JOURNAL OF BACTERIOLOGY, JUlY 1977, P. 133-135 Copyright © 1977 American Society for Microbiology
Vol. 131, No. 1 Printed in U.S.A.
Evidence for the Presence of Phosphoriboisomerase and Ribulose-1,5-Diphosphate Carboxylase in Extracts of Desulfovibrio vulgaris MARIA ALVAREZ AND LARRY L. BARTON* Department of Biology, University of New Mexico, Albuquerque, New Mexico 87131 Received for publication 31 March 1977
Cell extracts of Desulfovibrio vulgaris were found to incorporate 14CO2 into acid-stable products when ribose-5-phosphate or ribulose-1,5-diphosphate was used as a substrate. This C02 fixation required adenosine triphosphate and produced 3-phosphoglyceric acid as one of the products. The assimilation of C02 by pentose phosphates was unrelated to the pyruvate-C02 exchange reaction. The pyruvate-CO2 exchange did not require adenosine triphosphate, did not produce phosphorylated compounds, and, unlike the pentose phosphate system, required an acidic protein fraction for activity. RuDP, and 7 mg of dialyzed cell extract. In specified reactions, acidic proteins were removed from the cell extract by passing 10 ml of the extract through a diethylaminoethyl (DEAE)-cellulose column (1.5 by 4 cm) equilibrated at 1 mM Tris-hydrochloride, pH 8.0. After incubation at 37°C for 30 min with R-5-P or for 10 min with RuDP, the reaction was stopped with the addition of perchloric acid to a final concentration of 0.1%, and the resulting precipitate was removed by centrifugation. The acidified supernatant liquid was dried at 850C, and radioactivity was measured in a liquid scintillation counter (Beckman L5230) with a counting fluid composed of 0.4% 2,5diphenyloxazole, 70% toluene, and 30% Triton X100. MATERIALS AND METHODS The pyruvate-CO2 exchange reaction was examOrganism and cell-free preparation. Desulfovi- ined by using a modification of the procedure rebrio vulgaris NCIB 8303 was grown in the lactate- ported by Akagi (1). Pyruvate, 5 mM, was substisulfate medium described by Postgate (16). The cells tuted for pentose phosphates in the reaction, with were harvested from the early stationary phase by incubation at 37°C for 30 min. Protein was determined by the biuret method (10) centrifugation and suspended in 0.01 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, using bovine serum albumin fraction five as the pH 7.6, at 1 g of packed wet cells per 1 ml of buffer. protein standard. Activity of the reactions refers to The cell suspension was disrupted by a single pas- that amount of C02 converted to acid-stable matesage through a chilled French pressure cell at 10,000 rial and is expressed as 10-" mol of C02 fixed per mg lb/in2. A few crystals of deoxyribonuclease (Sigma) of extract protein per min of incubation. were added before centrifugation at 15,000 x g for 20 Product analysis. The acid-stable products of the min. The pellet, found to contain no C02-fixing ac- reactions were examined by ascending two-dimentivity, was discarded and the supernatant fraction sional chromatographic procedures using Whatman was dialyzed against 1 mM Tris-hydrochloride, pH no. 1 filter paper (28 by 28 cm) according to established procedures (2, 25). After about 35,000 cpm of 8.0, at 4°C for 18 h before being used. Enzyme assay procedures. CO2 fixation by the the reaction mixture was applied to the paper, it was reductive pentose phosphate cycle reaction was developed first in an acid solvent (methanol-88% based on published procedures (7, 11, 12, 21) in formic acid-water, 80:15:5, vol/vol) and then in the which the substrates of ribose-5-phosphate (R-5-P) basic solvent (methanol-ammonium hydroxide-waand RuDP were employed to indicate activity of ter, 60:10:30, vol/vol). Phosphorylated compounds phosphoriboisomerase and RuDP carboxylase, re- were visualized by established procedures (4), and spectively. The 2.0-ml reaction mixture contained labeled compounds were located by autoradiogra0.165 M Tris-hydrochloride, pH 8.0, 15 mM MgCl2, 5 phy. Radioactive areas on the chromatograms were mM adenosine 5'-triphosphate (ATP), 8 mM quantitated by the liquid scintillation system. The NaH'4C03 (5.4 Ci/mol), either 5 mM R-5-P or 1 mM verification of 3-phosphoglyceric acid (PGA) on 133
The sulfate-reducing bacteria are not capable of utilizing C02 as the sole source of carbon. Assimilation of C02 in growing cultures of Desulfovibrio has been reported to occur when acetate or yeast extract was present, but whether the CO2 was incorporated by pathways characteristic of autotrophic or heterotrophic organisms was not determined (20, 23). This study presents evidence for the presence of ribulose-1,5-diphosphate (RuDP) carboxylase and phosphoriboisomerase in extracts of D. vulgaris.
134
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ALVAREZ AND BARTON
ated CO2 fixation reaction appeared to be specific in that other nucleotides could not be substituted (Table 2). A small amount of activity was evident when adenosine diphosphate (ADP) was substituted for ATP. This may be attributed to ATP formation from ADP by enzymes in the cell extract. Removal of acidic proteins from the cell extract resulted in marked reduction of the pyruRESULTS reaction (Table 3), a result consistent Moderate levels of CO2 were fixed by cell vate-C02 exchange reactions (1, 19). with pyruvate-C02 extracts when R-5-P or RuDP was used as the reaction was only pentose phosphate-CO2 The carboxylation substrate. Table 1 shows a com- slightly affected by the DEAE-cellulose treatplete dependency on magnesium ion, and ATP initial activity 90% of the since almost ment, with maximal either activity was necessary for with R-5-P remained in the absence of the substrate. The stimulation by ATP was appar- acidic proteins. in the with increase 1 little ent at mM, very Product analysis revealed that with both R-5level of CO2 fixed when the concentration of containing ATP was increased from 2 to 5 mM. The ATP P14Cand RuDP several of the products One of the prodalso contained phosphate. requirement observed with RuDP is unexwas determined to be PGA; however, no plained at this time; however, it may be due to ucts PGA was produced in the pyruvate-C02 reacthe presence of a phosphatase, or the removal of tion (Table 4). In fact, none of the products of a reaction product may make ATP necessary. contained phospyruvate-C02 the Addition of NaF at 5 mM did not enhance the phate, even thoughexchange ortho- and pyrophosphate reaction but in fact had a slight inhibitory effect. The pH of reaction, the incubation time, were present in the cell extract as evidenced the concentration of substrates, and the from the paper chromatograms. Noteworthy amount of cell extracts employed in these reactions were determined to provide the greatest TABLE 2. Nucleotide specificity of R-5-P-associated CO2 fixation level of CO2 fixation. The testing of other compounds as carboxylaCm2 fixed (10-pt Addition to complete reaction mixtion substrates revealed that no CO2 fixation mol/mg of protein tures per min) occurred with formaldehyde, lithium lactate, or sodium acetate, and low levels of activity were ATP 20.8 detected when sodium salts of malate, fuma- Inosine triphosphate 1.9 4.2 rate, or formate were employed as substitutes Guanosine triphosphate 2.1 for the pentose phosphates. However, with so- Uridine triphosphate 2.2 dium pyruvate the magnitude of CO2 incorpo- Cytosine triphosphate 7.0 rated was about five to six times greater than Adenosine diphosphate 4.9 monophosphate with R-5-P but similar to that obtained with Adenosine 4.6 RuDP. The activity with pyruvate was attrib- None uted to the pyruvate-C02 exchange reaction (24) and not to CO2 fixation in the reductive TABLE 3. Resolution of two systems: ribose-5phosphate-CO2 and pyruvate-C02 exchange pentose cycle. ATP was not required for the pyruvate-C02 reaction, and concentrations of C02 fixed (10-i mol/mg of ATP greater than 2 mM resulted in marked Cell-extract used in reacprotein per min) tion mixture inhibition. R-5-P Pyruvate The requirement of ATP by the R-5-P-associchromatograms was established by co-chromatographic procedures. Nucleotides, sugar phosphates, and pyruvate were commercial preparations obtained from Sigma Chemical Co., St. Louis, Missouri. The radiochemical NaH'4CO3 was prepared from Ba'4CO3, which was purchased from ICN Pharmaceuticals, Inc., Cleveland, Ohio.
TABLE 1. Requirements for CO2 fixation in extracts of D. vulgaris Reaction
CO2 fixed (10-9 mol/mg of protein per min) R-5-P
Complete Minus Mg Minus ATP Minus pentose phosphate
18.5 0.5 4.5 6.8
Dialyzed extract Dialyzed plus DEAEtreated fraction
TABLE 4. Formation of PGA as a product of CO2
fixation
RuDP
101.3 3.0 43.0 11.1
19.0 16.8
105.3 2.8
Substrate
RuDP R-5-P
Pyruvate
Products as 3-PGA (%)
31.2 21.3 0
VOL. 131, 1977
C02 FIXATION BY DESULFOVIBRIO
was the observation that none of the products of the pyruvate-C02 exchange was detected in the radioautograms when either R-5-P or RuDP was the carboxylation substrate.
DISCUSSION Evidence for the presence of a part of the reductive pentose pathway in D. vulgaris is based on the use of RuDP or R-5-P as a carboxylation substrate with the resulting formation of PGA as a product. This pentose phosphate fixation of CO2 could account for the observation that the level of C02 assimilated by Desulfovibrio was higher than with heterotrophic bacteria (17). The fixation of C02 by way of the pentose reductive pathway presents a variety of problems for the Desulfovibrio. From the standpoint of bioenergetics (14, 22), the organisms would appear to be unable to support the ATP-dependent CO2 fixation; however, the Desulfovibrio are capable of fixing dinitrogen (18), which also is an energy-demanding activity. Although the formation of ATP coupled to electron flow may be significant in the energetics of growth (3), there may be a limited amount of energy for biosynthesis. This would not enable the Desulfovibrio to fix large amounts of CO2 or to use C02 as the sole carbon source with sulfate as the terminal electron acceptor. Although reductive power for C02 fixation would presumably come from reduced pyridine nucleotides, few reactions involving pyridine nucleotides have been described in Desulfovibrio (5, 6, 8, 9). Since the role of yeast extract and acetate promoted considerable interest in previous studies of C02 assimilation (13, 17, 20, 23), it would be of interest to pursue the mixotrophic character (15) of Desulfovibrio. ACKNOWLEDGMENTS Appreciation is expressed to H. D. Peck, Jr., for suggesting this project and to the Research Allocations of the University of New Mexico for financing part of this research. LITERATURE CITED 1. Akagi, J. M. 1967. Electron carriers for the phosphoroclastic reaction of Desulfovibrio desulfuricans. J. Biol. Chem. 242:2478-2483. 2. Bandurski, R. S., and B. Axehrod. 1951. The chromatographic identification of some biologically important phosphate esters. J. Biol. Chem. 193:405-411. 3. Barton, L. L., J. LeGall, and H. D. Peck, Jr. 1972. Oxidative phosphorylation in the obligate anaerobe Desulfovibrio gigas, p. 33-51. In A. S. Pietro and H. Gest (ed.), Horizons of bioenergetics. Academic Press Inc., New York.
135
4. Hanes, C. S., and F. A. Isherwood. 1949. Separation of the phosphoric esters on the filter paper chromatogram. Nature (London) 164:1107-1112. 5. Hatchikian, E. C. 1970. Menadione reductase from Desulfovibrio giggas. Biochim. Biophys. Acta 212:353355. 6. Hatchikian, E. C., and J. LeGall. 1970. Etude du metabolisme des acides dicarbozyliques et du pyruvate
chez les bacteries sulfato-reductrices. Etude de l'oxydation enzymatique du fumarate en acetate. Ann. Inst. Pasteur Paris 118:125-142. 7. Johnson, E. J., and H. D. Peck, Jr. 1965. Coupling of phosphorylation and carbon dioxide fixation in extracts of Thiobacillus thioparus. J. Bacteriol. 89:1041-1050.
8. LeGall, J. 1968. Purification partielle et etude de la nad: rubredoxine oxydo-reductase de D. gigas. Ann. Inst. Pasteur Paris 114:109-115. 9. LeGall, J., and J. R. Postgate. 1973. The physiology of sulfate-reducing bacteria. Adv. Microbiol. Physiol. 10:81-133. 10. Levin, R., and R. W. Brauer. 1951. The biuret reaction for the determination of proteins-an improved reagent and its application. J. Lab. Clin. Med. 38:474480. 11. MacEiroy, R. D., E. J. Johnson, and M. K. Johnson. 1968. Characterization of ribulose diphosphate carboxylase and phosphoribulokinase from Thiobacillus thioparus and Thiobacillus neopolitanus. Biochim. Biophys. Acta 127:310-316. 12. McFadden, B. A., and C. L. Tu. 1967. Regulation of autotrophic and heterotrophic carbon dioxide fixation in Hydrogenomonas facilis. J. Bacteriol. 93:887-893. 13. Mechalas, B. J., and S. C. Rittenberg. 1960. Energy coupling in Desulfovibrio desulfuricans. J. Bacteriol. 80:501-507. 14. Peck, H. D., Jr. 1962. Symposium on metabolism of inorganic compounds. V. Comparative metabolism of inorganic sulfur compounds in micro-organisms. Bacteriol. Rev. 26:67-94. 15. Postgate, J. 1959. Sulfate reduction by bacteria. Annu. Rev. Microbiol. 13:505-520. 16. Postgate, J. R. 1951. On the nutrition of Desulfovibrio desulphuricans. J. Gen. Microbiol. 5:714-724. 17. Postgate, J. R. 1965. Recent advances in the study of the sulfate-reducing bacteria. Bacteriol. Rev. 29:425441. 18. Riederer-Henderson, M., and P. W. Wilson. 1970. Nitrogen fixation by sulfate-reducing bacteria. J. Gen. Microbiol. 61:27-31. 19. Raeburn, S., and J. C. Rabinowitz. 1971. Pyruvate: ferredoxin oxidoreductase. II. Characteristics of the forward and reverse reactions and properties of the enzyme. Arch. Biochem. Biophys. 146:21-33. 20. Rittenberg, S. C. 1969. The roles of exogenous organic matter in the physiology of chemolithotrophic bacteria. Adv. Microbiol. Physiol. 3:159-196. 21. Scrutton, M. C. 1971. Assays of enzymes of CO2 metabolism. Methods Microbiol. 6A:479-544. 22. Senez, J. C. 1962. Some consideration of the energetics of bacterial growth. Bacteriol. Rev. 28:95-105. 23. Sorokin, Y. I. 1966. Role of carbon dioxide and acetate in biosynthesis of sulfate-reducing bacteria. Nature (London) 210:551-552. 24. Suh, B., and J. M. Akagi. 1966. Pyruvate-carbon dioxide exchange reaction of Desulfovibrio desulfuricans. J. Bacteriol. 91:2281-2285. 25. Wood, T. 1968. The detection and identification of intermediates of the pentose phosphate cycle and related compounds. J. Chromatogr. 35:352-361.
Vol. 131, No. 1 Printed in U.S.A.
Evidence for the Presence of Phosphoriboisomerase and Ribulose-1,5-Diphosphate Carboxylase in Extracts of Desulfovibrio vulgaris MARIA ALVAREZ AND LARRY L. BARTON* Department of Biology, University of New Mexico, Albuquerque, New Mexico 87131 Received for publication 31 March 1977
Cell extracts of Desulfovibrio vulgaris were found to incorporate 14CO2 into acid-stable products when ribose-5-phosphate or ribulose-1,5-diphosphate was used as a substrate. This C02 fixation required adenosine triphosphate and produced 3-phosphoglyceric acid as one of the products. The assimilation of C02 by pentose phosphates was unrelated to the pyruvate-C02 exchange reaction. The pyruvate-CO2 exchange did not require adenosine triphosphate, did not produce phosphorylated compounds, and, unlike the pentose phosphate system, required an acidic protein fraction for activity. RuDP, and 7 mg of dialyzed cell extract. In specified reactions, acidic proteins were removed from the cell extract by passing 10 ml of the extract through a diethylaminoethyl (DEAE)-cellulose column (1.5 by 4 cm) equilibrated at 1 mM Tris-hydrochloride, pH 8.0. After incubation at 37°C for 30 min with R-5-P or for 10 min with RuDP, the reaction was stopped with the addition of perchloric acid to a final concentration of 0.1%, and the resulting precipitate was removed by centrifugation. The acidified supernatant liquid was dried at 850C, and radioactivity was measured in a liquid scintillation counter (Beckman L5230) with a counting fluid composed of 0.4% 2,5diphenyloxazole, 70% toluene, and 30% Triton X100. MATERIALS AND METHODS The pyruvate-CO2 exchange reaction was examOrganism and cell-free preparation. Desulfovi- ined by using a modification of the procedure rebrio vulgaris NCIB 8303 was grown in the lactate- ported by Akagi (1). Pyruvate, 5 mM, was substisulfate medium described by Postgate (16). The cells tuted for pentose phosphates in the reaction, with were harvested from the early stationary phase by incubation at 37°C for 30 min. Protein was determined by the biuret method (10) centrifugation and suspended in 0.01 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, using bovine serum albumin fraction five as the pH 7.6, at 1 g of packed wet cells per 1 ml of buffer. protein standard. Activity of the reactions refers to The cell suspension was disrupted by a single pas- that amount of C02 converted to acid-stable matesage through a chilled French pressure cell at 10,000 rial and is expressed as 10-" mol of C02 fixed per mg lb/in2. A few crystals of deoxyribonuclease (Sigma) of extract protein per min of incubation. were added before centrifugation at 15,000 x g for 20 Product analysis. The acid-stable products of the min. The pellet, found to contain no C02-fixing ac- reactions were examined by ascending two-dimentivity, was discarded and the supernatant fraction sional chromatographic procedures using Whatman was dialyzed against 1 mM Tris-hydrochloride, pH no. 1 filter paper (28 by 28 cm) according to established procedures (2, 25). After about 35,000 cpm of 8.0, at 4°C for 18 h before being used. Enzyme assay procedures. CO2 fixation by the the reaction mixture was applied to the paper, it was reductive pentose phosphate cycle reaction was developed first in an acid solvent (methanol-88% based on published procedures (7, 11, 12, 21) in formic acid-water, 80:15:5, vol/vol) and then in the which the substrates of ribose-5-phosphate (R-5-P) basic solvent (methanol-ammonium hydroxide-waand RuDP were employed to indicate activity of ter, 60:10:30, vol/vol). Phosphorylated compounds phosphoriboisomerase and RuDP carboxylase, re- were visualized by established procedures (4), and spectively. The 2.0-ml reaction mixture contained labeled compounds were located by autoradiogra0.165 M Tris-hydrochloride, pH 8.0, 15 mM MgCl2, 5 phy. Radioactive areas on the chromatograms were mM adenosine 5'-triphosphate (ATP), 8 mM quantitated by the liquid scintillation system. The NaH'4C03 (5.4 Ci/mol), either 5 mM R-5-P or 1 mM verification of 3-phosphoglyceric acid (PGA) on 133
The sulfate-reducing bacteria are not capable of utilizing C02 as the sole source of carbon. Assimilation of C02 in growing cultures of Desulfovibrio has been reported to occur when acetate or yeast extract was present, but whether the CO2 was incorporated by pathways characteristic of autotrophic or heterotrophic organisms was not determined (20, 23). This study presents evidence for the presence of ribulose-1,5-diphosphate (RuDP) carboxylase and phosphoriboisomerase in extracts of D. vulgaris.
134
J. BACTERIOL.
ALVAREZ AND BARTON
ated CO2 fixation reaction appeared to be specific in that other nucleotides could not be substituted (Table 2). A small amount of activity was evident when adenosine diphosphate (ADP) was substituted for ATP. This may be attributed to ATP formation from ADP by enzymes in the cell extract. Removal of acidic proteins from the cell extract resulted in marked reduction of the pyruRESULTS reaction (Table 3), a result consistent Moderate levels of CO2 were fixed by cell vate-C02 exchange reactions (1, 19). with pyruvate-C02 extracts when R-5-P or RuDP was used as the reaction was only pentose phosphate-CO2 The carboxylation substrate. Table 1 shows a com- slightly affected by the DEAE-cellulose treatplete dependency on magnesium ion, and ATP initial activity 90% of the since almost ment, with maximal either activity was necessary for with R-5-P remained in the absence of the substrate. The stimulation by ATP was appar- acidic proteins. in the with increase 1 little ent at mM, very Product analysis revealed that with both R-5level of CO2 fixed when the concentration of containing ATP was increased from 2 to 5 mM. The ATP P14Cand RuDP several of the products One of the prodalso contained phosphate. requirement observed with RuDP is unexwas determined to be PGA; however, no plained at this time; however, it may be due to ucts PGA was produced in the pyruvate-C02 reacthe presence of a phosphatase, or the removal of tion (Table 4). In fact, none of the products of a reaction product may make ATP necessary. contained phospyruvate-C02 the Addition of NaF at 5 mM did not enhance the phate, even thoughexchange ortho- and pyrophosphate reaction but in fact had a slight inhibitory effect. The pH of reaction, the incubation time, were present in the cell extract as evidenced the concentration of substrates, and the from the paper chromatograms. Noteworthy amount of cell extracts employed in these reactions were determined to provide the greatest TABLE 2. Nucleotide specificity of R-5-P-associated CO2 fixation level of CO2 fixation. The testing of other compounds as carboxylaCm2 fixed (10-pt Addition to complete reaction mixtion substrates revealed that no CO2 fixation mol/mg of protein tures per min) occurred with formaldehyde, lithium lactate, or sodium acetate, and low levels of activity were ATP 20.8 detected when sodium salts of malate, fuma- Inosine triphosphate 1.9 4.2 rate, or formate were employed as substitutes Guanosine triphosphate 2.1 for the pentose phosphates. However, with so- Uridine triphosphate 2.2 dium pyruvate the magnitude of CO2 incorpo- Cytosine triphosphate 7.0 rated was about five to six times greater than Adenosine diphosphate 4.9 monophosphate with R-5-P but similar to that obtained with Adenosine 4.6 RuDP. The activity with pyruvate was attrib- None uted to the pyruvate-C02 exchange reaction (24) and not to CO2 fixation in the reductive TABLE 3. Resolution of two systems: ribose-5phosphate-CO2 and pyruvate-C02 exchange pentose cycle. ATP was not required for the pyruvate-C02 reaction, and concentrations of C02 fixed (10-i mol/mg of ATP greater than 2 mM resulted in marked Cell-extract used in reacprotein per min) tion mixture inhibition. R-5-P Pyruvate The requirement of ATP by the R-5-P-associchromatograms was established by co-chromatographic procedures. Nucleotides, sugar phosphates, and pyruvate were commercial preparations obtained from Sigma Chemical Co., St. Louis, Missouri. The radiochemical NaH'4CO3 was prepared from Ba'4CO3, which was purchased from ICN Pharmaceuticals, Inc., Cleveland, Ohio.
TABLE 1. Requirements for CO2 fixation in extracts of D. vulgaris Reaction
CO2 fixed (10-9 mol/mg of protein per min) R-5-P
Complete Minus Mg Minus ATP Minus pentose phosphate
18.5 0.5 4.5 6.8
Dialyzed extract Dialyzed plus DEAEtreated fraction
TABLE 4. Formation of PGA as a product of CO2
fixation
RuDP
101.3 3.0 43.0 11.1
19.0 16.8
105.3 2.8
Substrate
RuDP R-5-P
Pyruvate
Products as 3-PGA (%)
31.2 21.3 0
VOL. 131, 1977
C02 FIXATION BY DESULFOVIBRIO
was the observation that none of the products of the pyruvate-C02 exchange was detected in the radioautograms when either R-5-P or RuDP was the carboxylation substrate.
DISCUSSION Evidence for the presence of a part of the reductive pentose pathway in D. vulgaris is based on the use of RuDP or R-5-P as a carboxylation substrate with the resulting formation of PGA as a product. This pentose phosphate fixation of CO2 could account for the observation that the level of C02 assimilated by Desulfovibrio was higher than with heterotrophic bacteria (17). The fixation of C02 by way of the pentose reductive pathway presents a variety of problems for the Desulfovibrio. From the standpoint of bioenergetics (14, 22), the organisms would appear to be unable to support the ATP-dependent CO2 fixation; however, the Desulfovibrio are capable of fixing dinitrogen (18), which also is an energy-demanding activity. Although the formation of ATP coupled to electron flow may be significant in the energetics of growth (3), there may be a limited amount of energy for biosynthesis. This would not enable the Desulfovibrio to fix large amounts of CO2 or to use C02 as the sole carbon source with sulfate as the terminal electron acceptor. Although reductive power for C02 fixation would presumably come from reduced pyridine nucleotides, few reactions involving pyridine nucleotides have been described in Desulfovibrio (5, 6, 8, 9). Since the role of yeast extract and acetate promoted considerable interest in previous studies of C02 assimilation (13, 17, 20, 23), it would be of interest to pursue the mixotrophic character (15) of Desulfovibrio. ACKNOWLEDGMENTS Appreciation is expressed to H. D. Peck, Jr., for suggesting this project and to the Research Allocations of the University of New Mexico for financing part of this research. LITERATURE CITED 1. Akagi, J. M. 1967. Electron carriers for the phosphoroclastic reaction of Desulfovibrio desulfuricans. J. Biol. Chem. 242:2478-2483. 2. Bandurski, R. S., and B. Axehrod. 1951. The chromatographic identification of some biologically important phosphate esters. J. Biol. Chem. 193:405-411. 3. Barton, L. L., J. LeGall, and H. D. Peck, Jr. 1972. Oxidative phosphorylation in the obligate anaerobe Desulfovibrio gigas, p. 33-51. In A. S. Pietro and H. Gest (ed.), Horizons of bioenergetics. Academic Press Inc., New York.
135
4. Hanes, C. S., and F. A. Isherwood. 1949. Separation of the phosphoric esters on the filter paper chromatogram. Nature (London) 164:1107-1112. 5. Hatchikian, E. C. 1970. Menadione reductase from Desulfovibrio giggas. Biochim. Biophys. Acta 212:353355. 6. Hatchikian, E. C., and J. LeGall. 1970. Etude du metabolisme des acides dicarbozyliques et du pyruvate
chez les bacteries sulfato-reductrices. Etude de l'oxydation enzymatique du fumarate en acetate. Ann. Inst. Pasteur Paris 118:125-142. 7. Johnson, E. J., and H. D. Peck, Jr. 1965. Coupling of phosphorylation and carbon dioxide fixation in extracts of Thiobacillus thioparus. J. Bacteriol. 89:1041-1050.
8. LeGall, J. 1968. Purification partielle et etude de la nad: rubredoxine oxydo-reductase de D. gigas. Ann. Inst. Pasteur Paris 114:109-115. 9. LeGall, J., and J. R. Postgate. 1973. The physiology of sulfate-reducing bacteria. Adv. Microbiol. Physiol. 10:81-133. 10. Levin, R., and R. W. Brauer. 1951. The biuret reaction for the determination of proteins-an improved reagent and its application. J. Lab. Clin. Med. 38:474480. 11. MacEiroy, R. D., E. J. Johnson, and M. K. Johnson. 1968. Characterization of ribulose diphosphate carboxylase and phosphoribulokinase from Thiobacillus thioparus and Thiobacillus neopolitanus. Biochim. Biophys. Acta 127:310-316. 12. McFadden, B. A., and C. L. Tu. 1967. Regulation of autotrophic and heterotrophic carbon dioxide fixation in Hydrogenomonas facilis. J. Bacteriol. 93:887-893. 13. Mechalas, B. J., and S. C. Rittenberg. 1960. Energy coupling in Desulfovibrio desulfuricans. J. Bacteriol. 80:501-507. 14. Peck, H. D., Jr. 1962. Symposium on metabolism of inorganic compounds. V. Comparative metabolism of inorganic sulfur compounds in micro-organisms. Bacteriol. Rev. 26:67-94. 15. Postgate, J. 1959. Sulfate reduction by bacteria. Annu. Rev. Microbiol. 13:505-520. 16. Postgate, J. R. 1951. On the nutrition of Desulfovibrio desulphuricans. J. Gen. Microbiol. 5:714-724. 17. Postgate, J. R. 1965. Recent advances in the study of the sulfate-reducing bacteria. Bacteriol. Rev. 29:425441. 18. Riederer-Henderson, M., and P. W. Wilson. 1970. Nitrogen fixation by sulfate-reducing bacteria. J. Gen. Microbiol. 61:27-31. 19. Raeburn, S., and J. C. Rabinowitz. 1971. Pyruvate: ferredoxin oxidoreductase. II. Characteristics of the forward and reverse reactions and properties of the enzyme. Arch. Biochem. Biophys. 146:21-33. 20. Rittenberg, S. C. 1969. The roles of exogenous organic matter in the physiology of chemolithotrophic bacteria. Adv. Microbiol. Physiol. 3:159-196. 21. Scrutton, M. C. 1971. Assays of enzymes of CO2 metabolism. Methods Microbiol. 6A:479-544. 22. Senez, J. C. 1962. Some consideration of the energetics of bacterial growth. Bacteriol. Rev. 28:95-105. 23. Sorokin, Y. I. 1966. Role of carbon dioxide and acetate in biosynthesis of sulfate-reducing bacteria. Nature (London) 210:551-552. 24. Suh, B., and J. M. Akagi. 1966. Pyruvate-carbon dioxide exchange reaction of Desulfovibrio desulfuricans. J. Bacteriol. 91:2281-2285. 25. Wood, T. 1968. The detection and identification of intermediates of the pentose phosphate cycle and related compounds. J. Chromatogr. 35:352-361.
