4%) [2] YE yeast extract medium yeast extract (0.4%) [2] AMS acetate-mineral salt medium acetate (40 mM), HCO3 – (20 mM) [2] and this report   hexose- and ribose-grown medium sugar (hexose or ribose, 40 mM), yeast extract (0.02%) [2] and this report Non-autotrophic CO2 assimilation by H. modesticaldum It has been recognized that pyruvate is the preferred organic carbon source for heliobacteria and it can support both photoheterotrophic and chemotrophic growth [3]. Consistent with previous reports, our studies show that H. modesticaldum grows

better using pyruvate as carbon source compared to other organic carbon sources (Figure 2A), and the rate of cell growth corresponds SAHA HDAC to that of pyruvate consumption (Figure 2B). In contrast to CO2-enhanced growth of Chlorobaculum (Cba.) tepidum and other green sulfur bacteria [12], no difference in growth rate can be detected with or without 0.4% HCO3 – included in pyruvate-grown cultures (Figure 2B). Moreover, no growth can be detected with HCO3 – as the sole carbon source (Figure 2A). The lack of autotrophic growth in H. modesticaldum can be attributed to the lack of a gene encoding ATP citrate lyase (ACL) [1, 5], which catalyzes the cleavage of citrate to acetyl-CoA and oxaloacetate (OAA) Cell Cycle inhibitor and is one of

the key enzymes specific in the autotrophic CO2 fixation via the reductive (or reverse) tricarboxylic acid (rTCA) cycle [13–15]. To confirm the absence of an enzyme having ACL activity, we performed activity assays in cell-free extracts of H. modesticaldum and Cba. tepidum. The latter served as a positive control for ACL activity, which is documented in Cba. tepidum [16, 17]. Consistent with previous reports, the activity of ACL was clearly detected in cell free extracts of Cba. tepidum, but not in H. modesticaldum (Additional file 4: Figure S3). Additionally, the activity of citrate synthase,

catalyzing the formation of citrate from condensation of OAA and acetyl-CoA in the oxidative TCA cycle, also cannot be detected (data not shown). Alternatively, the genomic data suggest that certain non-autotrophic pathways may be PS341 available TCL for CO2 assimilation in H. modesticaldum [1]. The pckA gene (HM1_2773), encoding phosphoenolpyruvate (PEP) carboxykinase (PEPCK), has been annotated in the genome of H. modesticaldum. The activity of PEPCK (30 nmole/min•mg protein) was detected in cell-free extracts of H. modesticaldum and pckA is expressed, based on QRT-PCR analysis, in all of the growth conditions tested (Table 2 and Additional file 3: Table S1). Together, our experimental data indicate that H. modesticaldum uses PEPCK to assimilate CO2 and generates ATP via substrate-level phosphorylation (PEP + ADP + CO2 → OAA + ATP), in agreement with previously proposed carbon metabolic pathways in heliobacteria [1, 18].