Enzymatic Analysis of the Requirement for Sodium in Aerobic Growth of Salmonella typhimurium on Citrate

Transcription

1 JOURNAL OF BACTERIOLOGY, Aug. 1969, p American Society for Microbiology Vol. 99, No. 2 Printed in U.S.A. Enzymatic Analysis of the Requirement for Sodium in Aerobic Growth of Salmonella typhimurium on Citrate R. W. O'BRIEN, G. M. FROST, AND JOSEPH R. STERN Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio Received for publication 24 March 1969 Na+ was required for the aerobic growth of Salmonella typhimurium on citrate, but not on L-malate, glucose, or glycerol. The maximal growth rate and the maximal total growth occurred with 6 to 7 mm Na+. Nat could not be replaced by K+, NH4+, Li+, Rb+, or Cs+. Sonically treated extracts of citrate-grown cells contained the enzymes of the citrate fermentation pathway (citritase and oxalacetate decarboxylase) and all of the enzymes of the citric acid cycle. Thus, two separate routes of citrate catabolism appeared to be operational in the cells. Two discrete oxalacetate (OAA) decarboxylases were also demonstrated. One was of the "classic" type, being activated by Mn++ and inhibited by ethylenediaminetetracetate (EDTA). It was present in the cell sap. The second decarboxylase closely resembled the Na+activated OAA decarboxylase of citrate-grown Aerobacter aerogenes, whose growth also requires, or is increased, by Na+. This decarboxylase was EDTA-insensitive, specifically activated by Nat and inhibited by avidin, and it had a high affinity for OAA. It was induced by growth on citrate, but not L-malate or glycerol. It is suggested that the Nat requirement for growth reflects the need to activate this OAA decarboxylase as a component of the citrate fermentation pathway and that citrate catabolism via the citric acid cycle, which should be independent of Na+, is somehow dependent upon the activity of the Nat-activated enzyme. A study of the growth of Aerobacter aerogenes this organism was undertaken. This paper on citrate showed that under anaerobic conditions Na+ was specifically required for growth murium on citrate, unlike that of A. aerogenes, demonstrates that aerobic growth of S. typhi- (5). This was explained at the enzymatic level by specifically requires Na+ and that cells so grown the Na+ activation of the oxalacetate (OAA) possess all the enzymes of both the citric acid decarboxylase step of the citrate fermentation cycle and the citrate fermentation pathway, pathway initiated by the cleavage of citrate to including the Na+-activated OAA decarboxylase OAA and acetate by citritase. Under anaerobic first described in A. aerogenes by Stern (9). conditions (5), citrate catabolism via the citric acid cycle was blocked as a result of the repression MATERIALS AND METHODS of a-ketoglutarate dehydrogenase by anaerobiosis. Aerobic growth on citrate did not require Culture conditions and growth studies. S. typhimurium SL934, a wild-type prototrophic strain, was Na+, and in its absence citrate catabolism proceeded via the citric acid cycle which became kindly provided by B.A.D. Stocker. Growth studies were carried out on a citrate-salts medium (ph 7.0), functional as a consequence of aerobic induction as previously described (6). Cultures were grown in of a-ketoglutarate dehydrogenase (6). However, nephelometer flasks and aerated by shaking on a gyratory shaker (New Brunswick Scientific Co., New the total amount of aerobic growth (cell yield) was increased by the presence of Na+ which Brunswick, N.J.) at 37 C. Growth was determined by caused repression of ca-ketoglutarate dehydrogenase and activation of the OAA decarboxylase, Summerson colorimeter with no. 54 filter. A Klett measuring the turbidity of the culture in a Klettthereby switching aerobic citrate catabolism from reading of 100 corresponds to 390 pg of cells (dry the citric acid cycle to the fermentation pathway wt) per ml. For enzyme studies, cells were grown in 4-liter (6) batches of citrate medium containing M Na+. Ṡince Salmonella typhimurium has long been After shaking for 18 hr, the cells were harvested in a known to grow aerobically on citrate as sole refrigerated Sharples ultracentrifuge and were stored carbon source, a study of citrate catabolism in at -20 C. 395

2 396 O'BRIEN, FROST, AND STERN J. BACTERIOL. Preparation of cell extracts. Cell-free extracts were prepared by suspending frozen cells in three to four volumes of 0.05 M tris(hydroxymethyl)aminomethane (Tris)-chloride or 0.05 M potassium phosphate buffer (ph 7.4) and by disrupting the suspension for 2 min at 2 to 8 C with a Sonifier (Branson Instruments, Inc., Stamford, Conn.). The suspension was centrifuged for 20 min at 26,000 X g, and the supernatant fluid, which contained 15 to 20 mg of protein per ml, was used for all enzyme assays except succinate dehydrogenase. The 26,000 X g supernatant fluid was further centrifuged for 1 hr at 105,000 X g, and the pellet obtained was resuspended in 0.05 M Tris-chloride buffer (ph 7.4). This suspension was used for estimating succinate dehydrogenase activity. The pellet from the 26,000 X g centrifugation was washed three times with 4.0 ml of 0.05 M Tris-chloride buffer (ph 7.0) and was finally resuspended in 2.0 ml of the same buffer. This suspension of cell particles was assayed for OAA decarboxylase activity. Enzyme assays. All enzyme activities were measured by the methods described by O'Brien and Stern (5), except for OAA decarboxylase, which was assayed in Tris-chloride buffer (ph 7.0) according to the method of Stern (9). To correct for any contribution of added components (e.g., metals, cofactors) to the nonenzymic decarboxylation of OAA, a control without enzyme was always included. Phosphoenolpyruvate (PEP) synthase activity was measured at ph 8.0 and 30 C by following the adenosine triphosphate- and Mg++-dependent synthesis of PEP from pyruvate and adenosine triphosphate. Pyruvate kinase was determined at ph 8.0 and 30 C by measuring pyruvate formation from PEP and adenosine diphosphate. Other assays. The protein content of cell-free extracts was determined by the biuret method (3) and that of the cell particle preparations by the Folin method (4). Sodium and potassium analyses were done with an Internal Standard Flame Photometer (Baird-Atomic, Inc., Cambridge, Mass.). Residual citrate in the spent culture medium was determined by the method of Stern (8). RESULTS Growth experiments. The aerobic growth of S. typhimurium on citrate required the addition of Na+ because insignificant growth occurred in its absence (Fig. 1). The optimal Na+ concentration (7 to 10 mm) increased total growth about 35- fold. Na+ concentrations above 10 mm became inhibitory, and at 200 mm Na+ growth was decreased by 30%. Under anaerobic conditions, moderate growth on citrate was observed only after a lag period of 20 to 40 hr with untrained cells, and this growth was almost completely dependent upon added Na+. Na+ not only increased total aerobic growth but also increased the growth rate (Table 1). The growth rate (measured as doubling time) increased with increasing Na+ concentration and was maximum at 6 mm Na+. In this experiment, minimal growth occurred in the absence of added Na+, possibly as a result of Na+ adhering to cells of the inoculum. It was observed that growth was arithmetic in the absence of added Na+ and in presence of 0.5 and 1.0 mm Na+. At higher Na+ concentrations, growth was exponential. Equimolar Na+ added as citrate or chloride salts gave the same stimulation of growth on citrate as did sodium sulfate (Fig. 2), showing that z ci J ' '0.200 Na' ADDED (M) FIG. 1. Effect of sodium (added as sulfate) on the aerobic growth of S. typhimurium on citrate. Growing time, 16 hr. TABLE 1. Effect of Na+ concentration on rate and amount of growth NaCI addeda Doubling time After 12 hr Klett reading After 22 hrb mm min None 5AIC 43d c c a Inoculum was grown in sodium citrate basal medium, harvested in mid-log phase, washed twice in potassium citrate basal medium, and suspended in the latter. A 5-ml amount of suspension was added to 45 ml of potassium citrate medium containing enough NaCl to give the final Nat concentration indicated. Growth was measured at hourly intervals up to 12 hr and from 22 to 24 hr. b Maximal growth (turbidity) had occurred in all cases before the 22 hr reading, probably after 17 to 18 hrs. c Growth was arithmetic at these Na+ concentrations and exponential at higher Na+ concentrations. d Klett reading was 19 after inoculation.

3 VOL. 99, 1969 SODIUM IN GROWTH AND CITRATE CATABOLISM 397 the growth stimulation was due solely to the cation. This effect was specific for Na+, which could not be replaced by Li+, Rb+, or Cs+ (Fig. 2). Also, neither K+ nor NH4+, which were constituents of the basal medium, could replace Na+. Growth of S. typhimurium on glucose, glycerol, or L-malate did not require Na+, nor did addition of Na+ affect the growth rate. Enzyme profile of cell extracts. In A. aerogenes grown aerobically on citrate, O'Brien and Stern (6) showed that two pathways of citrate catabolism occur, the fermentation pathway and the citric acid cycle, and that Na+ could activate the first and repress the second. To examine potential pathways of citrate catabolism in S. typhimurium, cells were grown on citrate medium of the ionic concentration shown in Table 2, which [ z c] 200 w L50 w -J Y Ll,,KD,OrL;Ss TIME (hours) FIG. 2. Effect of different sodium salts and lithium, rubidium, and cesium chlorides on the aerobic growth of S. typhimurium on citrate. TABLE 2. S. typhimurium _ Nn-t A *@F1, ''vw2a tna citrate / AP aiv A/. Ionic composition of citrate medium and some growth parameters Determination Value Sodium concentration (M) Potassium concentration (M) Growing time (hr) Cell densitya... 1,350 Final ph of medium Citrate utilized (umole/ml).62.5b Molar growth yieldc a Expressed as micrograms (dry wt) per milliliter of medium. b Represents 100% utilization. c Expressed as micrograms (dry wt) of cells per micromole of citrate utilized. 0 v4 I; Di r. TABLE 3. Enzyme profile of S. typhimurium grown on citrate Enzyme Specific ~~~~~activitya Citritase OAA decarboxylase (no Nat) OAA decarboxylase (+ 20 mm... Na+) Citrate synthase Aconitase Isocitrate dehydrogenase a-ketoglutarate dehydrogenase Succinate dehydrogenase Fumarase Malate dehydrogenase Glutamate dehydrogenase (triphosphopyridine nucleotide-specific) Glutamate-OAA transaminase Lactate dehydrogenase L-Malic-triphosphopyridine nucleotide enzyme Reduced diphosphopyridine nucleotide oxidase Pyruvate kinase PEP synthase a Expressed as micromoles of substrate transformed per minute per milligram of protein. also lists several parameters of cell growth. The ph of the medium fell to 6.4 during aerobic growth. This contrasts with A. aerogenes in which the medium became alkaline (ph 7.9) under aerobic conditions of growth and acid (ph 6.5) only after anaerobic growth on citrate. The molar growth yield (21.6) was greater than the value 16.0 determined for A. aerogenes grown under similar conditions (6). Cells were extracted and assayed for the enzymes of the two pathways of citrate catabolism (Table 3). Both enzymes of the citrate fermentation pathway, citritase and OAA decarboxylase, and all of the enzymes of the citric acid cycle were present in significant amounts. The activity of glutamate dehydrogenase, which was specific for reduced nicotinamide adenine dinucleotide phosphate, was low, but that of glutamate-oaa transaminase was high. Both pyruvate kinase and PEP synthase were present. Glutamate-pyruvate transaminase could not be detected. Studies of OAA-decarboxylase. In A. aerogenes, the Na+ requirement for anaerobic growth on citrate (5) and the Na+ stimulation of total aerobic growth on citrate (6) could both be explained by the Na+ requirement of its OAA decarboxylase, which is a biotinoprotein (9). The observation that Na+ also stimulated the OAA decarboxylase activity of extracts of S. typhimurium grown on citrate (Table 3) prompted an investigation of this activity. The endogenous OAA de-

4 398 O'BRIEN, FROST, AND STERN J. BACTERIOL. carboxylase activity of the 26,000 X g supernatant fraction was inhibited 70% by 10 mm ethylenediaminetetraacetate (EDTA) and was stimulated 47% by 12 mm Na+ and 26% by 5 Mn++. Mg++ was inhibitory. The absolute increase in specific activity was essentially the same (0.27 to 0.34) when Na+ (12 mm) was added to the extract either alone or in the presence of EDTA, Mn++, or Mg++. The increases in decarboxylase activity due to Mn++ and Na+ were additive. These observations indicated that two separate OAA decarboxylases were present in the extract, one activated by Mn++ and the other by Na+. Optimal activity of the Mn++-activated enzyme occurred with 0.5 mm Mn++ (Table 5). Mn++ concentrations above 1 mm were inhibitory. Mg++ did not activate this enzyme, but it inhibited it at concentrations of 5 mm and above. The effect of Na+ concentration on the activity of the Na+-activated enzyme is shown in Fig. 3. As little as 1 mm added Na+ stimulated the decarboxylase, and 12 mm Na+ gave maximal activation. A Lineweaver-Burk analysis of the data in which the reciprocal of the corrected rate (observed rate less the residual rate in the absence of added Na+) of decarboxylase activity was plotted against the reciprocal of the Na+ concentration yielded a straight line. The calculated Km for added Na+ was 1 mm, the same value as that determined for the A. aerogenes OAA decarboxylase (9). The effect of avidin on OAA decarboxylase activity in extracts prepared either in Tris-chloride or phosphate buffer was examined (Table 6). Endogeneous OAA decarboxylase activity was less when measured in phosphate buffer than TABLE 4. Effect of Na+, Mg+, Mn++, and EDTA on OAA decarboxylase activity Addition Specific activity' Noneb EDTA Na Nat + EDTA Mg Mg+++ Na Mn-F Mn++ + Na a Expressed as micromole per minute per milligram of protein. b Reaction mixture contained: Tris-chloride buffer (ph 7.0), 100,umoles; potassium oxalacetate, 10,umoles; and cell extract, 3.7 mg of protein. EDTA (10 mm), Na2SO4 (6 mm), MgCl2 (5 mm), and MnC12 (5 mm) were added as indicated. Final volume was 1.0 ml. Incubation time was 10 min at 30 C. TABLE 5. Effect of varying concentrations of Mn++ and Mg++ on the activity of OAA decarboxylasea Concn of added cation Manganese Specific activity Magnesium None X X X X 10-s X 10-i X X X aexperimental conditions are the same as those described in Table 4. Protein content of the test system was 4.2 mg g F_I 0 _ 40 -Km = 0.97 X 10-3M < (-) 0.020IC Na ADDED (mm) FIG. 3. Effect of Na+ concentration on OAA decarboxylase activity. Inset: Lineweaver-Burk plot of the data. Rates have been corrected for endogenous rate in absence of added Na+. when measured in Tris-chloride buffer. In addition, the degree of Na+ stimulation was greater in phosphate buffer than in Tris-chloride, suggesting that phosphate inhibited the Mn++-activated decarboxylase by binding endogenous divalent cation. Avidin (0.6 unit) had no effect on endogenous decarboxylase activity regardless of the buffer used for cell extraction or incubation. When Na+ was added to the assay system, avidin inhhbition of the Na+-stimulated decarboxylase activity was observed only in the presence of

5 VOL. 99, 1969 SODIUM IN GROWTH AND CITRATE CATABOLISM phosphate buffer (Table 6). Pretreatment of avidin with excess biotin prevented the inhibition, showing that the latter was caused by avidin and not by impurities. EDTA caused a large inhibition of decarboxylase activity in all three types of extract. In the presence of EDTA the residual decarboxylase activity which is presumably due to the Na+-activated enzyme, was now sensitive to avidin even in Tris-chloride buffer. This suggested that an endogenous metal which reacted with phosphate or EDTA was preventing the binding of avidin to the Na+-dependent decarboxylase in extracts prepared in Tris-chloride buffer. The washed 26,000 X g particle fraction was devoid of the Mn++-activated decarboxylase (Table 7). It contained the Na+-activated de- TABLE 6. Effect of avidin on OAA decarboxylase activitya Additionb Pyruvatec Extract A Extract B Extract C None Avidin Avidin + biotin Nat Nat + avidin Nat + avidin + biotin EDTA... O Avidin + EDTA a Experimental conditions were the same as those described in Table 4. bavidin (0.6 unit), biotin (10,ug), Na2SO4 (6 mm), and EDTA (10 mm) were added as indicated. c Expressed as micromole per 10 min per milligram. Extract A was prepared and incubated in 0.05 M Tris-chloride (ph 7.0). Extract B was prepared in 0.05 M Tris-chloride buffer (ph 7.0) and incubated in potassium phosphate buffer (ph 7.0). Extract C was prepared and incubated in 0.05 M potassium phosphate buffer (ph 7.0). TABLE 7. Effect of Na+, avidin, and Mn+ on OAA decarboxylase activily of washed 26,000 X g particle fractiona Additionb Specific activity None Na Nat + avidin Nat + avidin + biotin Mn a Experimental conditions were the same as those described in Table 4. Protein content of the reaction system was 3.5 mg. bna2so4 (6 mm), avidin (0.6 unit), biotin (10,ug), and MnCl2 (1 mm) were added as indicated. 399 carboxylase which was 86% inhibited by 0.6 unit of avidin even though the particles had been prepared and incubated in Tris-chloride. Presumably, the factor that prevented avidin inhibition in the supernatant fraction was absent from the particle or had been removed by the washing. Again, pretreatment of the avidin with biotin abolished the inhibition. The Mn++-activated decarboxylase remained in the supernatant fluid after centrifugation of the 26,000 X g supernatant fluid at 150,000 X g for 2 hr, whereas the Na+-activated enzyme was found in both the supernatant and pellet fractions. When S. typhimurium was grown on L-malate or glycerol, only the Mn++-dependent OAA decarboxylase was present in cell extracts. The Na+activated enzyme could not be detected in soluble or particulate fractions. Thus, growth on citrate caused induction of the latter enzyme. DISCUSSION The specific Na+ requirement demonstrated for aerobic growth of S. typhimurium on citrate is manifested by an increase in both the growth rate and total growth. This Na+ requirement was in marked contrast to the nonrequirement of Na+ for the aerobic growth of A. aerogenes on citrate where added Na+ increased total growth without changing the growth rate (6). An attempt was made to determine the enzymatic locus of this requirement. Like A. aerogenes (6), aerobic growth of S. typhimurium on citrate induced the formation of a Na+-activated OAA decarboxylase as well as citritase, the two enzymes of the citrate fermentation pathway. Unlike A. aerogenes, aerobic growth in the presence of Na+ did not lead to repression of a-ketoglutarate dehydrogenase and subsequent blockage of the citric acid cycle. Thus, two independent and active pathways of citrate catabolism appeared to coexist in the cell, and since the citric acid cycle route should operate independently of Na+, the Na+ requirement for growth on citrate was puzzling. Since the Na+-activated decarboxylase was specifically induced by citrate and no other Na+requiring reaction is known, one possible explanation is that the decarboxylase may play an essential, perhaps permissive, role in the overall catabolism of citrate via the citric acid cycle as well as via the fermentation pathway. For example, the catabolism of citrate via the cycle as well as via fermentation (cleavage) presents the cell with the problem of removing excess C-4 compounds (Lmalate and oxalacetate), and OAA decarboxylase as well as L-malic enzyme may perform this function. The presence of a Mn++-dependent OAA de-

6 400 O'BRIEN, FROST, AND STERN J. BACTERIOL. carboxylase in the cell sap should also contribute to this function. Another possible explanation is as follows. Acetate is known to induce a-ketoglutarate dehydrogenase in Escherichia coli (1) and also has been shown to reverse the Na+ repression of a- ketoglutarate dehydrogenase in A. aerogenes (6). Acid products were formed from citrate despite aerobic conditions of growth, and acetic acid could be among them. Thus, accumulation of acetate in the cell as a result of the citritase activity may have prevented any Na+ repression of a-ketoglutarate dehydrogenase. This implies that citrate catabolism occurred mainly via the fermentation pathway to acetate, utilizing only Na+-activated decarboxylase, and then acetate was more slowly activated and oxidized via the citric acid cycle. Two separate OAA decarboxylases were found in citrate-grown cells. One was activated by Mn++, but not by Mg++, and was inhibited by EDTA. It remained in the supernatant fluid after centrifugation at 26,000 and 150,000 X g, and was absent from the particle fractions. Its properties closely resembled those of the OAA decarboxylase induced by growth of S. typhimurium LT2 in the presence of meso-tartrate under microaerophilic conditions (7). The second OAA decarboxylase was not inhibited by EDTA, but it was sensitive to avidin and required Na+ for activity. Its properties paralleled those of the unique Na+-activated, avidin-sensitive OAA decarboxylase described in A. aerogenes by Stern (9), except that in the 26,000 X g supernatant fraction avidin inhibition of the Na+-stimulated decarboxylase required the presence of phosphate or EDTA and did not occur in Tris-chloride buffer. Avidin inhibition of the Aerobacter decarboxylase, on the other hand, was evident in either buffer. The washed cell-particle fractions settling at 26,000 and 150,000 X g contained the Na+-activated decarboxylase but not the Mn++activated one. The Salmonella enzyme also appeared to be more readily solubilized into the 150,000 X g supernatant fraction by prolonged sonic treatment of the disrupted cell particles than was the Aerobacter OAA decarboxylase, which is bound to the cytoplasmic membrane (G. M. Frost and J. R. Stern, Fed. Proc. p. 586, 1968). The presence of two OAA decarboxylases, one EDTA-sensitive and the other EDTA-insensitive, explains the finding of Rosenberger (7) that the OAA decarboxylase activity induced by mesotartrate was only 60% inhibited by EDTA or by extensive dialysis to remove endogenous cations. The EDTA-insensitive activity may represent the Na+-activated decarboxylase. (Sodium OAA was the probable substrate.) Thus, meso-tartrate apparently induced both enzymes. S. typhimurium extracts possessed both PEP synthase, first described by Cooper and Kornberg (2) in E. coli, and pyruvate kinase. Thus, enzymatic mechanisms were present for converting pyruvate, derived from L-malate or OAA, to PEP, thereby initiating the synthesis of sugar phosphates and polysaccharide. PEP carboxylase, PEP carboxykinase, and pyruvate carboxylase were not detected in these extracts. ACKNOWLEDGMENTS We thank R. E. Eckel for performing the sodium and potassium analyses and Harriet Richardson for excellent technical assistance. This invcstigation was supported by grant GB-8078 from the National Science Foundation by Public Health Service grant AM00739 from the National Institute of Arthritis and Metabolic Diseases, and by the Cleveland Diabetes Fund and the Heart Association of Northeastern Ohio. LITERATURE CITED 1. Amarasingham, C. J., and B. D. Davis Regulation of a-ketoglutarate dehydrogenase formation in Escherichia coli. J. Biol. Chem. 240: Cooper, R. A., and H. L. Kornberg Net formation of phosphoenolpyruvate from pyruvate by Escherichia coil. Biochim. Biophys. Acta 104: Gomall, A. G., C. J. Bardawill, and M. M. David Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177: Layne, E Spectrophotometric and turbidimetric methods for measuring proteins, p In S. P. Colowick and N. 0. Kaplan (ed), Methods in enzymology, vol. 3. Academic Press Inc., New York. 5. O'Brien, R. W., and J. R. Stern 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 alternate pathways of aerobic citrate catabolism in Aerobacter aerogenes. J. Bacteriol. 99: Rosenberger, R. F Derepression of oxalacetate 4-carboxy-lyase synthesis in Salmonella typhimurium. Biochim. Biophys. Acta 122: Stern, 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. 9. Stern, J. R Oxalacetate decarboxylase of Aerobacter aerogenes. I. Inhibition by avidin and requirement for sodium ion. Biochemistry 6: