Citrate Metabolism in Aerobacter cloacae

Size: px

Start display at page:

Download "Citrate Metabolism in Aerobacter cloacae"

Alice Butler
5 years ago
Views:

JOURNAL OF BACrERIOLOGY, Sept. 1974, p. 661-665 Vol. 119, No. 3 Copyright 0 1974 American Society for Microbiology Printed in U.S.A. Citrate Metabolism in Aerobacter cloacae R. W.

1 JOURNAL OF BACrERIOLOGY, Sept. 1974, p Vol. 119, No. 3 Copyright American Society for Microbiology Printed in U.S.A. Citrate Metabolism in Aerobacter cloacae R. W. O'BRIEN AND JARMILA GEISLER Department of Biochemistry, The University of Sydney, Sydney, N.S. W., 2006, Australia Received for publication 6 May 1974 Growth of Aerobacter cloacae on citrate either anaerobically or aerobically did not require and was not stimulated by the presence of Na+ in the medium. Citrate was metabolized anaerobically via the fermentation pathway as evidenced by the (i) presence of oxalacetate decarboxylase, (ii) induction of citrate lyase, and (iii) repression of a-ketoglutarate dehydrogenase under anaerobic conditions. Thus, although all the other enzymes of the citric acid cycle were present in anaerobic cells, this pathway was not available for the metabolism of citrate. Citrate was metabolized aerobically via the citric acid cycle, since (i) citrate lyase but not oxalacetate decarboxylase was repressed and (ii) a-ketoglutarate dehydrogenase was induced under these conditions. The presence of Na+ in the medium did not lead to a repression of a-ketoglutarate dehydrogenase as in the case of Aerobacter aerogenes. The oxalacetate decarboxylase was a soluble, constitutive enzyme, not activated by Na+ nor inhibited by avidin. It was slightly inhibited by ethylenediaminetetraacetate but was not stimulated by Mg2+ or Mn2+. Thus, this enzyme differed markedly in its properties from the same enzyme found in citrate-grown A. aerogenes. The fermentation of citric acid by Aerobacter aerogenes was shown by Brewer and Werkman (3) and Dagley and Dawes (4) to proceed via a cleavage of citrate to oxalacetate (OAA) and acetate catalyzed by citrate lyase (EC ). The OAA was then decarboxylated to pyruvate and carbon dioxide. The OAA decarboxylase (EC ) in A. aerogenes NCTC 418 was subsequently shown by Stern (20) to be a sodium-dependent enzyme. Later work by O'Brien and Stern (14) demonstrated that anaerobic growth of this strain of A. aerogenes on citrate required the presence of Na+ and that failure to grow in the absence of Na+ could be attributed to a failure to activate the OAA decarboxylase. Aerobic growth of strain NCTC 418 on citrate did not require Na+, although growth was stimulated by its presence. Cells grown in the absence of Na+ possessed both the fermentation pathway enzymes (although the OAA decarboxylase was inactive because of the lack of Na+) and citric acid cycle enzymes, indicating that citrate was metabolized via the latter pathway. The presence of Na+ in the medium led to a repression of a-ketoglutarate dehydrogenase (EC ) and activation of the OAA decarboxylase, suggesting that metabolism of citrate now occurred via the fermentation pathway even in aerated cultures (15). A study of citrate metabolism in Aerobacter cloacae, results of which are reported in this paper, shows that both anaerobic and aerobic growth are independent of Na+. Anaerobic metabolism is effected via the fermentation pathway, whereas aerobic metabolism is effected via the citric acid cycle. The properties of the OAA decarboxylase in A. cloacae differ markedly from those of the same enzyme found in A. aerogenes. MATERIALS AND METHODS Growth of the organism. A. cloacae NCTC was grown on a medium containing (g/liter): citric acid, 12.0; KH2PO4, 2.0; MgSO4*7H2O, 0.4; and (NH4)2SO4, 1.0. The medium was neutralized to ph 7.0 with either NaOH or KOH and is designated sodium and potassium citrate medium, respectively. In some experiments, glucose, DL-lactate, or L-malate (1%) was substituted for citrate as sole carbon source. Anaerobic cultures were grown in 500-ml bottles filled to capacity, whereas aerobic cultures were grown in 500 ml of medium contained in a baffled 2-liter flask. A high rate of aeration was achieved by shaking the flasks on a rotary shaker at about 100 rpm. The growth temperature for both anaerobic and aerobic cultures was 35 C. Growth was monitored by measuring the absorbance of the culture at 680 nm using a Unicam SP600 spectrophotometer. The cells used for enzymic studies were grown for 16 h and were harvested by centrifugation at 16,000 x g for 15 min at 5 C Ṗreparation of cell extracts. The cells from 500 ml of culture were resuspended in 6 ml of 0.05 M tris(hydroxymethyl)aminomethane (Tris)-chloride buf- 661

662 O'BRIEN AND GEISLER J. BACTERIOL. fer (ph 7.4) and were ruptured by sonication for 6 x 30 s at 50 W with a Branson sonifier (Branson Sonic Power Co., Danbury, Conn.). Cell debris was removed by centrifugation at 27,000 x g for 15 min at 5 C, and the supernatant was used for enzyme assays.

2 662 O'BRIEN AND GEISLER J. BACTERIOL. fer (ph 7.4) and were ruptured by sonication for 6 x 30 s at 50 W with a Branson sonifier (Branson Sonic Power Co., Danbury, Conn.). Cell debris was removed by centrifugation at 27,000 x g for 15 min at 5 C, and the supernatant was used for enzyme assays. Enzyme assays. Citrate lyase was assayed spectrophotometrically by determining the rate of formation of pyruvate from citrate by coupling the reaction to OAA decarboxylase, lactate dehydrogenase, and reduced nicotinamide adenine dinucleotide (NADH) and measuring the decrease in absorbance at 340 nm. The addition of OAA decarboxylase was unnecessary because of the high endogeneous activity of this enzyme in the extracts. The incubation system contained (in micromoles in a final volume of 1 ml): Tris-chloride buffer, ph 7.0, 100; MgCl2, 10; sodium citrate, 5; NADH, 0.02; lactate dehydrogenase, 2 U; and cell extract (approximately 0.1 to 0.2 mg of protein). The reaction was initiated by the addition of the citrate. Citrate lyase was also assayed according to the procedure of O'Brien and Stern (14) as a check on the spectrophotometric assay. OAA decarboxylase activity was measured by determining the rate of formation of pyruvate from OAA by coupling the reaction to lactate dehydrogenase and NADH and measuring the decrease in absorbance at 340 nm. The incubation system contained (in micromoles in a final volume of 1 ml): Tris-chloride buffer, ph 7.0, 100; potassium OAA (ph 6.8 to 7.0), 5; NADH, 0.02; lactate dehydrogenase, 2 U; and cell extract (approximately 0.02 mg of protein). The reaction was initiated by the addition of the cell extract and was corrected for the non-enzymic decarboxylation of OAA. To test for the effect of avidin on OAA decarboxylase activity, 0.1 ml of cell extract (containing approximately 0.2 mg of protein) was mixed with an equal volume of avidin solution (20 U/ml) and incubated at room temperature for 10 min before addition to the reaction mixture. Citrate synthase (EC ) was assayed according to Weitzman (22). Aconitase (EC ) activity was measured by determining the rate of conversion of cis-aconitate to isocitrate by coupling the reaction to isocitrate dehydrogenase and nicotinamide adenine dinucleotide phosphate and measuring the increase in absorbance at 340 nm. The reaction mixture contained (in micromoles in a final volume of 1 ml): Tris-chloride buffer, ph 8.0, 100; MgCl,, 5; potassium cis-aconitate, 10; nicotinamide adenine dinucleotide phosphate, 0.5; isocitrate dehydrogenase, 5 U; and cell extract (approximately 0.05 mg of protein). Isocitrate dehydrogenase (EC ) was assayed according to O'Brien and Stern (14). a-ketoglutarate dehydrogenase activity was determined by measuring the rate of reduction of nicotinamide adenine dinucleotide at 340 nm in the presence of a-ketoglutarate and reduced coenzyme A according to Kaufman (11). Succinate dehydrogenase (EC ) was assayed by a modification of the method used for malate oxidase by Francis et al. (6). The reaction mixture contained (in micromoles in a final volume of 1.5 ml): potassium phosphate buffer, ph 7.2, 100; KCN (ph 8.5), 10; phenazine methosulfate, 1.6; dichlorophenolindophenol, 0.1; potassium succinate, 10; and cell extract (approximately 0.2 mg of protein). The rate of the reaction was determined by measuring the decrease in absorbance of dichlorophenolindophenol at 600 nm, and the activity was calculated according to Singer and Kearney (18). Fumarase (EC ) activity was determined by measuring the increase in absorbance at 250 nm due to the formation of fumarate from L-malate. The reaction mixture contained (in micromoles in a final volume of 1 ml): Tris-chloride buffer, ph 8.0, 100; potassium L-malate, 10; and cell extract (approximately 0.05 mg of protein). Activity was calculated on the basis of E,Mmarate of 2.44 x 106 cm2/mol. Malate dehydrogenase (EC ) and lactate dehydrogenase (EC ) activities were determined by measuring the rate of NADH oxidation by OAA and pyruvate, respectively, at 340 nm. The incubation system contained (in micromoles in a final volume of 1 ml): potassium phosphate buffer, ph 6.5, 100; NADH, 0.02; potassium OAA or potassium pyruvate, 5; and cell extract. Malate dehydrogenase activity was corrected for the endogeneous OAA decarboxylase and lactate dehydrogenase activities. All enzyme activities are expressed as micromoles of substrate converted or product formed per minute per milligram of protein. Spectrophotometric assays were performed at 21 C by using a Unicam SP800A spectrophotometer and were corrected for NADH oxidase activity where applicable. Protein was measured by the biuret method (7) using bovine plasma albumin as a standard. Citrate in the culture liquid was assayed according to Stem (19). Chemicals and enzymes. Substrates and enzymes used in the assays were obtained from Sigma Chemical Co., St. Louis, Mo. All other chemicals were of reagent grade. RESULTS Growth studies. The growth parameters shown in Table 1 indicate that A. cloacae grew equally well on citrate under anaerobic conditions in the presence or absence of Na+. Thus the initial and final absorbances of the cultures were virtually identical, and the utilization of citrate in each case was the same. O'Brien and Stern (15) have reported that Na+ stimulated the aerobic growth of A. TABLE 1. Growth parameters and ionic composition of anaerobic cultures Parameter Na+ citrate Medium K+ citrate Initial absorbance (680 nm) Final absorbance (680 nm) Growing time (h) Final ph of medium Citrate utilized (mg/ml) Na concn (M) K+ concn (M)

3 VOL. 119, 1974 CITRATE METABOLISM IN A. CLOACAE 663 aerogenes on citrate. However, our experiments show that the rate of growth of A. cloacae under aerobic conditions and the rate of utilization of citrate were almost identical when cells were grown on Na+ citrate or K+ citrate medium (Fig. 1). Thus, Na+ had no stimulatory effect on growth. Enzyme studies. Extracts prepared from cells grown anaerobically on Na+ or K+ citrate medium were assayed for the enzymes listed in Table 2. Both lots of cells contained citrate lyase, OAA decarboxylase, and all the enzymes of the citric acid cycle except a-ketoglutarate dehydrogenase. The activities of the enzymes in the two extracts were similar to one another. The figures quoted for citrate lyase were determined by the spectrophotometric assay (Materials and Methods) which gives higher values than the discontinuous assay of O'Brien and Stern (14). This is due to the fact that the rate of the reaction was linear only for about the first minute of the incubation. The discontinuous assay involves an incubation time of 5 min and thus gives a lower value. The enzyme profile of cells grown aerobically (Table 2) show that the change from anaerobic to aerobic conditions resulted in the repression of citrate lyase, but not OAA decarboxylase and Eos4 _0 _91 / *-< * , tUK T ME (hr) FIG. 1. Aerobic growth of A. cloacae and citrate utilization. A 10% inoculum of cells grown on potassium citrate medium was transferred into sodium citrate and potassium citrate medium, respectively. Symbols: 0, growth on potassium citrate medium; *, growth on sodium citrate medium; A, citrate utilization in potassium citrate medium; V, citrate utilization in sodium citrate medium. -14 TABLE 2. Enzyme profiles of A. cloacae grown anaerobically and aerobically on citrate Mediuma Enzyme Anaerobic Aerobic Na+ K+ Na+ K+ citrate citrate citrate citrate Citrate lyase N.D." N.D. Oxalacetate decarbox ylase Citrate synthase Aconitase Isocitrate dehydrogenase a-ketoglutarate dehy- N.D. N.D drogenase Succinate dehydrogenase Fumarase Malate dehydrogenase Lactate dehydrogenase a Values are expressed as micromoles of substrate transformed or product formed per minute per milligram of protein. b N.D., not detected. the induction of a-ketoglutarate dehydrogenase, thus making the citric acid cycle fully operational. The presence of Na+ in the medium did not result in repression of a-ketoglutarate dehydrogenase. Properties of OAA decarboxylase. The OAA decarboxylase of A. aerogenes NCTC 418 was shown by Stern (20) to be activated by Na+ but not by divalent metal, inhibited by avidin, and unaffected by ethylenediaminetetraacetate (EDTA). We examined the OAA decarboxylase present in dialyzed extracts prepared from cells of A. cloacae grown anaerobically and aerobically, and the results are shown in Table 3. The enzyme was clearly not stimulated by Na+ and only slightly inhibited by avidin. Interestingly, the activity of the enzyme was inhibited only to about 10% by 10 mm EDTA and was not stimulated by addition of 5 mm Mg2+ or Mn2+. Both metals proved to be somewhat inhibitory, with Mg2+ being the more effective inhibitor. Similar results were obtained on undialyzed cell extracts. The similarity of properties shown by the enzyme from anaerobic or aerobic cell extracts demonstrated that the enzyme species was the same in both types of cells. By the same token, the presence or absence of Na+ in the medium had no effect on the properties of the enzyme. The OAA decarboxylase of A. aerogenes was shown to be a particulate enzyme (G. M. Frost and J. R. Stern, Fed. Proc., p. 586, 1968). After centrifuging extracts of A. cloacae at 135,000 x g for 2 h, it was found that all the OAA decarboxylase activity remained in the supernatant, indicating that the enzyme was

4 664 O'BRIEN AND GEISLER J. BACTERIOL. soluble and not particulate. The OAA decarboxylase was a constitutive enzyme since it was present in cells grown aerobically on glucose, DL-lactate, or L-malate (Table 4). However, aerobic growth on citrate induced a higher activity of the enzyme (Tables 2 and 4). DISCUSSION These experiments demonstrate that A. cloacae NCTC can utilize citrate as a sole carbon source for either anaerobic or aerobic growth. In marked contrast to A. aerogenes NCTC 418, anaerobic growth was not dependent on Na+ (14) nor was aerobic growth stimulated by its presence (15). Eagon and Wilkerson (5) have observed that aerobic citrate uptake by A. aerogenes Uga-1 was potassium dependent and was considerably inhibited by Na+ at 0.15 M concentration. On the other hand, A. aerogenes NCTC 418 required Na+ for anaerobic uptake of citrate (J. R. Stern and D. S. Sachan, Fed. Proc., p. 932, 1970). The results observed with the growth studies of A. cloacae seem to indicate that the presence of Na+ at a concentration of 0.20 M did not inhibit citrate uptake under either anaerobic or aerobic conditions. The anaerobic dissimilation of citrate by TABLE 3. Effect of Na+, avidin, EDTA, Mg2+, and Mn2+ on oxalacetate decarboxylase activity Mediuma Addition Anaerobic Aerobic Na+ K+ Na+ K+ citrate citrate citrate citrate None' Na+ (20 mm) Avidin (0.2 U) EDTA (10 mm) Mg2+ (5 mm) Mn2+ (1 mm) Mn2+ (5 mm) a Activity is expressed as micromoles of oxalacetate decarboxylated per minute per milligram of protein. ' Extracts (1 ml) were dialyzed against 2 liters of 0.05 M Tris-chloride buffer (ph 7.4) for 24 h at 5 C. TABLE 4. Effect of carbon source on oxalacetate decarboxylase activity Carbon source Sp acta Glucose DL-Lactate L-Malate a Activity is expressed as micromoles of oxalacetate decarboxylated per minute per milligram of protein. A. aerogenes has been shown (4) to occur by cleavage of citrate to OAA and acetate, followed by decarboxylation of OAA to pyruvate and CO2. This is termed the citrate fermentation pathway. The enzyme profile for anaerobically grown A. cloacae indicates that this organism also uses the fermentation pathway for anaerobic catabolism of citrate, since extracts contained citrate lyase and OAA decarboxylase. The importance of the fermentation pathway was emphasized by the fact that the citrate lyase was induced under anaerobic conditions and was absent from aerobically grown cells and by the fact that the citric acid cycle was inoperative due to repression of a-ketoglutarate dehydrogenase. The absence of this enzyme is not suprising since its repression under anaerobic conditions has been observed in Escherichia coli (1, 8) and A. aerogenes (14). Thus, the citric acid cycle becomes in effect two biosynthetic pathways, one leading to the synthesis of glutamate (involving aconitase and isocitrate dehydrogenase) and the other leading to the synthesis of succinate (involving malate dehydrogenase, fumarase, and succinate dehydrogenase). O'Brien and Stern (15) observed that A. aerogenes used the citric acid cycle for the aerobic metabolism of citrate when cells were grown in the absence of Na+. When Na+ was present in the medium, the a-ketoglutarate dehydrogenase was repressed, and metabolism of citrate appeared to be carried out via the fermentation pathway. In the case of A. cloacae, no repression of the a-ketoglutarate dehydrogenase was observed in cells grown on the Na+ citrate medium, and metabolism of citrate proceeded via the citric acid cycle since the citrate lyase was repressed and a-ketoglutarate dehydrogenase was induced under aerobic conditions. Thus, the presence of oxygen shifted the metabolism of citrate from the fermentation pathway to that of the citric acid cycle. Wilkerson and Eagon (23) reported that A. aerogenes strain UGa-1 metabolized citrate aerobically, in a sodium-free medium, via the citric acid cycle. They found that this strain and two other strains were devoid of citrate lyase when grown aerobically. Villareal-Moguel and Ruiz-Herrera (21) observed similar results in other strains of A. aerogenes. A. cloacae therefore appears to be more similar physiologically to strains of A. aerogenes other than strain NCTC 418. The OAA decarboxylase of citrate-grown A. aerogenes has been reported as being a particulate enzyme, activated by Na+ but not by divalent metals, sensitive to avidin but not to EDTA, and specifically induced by either anaerobic or aerobic growth on citrate (20). The

VOL. 119, 1974 CITRATE METABOLISM IN A. CLOACAE 665 properties of the OAA decarboxylase of A. cloacae, on the other hand, are almost the complete reverse of those described for the A.

5 VOL. 119, 1974 CITRATE METABOLISM IN A. CLOACAE 665 properties of the OAA decarboxylase of A. cloacae, on the other hand, are almost the complete reverse of those described for the A. aerogenes enzyme. Thus the enzyme is soluble, not activated by Na+, Mg2+, or Mn2+, not inhibited by avidin, and only slightly inhibited by EDTA. In addition, the enzyme appears to be constitutive. The enzyme is, therefore, not of the same type as that found in A. aerogenes (20) and appears to be more closely related to the OAA decarboxylases observed in other bacteria (2, 9, 10, 12, 13, 16, 17). However, it also differs from these in not being stimulated by Mg2+ or Mn2+ and being only slightly inhibited by EDTA. This of course does not rule out the possibility that the enzyme contains a tightly bound metal activator which is not chelated by EDTA or removed by dialysis. Nevertheless, the enzyme may represent a new species of OAA decarboxylase. The function of the enzyme in cells grown anaerobically on citrate is easily discerned, but in the case of cells grown aerobically on citrate or other carbon sources its function remains to be elucidated. ACKNOWLEDGMENTS We thank Dorothy Jones, M.R.C. Microbial Systematics Unit, University of Leicester, England, for the provision of the culture and Andrew Muir for the Na+ and K+ analyses. This research was supported by a University of Sydney research grant. LITERATURE CITED 1. Amarasingham, C. J., and B. D. Davis Regulation of a-ketoglutarate dehydrogenase formation in Escherichia coli. J. Biol. Chem. 240: Benziman, M., and N. Heller Oxaloacetate decarboxylation and oxaloacetate-carbon dioxide exchange in Acetobacter xylinum. J. Bacteriol. 88: Brewer, C. R., and C. H. Werkman The anaerobic dissimilation of citric acid by Aerobacter indologenes. Enzymologia 6: Dagley, S., and E. A. Dawes Dissimilation of citric acid by bacterial extracts. Nature (London) 172: Eagon, R. G., and L. S. Wilkerson A potassiumdependent citric acid transport system in Aerobacter aerogenes. Biochem. Biophys. Res. Commun. 46: Francis, M. J. O., D. E. Hughes, H. L. Kornberg, and P. J. R. Phizackerley The oxidation of L-malate by Pseudomonas sp. Biochem. J. 89: Gomall, A. G., C. H. Bardawill, and M. M. David Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177: Gray, C. T., J. W. T. Wimpenny, and M. R. Mossman Regulation of metabolism in facultative bacteria. II. Effect of aerobiosis, anaerobiosis and nutrition on the formation of Krebs cycle enzymes in Escherichia coli. Biochim. Biophys. Acta 117: Herbert, D Oxalacetic decarboxylase and carbon dioxide assimilation in bacteria. Symp. Soc. Exp. Biol. 5: Horton, A. A., and H. L. Komberg Oxalacetate 4-carboxylyase from Pseudomonas ovalis Chester. Biochim. Biophys. Acta 89: Kaufman, S a-ketoglutaric dehydrogenase system and phosphorylating enzyme from heart muscle, p In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 1. Academic Press Inc., New York. 12. Krampitz, L. O., and C. H. Werkman The enzymic decarboxylation of oxaloacetate. Biochem. J. 35: O'Brien, R. W., G. M. Frost, and J. R. Stem Enzymatic analysis of the requirement for sodium in aerobic growth of Salmonella typhimurium on citrate. J. Bacteriol. 99: O'Brien, R. W., and J. R. Stem Requirement for sodium in the anaerobic growth of Aerobacter aerogenes on citrate. J. Bacteriol. 98: O'Brien, R. W., and J. R. Stern Role of sodium in determining altemate pathways of aerobic citrate catabolism in Aerobacter aerogenes. J. Bacteriol. 99: Plaut, G. W. E., and H. A. Lardy, The oxaloacetate decarboxylase of Azotobacter vinelandii. J. Biol. Chem. 180: Rosenberger, R. F Derepression of oxaloacetate 4-carboxylyase synthesis in Salmonella typhimurium. Biochim. Biophys. Acta 122: Singer, R. P., and E. B. Kearney Determination of succinic dehydrogenase activity, p In D. Glick (ed.), Methods of biochemical analysis, vol. 4. Interscience Publishers Inc., New York. 19. Stem, J. R Assay of tricarboxylic acids, p In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 3. Academic Press Inc., New York. 20. Stem, J. R Oxalacetate decarboxylase of Aerobacter aerogenes. I. Inhibition by avidin and requirement for sodium ion. Biochemistry 6: Villareal-Moguel, E. I., and J. Ruiz-Herrera Induction and properties of the citrate transport system in Aerobacter aerogenes. J. Bacteriol. 98: Weitzman, P. D. J Citrate synthase from Escherichia coli, p In J. M. Lowenstein (ed.), Methods in enzymology, vol. 13. Academic Press Inc., New York. 23. Wilkerson, L. S., and R. G. Eagon Transport of citric acid by Aerobacter aerogenes. Arch. Biochem. Biophys. 149:

Role of Sodium in Determining Alternate Pathways of Aerobic Citrate Catabolism in Aerobacter aerogenes

JOURNAL OF BACTERIOLOGY, Aug. 1969, p. 389-394 Copyright 1969 American Society for Microbiology Vol. 99, No. 2 Printed in U.S.A. Role of Sodium in Determining Alternate Pathways of Aerobic Citrate Catabolism