Molecular analysis of the anaerobic succinate degradation pathway in Clostridium kluyveri

Abstract

The gram-positive anaerobic bacterium Clostridium kluyveri (3) ferments ethanol and acetate to butyrate, caproate, and molecular hydrogen (7). ATP, required for growth, is gained by substrate-level phosphorylation from acetyl phosphate, and the quantity is proportional to the amount of hydrogen produced (46, 50). Investigations of additional metabolic abilities revealed that this organism can utilize crotonate, vinylacetate, and 4-hydroxybutyrate as substrates (4, 5) and is able to ferment the unusual substrate combination of succinate plus ethanol (27). A pathway was proposed, one in which succinate is first activated and then reduced by a two-step reaction to give 4-hydroxybutyrate, which is then further metabolized to crotonyl-coenzyme A (CoA) ( Fig. 1) (27). In a previous study, we discussed enzymes involved in the anaerobic breakdown of succinate by C. kluyveri, specifically, a succinyl-CoA:CoA transferase, a CoA-and NADP ϩ -dependent succinate-semialdehyde dehydrogenase, and a 4-hydroxybutyrate dehydrogenase (49). Wolff et al. (55) independently confirmed these data by 13 C-nuclear magnetic resonance studies as well as enzymatic investigations on the dehydrogenases. 4-Hydroxybutyryl-CoA dehydratase, which catalyzes the last step of the succinatespecific pathway, the dehydration and isomerization of 4-hydroxybutyryl-CoA to crotonyl-CoA, was recently identified, purified, and characterized (45). We present here some molecular aspects of this pathway, including the cloning, sequencing, and heterologous expression of a C. kluyveri DNA region which encodes a succinyl-CoA:CoA transferase, the succinatesemialdehyde dehydrogenase, and the 4-hydroxybutyrate dehydrogenase. MATERIALS AND METHODSBacterial strains, plasmids, media, and growth conditions. C. kluyveri (DSM 555) was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany. Escherichia coli JM109 (58) and the pBluescript SK vector (Stratagene, San Diego, Calif.) were from the laboratory collection. C. kluyveri cells used for DNA isolation were grown at 37ЊC under strictly anaerobic conditions on ethanol (300 mM) and succinate (100 mM) as previously described (49). Cells for RNA preparation were cultured in the same medium except that succinate was replaced by acetate (100 mM). E. coli cultures were routinely grown at 30ЊC in Luria-Bertani (LB) medium (42) on a rotary shaker. Tetrazolium indicator plates (6) containing 4 g of 4-hydroxybutyrate per liter were employed for the screening procedure (oxidation of 4-hydroxybutyrate). Utilization of 4-hydroxybutyrate (4 g/liter) as a carbon source for recombinant E. coli clones was investigated in M9 medium (42), supplemented with a small amount of yeast extract (0.2 g/liter), MgSO 4 (2 mM), and CaCl 2 (0.1 mM). Ampicillin (75 mg/liter) was added to the media for E. coli as a selection marker, when needed.Nucleic acids isolation and recombinant DNA techniques. Chromosomal DNA from C. kluyveri was isolated by the method of Saito and Miura (41). Total RNA from C. kluyveri...

Evidence for the bacterial origin of genes encoding fermentation enzymes of the amitochondriate protozoan parasite Entamoeba histolytica

“…Like the E. histolytica ADHE, the G. lamblia ADHE lacked mitochondrion-or hydrogenosome-targeting sequences at its amino terminus (49). The amino half of G. lamblia ADHE was more similar to the CoA-and NADP ϩ -dependent succinate semialdehyde dehydrogenase (encoded by sucD) of Clostridium kluyveri (32%) than to the CoA-independent aldehyde dehydrogenase of E. histolytica, yeasts, and eubacteria (all with Ͻ20% identity) (33,54,69). Throughout the amino half of the G. lamblia ADHE were numerous residues conserved in CoAindependent aldehyde dehydrogenases, including Glu and Cys residues that may be involved in catalysis.…”

Malonyl-Coenzyme A Reductase from Chloroflexus aurantiacus , a Key Enzyme of the 3-Hydroxypropionate Cycle for Autotrophic CO 2 Fixation

“…The oxidation of a primary alcohol via its aldehyde to the CoA thioester of the corresponding acid and the reverse reaction are common reactions in metabolism. In bacteria, such reactions play a role in the energy metabolism of many fermentative bacteria (5,6,26,27). So far, reduction of malonyl-CoA or oxidation of 3-hydroxypropionate to malonyl-CoA has not been reported because such reactions do not occur in the energy metabolism of any fermenting bacteria.…”

“…Malonyl-CoA reductase also forms a homodimer, but its subunit is much larger; the reason for this is unknown. In contrast, the NADPH-dependent reduction of succinylCoA to 4-hydroxybutyrate via succinate semialdehyde in Clostridium kluyveri is catalyzed by the combined action of two separate proteins, a CoA-acylating succinate semialdehyde dehydrogenase and a 4-hydroxybutyrate dehydrogenase (26,27). The genes coding for these enzymes constitute an operon.…”

“…The genes coding for these enzymes constitute an operon. Under cellular conditions, two semialdehyde dehydrogenase subunits and two alcohol dehydrogenase subunits seem to form a tetrameric enzyme complex of about 200 kDa (27).…”

The genome ofClostridium kluyveri, a strict anaerobe with unique metabolic features

“…Two specific CoA transferases (Cat1 and Cat2), NAD-dependent succinate semialdehyde dehydrogenase (SucD), NAD-dependent 4-hydroxybutyrate dehydrogenase (4Hbd), and 4-hydroxybutyryl-CoA dehydratase/isomerase (AbfD) have been characterized (42). The genes for these enzymes are clustered (SI Table 2), and their expression is induced by succinate (43,44). Enoate Reduction.…”