During the exponential growth phase, high PPi levels (approximately

4 ± 2 mM) and relatively low ATP levels (0.43 ± 0.07 mM) were found, and the PPi/ATP ratio decreased 13-fold when the cells entered the stationary phase. Pyruvate kinase activity appeared to be allosterically affected by PPi. Altogether, these findings suggest an important role for PPi in the central energy metabolism of C. saccharolyticus. The extremely thermophilic and strictly anaerobic bacterium Caldicellulosiruptor saccharolyticus belongs to the class of the Clostridia. This bacterium has potential for industrial applications because R788 clinical trial of its ability (1) to produce high hydrogen levels (de Vrije et al., 2007), (2) to grow on complex lignocellulosic material (Ivanova et al.,

2008; de Vrije et al., 2009) and (3) to cometabolize a number of monosaccharides without revealing any form of carbon catabolite repression (van de Werken et al., 2008; VanFossen et al., 2009). For these reasons, C. saccharolyticus recently became the subject of various research projects focusing on renewable energy production (van Niel et al., 2002; Ivanova et al., 2008; de Vrije et al., 2009). The classical Embden–Meyerhof (EM) pathway is the main route of glycolysis in this organism (de Vrije et al., 2007), and analysis of the C. saccharolyticus genome sequence has revealed the presence of all the EM-pathway enzymes (van de Werken et al., 2008). However, the authors of this study indicated further that the C. saccharolyticus genome contains genes coding for an inorganic Ruxolitinib ic50 pyrophosphate (PPi)-dependent pyruvate phosphate dikinase (PPDK) in addition to the pyruvate kinase (PK). Genes coding for typical gluconeogenic enzymes such as pyruvate water dikinase (or PEP synthase) and fructose bisphosphatase next are absent (van de Werken et al., 2008). Interestingly, recent studies on the acetate–lactate metabolic shift in C. saccharolyticus revealed that PPi is a strong modulator of the lactate dehydrogenase (LDH) (Willquist & van Niel, 2010). These observations motivated us to investigate

the role of PPi in the energy metabolism of C. saccharolyticus. PPi-dependent reactions have regularly been described for plants and primitive eukaryotes (Heinonen, 2001). However, little is known about PPi dependency in heterotrophic prokaryotes. Caldicellulosiruptor saccharolyticus DSM 8903 (Rainey et al., 1994) was purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen. For the enzyme and nucleotide measurements, cell extracts (CEs) were prepared from C. saccharolyticus cells, which were cultured batchwise in pH-controlled reactors and in a medium as described previously (van de Werken et al., 2008; Willquist et al., 2009), using glucose as a carbon source (4 g L−1 for the determination of enzyme levels and 10 g L−1 for the determination of nucleotide levels). For the determination of nucleotide levels, the working volume was 1.7 L to minimize the effect of sampling on the culture.