The additional reduced Fd produced via PFO must then be reoxidized using Fd-dependant or bifurcating H2ases. Accordingly, expression of bifurcating H2ase Cthe_0428-0430 increases >1.5-fold in stationary phase. While both bifurcating H2ases (Cthe_0428-0430 and Cthe_0340-342) contain selleck chemicals various upstream regulatory elements including phosphatases, kinases, and/or PAS/PAC sensors potentially capable of regulating transcription

in response to H2 levels or redox changes via a two-component regulatory system as in Ralstonia eutropha[17, 91, 92], only Cthe_0428-0430 expression changed under the conditions tested. Regulation of a NAD(H)-dependent Fe-only H2ase containing an upstream histidine and serine/threonine protein kinase has selleck kinase inhibitor also been reported in Ta. tencongensis, in which a fourfold decrease in NAD(H)-dependent H2ase activity was accompanied by an increase in AldH and ADH activities in response to high H2 partial pressures [19]. Providing that NADH/NAD+ ratios increase during

transition from exponential to stationary phase as in C. cellulolyticum and Ca. saccharolyticus, the observed increase in select ADHs [AdhE (Cthe_0423), Cthe_0101, glutamyl reductase (Cthe_1863), and groES (Cthe_0388)] during stationary phase may help C. thermocellum reoxidize NADH and concomitantly produce ethanol, GNA12 which explains the observed inversion of acetate-to-ethanol ratio. A similar mechanism of increasing expression of select ADHs

to dispose of reducing equivalents during growth and ethanol accumulation is employed by Thermoanaerobacter species [93]. Surprisingly, we observed a 2.4-fold increase in acetate kinase expression in stationary phase despite having lower acetate to ethanol ratios. This differs from the mRNA expression profiles on cellulose reported by Raman et al.[37]. However, 4-plex 2D-HPLC-MS/MS did not detect the presence of PTA required for production of acetyl-P, and thus changes in expression profiles of PTA in response to growth phase could not be determined. Energy generation and pyrophosphate (PPi) metabolism In addition to substrate level phosphorylation mediated by 1,3-phosphoglycerate kinase, pyruvate phosphate dikinase, phosphoenolpyruvate carboxykinase, acetate kinase, and acetate thiokinase (see above), ATP can also be generated using ATP synthase powered by a proton motive force (PMF). While two types of ATP synthases were detected, including the F-type (Cthe_2602-2609) and the V-type (Cthe_2262-2269), overall expression of the latter was higher ( Additional file 2). Expression of both ATP synthases was generally consistent throughout growth.