Ammonia-oxidizing archaea use the most energy efficient aerobic pathway for CO2 fixation Martin Könneke, Daniel M. Schubert, Philip C. Brown, Michael Hügler, Sonja Standfest, Thomas Schwander, Lennart Schada von Borzyskowski, Tobias J. Erb, David A. Stahl, Ivan A. Berg Supporting Information: Supplementary Appendix SI Text Phylogenetic analysis of the proteins involved in the HP/HB cycle in N. maritimus. The enzymes of the HP/HB cycle with unequivocally identified genes consisting of more than 200 amino acids were used for the phylogenetic analysis (Table 1). The genes for biotin carrier protein (Nmar_0274, 140 amino acids), small subunit of methylmalonyl-CoA mutase (Nmar_0958, 140 amino acids) and methylmalonyl-CoA epimerase (Nmar_0953, 131 amino acids) were not analyzed, because their small size prevents reliable phylogenetic tree construction. The genes for acetyl-CoA/propionyl-CoA carboxylase and methylmalonyl-CoA carboxylase homologues can be found in autotrophic Thaumarchaeota and aerobic Crenarchaeota as well as in some heterotrophic Archaea (SI Appendix, Figs. S7-S9, Table S4). Although these enzymes are usually regarded as characteristic enzymes of the HP/HB cycle, they also participate in various heterotrophic pathways in Archaea, e.g. in the methylaspartate cycle of acetate assimilation in haloarchaea (1), propionate and leucine assimilation (2, 3), oxaloacetate or methylmalonyl-CoA decarboxylation (4, 5), anaplerotic pyruvate carboxylation (6). In all three trees (SI Appendix, Figs. S7-S9) the crenarchaeal enzymes tend to cluster with thaumarchaeal ones, but there appears to be no special connection between aerobic Crenarchaeota (Sulfolobales) and Thaumarchaeota sequences. Thaumarchaeal 4-hydroxybutyryl-CoA dehydratase genes form a separate cluster closely related to bacterial sequences; the bacterial enzymes are probably involved in aminobutyrate fermentation (Fig. 2D and SI Appendix, Fig. S6). The aerobic Crenarchaeota (Sulfolobales) sequences are closely related to those of other autotrophic Crenarchaeota possessing the dicarboxylate/4-hydroxybutyrate cycle, but only distantly related to the sequences from Thaumarchaeota (Fig. 2D and SI Appendix, Fig. S6). This suggests an independent emergence of the 4-hydroxybutyryl-CoA dehydratase gene in Thaumarchaeota and autotrophic Crenarchaeota. N. martitimus 3-hydroxybutyryl-CoA dehydrogenase Nmar_1028 is homologous to the 3-hydroxybutyryl-CoA dehydrogenase domain of archaeal bifunctional (fusion) crotonyl-CoA hydratase/3-hydroxybutyryl-CoA dehydrogenase (7). This protein is not specific for autotrophic species and is present in most of Archaea. Interestingly, the Thaumarchaeota ancestor seems to have lost the crotonyl-CoA dehydratase domain of this protein, whereas the dehydrogenase domain remained untouched. Aerobic Crenarchaeota have the “full” version of the protein, and no special connection between crenarchaeal and thaumarchaeal proteins could be seen (SI Appendix, Fig. S13). Neither the ADP-forming 3-hydroxypropionyl-CoA nor 4-hydroxybutyryl-CoA synthetases from N. maritimus are homologous to the corresponding AMP-forming synthetases from M. sedula (7-10) (Figs. 2A and 2B, SI Appendix, Figs. S10 and S11). The homologous proteins are distributed in euryarchaeal and bacterial genomes, but not in Crenarchaeota. The N. maritimus 3-hydroxypropionyl-CoA dehydratase/crotonyl-CoA hydratase Nmar_1308 shares high sequence identity with bacterial enzymes (Fig. 2C and SI Appendix, Fig. S12), mainly to those from Firmicutes, and is thus an obvious example of lateral gene transfer. The genes for reductases of the cycle (malonyl-CoA, malonic semialdehyde, acryloyl- CoA, succinyl-CoA and succinyl-CoA reductases) functioning in N. maritimus are not known. The BLASTP search using the corresponding proteins of M. sedula as queries against the genome of N. maritimus revealed the absence of closely related proteins (SI Appendix, Table S3). Furthermore, the best hits outside of aerobic Crenarchaeota (Sulfolobales) were (heterotrophic) Bacteria, Archaea and Eukarya, but never Thaumarchaeota. Therefore, N. maritimus probably uses unrelated enzymes responsible for the corresponding transformations in the HP/HB cycle. This again lends support to the hypothesis that the autotrophic cycles evolved independently. Taken together, our phylogenetic analysis indicates the absence of any specifical relationship between the proteins involved in the HP/HB cycle in Crenarchaeota and in Thaumarchaeota. This indicates that this pathway evolved independently in the ancestors of Thaumarchaeota and aerobic Crenarchaeota. Importantly, the corresponding proteins are present in all currently sequenced thaumarchaeal genomes, thus suggesting their potential for autotrophic growth using the HP/HB cycle. Comparison of ATP requirements for the synthesis of central metabolic precursors. The ATP costs for the different aerobic autotrophic CO2 fixation pathways (Table 3) were calculated as followed. Calvin-Benson cycle. The synthesis of the main product of the cycle, glyceraldehyde 3- phosphate, requires 9 ATP. Its further conversion to phosphoenolpyruvate via classical reactions of glycolysis leads to reimbursement of an ATP equivalent (8 ATP). Oxaloacetate is produced in phosphoenolpyruvate carboxylase reaction (no additional ATP is required, i.e. 8 ATP). Pyruvate formation from phosphoenolpyruvate in pyruvate kinase reaction leads to the release of one ATP equivalent (7 ATP for 1 pyruvate). Acetyl-CoA is synthesized through pyruvate dehydrogenase reaction (7 ATP). Glutamate precusor 2-oxoglutarate is synthesized from acetyl-CoA and oxaloacetate via citrate synthase, aconitase and isocitrate dehydrogenase reactions (15 ATP). Note that the extra costs of the oxygenase side reaction of the key enzyme of the Calvin-Benson cycle (ribulose-1,5-bisphosphate carboxylase) are not taken into account. 3-Hydroxypropionate bi-cycle. The synthesis of the main product of the cycle, pyruvate, costs 7 ATP. Acetyl-CoA is synthesized through pyruvate dehydrogenase or pyruvate:acceptor oxidoreductase reaction (7 ATP). Pyruvate conversion to phosphoenolpyruvate proceeds via pyruvate phosphate dikinase reaction (9 ATP) (11, 12). Oxaloacetate is produced from phosphoenolpyruvate via phosphoenolpyruvate carboxylase (9 ATP). 2-Oxoglutarate synthesis proceeds from acetyl-CoA and oxaloacetate via citrate synthase, aconitase and isocitrate dehydrogenase reactions (16 ATP). Note that the fate of a pyrophosphate molecule synthesized in the propionyl-CoA synthase reaction is not known. It is either hydrolyzed by soluble pyrophosphatase, or the energy could be partly recovered through the action of proton-translocating pyrophosphatase. In any case, it does not significantly change the ATP costs of the pathway. M. sedula HP/HB cycle. The main product of the cycle is acetyl-CoA (6 ATP). An additional half-turn of the cycle lead to the formation of succinyl-CoA (10 ATP), which is converted to oxaloacetate either via succinyl-CoA synthetase, succinate dehydrogenase, fumarase and malate dehydrogenase (9 ATP), or via succinyl-CoA reductase and then via succinic semialdehyde dehydrogenase, succinate dehydrogenase, fumarase and malate dehydrogenase (10 ATP) (13). Phosphoenolpyruvate synthesis requires phosphoenolpyruvate carboxykinase activity (9 or 10 ATP, depending on the pathway of succinyl-CoA conversion into oxaloacetate), and pyruvate is synthesized from malate via malic enzyme (9 or 10 ATP, depending on the pathway of succinyl-CoA conversion into malate). 2-Oxoglutarate synthesis proceeds from acetyl-CoA and oxaloacetate (15 or 16 ATP). Note that the fate of two pyrophosphate molecules synthesized in the cycle (in 3- hydroxypropionyl-CoA and 4-hydroxybutyryl-CoA synthetase reactions) is not known. It is probably hydrolyzed by soluble or proton-translocating pyrophosphatase. In the last case, one proton/pyrophosphate would be translocated through the membrane, making the pathway slightly more energetically efficient. N. maritimus HP/HB cycle. The main product of the cycle is acetyl-CoA. Since the synthetases in the cycle produce ADP instead of AMP, the synthesis of one acetyl-CoA costs 4 ATP. An additional half-turn of the cycle leads to the formation of succinyl-CoA (7 ATP), which is then converted to oxaloacetate via succinyl-CoA synthetase, succinate dehydrogenase, fumarase and malate dehydrogenase (6 ATP, see Fig. 1, SI Appendix, Table S1). Since phosphoenolpyruvate carboxykinase Nmar_0392 is the only gene in the genome capable of catalyzing C4 conversion to C3 (malic enzyme as well as phosphoenolpyruvate carboxylase genes cannot be identified), phosphoenolpyruvate synthesis requires 7 ATP. The genome of N. maritimus possesses a gene encoding pyruvate:phosphate dikinase (Nmar_0951), whereas the genes for other genes capable of catalyzing phosphoenolpyruvate/pyruvate conversion cannot be identified. Therefore, pyruvate formation from phosphoenolpyruvate leads to the release of two ATPs (5 ATP). 2- Oxoglutarate synthesis proceeds from acetyl-CoA and oxaloacetate (10 ATP). For the schemes of M. sedula and N. maritimus central carbon metabolism, see Fig. 1. The values for the amount of ATP required for the synthesis of 1 g of the cell biomass (Table 3) are designated here as the amount of ATP (in mole) required for the formation of the central metabolic precursors acetyl-CoA, pyruvate, phosphoenolpyruvate, oxaloacetate and 2-oxoglutarate necessary for the synthesis of 1 g of dry cells. The amounts of the precursors required for the biosynthesis