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

Transcription

1 JOURNAL OF BACTERIOLOGY, Aug. 1969, p 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 in Aerobacter aerogenes R. W. O'BRIEN AND JOSEPH R. STERN Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio Received for publication 24 March 1969 In contrast to the absolute Na+ requirement for anaerobic growth of Aerobacter aerogenes on citrate as sole carbon source, aerobic growth of this microorganism did not require the presence of Na+. However, Na+ (optimal concentration, 10 mm) did increase the maximal amount of aerobic growth by 60%, even though it did not change the rate of growth. This increase in growth was specifically affected by Na+, which could not be replaced by K+, NH+, Li+, Rb+, or Cs+. Enzyme profiles were determined in A. aerogenes cells grown aerobically on citrate in media of varying cationic composition. Cells grown in Na+-free medium possessed all the enzymes of the citric acid cycle including a-ketoglutarate dehydrogenase, which is repressed by anaerobic conditions of growth. The enzymes of the anaerobic citrate fermentation pathway, citritase and oxalacetate decarboxylase, were also present in these cells, but this pathway of citrate catabolism was effectively blocked by the absence of Na+, which is essential for the activation of the oxalacetate decarboxylase step. Thus, in Na+-free medium, aerobic citrate catabolism proceeded solely via the citric acid cycle. Addition of 10 mm Nat to the aerobic citrate medium resulted in the activation of oxalacetate decarboxylase and the repression of a-ketoglutarate dehydrogenase, thereby diverting citrate catabolism from the (aerobic) citric acid cycle mechanism to the fermentation mechanism characteristic of anaerobic growth. The further addition of 2% potassium acetate to the medium caused repression of citritase and derepression of a-ketoglutarate dehydrogenase, switching citrate catabolism back into the citric acid cycle. The pathway of the anaerobic fermentation of citrate by cell suspensions of Aerobacter indologenes (3) and by cell-free extracts of A. aerogenes (4) involves the cleavage of citrate to oxalacetate (OAA) and acetate followed by decarboxylation of OAA to pyruvate. Stern (7) showed that the enzymes concerned, citritase and OAA decarboxylase, were specifically induced by anaerobic growth on citrate and that OAA decarboxylase was a biotinoprotein which specifically required Na+ for activity. This observation prompted an examination of the role of cations in the growth of A. aerogenes. O'Brien and Stern (6) showed that Na+ was essential for anaerobic growth on citrate and that failure of A. aerogenes to utilize citrate anaerobically in the absence of Na+ was explained in enzymatic terms by the requirement of Na+ for activation of the OAA decarboxylase step of the fermentation pathway and by the absence of an alternate pathway of citrate catabolism. Thus, under anaerobic conditions, all the enzymes of the citric acid cycle except a-ketoglutarate dehydrogenase were present in Na+sufficient cells and the role of the incomplete cycle was restricted to the biosynthesis of glutamate and succinate. Stern (7) also noted that citritase and OAA decarboxylase, as well as many enzymes of the citric acid cycle, were also induced during aerobic growth of A. aerogenes on citrate and suggested that citrate catabolism under aerobic conditions might proceed largely via the fermentation pathway. This paper describes the role of Na+ in determining alternate pathways of aerobic citrate catabolism. Sodium, though not required for growth, increased the maximal amount of growth at low concentrations and inhibited it at higher concentrations. Its presence activated the citrate fermentation pathway and repressed citrate oxidation via the citric acid cycle. MATERIALS AND METHODS A. aerogenes NCTC 418 was used in all experiments. The methods and basal media employed for studying the effect of Na+ and K+ on growth were those described previously for anaerobic growth studies (6), except that the sodium sulfate concentration of the 389

2 390 O'BRIEN AND STERN J. BACTERIOL. basal K+-free medium was decreased to 0.7 g/liter. Cultures were grown in nephelometer flasks and aerated by shaking on a gyratory shaker (New Brunswick Scientific Co., New Brunswick, N.J.) at 37 C. Growth was determined by measuring the turbidity of the culture in a Klett-Summerson colorimeter with a no. 54 filter. A Klett reading of 100 corresponds to 500,ug of cells (dry wt) per ml. For enzymatic studies, the cells were grown in 4- liter batches on the Na+-free basal medium containing the levels of sodium and potassium indicated in Table 1. They were harvested in a refrigerated Sharples Ultracentrifuge. The unwashed cells were stored at -20 C. The preparation of cell-free extracts, the assay of enzymes, and the determination of protein, citrate, K+, and Na+ were carried out as previously described (6). Phosphoenolpyruvate (PEP) synthase activity was measured at ph 8.0 and 30 C as the adenosine triphosphate and Mg++-dependent synthesis of PEP from pyruvate. Pyruvate kinase was determined at ph 8.0 and 30 C by measuring pyruvate formation from PEP and adenosine diphosphate. RESULTS Growth studies. The effect of Na+ (added as sodium sulfate) on the aerobic and anaerobic growth of A. aerogenes on citrate is shown in Fig. 1. Whereas anaerobic growth required Na+, as previously reported (6), aerobic growth occurred in the absence of Na+. However, as the concentration of Na+ in the medium was increased, the maximal amount of growth increased up to a concentration of 7 to 10 mm Na+. Concentrations of Na+ above 10 mm caused a decrease in maximal aerobic growth, and at the highest concentration (200 mm) growth was little more than that observed in the absence of added Na+. The amount of aerobic growth, both in the absence of Na+ and in the presence of excess Na+, was slightly greater than the maximal anaerobic growth. O'Brien and Stern (6) showed that under anaerobic conditions Na+ increased both the growth rate and the maximal amount of growth on citrate. The doubling time of A. aerogenes cells grown aerobically on citrate was determined in several experiments to be 61 min in the absence of Na+ and 62 min in 10 mm Na+ (cf. Fig. 2). These times are not significantly different from each other or from the doubling time of 64 min reported (6) for cells grown anaerobically on citrate in the presence of an optimal concentration of Na+ (100 mm). Thus, under aerobic conditions Na+ specifically increased the amount of growth (cell yield) without affecting the rate of growth. The increase in maximal aerobic growth was specifically an action of Na+ and was independent of the anion, since 10 mm Na+ added as the citrate, chloride, or sulfate gave the same increase in growth relative to that observed in Na+-free medium (Fig. 2). Li+, Rb+, or Cs+ at a concentration of 10 mm failed to increase total growth when added to Na+-free medium and thus were unable to replace Na+. Indeed, Li+ had a small inhibitory effect on growth, whereas Cs+ exerted some inhibition after 7 hr of growth (Fig. 2); nor could Na+ be replaced by K+ or NH4+, which were constituents of the Na+-free medium. Also, ao 250 A_O AEROBIC 200 A. aerogenes No2SO ANAEROBIC C / FIG. 1. Effect ofsodium concentration on the aerobic and anaerobic growth of A. Aerogenes on citrate. Growth was measured after 16 hr at 37 C. Sodium was added as sodium sulfate to basal medium which contained potassium and ammonium ions Ir 200 -i 150 y A. aerogenes ) Na2SO4 A NoCl A No citrate /Z x RbCl a CsCI olico o No Noe *N a 0 /0 Rbll~ TIME (hours) FIG. 2. Effect of different sodium salts and monovalent cations on the aerobic growth of A. aerogenes on citrate. All cations were added at 10 mm concentration to basal medium. A semilogarithmic plot of these data showed no significant difference in the doubling times with or without Na+ during exponential growth. This was verified in several such experiments. A e :e;li 0

3 VOL. 99, 1969 SODIUM REGULATION OF AEROBIC CITRATE CATABOLISM mm Na+ could not replace a K+ requirement for aerobic growth (Fig. 3). Although slight growth occurred in the absence of added K+, growth was proportional to the concentration of added K+ and reached a maximum at 0.15 mm K+ with the Na+ concentration at 10 mm (Fig. 3). O'Brien and Stern (6) have shown that K+ was also required for anaerobic growth of A. aerogenes on citrate, the optimal concentration also being 0.15 mm. Enzyme profiles in cell extracts. In an attempt to explain the dual actions of Na+ in both increasing and inhibiting maximal aerobic growth, a comparative study was made of activities of the enzymes of the citrate fermentation pathway and of the citric acid cycle in extracts of cells grown in media containing different concentrations of Na+ and K+. Cells were grown in media containing no Na+ (medium A), in a Na+ concentration causing the maximal increase of aerobic growth (medium B), and in a high Na+ concentration which suppressed this increase aerobically, although causing maximal growth under anaerobic conditions (medium C). Cells were also grown in a standard medium (D) routinely used for previous investigations of the metabolism of A. aerogenes (5, 7). This medium had a high Na+ concentration but a much lower content of K+, although far above the optimal K+ requirement for growth. The ionic composition and growth parameters of each medium are summarized in Table 1. In the absence of Na+, the rate of utilization of citrate was slower than in its presence. After 24 hr of growth, 300 AA aerogenes 250 / Z 200 ; w 150 5Y j, - K4 ADDED (10-5 M ) FIG. 3. Effect of potassium on the aerobic growth of A. aerogenes on citrate in the presence of 10 mm Na+. Growth was measured after 16 hr at 37 C. TABLE 1. Ionic composition of media and some growth parameters E~ ~ y i IC }W r, A , B , C , Dc d 20.0 a Expressed as micrograms (dry wt) per milliliter of medium. b Expressed as micrograms (dry wt) of cells per micromole of citrate utilized. c Medium used by Stern (7). d Complete citrate utilization only 80% of the citrate was utilized in the Na+free medium, whereas complete utilization occurred within 16 to 22 hr in media B, C, and D. However, the molar growth yield was greatest in the absence of Na+. The specific activities of various enzymes extracted from cells grown in each medium are hsted in Table 2. The following significant differences in the enzyme profiles were evident. In cells grown on the Na+-free medium A, OAA decarboxylase activity was essentially absent unless Na+ was added to the assay system. However, with cells grown in media containing Na+, the amount of Na+ adhering to the unwashed cells and carried over, after disruption, into the assay system was sufficient to cause partial activation of the decarboxylase in the case of cells from medium B and complete activation in the case of cells from media C and D. The Na+ concentration of medium B, 12 mm, was exactly that required to saturate OAA decarboxylase in vitro (7), and from the enzyme activity without added Na+ one can calculate from the Na+ dependency curve (7) that 0.6 mm Na+ was carried over from medium B to the assay system. The OAA decarboxylase in cells from the Na+-free medium A and the optimal Na+ medium B had the same properties as the decarboxylase described by Stern (7) in cells grown in medium D. In each case, the enzyme was activated by Na+ and inhibited about 90% by avidin (0.5 unit). The OAA decarboxylase in dialyzed extracts of cells from medium C exhibited these same properties. These results showed that the induction of OAA decarboxylase by citrate was independent of the presence of its activator, sodium ion. Citritase was present in all types of cell, although its activity was highest in the high Na+-high K+

4 392 O'BRIEN AND STERN J. BACTERIOL. TABLE 2. Effects of cations on enzyme profiles of A. aerogenes grown on citrate Enzyme Mediaa Citritase OAA decarboxylase (no Na+)... OAA decarboxylase (+ 20 mm Na+) Citrate synthetase Aconitase Isocitrate dehydrogenase a-ketoglutarate dehydrogenaseb Succinate dehydrogenase Fumarase Malate dehydrogenase Glutamate dehydrogenase (triphosphopyridine nucleotide-specific)... Glutamate-OAA transaminase... Lactate dehydrogenase... L-Malic-triphosphopyridine nucleotide enzyme NADH2-oxidase Pyruvate kinase PEP synthase A B c a Values are expressed as micromoles of substrate transformed per minute per milligram of protein. b For media A, B, and C, the specific activity of a-ketoglutarate dehydrogenase was less than 0.002, the lowest detectable rate. medium C. Its activity in vitro was not affected by Na+. Cells grown in the absence of Na+ possessed a-ketoglutarate dehydrogenase activity, whereas cells grown in the presence of Na+ were apparently devoid of this enzyme. The a-ketoglutarate dehydrogenase required Mg++, coenzyme A, and nicotinamide adenine dinucleotide for its activity and was not inhibited by 20 mm Na+ under the conditions of assay. Thus, the presence of Na+ in the medium repressed the formation of this enzyme. Repression of ca-ketoglutarate dehydrogenase was also observed during anaerobic growth of A. aerogenes on citrate (6). All the other enzymes of the citric acid cycle were detected in all of the cells tested. A comparison of enzyme activities in extracts from cells grown in Na+-free medium versus optimal Na+ medium indicated that in the absence of Na+ the activities of citrate synthetase, isocitrate, malate, lactate, and glutamate dehydrogenases had increased, succinate dehydrogenase activity had decreased, and the other enzymes were little changed. However, with cells from the high Na+ medium C, all citric acid cycle enzymes had lower activities than those observed in cells from the optimal Na+ medium B. An inverse relationship was apparent in the specific activities of citritase and citrate synthetase. Under growth conditions where the activity of one was increased, the activity of the other was decreased, and vice versa. This inverse relationship was also observed during anaerobic growth on citrate (6). In cells grown on medium D (high Na+, low K+), the activities of citritase, aconitase, and fumarase fell considerably compared to the activities of cells grown on medium C (high Na+, high K+). Decreases in these same enzymes had also occurred during anaerobic growth on citrate when the K+ level of high Na+ medium was dropped from 0.26 to M (6). These K+ concentrations are both well above the optimal concentration required for cell division, and they imply an additional effect of high K+ concentrations on the synthesis of these enzymes. In cells from media B and C, malate dehydrogenase activity was severely repressed. Glutamate dehydrogenase activity (triphosphopyridine nucleotide-specific) was extremely low in all cells and glutamate-oxalacetate transaminase was quite high in all the cells. Transaminase, rather than the dehydrogenase, would appear to be involved in glutamate biosynthesis. Glutamate-pyruvate transaminase was not detected in any of the cells. Acetate induction of a-ketoglutarate dehydrogenase. Amarasingham and Davis (1) showed that aerobic growth of Escherichia coli on acetate or glutamate resulted in the induction of a-ketoglutarate dehydrogenase. When potassium acetate was added to citrate medium containing 10 mm Na+, concentrations of acetate greater than 0.5 % caused inhibition of the aerobic growth of A. aerogenes (Fig. 4). Acetate itself was poorly utilized as a carbon source for growth by these untrained cells, as first noted by Baskett and Hinshelwood (2). Enzyme profiles of cells grown in the presence of various concentrations of acetate added to sodium citrate medium B were examined (Table 3). The activity of a-ketoglutarate dehydrogenase in cells grown in the presence of 0.5% acetate was greater than that in cells grown in Na+-free citrate medium. However, increasing the concentration of acetate in the medium resulted in decreased a-ketoglutarate dehydrogenase activity. When the cells were grown aerobically on 0.5% potassium L-glutamate as carbon source with 10 mm Na+ in the medium, the specific activity of a-ketoglutarate dehydrogenase was Thus,

5 VOL. 99, 1969 SODIUM REGULATION OF AEROBIC CITRATE CATABOLISM 393 z [ ct w cr 2001 w -J y 250[ 150I 100[ A. aerogenes (with 0.01 M Na+) o K citrate *,. +0.5% K Ac A, % K Ac A % K Ac o 1% K Ac Enzyme Citritase OAA decarboxylasee. 0.4 a-ketoglutarate dehydrogenase Malate dehydrogenase L-Malic-triphosphopyridine nucleotide enzyme their activities in medium B alone. The activities of other citric acid cycle enzymes were essentially unchanged by acetate addition from those in medium B and are not recorded. DISCUSSION / 7/ These experiments demonstrated that A. aerogenes can utilize citrate for aerobic growth in the / / absence of Na+, in striking contrast to the Na+,// requirement observed when it was grown on / ^/citrate under anaerobic conditions (6).However, added Na+ (7 to 10 mm) did increase the maximal amount of aerobic growth about 60% without / changing the rate of growth. Increasing the concentration of Na+ above 10 mm gradually suppressed this action. At 200 mm Na+, a concentration that results in maximal anaerobic growth, the 50[ increase of maximal aerobic growth was only 9% As with anaerobic growth, the action of Na+ was TIME (hours) highly specific, and Na+ could not be replaced FIG. 4. Effect of potassium acetal bv K+. NH4+, Li+, Rb+, and Cs+. growth of A. aerogenes on citrate in the Determination of presence of enzyme profiles showed that 10 mm Na+. cells grown aerobically on citrate in Na+-free medium possessed all the enzymes of the citric TABLE 3. Enzyme activities of celhi extracts of A. acid cycle, thereby providing an alternate pathr aerogenes grown in presence of Na+, citrate, way of citrate catabolism. Such an alternate route anid acetatea was essential for aerobic growth since, even though the enzymes citritase and OAA decardditions to citrate boxylase were also present, this fermentation medium pathway of citrate catabolism was effectively 7o 1.0% 2.0% blocked by the absence of Na+ required to acti- 0.5~ Aceta Lte Acetate Acetate vate the decarboxylase. This explains the lack of a Na+ requirement for growth under aerobic con- ) ditions in which oxygen, by causing induction of a-ketoglutarate dehydrogenase, permits the operation of the citric acid cycle. Repression of the dehydrogenase during anaerobic growth (6).171 eliminates this alternate pathway of citrate catabolism so that anaerobic then becomes dependent on the Na+-requiring growth citrate a Cells were harvested after 16 hr. fermentation pathway. b Values are expressed as miciromole of sub- The addition of Na+ to the medium caused two strate transformed per minute p er milligram of important changes in the enzyme proffle of protein. aerobic cells. First, it activated the OAA dec In the presence of 20 mm Na- +- carboxylase and, thus, the citrate fermentation pathway. Second, it resulted in the repression of Na+ did not prevent induction ( Df the dehydro- ca-ketoglutarate dehydrogenase. Thus, the citric genase by supplemental acetate o] r during growth acid cycle was blocked and relegated to a biosynthetic role, leaving the fermentation pathway on L-glutamate. As the concentration of acetatte in the sodium as the sole route of citrate catabolism. In effect, citrate medium was increased, the activity of the addition of Na+ converted aerobic citrate citritase fell markedly. OAA decarboxylase ac- catabolism from an aerobic mechanism, the citric tivity, although unaffected by 0. 5 or 1.0% ace- acid cycle, to the anaerobic fermentation mecha- of 2.0% nism. The latter mechanism utilized citrate more tate, was decreased in the presence acetate. Increasing the concentration of added rapidly than did the oxidative citric acid cycle acetate resulted in increased levels of malate (Table 1) and increased the maximal amount of dehydrogenase and L-malic enzyrne compared to growth (Fig. 1, 2).

6 394 O'BRIEN AND STERN J. BACTERIOL. Sodium repression of a-ketoglutarate dehydrogenase explains the earlier observation of Dagley and Dawes (5) that addition of citrate to resting cells of A. aerogenes which had been grown aerobically on sodium citrate medium D resulted in a large accumulation of a-ketoglutarate. It is noteworthy that the maximal increase in aerobic growth occurred at a Na+ concentration (about 10 mm) just sufficient to cause maximal stimulation of OAA decarboxylase in vitro (7), whereas maximal anaerobic growth required 150 mm Na+. This suggested that either access or transport of Na+ to the decarboxylase, which is bound to the cytoplasmic membrane (G. M. Frost and J. R. Stern, Fed. Proc., p. 586, 1968), was more efficient under aerobic conditions. The mechanism of Na+ repression of a-ketoglutarate dehydrogenase during aerobic growth on citrate appears to be indirect and a consequence of Na+ activating the citrate fermentation pathway, which may generate a repressor molecule. Thus, Na+ did not inhibit the dehydrogenase nor did it prevent its induction during growth on L-glutamate. Moreover, Na+ repression of a-ketoglutarate dehydrogenase was reversed by addition of 0.5% potassium acetate to the citrate medium, so that citrate catabolism was able to proceed via the oxidative as well as the fermentation pathway. This metabolic change did not affect the growth rate (Fig. 4). However, when 2% acetate was added to the medium, citritase was severely repressed, which effectively blocked the fermentation pathway and restricted citrate catabolism to the citric acid cycle, duplicating the metabolic pattern already seen in Na+free medium. Significantly, growth curves determined in Na+-free medium and in Na+ medium supplemented with 2% acetate, conditions where citrate catabolism is essentially limited to the citric acid cycle, were identical. One may conclude that during aerobic growth in low Na+ medium B, citrate fermentation caused little or no acetate to accumulate in the cell; otherwise, a-ketoglutarate dehydrogenase would have been induced. Under anaerobic conditions complete fermentation of the citrate (1.2%o in the medium) would result in the accumulation of 0.55% acetate (3). Failure to accumulate acetate may account for the very low activities of malic dehydrogenase in cells grown in media B and C, since supplemental acetate induced malic dehydrogenase and also L-malic enzyme. One consequence of the utilization of citrate as sole carbon source for growth is that its catabolism via the citric acid cycle or the fermentation pathway presents the cell with the problems of removing excess C-4 compounds (i.e., L- malate and OAA) and of generating PEP for the biosynthesis of sugar phosphates and polysaccharide. L-Malic enzyme and OAA decarboxylase provide the means of converting C-4 compounds to pyruvate, and the extracts were found to contain PEP synthase as well as pyruvate kinase. ACKNOWLEDGMENTS We thank R. E. Eckel for performing the sodium and potassium analyses and Harriet Richardson for excellent technical assistance. This investigation was supported by grant GB-4716 from the National Science Foundation. LITERATURE CITED 1. Amarasingham, C. J., and B. D. Davis Regulation of a-ketoglutarate dehydrogenase formation in Escherichia coil. J. Biol. Chem. 240: Baskett, A. C., and C. Hinshelwood The utilization of carbon sources by Bact. lactic aerogenes. I. General survey. Proc. Roy. Soc. Ser. B. Biol. Sci. 136: Brewer, C. R., and C. H. Werkman The anaerobic dissimilation of citric acid by Aerobacter indologenes. Enzymol. Acta Biocatal. 6: Dagley, S., and E. A. Dawes Dissimilation of citric acid by bacterial extract. Nature 172: Dagley, S., and E. A. Dawes Citric acid metabolism of Aerobacter aerogenes. J. Bacteriol. 66: O'Brien, R. W., and J. R. Stern Requirement for sodium in the anaerobic growth of Aerobacter aerogenes on citrate. J. Bacteriol. 98: Stern, J. R Oxalacetate decarboxylase of Aerobacter aerogenes. I. Inhibition by avidin and requirement for sodium ion. Biochemistry 6: