Studies on the Mechanism of Acetate Oxidation by Bacteria

Abstract

In a previous publication (1) evidence was presented impficating succinate and pyruvate quantitatively in the oxidative breakdown of acetic acid to CO2 and water by acetate-adapted strains of Escherichia coli, while a-ketoglutaric acid was eliminated as a possible intermediate. In addition, fumarate, malate, and oxalacetate have also been shown, qualitatively at least, to participate as intermediates in the oxidation of the C2 fatty acid. However, the occurrence of an unexpected side reaction, involving appreciable reduction of C4-dicarboxylic acid products of succinate oxidation back to succinate, complicated the interpretations of most experiments performed using carrier malate, fumarate, or oxalacetate.In this paper detailed data are presented which were obtained when 2-C 14-acetate was incubated in the presence of fumarate, malate, 0xalacetate, and a-ketoglutarate. Further, conditions will be described which reduce the unexpected reduction of the above mentioned C4-dicarboxylic acids back to succinate. From chemical analysis of the residual acids, CO~, and cell material, it was possible to show that when an appreciable fraction of acetate, as well as other substrates, was metabolized under high oxygen tension, there were incorporation and distribution of labeled carbon into fumarate and malate in the amount to be expected on the basis of a Thunberg-Knoop condensation cycle, whereas there was no comparable incorporation into a-ketoglutarate. These results plus those already published (1, 2) appear to exclude a tricarboxylic acid cycle as a major pathway for acetate oxidation in E. coll. Methods and MaterialsThe same strain of Esckerichia coli (E26) was used in these experiments as in the previous work. For all experiments cells were grown with constant aeration at 30°C. in a medium containing 1.5 per cent anhydrous sodium acetate, 0.4 per cent ammonium sulfate, 0.8 per cent KH2PO,, 0.07 per cent tryptone, and 20 per cent tap water (for 785

Studies on the Mechanism of Acetate Oxidation by Bacteria

“…The major datum in this table is the 1.92-fold dilution of acetate resulting from turnover with endogenous substances. Similar results were obtained with acetate-grown E. coli (Ajl and Kamen, 1951) where acetic acid appears to be oxidized by a Knoop-Thunberg condensation mechanism. Citrate-grown Aerobacler, however, showed little or no dilution of acetate as would be expected on the basis of the conventional tricarboxylic acid cycle (Ajl and Wong, 1951).…”

“…Further, acetate-grown Aerobavter showed a heightened activity on succinate, fumarate, malate, oxalacetate, and pyruvate, but not on a-ketoglutarate, cis-aconitate, or citrate. Similar results were obtained with E. coli (Ajl, 1950;Ajl and Kamen, 1951). The suggestion was put forth (Ajl and Wong, 1951), therefore, that A. aerogenes, when grown on acetic acid, may oxidize the C~-fatty acid in a manner similar to that of E. coll.…”

“…A difference between the two organisms can, however, be detected. In the case of E. coli (Ajl and Kamen, 1951), a high endogenous production of succinate from more oxidized C4dicarboxylic acids always took place in our Warburg cups. This resulted in entrapment of most of the activity in succinate, regardless of whether fumarate, malate, or oxalacetate was the carrier.…”

“…In paper IV of this series (Ajl and Wong, 1951), it was shown that citrategrown Aerobacter aerogenes may utilize simultaneously a tri-and dicarboxylic acid cycle for acetic acid oxidation. From adaptation data, however, it appeared that acetate-adapted Aerobacter behaves more like Escherichia coli; i.e., breaks down acetate d a a pathway best reconciled with a Knoop-Thunberg condensation mechanism (Ajl, 1950;Ajl and Kamen, 1951). The adaptation experiments revealed that whereas citrate-grown cells adapted themselves to oxidize acetate rapidly, the reverse was not altogether true; i.e., acetate-grown Aerobacter was not adapted to oxidize citrate rapidly.…”

Factors affecting Oxidation of Acetate by Bacterium coli

“…test-tubes in order to diminish the reduction of C,-dicarboxylic acids back to succinate (Ajl, 1951a). The CO, evolved was caught in alkali.…”