Preparation of cell-free extracts and the enzymes involved in fatty acid metabolism in Syntrophomonas wolfei

Abstract

Syntrophomonas wolfei is an anaerobic fatty acid degrader that can only be grown in coculture with H2-using bacteria such as Methanospirillum hungatei. Cells of S. wolfei were selectively lysed by lysozyme treatment, and unlysed cells of M. hungatei were removed by centrifugation. The cell extract of S. wolfei obtained with this method had low levels of contamination by methanogenic cofactors. However, lysozyme treatment was not efficient in releasing S. wolfei protein; only about 15% of the L-3-hydroxyacyl-coenzyme A (CoA) dehydrogenase activity was found in the lysozyme supernatant. Cell extracts of S. wolfei obtained with this method had high specific activities of acyl-CoA dehydrogenase, enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase. These activities were not detected in cell extracts of M. hungatei grown alone, confirming that these activities were present in S. wolfei. The acyl-CoA dehydrogenase activity was high when a C4 but not a C8 or C16 acyl-CoA derivative served as the substrate. S. wolfei cell extracts had high CoA transferase specific activities and no detectable acyl-CoA synthetase activity, indicating that fatty acid activation occurred by transfer of CoA from acetyl-CoA. Phosphotransacetylase and acetate kinase activities were detected in cell extracts of S. wolfei, indicating that S. wolfei is able to perform substrate-level phosphorylation.The complete anaerobic degradation of organic matter to CH4 and CO2 involves the concerted action of four major metabolic groups of bacteria (9,27,45). First, fermentative bacteria hydrolyze the primary substrate polymers such as polysaccharides and proteins and ferment the products mainly to volatile fatty acids, C02, and CH4. The H2-producing acetogenic bacteria degrade propionate and longer-chain fatty acids and some aromatic acids to acetate, H2, and sometimes CO2. The methanogenic bacteria use H2 to reduce CO2 to CH4, and some species cleave acetate to CO2 and CH4. A fourth group of bacteria, H2-using acetogens, produces acetate and some butyrate from H2/CO2, methanol, CO, and methoxy moieties of some aromatic compounds (1, 2, 38).Propionate and longer-chain acids are important intermediates in the complete degradation of organic matter to CO2 and CH4 (21,23,25), and the degradation of these compounds is often the rate-limiting step in methane fermentation (26). Only recently have the bacteria responsible for the degradation of these compounds been isolated in coculture with H2-using bacteria (7,29,32 of fatty acids with H2 production is energetically unfavorable unless the H2 concentration is maintained at a very low level by the H2-using bacterium (9, 27). Because of this, the anaerobic fatty acid-degrading syntrophic bacteria can only be grown in coculture with H2-using bacteria.S. wolfei does not use any other common bacterial energy source or combination of electron donor and acceptor that would enable it to grow in pure culture (28,29). Growth of S. wolfei in coculture with an H2-using sulfate reducer or met...

Evidence of reversed electron transport in syntrophic butyrate or benzoate oxidation by Syntrophomonas wolfei and Syntrophus buswellii

“…Seventy-five percent of the total activity of butyryl-CoA dehydrogenase of crude Frenchpress extract was released by this method. Similar studies with lysozyme as the selective bacteriolytic agent resulted in a release of only 15.4% of total 3-hydroxybutyryl-CoA dehydrogenase activity (Wofford et al 1986). Fluorescence measurements after mutanolysin treatment of M.…”

“…Obviously, hydrogen and acetate accumulate to a concentration that corresponds thermodynamically with the equivalent of one-third of an ATP unit, which has been defined as the minimum amount of energy required to make ATP (Schink 1990(Schink , 1992. This is the amount of energy remaining from 1 ATP formed by substrate-level phosporylation in the acetate kinase reaction (Wofford et al 1986;Schink 1992) if two-thirds of this ATP unit are invested into translocation of two protons across the cytoplasmic membrane. Two protons could shift the redox potential for two electrons by 165 mV and thus cover exactly the gap between the redox potentials of butyryl-CoA dehydrogenase (-125 mV) and 2 H+/H2 at 10 -~ atm (-295 mV).…”

Metabolic pathways and energetics of the acetone-oxidizing, sulfate-reducing bacterium, Desulfobacterium cetonicum

“…3-Hydroxybutyrate dehydrogenase (EC 1.1.1.30), 3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.35/36/157), and isopropanol dehydrogenase (EC 1.1.1.80) were all tested with NADH or NADPH as described by . 3-Enoyl-CoA hydratase (EC42.1.17/55), omitting BSA and increasing NAD + to 2 mM, and acetyl-CoA acetyltransferase (EC 2.3.1.9) were assayed after Wofford et al (1986). Carbon monoxide dehydrogenase (EC 1.2.99.2) was measured as described by Diekert and Thauer (1978), and formate dehydrogenase (EC 1.2.99.-) was assayed in the same way except under N 2 with the addition of 10mM sodium formate.…”

Pathway of anaerobic poly-?-hydroxybutyrate degradation byIlyobacter delafieldii

“…Acetyl-CoA acetyltransferase (3-ketoacyl-CoA thiolase, EC 2.3.1.9), enoyl-CoA hydratase (EC 4.2.1.17/55), acyl-CoA dehydrogenase with butyryl-CoA (EC 1.3.99.3) and butyryl-CoA:acetate CoA transferase (EC 2.8.2.8), were assayed as described by Wofford et al (1986). Hydrogenase (EC 1.18.99.1) was assayed with methylviologen by the method of Badziong & Thauer (1980).…”