availability likely plays important role on H2 production, as well as nitrogen and carbon flux, in H. modesticaldum. Figure 8 Summary of energy metabolism of H. modesticaldum during phototrophic and chemotrophic growth described in this report. Bold curves and lines represent the proposed major pathways during phototrophic (shown in blue) and chemotrophic (shown in green) growth.