JOURNAL OF BACTERIOLOGY, Dec. 1975, p. 1084-1088 Copyright 0 1975 American Society for Microbiology
Vol. 124, No. 3 Printed in U.S.A.
Induction of Citrate Lyase in Enterobacter cloacae Grown Under Aerated Conditions and Its Effect on Citrate Metabolism R. W. O'BRIEN Department of Biochemistry, The University of Sydney, Sydney, N.S.W., 2006, Australia Received for publication 8 August 1975
Growth of Enterobacter cloacae on K+ citrate under aerated conditions (no detectable oxygen tension in the medium even though it was aerated) was slower (mean generation time, 130 min) than under aerobic conditions (mean generation time, 72 min), but with a faster utilization of citrate, resulting in a molar growth yield of 10.6 g (dry weight) of cells per mol of citrate utilized versus 40 g (dry weight) of cells per mol of citrate utilized for aerobic growth. The rapid utilization of citrate under aerated conditions was apparently due to the induction of citrate lyase and was supported by the finding that cells excreted acetate and a small amount of oxalacetate under aerated conditions, but not under aerobic conditions when the cells were devoid of citrate lyase activity. The activity of oxalacetate decarboxylase in aerated cells was slightly lower than in aerobic cells, indicating that little of the oxalacetate produced by the citrate lyase was metabolized by the decarboxylase. Oxalacetate was probably metabolized by malate dehydrogenase, previously shown to be present in anaerobic and aerobic cells. Thus, about 70% of the citrate was cleaved by the citrate lyase, resulting in little or no production of energy for growth. The remaining citrate was metabolized via the citric acid cycle under aerated conditions, since the cells contained a-ketoglutarate dehydrogenase at the same level as in aerobically grown cells. The presence of the other enzymes of the cycle was shown in earlier studies.
The anaerobic catabolism of citrate by Aerobacter indologenes (1), Klebsiella aerogenes (3), and Aerobacter cloacae (10) proceeds via the cleavage of citrate to oxalacetate (OAA), catalyzed by citrate lyase, and decarboxylation of OAA to pyruvate, catalyzed by OAA decarboxylase. This is termed the fermentation pathway. In the case of K. aerogenes, NCTC 418, Na+ is essential for the anaerobic growth of the organism (12) and for the activation of the OAA decarboxylase (14). Anaerobic growth of A. cloacae, on the other hand, is independent of Na + and the OAA decarboxylase does not require any metal ion activator (10). The aerobic catabolism of citrate by both K. aerogenes and A. cloacae proceeds via the citric acid cycle and is independent of Na+ (8, 10, 15). Under aerated conditions (defined as those under which no dissolved oxygen could be measured in the medium even though the culture was aerated), K. aerogenes metabolized citrate via the citric acid cycle in the absence of Na+. In the presence of Na+ and under aerated (but not aerobic) conditions the enzymes of the fermentation pathway were induced (9), thus enabling the organism to metabolize citrate via
both the fermentation pathway and the citric acid cycle (8). A study of the effect of aeration on the growth of citrate metabolism by Enterobacter cloacae was undertaken to determine whether this organism behaved in the same way as K. aerogenes. The results indicate that the citrate lyase, but not OAA decarboxylase, was induced under aerated conditions and as a consequence a considerable amount of energy was lost by citrate being cleaved by the citrate lyase.
MATERIALS AND METHODS Growth of the organism. E. cloacae NCTC 10005 was grown on Na+ citrate anid K+ citrate medium as described by O'Brien and Geisler (10). Anaerobic cultures were grown in 1-liter bottles filled to capacity with medium rendered anaerobic by the addition of sterile sodium dithionite. Aerobic and aerated cultures were grown in a growth vessel fitted with a Clark-type oxygen electrode (11). For aerated cultures the rate of the air flow was continuously adjusted throughout the whole of the growth period to maintain a zero oxygen tension. For aerobic cultures the rate of the air flow was adjusted to give a dissolved oxygen concentration of 50 to 100 AM and was recorded on a 1084
VOL. 124, 1975
1085
CITRATE LYASE INDUCTION IN E. CLOACAE
Heath-built Servo Recorder model EU-20B. The growth temperature for all cultures was 35 C. Growth was monitored by measuring the absorbance of the culture at 680 nm using a Unicam SP600 spectrophotometer, and the dry weight of organisms per milliliter of culture was determined from a calibration curve obtained by drying samples at 100 C. Samples of the culture were collected at intervals, clarified by centrifugation in a bench centrifuge at 5 C, and assayed immediately for OAA and pyruvate. The remainder of the samples was stored at -20 C until assayed for citrate and acetate. At the end of the exponential growth phase, the cells were harvested by centrifugation at 16,000 x g for 15 min at 5 C and were stored at -20 C for 16 h. Preparation of cell extracts and enzyme assays. The frozen cells were thawed in 0.05 M tris(hydroxymethyl)aminomethane-chloride buffer, pH 7, and extracts were prepared according to the method of O'Brien and Geisler (10). Citrate lyase (EC 4.1.3.6) activity was assayed spectrophotometrically by determining the rate of formation of OAA from citrate by coupling the reaction to malate and lactate dehydrogenases. The latter enzyme was added to measure any pyruvate formed from OAA by either enzymic or non-enzymic decarboxylation., The incubation contained (in a final volume of 1 ml): tris(hydroxymethyl)aminomethanehydrochloride buffer, pH 7, 100 umol; MgCl,, 10 gmol; sodium citrate, 5 jmol; reduced nicotinamide adenine dinucleotide, 0.02 Amol; malate dehydrogenase (EC 1.1.1.37), 1 U; lactate dehydrogenase (EC 1.1.1.27), 1 U; and cell extract. OAA decarboxylase (EC 4.1.1.3) activity was measured by determining the rate of formation of pyruvate from OAA using the discontinuous assay procedure of Stern (14). a-Ketoglutarate dehydrogenase (EC 1.2.4.2) activity was determined according to Kaufman (6). Spectrophotometric assays were performed at 25 C using a Varian Techtron model 635 spectrophotometer and were corrected for reduced nicotinamide adenine dinucleotide-oxidase activity where applicable. Assay procedures. The following compounds in the spent culture liquor were measured according to the cited methods: citrate by the acetic anhydridepyridine method (7); acetate (13); pyruvate (2); and OAA (5). Protein was measured by the biuret method using bovine plasma albumin as a standard (4). Chemicals and enzymes. Substrates, cofactors, and enzymes used in the assay procedures were obtained from Sigma Chemical Co., St. Louis, Mo. All other chemicals were of reagent grade. RESULTS Growth studies. The rate of growth of E.
cloacae on K + citrate medium under aerobic and aerated conditions is shown in Fig. 1. Growth was slower under aerated conditions (mean generation time, 130 min) than under aerobic conditions (mean generation time, 72 min) (Table 1). However, under aerobic conditions the cells entered the stationary phase, with a considerable amount of the citrate re-
E
0
0
E
ui
z
co
I-
u
0 (A
I-
41 I
4 x
o0
TIME hr
FIG. 1. Effect of aerated and aerobic growth of E. cloacae on oxalacetate excretion and citrate utilization. Symbols: 0, aerated growth; 0, aerobic growth; 0, citrate utilization under aerated conditions; *, citrate utilization under aerobic conditions; A, OAA excretion under aerated conditions. TABLE 1. Growth parameters of cultures of E. cloacae grown under different conditions on K+ citrate Growth conditions Parameter
Anaerobic Aerated
Initial absorbance (680 nm) Final absorbance (680 nm) Growing time (h) Mean generation time (min) Initial pH Final pH Citrate utilized (rmol/ ml)a Acetate excreted (Umol/ml)a Cell density (jig of
cells/ml)a Molar growth yield (g of cells/mol of cit-
Aerobic
0.06
0.14
0.16
0.47
1.10
1.40
23
7 130
5 72
7.0 6.6 27.4
6.9 7.3 55.5
6.9 8.5 18.8
45.6
40.2
0.0
250
9.1
587 10.6
753 40.0
rate utilized) a Measured as the difference in the culture at the time of inoculation and at the time of harvesting.
maining in the medium. This premature slowing of growth may have been due to excretion of inhibitory metabolites, as shown by the high pH (8.1) of the culture at the end of exponential
1086
O'BRIEN
J. BACTERIOL.
growth (Table 1). This suggestion is supported by the finding that cells grown in aerated medium almost completely utilized the citrate without the pH rising above 7.3 (Fig. 1; Table 1). A similar behavior was observed when the cells were grown on Na+ citrate under aerobic and aerated conditions. The molar growth yield of the organism when grown under aerobic conditions, on K+ citrate, was 40 g (dry weight) of cells per mol of citrate utilized. Under aerated conditions, this value fell to about 11 g (dry weight) of cells per mol of citrate utilized (Table 1). This dramatic fall in cell yield paralleled the very much increased rate of utilization of citrate under aerated, as compared to aerobic, conditions (Fig. 1) and was not much greater than the yield obtained when the cells were grown anaerobically (Table 1). Enzyme studies. To maintain a correlation between growth and metabolism, the cultures used in the growth studies were harvested and extracts were prepared from the cells. The extracts were assayed for citrate lyase, OAA decarboxylase, and a-ketoglutarate dehydrogenase. These enzymes are probably the key ones in determining the pathway for the metabolism of citrate (10). The other enzymes of the citric acid cycle have been detected in both anaerobically and aerobically grown cells of E. cloacae (10) and were therefore not assayed in the present study. The results of the enzyme assays for K+ citrate cultures are shown in Table 2. Similar results (not shown) were observed when the organism was grown on Na+ citrate medium. Cells grown under anaerobic conditions contained citrate lyase and OAA decarboxylase but not a-ketoglutarate dehydrogenase, whereas aerobically grown cells contained OAA decarboxylase and a-ketoglutarate dehydrogenase, but not citrate lyase, thus confirming the previTABLE 2. Enzyme activities of E. cloacae grown under different conditions on K+ citrate Growth conditionsa Enzvme
Citrate lyase OAA decarboxylase a-Ketoglutarate dehydrogenase
Anaerobic
Aerated
Aerobic
0.45 0.20 ND
0.56 0.06 0.082
0.14 0.096
NDb
aValues are expressed as micromoles of substrate transformed or product formed per minute per milligram of protein. bND, Not detected. The lower limit of detection was 0.001.
ous results of O'Brien and Geisler (10). However, the activities for OAA decarboxylase reported in this paper are considerably lower than those reported earlier (10). The assay procedure used in this study was a discontinuous one and was not subject to interference by malate dehydrogenase as was the procedure used by O'Brien and Geisler (10). Thus, the values quoted here are probably more realistic. The properties of the OAA decarboxylase have been re-examined and are the same as those previously reported (10). Citrate lyase, OAA decarboxylase, and a-ketoglutarate dehydrogenase were present in cells grown under aerated conditions. The activity of citrate lyase was similar to that found in anaerobic cells. The activity of the OAA decarboxylase did not increase to that of the anaerobic cells but instead fell to a value slightly lower than that in aerobic cells. The activity of a-ketoglutarate dehydrogenase in aerated cells remained about the same as that in aerobic cells. Excretion of metabolites. Since the enzymic analyses indicated that anaerobic and aerated, but not aerobic, cells contained citrate lyase, the presence of acetate in the medium would be an indication of whether or not the enzyme was involved in citrate catabolism. Samples were taken from the culture at the time of inoculation and the time of harvesting and were assayed for acetate (Table 1). No acetate was excreted by aerobic cells. The amount excreted by anaerobic cells was of the order of 1.7 mol/mol of citrate utilized. This value was similar to that obtained when K. aerogenes was grown anaerobically on citrate (1, 8). When E. cloacae was grown under aerated conditions, the excretion of acetate fell to about 0.7 mol/mol of citrate utilized. This value was considerably lower than the value of 1.2 for K. aerogenes when it was grown under aerated conditions on Na+ citrate (8). Further evidence that citrate was metabolized by citrate lyase was provided by the finding that aerated cells excreted oxalacetate into the medium (Fig. 1). The amount excreted was small, reaching a concentration of slightly over 1 Mmol/ml of culture at the end of the growth period. A small amount of pyruvate (about 0.3 imol/ml of culture) was also detected. This concentration remained essentially constant throughout the growth period. No OAA or pyruvate was excreted by the anaerobic or aerobic cells. A search for other metabolites indicated that trace amounts of lactate and succinate but not malate were excreted by the cells, irrespective of the growth conditions. A
VOL. 124, 1975
CITRATE LYASE INDUCTION IN E. CLOACAE
1087
similar excretion of metabolites occurred when activity of OAA decarboxylase than that observed in aerobic cells, where the fermentation the organism was grown on Na+ citrate. pathway does not function, and (ii) the excretion of a small amount of OAA, which did not DISCUSSION occur with anaerobic or aerobic cultures. A The results of the growth studies clearly comparison of these results with those observed indicate that aerated growth of E. cloacae on with K. aerogenes, when grown under aerated K+ citrate was slower than aerobic growth, conditions in the presence of Na+, also points to presumably because of oxygen limitation. How- the fermentation pathway being inoperative in, ever, the rate of citrate utilization under E. cloacae. In the case of K. aerogenes, both aerated conditions was considerably greater enzymes of the fermentation pathway were than under aerobic conditions, despite the induced, enabling the organism to utilize both slower growth rate, and led to a much lower fermentation and the citric acid cycle for energy molar growth yield. The presence of Na+ in the production and thus achieving a higher molar medium did not affect the behavior of the growth yield and greater excretion of acetate organism in the same way as observed with K. than observed with E. cloacae (8, 9). The OAA produced by the citrate lyase activaerogenes, where both the growth rate and citrate utilization increased when Na+ was ity was probably metabolized by malate dehyadded to a K+ citrate culture growing under drogenase, which is present in high activity in anaerobic and aerobic cells (10), since no signifaerated conditions (8, 9). The rapid utilization of citrate under aerated icant quantities of OAA, or metabolites which conditions was paralleled by the induction of could be derived from OAA (malate, succinate, citrate lyase. Evidence that the enzyme partici- pyruvate, or lactate), were found in the spent pated in citrate catabolism was shown by the medium. excretion of acetate by the aerated cells but not ACKNOWLEDGMENTS by aerobic cells, which were devoid of citrate I thank Dorothy Jones, Microbial Systematics Unit, Unilyase activity. Citrate lyase was induced in E. of Leicester, England, for the provision of the culture. cloacae in both the presence and absence of versity This research was supported by a University of Sydney Na+, which contrasts with the behavior of K. research grant. aerogenes where the enzyme was induced in aerated cells only in the presence of Na+ (9). LITERATURE CITED If the acetate excreted by the aerated cells 1. Brewer, C. R., and C. H. Werkman. 1939. The anaeroresulted fom the operation of the fermentation bic dissimilation of citric acid by Aerobacter indologenes. Enzymologia 6:273-281. pathway, then the molar growth yield should T., R. Czok, W. Lamprecht, and E. Latzko. 1963. have been 26.6 g (dry weight) of cells per mol of 2. Bucher, Pyruvate, p. 253-262. In H. U. Bergmeyer (ed.), citrate utilized, basing the calculation on the Methods of enzymatic analysis. Academic Press Inc., amount of acetate excreted by anaerobic cells, New York. where the fermentation pathway alone was 3. Dagley, S., and E. A. Dawes. 1953. Dissimilation of citric acid by bacterial extracts. Nature (London) 172: operative (10). However, the value obtained for 345-346. the molar growth yield of the aerated cells was 4. Gornall, A. G., C. H. Bardawill, and M. M. David. 1949. only 10.6 g (dry weight) of cells per mol of Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177:751-766. citrate utilized, which corresponds closely to the H. J., and M. Reim. 1963. Oxaloacetate, p. value of 11.0 which would be obtained if the 5. Hohorst, 335-339. In H. U. Bergmeyer (ed.), Methods of enacetate resulted from the citrate lyase reaction zymatic analysis. Academic Press Inc., New York. alone, with the remaining citrate being metabo- 6. Kaufman, S. 1955. a-Ketoglutaric dehydrogenase system and phosphorylating enzyme from heart muscle, p. lized via the citric acid cycle. These results 714-722. In S. P. Colowick and N. 0. Kaplan (ed.), indicate that energy for the growth of the Methods in enzymology, vol 1. Academic Press Inc., organism under aerated conditions came mainly New York. from the metabolism of some 27% of the citrate 7. Lowenstein, J. M. 1969. Chemical methods for citrate and aconitate, p. 573-576. In J. M. Lowenstein (ed.), via the citric acid cycle. Therefore, the rapid Methods in enzymology, vol 13. Academic Press Inc., utilization of citrate, combined with the less New York. efficient growth, under aerated conditions is 8. O'Brien, R. W. 1975. Effect of aeration and sodium on the attributable to a large amount of the substrate metabolism of citrate by Klebsiella aerogenes. J. Bacteriol. 122:468-473. being diverted from the citric acid cycle by the R. W. 1975. Induction by sodium of the citrate action of citrate lyase. Further evidence that the 9. O'Brien, fermentation enzymes in Klebsiella aerogenes. FEBS fermentation pathway was largely inoperative Lett. 53:61-63. beyond the citrate lyase reaction was shown by 10. O'Brien, R. W., and J. Geisler. 1974. Citrate metabolism in Aerobacter cloacae. J. Bacteriol. 119:661-665. (i) the failure of aerated cells to induce a higher
1088
O'BRIEN
11. O'Brien, R. W., and J. G. Morris. 1971. Oxygen and the growth and metabolism of Clostridium acetobutylicum. J. Gen. Microbiol. 68:307-318. 12. O'Brien, R. W., and J. R. Stern. 1969. Requirement for
sodium in the anaerobic growth of Aerobacter 4. Bacteriol. 98:388-393. 13. Rose, I. A. 1955. Acetate kinase of bacteria (acetokinase), p. 591-595. In S. P. Colowick and N. 0. Kaplan (ed.), aerogenes.
J. BACTERIOL. Methods in enzymology, vol. 1. Academic Press Inc., New York. 14. Stern, J. R. 1967. Oxalacetate decarboxylase of Aerobacter aerogenes. I. Inhibition by avidin and requirement for sodium ion. Biochemistry 6:3545-3551. 15. Wilkerson, L. S., and R. G. Eagon. 1972. Transport of citric acid by Aerobacter aerogenes. Arch. Biochem. Biophys. 149:209-221.
Vol. 124, No. 3 Printed in U.S.A.
Induction of Citrate Lyase in Enterobacter cloacae Grown Under Aerated Conditions and Its Effect on Citrate Metabolism R. W. O'BRIEN Department of Biochemistry, The University of Sydney, Sydney, N.S.W., 2006, Australia Received for publication 8 August 1975
Growth of Enterobacter cloacae on K+ citrate under aerated conditions (no detectable oxygen tension in the medium even though it was aerated) was slower (mean generation time, 130 min) than under aerobic conditions (mean generation time, 72 min), but with a faster utilization of citrate, resulting in a molar growth yield of 10.6 g (dry weight) of cells per mol of citrate utilized versus 40 g (dry weight) of cells per mol of citrate utilized for aerobic growth. The rapid utilization of citrate under aerated conditions was apparently due to the induction of citrate lyase and was supported by the finding that cells excreted acetate and a small amount of oxalacetate under aerated conditions, but not under aerobic conditions when the cells were devoid of citrate lyase activity. The activity of oxalacetate decarboxylase in aerated cells was slightly lower than in aerobic cells, indicating that little of the oxalacetate produced by the citrate lyase was metabolized by the decarboxylase. Oxalacetate was probably metabolized by malate dehydrogenase, previously shown to be present in anaerobic and aerobic cells. Thus, about 70% of the citrate was cleaved by the citrate lyase, resulting in little or no production of energy for growth. The remaining citrate was metabolized via the citric acid cycle under aerated conditions, since the cells contained a-ketoglutarate dehydrogenase at the same level as in aerobically grown cells. The presence of the other enzymes of the cycle was shown in earlier studies.
The anaerobic catabolism of citrate by Aerobacter indologenes (1), Klebsiella aerogenes (3), and Aerobacter cloacae (10) proceeds via the cleavage of citrate to oxalacetate (OAA), catalyzed by citrate lyase, and decarboxylation of OAA to pyruvate, catalyzed by OAA decarboxylase. This is termed the fermentation pathway. In the case of K. aerogenes, NCTC 418, Na+ is essential for the anaerobic growth of the organism (12) and for the activation of the OAA decarboxylase (14). Anaerobic growth of A. cloacae, on the other hand, is independent of Na + and the OAA decarboxylase does not require any metal ion activator (10). The aerobic catabolism of citrate by both K. aerogenes and A. cloacae proceeds via the citric acid cycle and is independent of Na+ (8, 10, 15). Under aerated conditions (defined as those under which no dissolved oxygen could be measured in the medium even though the culture was aerated), K. aerogenes metabolized citrate via the citric acid cycle in the absence of Na+. In the presence of Na+ and under aerated (but not aerobic) conditions the enzymes of the fermentation pathway were induced (9), thus enabling the organism to metabolize citrate via
both the fermentation pathway and the citric acid cycle (8). A study of the effect of aeration on the growth of citrate metabolism by Enterobacter cloacae was undertaken to determine whether this organism behaved in the same way as K. aerogenes. The results indicate that the citrate lyase, but not OAA decarboxylase, was induced under aerated conditions and as a consequence a considerable amount of energy was lost by citrate being cleaved by the citrate lyase.
MATERIALS AND METHODS Growth of the organism. E. cloacae NCTC 10005 was grown on Na+ citrate anid K+ citrate medium as described by O'Brien and Geisler (10). Anaerobic cultures were grown in 1-liter bottles filled to capacity with medium rendered anaerobic by the addition of sterile sodium dithionite. Aerobic and aerated cultures were grown in a growth vessel fitted with a Clark-type oxygen electrode (11). For aerated cultures the rate of the air flow was continuously adjusted throughout the whole of the growth period to maintain a zero oxygen tension. For aerobic cultures the rate of the air flow was adjusted to give a dissolved oxygen concentration of 50 to 100 AM and was recorded on a 1084
VOL. 124, 1975
1085
CITRATE LYASE INDUCTION IN E. CLOACAE
Heath-built Servo Recorder model EU-20B. The growth temperature for all cultures was 35 C. Growth was monitored by measuring the absorbance of the culture at 680 nm using a Unicam SP600 spectrophotometer, and the dry weight of organisms per milliliter of culture was determined from a calibration curve obtained by drying samples at 100 C. Samples of the culture were collected at intervals, clarified by centrifugation in a bench centrifuge at 5 C, and assayed immediately for OAA and pyruvate. The remainder of the samples was stored at -20 C until assayed for citrate and acetate. At the end of the exponential growth phase, the cells were harvested by centrifugation at 16,000 x g for 15 min at 5 C and were stored at -20 C for 16 h. Preparation of cell extracts and enzyme assays. The frozen cells were thawed in 0.05 M tris(hydroxymethyl)aminomethane-chloride buffer, pH 7, and extracts were prepared according to the method of O'Brien and Geisler (10). Citrate lyase (EC 4.1.3.6) activity was assayed spectrophotometrically by determining the rate of formation of OAA from citrate by coupling the reaction to malate and lactate dehydrogenases. The latter enzyme was added to measure any pyruvate formed from OAA by either enzymic or non-enzymic decarboxylation., The incubation contained (in a final volume of 1 ml): tris(hydroxymethyl)aminomethanehydrochloride buffer, pH 7, 100 umol; MgCl,, 10 gmol; sodium citrate, 5 jmol; reduced nicotinamide adenine dinucleotide, 0.02 Amol; malate dehydrogenase (EC 1.1.1.37), 1 U; lactate dehydrogenase (EC 1.1.1.27), 1 U; and cell extract. OAA decarboxylase (EC 4.1.1.3) activity was measured by determining the rate of formation of pyruvate from OAA using the discontinuous assay procedure of Stern (14). a-Ketoglutarate dehydrogenase (EC 1.2.4.2) activity was determined according to Kaufman (6). Spectrophotometric assays were performed at 25 C using a Varian Techtron model 635 spectrophotometer and were corrected for reduced nicotinamide adenine dinucleotide-oxidase activity where applicable. Assay procedures. The following compounds in the spent culture liquor were measured according to the cited methods: citrate by the acetic anhydridepyridine method (7); acetate (13); pyruvate (2); and OAA (5). Protein was measured by the biuret method using bovine plasma albumin as a standard (4). Chemicals and enzymes. Substrates, cofactors, and enzymes used in the assay procedures were obtained from Sigma Chemical Co., St. Louis, Mo. All other chemicals were of reagent grade. RESULTS Growth studies. The rate of growth of E.
cloacae on K + citrate medium under aerobic and aerated conditions is shown in Fig. 1. Growth was slower under aerated conditions (mean generation time, 130 min) than under aerobic conditions (mean generation time, 72 min) (Table 1). However, under aerobic conditions the cells entered the stationary phase, with a considerable amount of the citrate re-
E
0
0
E
ui
z
co
I-
u
0 (A
I-
41 I
4 x
o0
TIME hr
FIG. 1. Effect of aerated and aerobic growth of E. cloacae on oxalacetate excretion and citrate utilization. Symbols: 0, aerated growth; 0, aerobic growth; 0, citrate utilization under aerated conditions; *, citrate utilization under aerobic conditions; A, OAA excretion under aerated conditions. TABLE 1. Growth parameters of cultures of E. cloacae grown under different conditions on K+ citrate Growth conditions Parameter
Anaerobic Aerated
Initial absorbance (680 nm) Final absorbance (680 nm) Growing time (h) Mean generation time (min) Initial pH Final pH Citrate utilized (rmol/ ml)a Acetate excreted (Umol/ml)a Cell density (jig of
cells/ml)a Molar growth yield (g of cells/mol of cit-
Aerobic
0.06
0.14
0.16
0.47
1.10
1.40
23
7 130
5 72
7.0 6.6 27.4
6.9 7.3 55.5
6.9 8.5 18.8
45.6
40.2
0.0
250
9.1
587 10.6
753 40.0
rate utilized) a Measured as the difference in the culture at the time of inoculation and at the time of harvesting.
maining in the medium. This premature slowing of growth may have been due to excretion of inhibitory metabolites, as shown by the high pH (8.1) of the culture at the end of exponential
1086
O'BRIEN
J. BACTERIOL.
growth (Table 1). This suggestion is supported by the finding that cells grown in aerated medium almost completely utilized the citrate without the pH rising above 7.3 (Fig. 1; Table 1). A similar behavior was observed when the cells were grown on Na+ citrate under aerobic and aerated conditions. The molar growth yield of the organism when grown under aerobic conditions, on K+ citrate, was 40 g (dry weight) of cells per mol of citrate utilized. Under aerated conditions, this value fell to about 11 g (dry weight) of cells per mol of citrate utilized (Table 1). This dramatic fall in cell yield paralleled the very much increased rate of utilization of citrate under aerated, as compared to aerobic, conditions (Fig. 1) and was not much greater than the yield obtained when the cells were grown anaerobically (Table 1). Enzyme studies. To maintain a correlation between growth and metabolism, the cultures used in the growth studies were harvested and extracts were prepared from the cells. The extracts were assayed for citrate lyase, OAA decarboxylase, and a-ketoglutarate dehydrogenase. These enzymes are probably the key ones in determining the pathway for the metabolism of citrate (10). The other enzymes of the citric acid cycle have been detected in both anaerobically and aerobically grown cells of E. cloacae (10) and were therefore not assayed in the present study. The results of the enzyme assays for K+ citrate cultures are shown in Table 2. Similar results (not shown) were observed when the organism was grown on Na+ citrate medium. Cells grown under anaerobic conditions contained citrate lyase and OAA decarboxylase but not a-ketoglutarate dehydrogenase, whereas aerobically grown cells contained OAA decarboxylase and a-ketoglutarate dehydrogenase, but not citrate lyase, thus confirming the previTABLE 2. Enzyme activities of E. cloacae grown under different conditions on K+ citrate Growth conditionsa Enzvme
Citrate lyase OAA decarboxylase a-Ketoglutarate dehydrogenase
Anaerobic
Aerated
Aerobic
0.45 0.20 ND
0.56 0.06 0.082
0.14 0.096
NDb
aValues are expressed as micromoles of substrate transformed or product formed per minute per milligram of protein. bND, Not detected. The lower limit of detection was 0.001.
ous results of O'Brien and Geisler (10). However, the activities for OAA decarboxylase reported in this paper are considerably lower than those reported earlier (10). The assay procedure used in this study was a discontinuous one and was not subject to interference by malate dehydrogenase as was the procedure used by O'Brien and Geisler (10). Thus, the values quoted here are probably more realistic. The properties of the OAA decarboxylase have been re-examined and are the same as those previously reported (10). Citrate lyase, OAA decarboxylase, and a-ketoglutarate dehydrogenase were present in cells grown under aerated conditions. The activity of citrate lyase was similar to that found in anaerobic cells. The activity of the OAA decarboxylase did not increase to that of the anaerobic cells but instead fell to a value slightly lower than that in aerobic cells. The activity of a-ketoglutarate dehydrogenase in aerated cells remained about the same as that in aerobic cells. Excretion of metabolites. Since the enzymic analyses indicated that anaerobic and aerated, but not aerobic, cells contained citrate lyase, the presence of acetate in the medium would be an indication of whether or not the enzyme was involved in citrate catabolism. Samples were taken from the culture at the time of inoculation and the time of harvesting and were assayed for acetate (Table 1). No acetate was excreted by aerobic cells. The amount excreted by anaerobic cells was of the order of 1.7 mol/mol of citrate utilized. This value was similar to that obtained when K. aerogenes was grown anaerobically on citrate (1, 8). When E. cloacae was grown under aerated conditions, the excretion of acetate fell to about 0.7 mol/mol of citrate utilized. This value was considerably lower than the value of 1.2 for K. aerogenes when it was grown under aerated conditions on Na+ citrate (8). Further evidence that citrate was metabolized by citrate lyase was provided by the finding that aerated cells excreted oxalacetate into the medium (Fig. 1). The amount excreted was small, reaching a concentration of slightly over 1 Mmol/ml of culture at the end of the growth period. A small amount of pyruvate (about 0.3 imol/ml of culture) was also detected. This concentration remained essentially constant throughout the growth period. No OAA or pyruvate was excreted by the anaerobic or aerobic cells. A search for other metabolites indicated that trace amounts of lactate and succinate but not malate were excreted by the cells, irrespective of the growth conditions. A
VOL. 124, 1975
CITRATE LYASE INDUCTION IN E. CLOACAE
1087
similar excretion of metabolites occurred when activity of OAA decarboxylase than that observed in aerobic cells, where the fermentation the organism was grown on Na+ citrate. pathway does not function, and (ii) the excretion of a small amount of OAA, which did not DISCUSSION occur with anaerobic or aerobic cultures. A The results of the growth studies clearly comparison of these results with those observed indicate that aerated growth of E. cloacae on with K. aerogenes, when grown under aerated K+ citrate was slower than aerobic growth, conditions in the presence of Na+, also points to presumably because of oxygen limitation. How- the fermentation pathway being inoperative in, ever, the rate of citrate utilization under E. cloacae. In the case of K. aerogenes, both aerated conditions was considerably greater enzymes of the fermentation pathway were than under aerobic conditions, despite the induced, enabling the organism to utilize both slower growth rate, and led to a much lower fermentation and the citric acid cycle for energy molar growth yield. The presence of Na+ in the production and thus achieving a higher molar medium did not affect the behavior of the growth yield and greater excretion of acetate organism in the same way as observed with K. than observed with E. cloacae (8, 9). The OAA produced by the citrate lyase activaerogenes, where both the growth rate and citrate utilization increased when Na+ was ity was probably metabolized by malate dehyadded to a K+ citrate culture growing under drogenase, which is present in high activity in anaerobic and aerobic cells (10), since no signifaerated conditions (8, 9). The rapid utilization of citrate under aerated icant quantities of OAA, or metabolites which conditions was paralleled by the induction of could be derived from OAA (malate, succinate, citrate lyase. Evidence that the enzyme partici- pyruvate, or lactate), were found in the spent pated in citrate catabolism was shown by the medium. excretion of acetate by the aerated cells but not ACKNOWLEDGMENTS by aerobic cells, which were devoid of citrate I thank Dorothy Jones, Microbial Systematics Unit, Unilyase activity. Citrate lyase was induced in E. of Leicester, England, for the provision of the culture. cloacae in both the presence and absence of versity This research was supported by a University of Sydney Na+, which contrasts with the behavior of K. research grant. aerogenes where the enzyme was induced in aerated cells only in the presence of Na+ (9). LITERATURE CITED If the acetate excreted by the aerated cells 1. Brewer, C. R., and C. H. Werkman. 1939. The anaeroresulted fom the operation of the fermentation bic dissimilation of citric acid by Aerobacter indologenes. Enzymologia 6:273-281. pathway, then the molar growth yield should T., R. Czok, W. Lamprecht, and E. Latzko. 1963. have been 26.6 g (dry weight) of cells per mol of 2. Bucher, Pyruvate, p. 253-262. In H. U. Bergmeyer (ed.), citrate utilized, basing the calculation on the Methods of enzymatic analysis. Academic Press Inc., amount of acetate excreted by anaerobic cells, New York. where the fermentation pathway alone was 3. Dagley, S., and E. A. Dawes. 1953. Dissimilation of citric acid by bacterial extracts. Nature (London) 172: operative (10). However, the value obtained for 345-346. the molar growth yield of the aerated cells was 4. Gornall, A. G., C. H. Bardawill, and M. M. David. 1949. only 10.6 g (dry weight) of cells per mol of Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177:751-766. citrate utilized, which corresponds closely to the H. J., and M. Reim. 1963. Oxaloacetate, p. value of 11.0 which would be obtained if the 5. Hohorst, 335-339. In H. U. Bergmeyer (ed.), Methods of enacetate resulted from the citrate lyase reaction zymatic analysis. Academic Press Inc., New York. alone, with the remaining citrate being metabo- 6. Kaufman, S. 1955. a-Ketoglutaric dehydrogenase system and phosphorylating enzyme from heart muscle, p. lized via the citric acid cycle. These results 714-722. In S. P. Colowick and N. 0. Kaplan (ed.), indicate that energy for the growth of the Methods in enzymology, vol 1. Academic Press Inc., organism under aerated conditions came mainly New York. from the metabolism of some 27% of the citrate 7. Lowenstein, J. M. 1969. Chemical methods for citrate and aconitate, p. 573-576. In J. M. Lowenstein (ed.), via the citric acid cycle. Therefore, the rapid Methods in enzymology, vol 13. Academic Press Inc., utilization of citrate, combined with the less New York. efficient growth, under aerated conditions is 8. O'Brien, R. W. 1975. Effect of aeration and sodium on the attributable to a large amount of the substrate metabolism of citrate by Klebsiella aerogenes. J. Bacteriol. 122:468-473. being diverted from the citric acid cycle by the R. W. 1975. Induction by sodium of the citrate action of citrate lyase. Further evidence that the 9. O'Brien, fermentation enzymes in Klebsiella aerogenes. FEBS fermentation pathway was largely inoperative Lett. 53:61-63. beyond the citrate lyase reaction was shown by 10. O'Brien, R. W., and J. Geisler. 1974. Citrate metabolism in Aerobacter cloacae. J. Bacteriol. 119:661-665. (i) the failure of aerated cells to induce a higher
1088
O'BRIEN
11. O'Brien, R. W., and J. G. Morris. 1971. Oxygen and the growth and metabolism of Clostridium acetobutylicum. J. Gen. Microbiol. 68:307-318. 12. O'Brien, R. W., and J. R. Stern. 1969. Requirement for
sodium in the anaerobic growth of Aerobacter 4. Bacteriol. 98:388-393. 13. Rose, I. A. 1955. Acetate kinase of bacteria (acetokinase), p. 591-595. In S. P. Colowick and N. 0. Kaplan (ed.), aerogenes.
J. BACTERIOL. Methods in enzymology, vol. 1. Academic Press Inc., New York. 14. Stern, J. R. 1967. Oxalacetate decarboxylase of Aerobacter aerogenes. I. Inhibition by avidin and requirement for sodium ion. Biochemistry 6:3545-3551. 15. Wilkerson, L. S., and R. G. Eagon. 1972. Transport of citric acid by Aerobacter aerogenes. Arch. Biochem. Biophys. 149:209-221.
