Ethanol-acetate fermentation of C. kluyveri. For simplification purposes it was assumed that only butyrate, acetic acid, and H₂ rather than caproate are formed as fermentation products. Inset shows the two membrane-associated, energy-converting enzyme complexes involved in the fermentation: ferredoxin:NAD oxidoreductase (RnfA-E) and ATP synthase (AtpA-I). For the other cytoplasmic enzymes involved see SI Table 2. The clostridial ferredoxin with two [4Fe4S] clusters accepts two electrons upon reduction (28). In the scheme it is assumed that 7 of the 10 NADH required for the reduction of 5 crotonyl-CoA to 5 butyryl-CoA (see text and reaction 5) are generated during ethanol oxidation to acetyl-CoA (gray background) and that three are generated via NAD⁺ reduction with Fd_red²⁻ (orange background). It has been omitted that some of the Fd_red²⁻ generated during crotonyl-CoA reduction is also used for the regeneration of NADPH, which is required for acetoacetyl-CoA reduction. The yellow area indicates the microcompartment, and the shaded area highlights the steps proposed to be involved in the catalysis of reaction 5 (see text) by the butyryl-CoA dehydrogenase complex composed of butyryl-CoA dehydrogenase (Bcd) and the electron transport flavoproteins (EtfAB). Bcd, EtfA, and EtfB each contain FAD.

CDS clusters predicted to encode nonribosomal peptide synthetase and polyketide synthase-nonribosomal peptide synthetase hybrids. A, C, Cy, E, and T are domains of nonribosomal peptide synthetases: A, adenylation domain; C, condensation domain; Cy, cyclization domain; E, epimerization domain; T, thiolation domain. ACP, AT, KR, and KS are domains of polyketide synthases: ACP, acyl carrier protein domain; AT, acyltransferase domain; KR, ketoreductase domain; KS, ketosynthase domain. M, TE, and Sfp are domains occurring in both types: M, methyltransferase domain; TE, thioesterase domain; Sfp, phosphopantetheinyl transferase domain.

Circular representation of the chromosome and plasmid of C. kluyveri. Genes encoded on the forward and reverse strands of the chromosome (outer two rings) are color-coded by red and dark blue, respectively. Predicted prophages are colored in green, and the sets of genes predicted to encode polyketide/nonribosomal peptide synthetases are indicated in yellow. The next inner ring shows the position of putative alien genes in gray. On the fourth ring the deviation from the average G+C content is plotted. Significant deviations are due to ribosomal RNA clusters (black arrows) and putative alien genes. Encoded transfer RNAs are indicated as black bars on the fifth ring. The plasmid map shows genes on the forward and reverse strands, color-coded in red and dark blue. The most inner ring shows the deviation from the average G+C content of the plasmid. Furthermore, the phage-terminase containing an intein is indicated in light blue. The intein was verified by heterologous production of the terminase in Escherichia coli (data not shown).

Effects of protonophore and F₁F_o-ATPase inhibitor on H₂ and fatty acid formation by cell suspensions of C. kluyveri. Shown is the effect of protonophore TCS and the F₁F_o-ATPase inhibitor DCCD on hydrogen formation (A) and fatty acid formation (B). One mole of fatty acid corresponds to 1 mol of butyrate or 0.5 mol of caproate. For details on assay mixture composition see Materials and Methods. Open circles represent the control without TCS and DCCD (41).