TRICARBOXYLIC ACID CYCLE ACTIVITY IN ACETOBACTER PASTEURIANUM' TSOO E. KING, ELWELL H. KAWASAKI, AND VERNON H. CHELDELIN Department of Chemistry and Science Research Institute, Oregon State College, Corvallis, Oregon Received for publication March 14, 1956 Previous papers from this laboratory (King and Spectrophotometric determinations of re- Cheldelin, 1952, 1952a, 1954; Hauge et al., duced pyridinenucleotides were performed in a 1955, 1955a) have reported on the oxidation of Beckman Model B spectrophotometer with carbohydrates in Acetobacter suboxydans. Neither 1-cm2 Corex cell at X = 340 my. A Beckman resting nor disintegrated cells can oxidize acetate Model DU spectrophotometer with 1-cm2 silica or other intermediates of the tricarboxylic acid cells was used at wave lengths below 300 m,u. cycle. These findings have recently been con- Oxygen consumption and CO2 production were firmed in another laboratory (Rao and Gunsalus, determined by the conventional method in a 1955). From the comparative biochemical view- Warburg apparatus. CO2 retention was corrected point, it is of interest to study the oxidative by using the effective Bunsen coefficient at pH behavior of other species of the genus Aceto- 6.0. Protein was determined by the biuret method bacter. Results reported in this paper demonstrate as in a previous paper (King and Cheldelin, that Acetobacter pasteurianum, in contrast to A. 1954). Lipoic acid was determined by a mano- suboxydans, can oxidize all the intermediates of metric method with Streptococcus faecalis strain the Krebs cycle. Other evidence also indicates 10 Cl (Gunsalus et at., 1952). The organism was the functioning of this cycle in A. pasteurianum. kindly supplied by Drs. I. C. Gunsalus and D. J. O'Kane. Citrate was determined by a penta- METHODS AND MATERIALS bromoacetone method (Perlman et al., 1944). Acetobacter pasteurianum ATCC 6033 was used. Cells were grown, harvested and lyophilized as RESULTS reported previously (King and Cheldelin, 1954) Oxidation of tricarboxylic acid cycle intermedi- for A. suboxydans. The lyophilized whole cells ates. Resting cells of A. pasteurianum oxidized will be referred to in this paper as "resting cells." all Krebs cycle intermediates completely as Lyophilized cells were disintegrated in a 10-kc shown in table 1. The endogenous oxidation was Raytheon sonic oscillator as described in a very small. The initial rate, as well as the time previous paper (King and Cheldelin, 1956). The required for complete oxidation, varied with the product will be referred to as "cell homogenate." compounds, probably largely because of per- The soluble fraction was obtained by centrifuging meability differences. disintegrated cell mixture for 2-3 hr at about Citrate oxidation by resting cells varied among 22,000 X G. different batches of resting cells, with some cells DL-a-Lipoic acid was kindly supplied by Dr. showing practically no oxidation. However, Lester J. Reed. All other chemicals were ob- preparations of cell homogenates oxidized citrate tained commercially and used without further readily and completely to CO2 and water as purification. DL-Isocitric lactone was freshly shown by manometric determinations. hydrolyzed before use in a 5 per cent excess of the These data suggested the overall operation of calculated amount of KOH in a boiling water the tricarboxylic acid cycle in A. pasteurianum. bath for 15 min, then adjusted to pH 6.0. Formation of malate from fumarate. Conversion of fumarate to 1 Supported by the Nutrition Foundation, malate was demonstrated by Swift and Company, and the Division of Research measuring the latter compounds polarimetrically Grants, U. S. Public Health Service. Published as its molybdate complex (Wille, 1941). When with the approval of the Monographs Publica- 1.65 mmoles fumarate were incubated in the cations Committee, Research Paper No. 296, presence of 30 mg resting cells in a total volume School of Science, Department of Chemistry. of 10-ml of 0.1-M phosphate buffer, pH 6.0, under 418 1956] TRICARBOXYLIC ACID IN A. PASTEURIANUM 419

TABLE 1 Oxidation of tricarboxylic acid cycle intermediates by Acetobacter pasteurianum alcte Oxygen 1.2 - Consumption CO2 Formed Substrate Time Qo2 Calcu- Calcu- 1.0 - Found lated Found lated a Xcitrate min- atoms/ sub- molesl sub- 0.8 _ uies mlolecule strate inole strate 0._ Acetate .... 200 4.0 4 1.9 2 4.5 Pyruvate .... 200 4.8 5 2.8 3 13.0 0.6! 4s U I _ Oxalacetate.. 400 5.1 5 3.6 4 6.8 --O-- 2- 4 6 14 16 T ( iu 1s Citrate ...... (see 9 6 0.2 TIME (minutes) text) Figure 1. Fumarate and cis-aconitate formation cis-Aconi- from malate and citrate in Acetobacter pasteuria- tate.. 500 8.4 9 4.5 6 num. Each cuvette contained 80 smoles phosphate Isocitrate .... 450 11.5 9 6.0 6 buffer and 20-y (in terms of protein) of the soluble a-Ketoglu- fraction of a cell free extract of A. pasteurianum; tarate ..... 400 8.0 8 4.9 5 pH 6.0; total volume 2.6 ml. At zero time 0.1 ml of Succinate .... 300 7.3 7 3.9 4 O.1-M malate or citrate was added. Both enzyme Fumarate .... 300 6.0 6 4.1 4 and substrate blanks were zero. L-Malate.... 400 5.8 6 3.6 4 teins also show high ultraviolet absorption, the The system contained 10 ,umoles substrate concentration of enzymes was kept low. The (except isocitrate, see below), 10 jsmoles MgC92, results are shown in figure 1. In the presence of 160 ,moles phosphate buffer, and 20 mg of resting cells; and either 0.2 ml 10 per cent KOH or 0.2 ml a great excess of substrates, the formation of water in center well. Total volume, 2.8 ml; pH fumarate and cis-aconitate from malate and 6.0; temperature, 29 C. The values of oxygen con- citrate, respectively, increased in a nearly linear sumption and of CO2 formation are net values, fashion with time. corrected for endogenous oxidation. The times Citrate formation. Citrate was rapidly formed listed are the maximum for complete oxidation. 20 from cis-aconitate in the presence of A. pasteuria- ,umoles DL-isocitrate were used but the calculation num resting cells, but the rate of condensation of was made on 10 jAmoles, assuming that only the acetate or pyruvate with oxalacetate to give natural isomer was oxidized. citrate was very slow. Addition of adenosine For determination of oxygen, mg Qo2 (,uatoms triphosphate, BaCl2, or cells/hr during the first 45 min), the system con- (ATP)2, monofluoro- tained 100 ,umoles substrate (200 ,umoles iso- acetate did not increase the citrate formation. citrate), 20,umoles MgCl2, 160 ,umoles buffer, and Oxalacetate alone under anaerobic conditions 4 mg resting cells; other conditions were as de- also yielded citrate due to the presence of scribed above. oxalacetate carboxylase. The slower formation of citrate from pyruvate nitrogen for 16 hr, an optical rotation of aD = and oxalacetate might be due to several reasons. 4.16° was observed in a 2-dm polarimeter tube. When Krebs cycle intermediates were oxidized Readings were made after addition of 1.0 ml in the presence of triphenyl tetrazolium chloride, glacial acetic acid and 9 ml of 20 per cent am- cis-aconitate and citrate were utilized by far monium molybdate. Under the same conditions the most rapidly (up to 4 times as rapidly as no optical activity was observed by omission of fumarate, 15 times as rapidly as succinate). either cells or fumarate. This experiment quali- most conversion of all was the aconi- tatively demonstrated the presence of fumarase The rapid in the cells. 2 The following abbreviations were used in this Formation of fumarate and cis-aconitate from paper: ATP = adenosine triphosphate; CoA = L-malate and citrate. The presence of fumarase coenzyme A; DPN = diphosphopyridinenucleo- and cis-aconitase was also demonstrated in the tide; DPNH = reduced DPN; DPT = diphos- soluble fraction from A. pasteurianum by Racker's phothiamine; TPN = triphosphopyridinenucleo- spectrophotometric method (1950). Since pro- tide; Tris = tris (hydroxymethyl) aminomethane. 420 KING, KAWASAKI, AND CHELDELIN [VOL. 72 TABLE 2 oxidation was measured by the formation of Oxidations of a-ketoglutarate and pyruvate by the DPNH, as shown in table 2. Attempts to remove dialyzed soluble fraction of Acetobacter lipoic acid by alumina treatment as described by pasteurianum Seaman (1954) failed to demonstrate its require- AE. o ment. However, manometric assay (Gunsalus et al., 1952) of treated samples with Streptococcus a-Ketoglu- Pyruvate tarate faecalis showed that less than 20 per cent of the factor was removed. Apparently lipoic acid is Complete system ...... 1.042 0.485 tightly bound to the proteins in this organism Complete system + 10 m,ug in contrast to Escherichia coli. These results lipoic acid ...... 1.005 0.493 clearly show that these oxidations in A. pasteuria- No CoA ...... 0.093 0.105 num are different from those in A. suboxydans. No DPT ...... 0.173 0.085 No glutathione ...... 0.111 0.055 DISCUSSION No phosphate ...... 0.930 0.470 No substrate ...... 0.050 0.050 The results presented above have demon- No enzyme 0...... |.010 0.000 strated the occurrence of tricarboxylic acid cycle reactions in A. pasteurianum. It is interesting to The complete systems contained 50 - CoA, 500 y note also that the cytochrome spectra of this DPT, lmoles10 glutathione, 5,Amoles MgCl2, 200 organism are different from those of A. sub- /,moles tris buffer, 2 mg DPN, 0.1 ml dialyzed oxydans in intact cells (Smith, 1954; Castor cell-free extract containing 1.1 mg protein, and 10 ,umoles phosphate. Total volume, 2.9 ml; pH 8.0; and Chance, 1955) and in soluble and solubilized temperature 18-20 C. The reaction was started at particulate fractions (King and Cheldelin, 1956a). zero time by addition of 10 ,umoles a-ketoglu- Thus, the biochemical disparity between these tarate or pyruvate in 0.1 ml. AE340 values are two organisms, with respect to substrate oxi- optical densities at the end of 5 min. dizability, terminal pathways and electron transfer, contrasts strongly with their syste- tase reaction. On the contrary, the high activity matic (taxonomic) similarity. of oxalacetate decarboxylase (which can also function anaerobically) might deplete the supply ACKNOWLEDGMENT of oxalacetate for citrate synthesis. The authors are indebted to Mary K. Devlin Malonate inhibition of succinate oxidation. and Margaret G. Thome for technical assistance The influence of malonate was tested on succinate with various phases of this study. oxidation. The degree of inhibition varied with the ratio of the inhibitor to the substrate. SUMMARY About 50 per cent inhibition was observed when Qualitative and quantitative evidence has 200 inmoles of malonate were added to a system been presented for extensive tricarboxylic acid containing 50,imoles of succinate in the presence cycle activity in Acetobacter pasteurianum. The of resting cells. oxidative behavior of this organism contrasts Pyruvate and a-ketoglutarate oxidation. Oxida- strongly with that of Acetobacter suboxydans in tions of pyruvate and a-ketoglutarate by several respects, including substrate oxidizability, animal tissues and certain other microorganisms terminal pathways, and electron transfer. require CoA and lipoic acid (Gunsalus et al. 1955, Weinhouse, 1954). In A. suboxydans REFERENCES pyruvate is first decarboxylated to acetaldehyde CASTOR, L. N. AND CHANCE, B. 1955 Photo- (King and Cheldelin, 1954a), which is in turn chemical action spectra of carbon monoxide- oxidized to acetate in the presence of a TPN- or inhibited respiration. J. Bio. Chem., 217, a DPN-linked acetaldehyde dehydrogenase 453-465. (King and Cheldelin, 1956), with no demonstrable GUNSALUS, I. C., HORECKER, B. L., AND WOOD, W. CoA requirement. When these oxidations were A. 1955 Pathways of carbohydrate metab- olism in microorganisms. Bacteriol. Revs., studied in the soluble fraction of A. pasteuria- 19, 79-128. num, it was found that both were DPN specific, GUNSALUS, I. C., DOLIN, M. I., AND STRUGLIA, L. and that CoA and DPT were required. Each 1952 Pyruvic acid metabolism. III. A mano- 1956] TRICARBOXYLIC ACID IN A. PASTEURIANUM 421

metric assay for pyruvate oxidation factor. KING, T. E. AND CHELDELIN, V. H. 1956a Solu- J. Biol. Chem., 194, 849-857. bilization and cytochrome(s) of the particu- HAUGE, J. G., KING, T. E., AND CHELDELIN, V. H. lates from Acetobacter suboxydans. Federa- 1955 Alternate conversions of glycerol to di- tion Proc., 15, 288. hydroxyacetone in Acetobacter suboxydans. PERLMAN, D., LARDY, H. A., AND JOHNSON, M. J. J. Biol. Chem., 214, 1-9. 1944 Determination of citric acid in fermen- HAUGE, J. G., KING, T. E., AND CHELDELIN, V. H. tation media and biological materials. Ind. 1955a Oxidation of dihydroxyacetone via the Eng. Chem., Anal. Ed., 16, 515-516. pentose cycle in Acetobacter suboxydans. J. RACKER, E. 1950 Spectrophotometric measure- Biol. Chem., 214, 11-26. ments of enzymatic formation of fumaric and KING, T. E. AND CHELDELIN, V. H. 1952 Oxida- cis-aconitic acids. Biochim. et Biophys. tive dissimilation in Acetobacter suboxydans. Acta, 4, 211-214. J. Biol. Chem., 198, 127-133. RAO, M. R. R. AND GUNSALUS, I. C. 1955 Mech- KING, T. E. AND CHELDELIN, V. H. 1952a Phos- anism of pyruvate metabolism: Acetobacter phorylative and non-phosphorylative oxida- aceti and Acetobacter suboxydans. Federa- tion in Acetobacter suboxydans. J. Biol. tion Proc., 14, 267. Chem., 198, 135-141. SEAMAN, G. R. 1954 D)iscussion: Removal of KING, T. E. AND CHELDELIN, V. H. 1954 Oxida- thioctic acid from enzyme proteins. Federa- tions in Acetobacter suboxydans. Biochim. et tion Proc., 13, 731-733. Biophys. Acta, 14, 108-116. SMITH, L. 1954 Bacterial cytochromes. Bac- KING, T. E. AND CHELDELIN, V. H. 1954a Py- teriol. Revs., 18, 106-130. ruvic carboxylase of Acetobacter sutboxydans. WEINHOUSE, S. 1954 Carbohydrate metabolism. J. Biol. Chem., 208, 821-831. Ann. Rev. Biochem., 23, 125-176. KING, T. E. AND CHELDELIN, V. H. 1956 Oxida- WILLE, F. 1941 Die Methoden der Fermentfor- tion of acetaldehyde by Acetobacter suboxy- schung, IV, p. 2569. Edited by E. Baumann dans. J. Biol. Chem., 220, 177-191. and K. Myrback. Georg Thieme, Leipzig.