Clostridium kluyveri is unique among the clostridia; it grows anaerobically on ethanol and acetate as sole energy sources. Fermentation products are butyrate, caproate, and H2. We report here the genome sequence of C. kluyveri, which revealed new insights into the metabolic capabilities of this well studied organism. A membrane-bound energy-converting NADH:ferredoxin oxidoreductase (RnfCDGEAB) and a cytoplasmic butyryl-CoA dehydrogenase complex (Bcd/EtfAB) coupling the reduction of crotonyl-CoA to butyryl-CoA with the reduction of ferredoxin represent a new energy-conserving module in anaerobes. The genes for NAD-dependent ethanol dehydrogenase and NAD(P)-dependent acetaldehyde dehydrogenase are located next to genes for microcompartment proteins, suggesting that the two enzymes, which are isolated together in a macromolecular complex, form a carboxysome-like structure. Unique for a strict anaerobe, C. kluyveri harbors three sets of genes predicted to encode for polyketide/nonribosomal peptide synthetase hybrides and one set for a nonribosomal peptide synthetase. The latter is predicted to catalyze the synthesis of a new siderophore, which is formed under iron-deficient growth conditions. butyryl-CoA dehydrogenase ͉ electron transfer flavoproteins ͉ genome sequence ͉ Rnf-dependent energy conservation

Cell extracts of butyrate-forming clostridia have been shown to catalyze acetyl-coenzyme A (acetyl-CoA)-and ferredoxin-dependent formation of H 2 from NADH. It has been proposed that these bacteria contain an NADH:ferredoxin oxidoreductase which is allosterically regulated by acetyl-CoA. We report here that ferredoxin reduction with NADH in cell extracts from Clostridium kluyveri is catalyzed by the butyryl-CoA dehydrogenase/Etf complex and that the acetyl-CoA dependence previously observed is due to the fact that the cell extracts catalyze the reduction of acetyl-CoA with NADH via crotonyl-CoA to butyryl-CoA. The cytoplasmic butyryl-CoA dehydrogenase complex was purified and is shown to couple the endergonic reduction of ferredoxin (E 0 ‫؍‬ ؊410 mV) with NADH (E 0 ‫؍‬ ؊320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (E 0 ‫؍‬ ؊10 mV) with NADH. The stoichiometry of the fully coupled reaction is extrapolated to be as follows: 2 NADH ؉ 1 oxidized ferredoxin ؉ 1 crotonyl-CoA ‫؍‬ 2 NAD ؉ ؉ 1 ferredoxin reduced by two electrons ؉ 1 butyryl-CoA. The implications of this finding for the energy metabolism of butyrate-forming anaerobes are discussed in the accompanying paper.Clostridium kluyveri is a strictly anaerobic gram-positive endospore-forming bacterium (2). Among the clostridia this organism is unique in fermenting ethanol and acetate to butyrate, caproate, and H 2 (38, 49) and in deriving a large portion (30%) of its cell carbon from CO 2 (52). Both its energy metabolism and its pathways of biosynthesis have therefore been the subject of many investigations (26, 39a). In particular, understanding the energy metabolism of C. kluyveri remains a challenge for microbiologists, and one of the pertinent questions is how this organism generates H 2 (36, 39a, 48).

The photophysics of some newly prepared N-arylphenanthridinone derivatives have been investigated. It has been demonstrated how the luminescence properties are influenced by the size of the aromatic ring system. It has been shown that the replacement of the phenyl group in N-phenyphenanthridinone (PP) by an alpha-naphthyl or beta-naphthyl group (alphaNP and betaNP, respectively), influences the fluorescence spectra very differently. For alphaNP, the long-wavelength (LW) emission, which is well observable in case of PP, disappears, while for betaNP, the intensity of LW emission increases compared to the short-wavelength (SW) fluorescence. The rotation of the alpha-naphthyl group to the coplanar geometry, which is a requirement of the formation of the LW state, is strongly hindered, resulting in the lack of LW emission. In respect of steric hindrance, the beta-napthyl group is similar to phenyl, however, it decreases the energy of the LW state more as a consequence of its better electron donating character and the more extended conjugation of the coplanar system. This causes the increase of the LW/SW fluorescence ratio. The benzo-fusing on the phenanthridinone moiety results in a 6-7 kcal mol(-1) decrease in the SW singlet energy, however, surprisingly the LW state energy also decreases in almost the same manner. The phenomenon shows that the entire benzo-phenanthridinone group is strongly involved in both transitions. As a consequence, the benzo-fused N-aryl derivatives also show dual luminescence. The dipole moments of the LW state of betaNP and betaNBiP (6-naphthalen-2-yl-6H-benzo[i]phenanthridin-5-one) proved to be bigger by 30 and 50%, than that of the SW state, respectively. MO calculation indicates that in the SW --> LW reaction not only the size but the direction of the excited state dipole also changes significantly. In apolar solvents, the dominant deactivation process of the examined molecules is intersystem crossing. In polar solvents, where the LW emission energy is smaller, internal conversion becomes more significant than the other processes, resulting in low fluorescence yield.