saccharolyticus (van de Werken et al., 2008), including two genes that were annotated to code for a PFK (Csac_1830 and Csac_2366). A sequence alignment of these kinases against PFK-A family members (data not shown) and a more detailed analysis of their amino acid Cell Cycle inhibitor sequences (Fig. 1), revealed that Csac_1830 belongs to the B1 group and contains the typical ATP-dependent PFK amino acid combination G104 and G124 (Chi & Kemp, 2000; Bapteste et al., 2003). On the

other hand, Csac_2366 belongs to the B2 group and contains the typical PPi-dependent PFK amino acid residues D104 and K124 (Chi & Kemp, 2000; Moore et al., 2002). These results strongly suggest that Csac_1830 is an ATP-dependent PFK and that Csac_2366 is a PPi-dependent PFK. The genome also contains the genes encoding a PK (Csac_1831) and a PPDK (Csac_1955), which are both able to perform the catabolic conversion of PEP to pyruvate (van de Werken et al., 2008). PPDK catalyzes a PPi-dependent reaction, whereas PK requires ADP (Fig. 2a). The anticipated PPi dependence of the specified glycolytic steps prompted us to seek other PPi-dependent reactions. The C. saccharolyticus genome lacks any homolog to cytosolic pyrophosphatases (COG0221, COG1227) (Bacillus type; Shintani et al., 1998). Instead, ABT-199 research buy it was found to contain a gene coding

for a V-type proton-pumping membrane-bound pyrophosphatase (H+-PPase, COG3808, Csac_0905). The anticipated H+-PPase has 14 predicted membrane

helices and is expected to be an integral membrane protein. Sequence-based phylogenetic analysis of the H+-PPase revealed Silibinin it as a member of the K+-insensitive group of the H+-PPase protein family (data not shown; Serrano et al., 2004). To confirm the genome sequence-based predictions, we performed enzyme assays on crude CEs. The activities of PK, PPDK, PPi-PFK, ATP-PFK and membrane-bound PPase could all be detected in CEs of C. saccharolyticus (Table 1). Under the specified assay conditions, the average PPDK activity (0.4 U mg−1) was twice the PK activity. For the ATP- and PPi-dependent PFKs, no significant difference was observed in the activity levels (∼0.05 U mg−1). In line with the presence of a membrane-associated pyrophosphatase, high levels of PPase activity (∼0.15 U mg−1) could be demonstrated in the membrane fraction, while the membrane-free CE barely showed PPase activity. Whether the membrane-bound PPase in C. saccharolyticus couples pyrophosphatase activity to the translocation of protons across the membrane remains to be investigated. The presence of PPi-dependent enzymes in the central metabolic pathway suggested the involvement of PPi as an energy carrier in the metabolism of C. saccharolyticus. Therefore, the levels of both ATP and PPi were determined during growth (Fig. 3). The PPi levels were relatively high (approximately 4 ± 2 mM), while the ATP levels were remarkably low (0.43 ± 0.07 mM).