JOURNAL OF BACrERIOLOGY, May 1974, p. 590-597 Copyright i 1974 American Society for Microbiology Vol. 118, No. 2 Printed in U.S.A. Regulation of Alanine Dehydrogenase in Bacillus licheniformis S. M. McCOWEN AND P. V. PHIBBS, JR. Department of Microbiology, Virginia Commonwealth University, Health Sciences Division, Richmond, Virginia 23298 Received for publication 28 January 1974 Cell extracts of Bacillus licheniformis were found to contain nicotinamide adenine dinucleotide (NAD)-dependent L-alanine dehydrogenase (ADH) (L- alanine:NAD oxidoreductase, EC 1.4.1.1)'. High specific activities (3.5 to 6.0 IU/mg of protein) were found in extracts of cells throughout growth cycles only when L-alanine served as the primary source of carbon or carbon and nitrogen. Specific activities were minimal (0.02 to 0.04 IU/mg of protein) during growth on' glucose, but increased at least sevenfold during the first 5 h of postlogarithmic- phase metabolism. Addition of 10 mM glucose to cultures during logkrithmic- phase growth on L-alanine resulted in a rapid decrease in enzyme activity. Addition of 20 mM L-alanine to cells near the completion of log-phase growth on glucose resulted in a 20-fold increase in ADH specific activity during less than one cell generation. Extracts of postlogarithmic-phase cells cultured on glucose, malate, L-glutamate, or Casamino Acids contained intermediate levels of ADH activity. The enzyme was partially purified from crude extracts of B. lichenifor- mis, and apparent kinetic constants were estimated. A role for ADH in the catabolism of L-alanine to pyruvate during vegetative growth on L-alanine and during sporulation of cells cultured on glucose is proposed on the basis of these experimental results. The energy and the carbon skeletons neces- sary for synthesis of spore structural compo- nents in Bacillus licheniformis are thought to be derived from endogenous amino acids and from compounds released by lysed cells into the medium during growth (16). L-Glutamate and L-alanine have been shown to comprise 60 to 90%o of the endogenous amino acid pool in vegetative and sporulating cells of this organism (1). The total, intracellular pool of amino acids increases during exponential growth and then rapidly decreases during postexponential-phase metabolism (4). The relative proportion of ala- nine in the intracellular amino acid pool is low during exponential growth, but increases in postexponential-phase cells to become the major constituent of the pool at a level of 40% (4). However, the proportion of alanine in the total (intracellular plus extracellular) amino acid pool decreases significantly during postex- ponential-phase metabolism (4). There is considerable experimental evidence that a functional tricarboxylic acid cycle is necessary for sporulation in B. subtilis (6, 15). The mechanism of entry of amino acid carbon skeletons into the tricarboxylic acid cycle for the generation of energy and for initiation of gluconeogenesis is not clear. L-Alanine dehydro- genase (ADH) (EC 1.4.1.1) may be a key enzyme in the catabolism of L-alanine to pyru- vate and may be required for further catabolism of alanine by the tricarboxylic acid cycle. McCormick and Halvorson (10) have reported minimal levels of ADH activity during sporula- tion in B. cereus, whereas Buono et al. (2) observed that ADH activity was high during vegetative growth and increased more than twofold during sporulation in this organism. O'Connor and Halvorson (12) demonstrated that ADH from B. cereus deaminated L-alanine to form pyruvate, although equilibrium favored the formation of alanine. Hong et al. (7) de- tected ADH activity in vegetative cell extracts of B. subtilis, B. mycoides, B. megaterium, B. brevis, and B. anthracoides. ADH has been partially purified from cell extracts of B. subtilis (14, 15) and B. cereus (11, 12). We report evidence for nicotinamide ade- nine dinucleotide (NAD)-dependent ADH activ- ity in B. licheniformis. Some kinetic properties of the partially purified enzyme from B. licheni- formis and the regulation of synthesis of this en- zyme during growth and sporulation will be de- scribed. (These results are from an M.S. thesis submitted by S. M. McCowen to the graduate faculty of Virginia Commonwealth University, 59() on February 17, 2020 by guest http://jb.asm.org/ Downloaded from
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JOURNAL OF BACrERIOLOGY, May 1974, p. 590-597Copyright i 1974 American Society for Microbiology
Vol. 118, No. 2Printed in U.S.A.
Regulation of Alanine Dehydrogenase in Bacillus licheniformisS. M. McCOWEN AND P. V. PHIBBS, JR.
Department of Microbiology, Virginia Commonwealth University, Health Sciences Division,Richmond, Virginia 23298
Received for publication 28 January 1974
Cell extracts of Bacillus licheniformis were found to contain nicotinamideadenine dinucleotide (NAD)-dependent L-alanine dehydrogenase (ADH) (L-alanine:NAD oxidoreductase, EC 1.4.1.1)'. High specific activities (3.5 to 6.0IU/mg of protein) were found in extracts of cells throughout growth cycles onlywhen L-alanine served as the primary source of carbon or carbon and nitrogen.Specific activities were minimal (0.02 to 0.04 IU/mg of protein) during growth on'glucose, but increased at least sevenfold during the first 5 h of postlogarithmic-phase metabolism. Addition of 10 mM glucose to cultures during logkrithmic-phase growth on L-alanine resulted in a rapid decrease in enzyme activity.Addition of 20 mM L-alanine to cells near the completion of log-phase growth on
glucose resulted in a 20-fold increase in ADH specific activity during less thanone cell generation. Extracts of postlogarithmic-phase cells cultured on glucose,malate, L-glutamate, or Casamino Acids contained intermediate levels of ADHactivity. The enzyme was partially purified from crude extracts of B. lichenifor-mis, and apparent kinetic constants were estimated. A role for ADH in thecatabolism of L-alanine to pyruvate during vegetative growth on L-alanine andduring sporulation of cells cultured on glucose is proposed on the basis of theseexperimental results.
The energy and the carbon skeletons neces-sary for synthesis of spore structural compo-nents in Bacillus licheniformis are thought to bederived from endogenous amino acids and fromcompounds released by lysed cells into themedium during growth (16). L-Glutamate andL-alanine have been shown to comprise 60 to90%o of the endogenous amino acid pool invegetative and sporulating cells of this organism(1). The total, intracellular pool of amino acidsincreases during exponential growth and thenrapidly decreases during postexponential-phasemetabolism (4). The relative proportion of ala-nine in the intracellular amino acid pool is lowduring exponential growth, but increases inpostexponential-phase cells to become themajor constituent of the pool at a level of 40%(4). However, the proportion of alanine in thetotal (intracellular plus extracellular) aminoacid pool decreases significantly during postex-ponential-phase metabolism (4).There is considerable experimental evidence
that a functional tricarboxylic acid cycle isnecessary for sporulation in B. subtilis (6, 15).The mechanism of entry of amino acid carbonskeletons into the tricarboxylic acid cycle forthe generation of energy and for initiation ofgluconeogenesis is not clear. L-Alanine dehydro-
genase (ADH) (EC 1.4.1.1) may be a keyenzyme in the catabolism of L-alanine to pyru-vate and may be required for further catabolismof alanine by the tricarboxylic acid cycle.McCormick and Halvorson (10) have reportedminimal levels of ADH activity during sporula-tion in B. cereus, whereas Buono et al. (2)observed that ADH activity was high duringvegetative growth and increased more thantwofold during sporulation in this organism.O'Connor and Halvorson (12) demonstratedthat ADH from B. cereus deaminated L-alanineto form pyruvate, although equilibrium favoredthe formation of alanine. Hong et al. (7) de-tected ADH activity in vegetative cell extractsof B. subtilis, B. mycoides, B. megaterium, B.brevis, and B. anthracoides.ADH has been partially purified from cell
extracts of B. subtilis (14, 15) and B. cereus (11,12). We report evidence for nicotinamide ade-nine dinucleotide (NAD)-dependent ADH activ-ity in B. licheniformis. Some kinetic propertiesof the partially purified enzyme from B. licheni-formis and the regulation of synthesis of this en-zyme during growth and sporulation will be de-scribed. (These results are from an M.S. thesissubmitted by S. M. McCowen to the graduatefaculty of Virginia Commonwealth University,
Richmond, 1972. Portions of these results werepresented at the Annual Meeting of the Ameri-can Society for Microbiology, 1973.)
MATERIALS AND METHODSGrowth conditions. B. licheniformis strain A-5
was obtained from R. W. Bernlohr, University ofMinnesota, Minneapolis. The organism was culturedin a minimal salts (T-salts) medium composed of 65mM potassium phosphate (pH 7.0), 0.61 mM MgSO4,0.62 mM MgCl2, 5.6 gM MnCl,, and 0.34 mM CaCi2and prepared as described previously (13). Unlessotherwise specified, the concentrations of the variouscarbon and nitrogen sources were as follows: glucose,15 mM; malate (Na+), 20 mM; NH4Cl, 10 mM;L-glutamate (Na+), 50 mM; Casamino Acids, 0.3%;L-alanine (Na+) 40 mM; plus pyruvate (Na+), 2 mM.Strain A-5 grew very poorly,when 40 mM L-alaninewas employed as the sole source of carbon andnitrogen. Good growth (both rate and extent) wasachieved upon the addition of either 2 mM pyruvateor 5 mM L-glutamate to the medium. Pyruvate (2mM) alone did not support growth of the organism.Therefore, T-salts medium always contained 2 mMpyruvate when L-alanine served as the primary sourceof carbon and nitrogen in these experiments.
Starter cultures werxe prepared by the germinationof approximately 4 x 107 spores per ml of theappropriate T-salts medium, followed by growth at37 C for 16 h. One-liter volumes of medium in 2.8-literFembach flasks were inoculated with 50 ml from astarter culture grown in homologous medium, andincubated aerobically at 37 C on a rotary shaker. Cellgrowth was measured with a Bausch and Lomb Spec-tronic 20 colorimeter at a wavelength of 540 nm.Disappearance of D-glucose from the medium duringgrowth of the organism on T-salts-glucose-ammoniummedium was measured enzymatically (Glucostat En-zyme Reagent, Worthington Chemicals, Freehold,N.J.). The formation of heat-resistant spores in cul-tures was determined by plating appropriate dilutionsof pasteurized (65 C, 2 h) culture samples on nutrientagar.Enyme assay. ADH (EC 1.4.1.1) activity was
determined spectrophotometrically by measuring theoxidation of reduced NAD (NADH) at a wavelengthof 340 nm. Unless otherwise specified, the standardreaction mixture contained the following in a totalvolume of 3.0 ml: tris(hydroxymethyl)aminomethane(Tris)-hydrochloride (pH 8.0), 50 mM; 2-mercapto-ethanol (2-ME), 5 mM; sodium pyruvate, 16.7 mM;NH4C1, 80 mM; NADH 0.15 mM; and cell extract.Reactions were initiated by the addition of NH4C1,and the initial velocity was measured with a Gilfordmodel 2400 multiple recording spectrophotometer at22 C. Specific activities are reported as intemationalenzyme units (IU) per milligram of extract protein(micromoles of NADH oxidized per minute per milli-gram of protein at 22 C). Dialyzed 105,000 x gsupernatant fractions of cells ruptured by sonic oscil-lation were employed as "crude extracts" throughoutthis study. Under these standard conditions, theinitial velocity of the reaction was directly propor-
tional to protein concentration. Initial velocities forthe reverse reaction (oxidative deamination of Lala-nine) were determined by replacing pyruvate, NH4Cl,and NADH with 35 mM L-alanine and 0.3 mM NAD+in the standard reaction mixture. Protein concentra-tion in extracts was estimated by the method of Lowryet al. (9), with crystalline bovine serum albumin asthe standard.Enzyme purification. B. licheniformis was cul-
tured in T-salts medium containing 40 mM L-alanineplus 2 mM pyruvate, and late exponential-phase cellswere chilled with ice and harvested. Cells from 4 litersof culture were washed twice with 500 ml of ice-cold0.1 M Tris-hydrochloride (pH 8.0) containing 10 mM2-ME. All steps of the enzyme purification wereconducted at 0 to 5 C unless otherwise stated. Thepacked, washed cells were suspended in 20 to 30 ml offresh buffer and ruptured with a Biosonik Type Imsonic oscillator. Two portions of the suspension (10-to 15-ml each) were each subjected to five 20-s sonictreatments with 30-s intervals between each burst tominimize warming of the extract. The suspension ofruptured cells was centrifuged at 105,000 x g for 2 h ina Beckman L2-65B preparative ultracentrifuge, andthe supernatant fraction (crude extract) was col-lected.
Solid (NHJ),SO4 was added to the crude extractwith constant stirring until the concentration reached45% of saturation. After 1 h of stirring, the suspensionw#s centrifuged at 35,000 x g for 20 min and thesupernatant fraction (Sup I), which contained essen-tially all of the ADH activity, was collected. Solid(NHj),SO4 was added to the SUP I fraction until theconcentration reached 75% of saturation. After 1 h ofstirring, the precipitated protein (Pel II) was collectedby centrifugation at 35,000 x g for 30 min, suspendedin 5 ml of 0.1 M Tris-hydrochloride (pH 8.0) contain-ing 10 mM 2-ME, and dialyzed for 10 h against 1,500ml of the same ice-cold buffer.A column (2.5 by 23 cm) of diethylaminoethyl-cel-
lulose (Whatman 52) was prepared according toWhatman specifications and equilibrated with 0.1 MTris-hydrochloride (pH 8.3) containing 50 mM NaCl.The dialyzed Pel II fraction was applied to thecolumn, followed by 10 ml of the equilibration buffer,and eluted with an increasing linear gradient of NaCl(0.06 to 0.62 M) contained in 0.1 M Tris-hydrothlo-ride (pH 8.3). Fractions of eluate (110 fractions of6 ml each) were collected at a flow rate of 0.7 ml/min.The recovered enzyme activity was contained in the18 ml of eluate in fractions 25, 26, and 27. Thesefractions were pooled and protein was precipitated byaddition of solid (NHJ,)SO, to 75% of saturation. Theprecipitate was collected by centrifugation at 35,000x g for 20 min, suspended in 3 ml of 0.1 MTris-hydrochloride (pH 8.0) containing 10mM 2-ME,and dialyzed for 10 h against 1,500 ml of the samebuffer. The enzyme preparation was assayed immedi-ately (19 IU/mg of protein) and then stored at -20 C.The enzyme had a half-life of approximately 17 dayswhen stored under these conditions. A summary of thepurification scheme is shown in Table 1.
Regulation of ADH activity in growing ceols.Cells of B. licheniformis were grown in flasks of
aEnzyme obtained from ultrasonic extracts of lateexponential-phase cells of B. lichertiformis cultured inT-salts-L-alanine medium.
"Standard reaction conditions were employed.Specific activity expressed as international units permilligram of protein.
medium containing either 40 mM L-alanine plus 2mM pyruvate or 15 mM glucose plus 10 mM NH4Cl asthe source of carbon, nitrogen, and energy. At a pointduring the late exponential phase of growth, eitherglucose or L-alanine was added and the effect of theseadditions on the specific activity of ADH was deter-mined. Samples of 100 to 200 ml were withdrawn fromeach culture flask at the time of glucose or alanineaddition and at subsequent points during the growthcycle. The cells were chilled in ice, harvested in arefrigerated centrifuge, washed once, and suspendedin 3 to 5 ml of 0.1 M Tris-hydrochloride (pH 8.0) plus10 mM 2-ME. These suspensions of washed cells werefrozen overnight, and dialyzed crude extracts (105,000x g supernatant fractions) were prepared on thefollowing day as described above.ADH activity in extracts of cells cultured on
selected carbon and nitrogen sources. Cells of B.licheniformis were grown in flasks of T-salts mediumcontaining one of the following carbon and nitrogensources: 15 mM glucose plus 10 mM NH4Cl, 20 mMmalate plus 10 mM NH4Cl, 50 mM L-glutamate, 40mM L-alanine plus 2 mM pyruvate, or 0.3% CasaminoAcids. Samples (300 ml) were harvested from eachculture at a point during the mid-exponential phase ofgrowth and at another point 5 h after the completionof exponential growth. The cells were harvested andADH specific activities were determined in the crudeextracts (105,000 x g supernatant fractions) as de-scribed above.
Chemicals. The following chemicals were obtainedfrom P-L Biochemicals, Milwaukee, Wis.: NAD+,NADH, NADP+, NADPH. L-Malic acid and sodiumpyruvate (dimer-free) were obtained from SigmaChemical Co., St. Louis, Mo. L-Glutamate was pur-chased from Calbiochem, Los Angeles, Calif. Cas-amino Acids was a "certified" preparation obtainedfrom Difco Laboratories, Detroit, Mich. Enzymegrade (NH4)2SO4 used during enzyme purificationwas obtained from Mann-Schwartz Research Corp.,Orangeburg, N.J. L-Alanine (lot 41940) was purchasedfrom General Biochemicals, Chagrin Falls, Ohio, andlot number 712-1 was purchased from Eastman Or-ganic Chemicals. All other reagents and chemicals
used were of highest purity available from J. T. BakerChemical Co.
RESULTSCharacterization of partially purified
ADH. The reductive amination of pyruvate bypartially purified ADH and in crude extractswas shown to be highly specific for NADH ascofactor and was NH+4-dependent. NADPHwould not replace NADH. There was no detect-able oxidation of NADH when either pyruvateor NH,Cl was omitted from the reaction mix-ture and pyruvate could not be replaced with2-oxoglutarate. The oxidative deamination ofL-alanine. was NAD+-dependent, and NAD+could not be replaced with NADP+. D-Alaninewas not a substrate for ADH but caused stronginhibition of the oxidative deamination of L-ala-nine (Table 2). The enzyme was inactive belowpH 6.5 and above pH 9.9. Maximal activityoccured in the range of pH 7.9 to 9.1. Thereductive amination of pyruvate was preferredas the standard assay reaction because theinitial velocity remained constant for a longerperiod of time and the initial velocity of amina-tion was greater than that of deamination, thusallowing a more precise determination of en-zyme activity.The apparent kinetic constants of ADH in
crude extracts 'and of partially purified ADHwere estimated by constructing Woolf plots (3)of saturation curves for NADH, ammonium,pyruvate, L-alanine, and NAD+. Each substrateexhibited saturation kinetics which conformedwith the Michaelis-Menten equation. The ap-parent Km values were estimated from theseplots and found to remain essentially un-changed during enzyme purification (Table 3).The Km value for pyruvate was more than10-fold lower than that for L-alanine. Maximalvelocity (Vmax) values for the reductive amina-tion reaction were approximately 20-fold higherthan Vmax values for the oxidative deamination
TABLE 2. Inhibitory effects of D-alanine on theoxidative deamination of L-alanine in crude extractsa
L-Alanine Sp act in presence of D-alanine(mM) None 2mM 4mM 15 mM
2.5 0.09 NRb NR NR5.0 0.13 0.08 0.02 NR10.0 0.18 0.14 0.07 NR15.0 0.22 0.18 0.08 NR
aStandard assay conditions were employed. Spe-cific activity is expressed as international units permilligram of protein.
TABLE 3. Apparent Michaelis-Menten constants forsubstrates of L-alanine dehydrogenase
Kma (mM)Substrate
Crude extractb Partiallypue -
Pyruvate 0.40 0.37NH4+ 14.0 12.0NADH 0.015 0.025NADPH NRC NRL-Alanine 5.0 5.0NAD+ 0.080 0.045NADP+ NR NR
a Values were estimated from Woolf plots of satura-tion curves.
'Source of ADH activity.c NR, No detectable reaction over broad concentra-
tion range.
reaction in both crude and partially purifiedpreparations.Because it was suspected that ADH in B.
licheniformis may serve a catabolic role in themetabolism of L-alanine, the sensitivity of theoxidative deamination reaction to reactionproducts was investigated. Double-reciprocalplots of L-alanine saturation curves showed noapparent inhibition of the oxidative deamina-tion of L-alanine in the presence of 0.68 mMpyruvate (Fig. 1B). This concentration of pyru-vate was approximately twice the estimated Km(0.37 mM) for pyruvate. Ammonium concentra-tions of 6.7 mM (Km = 12 mM) caused noinhibition of L-alanine deamination (Fig. 1A).However, inhibition did occur in the presence ofa much higher concentration (33 mM) ofNH4Cl. This inhibition appeared to be of anon-competitive nature.Reductive amination of pyruvate by partially
purified ADH was inhibited by the reactionproduct L-alanine (Table 4). At saturating con-centrations of pyruvate (16.7 mM), the reactionwas inhibited 25% by 8.3 mM L-alanine. Thereaction was nearly totally inhibited by thesame concentration of L-alanine when the initialconcentration of pyruvate was 0.33 mM (Km =0.37 mM).Regulation of ADH activity in growing
cells. The specific activity of ADH was deter-mined in crude extracts of mid-exponentialphase and 5-h postexponential-phase cells of B.licheniformis when grown on selected carbonand nitrogen sources. The results are reportedas the average values obtained from at leastthree separate determinations (Table 5). Thehighest specific activities (3.5 to 6.0 IU/mg ofprotein) occurred in extracts of cells cultured onL-alanine. Crude extracts of cells cultured on
-0.2
4
-0.2
A
0 0.2 0.4 0.6I/mM ALANINE
B
0 0.2 0.4 0.6I/mM ALANINE
FIG. 1. Lineweaver-Burk double-reciprocal plots ofL-alanine saturation curves of partially purified L-alanine dehydrogenase in the presence of variousconcentrations of reaction products. (A) L-Alaninesaturation in the presence of 6.7 mM NH4CI (-), 33mM NH4CI (0), and no NH4CI (a). (B) L-Alaninesaturation in the presence of 0.33 mM pyruvate (A),0.68 mM pyruvate (0), and no pyruvate (0).
TABLE 4. Inhibition of reductive amination ofDvruvate bv L-alaninea
°Dialyzed 105,000 x g supernatant fractions ofcells ruptured by sonic oscillation were employed ascrude extracts. Standard reaction conditions wereemployed.
I Specific activities expressed as international unitsper milligram of protein and are average valuesdetermined in three separate experiments.
cData from single determination.
Casamino Acids contained at least fivefold lessactivity than those cultured on L-alanine. Ex-tracts from cells cultured on L-glutamate con-tained less than one-tenth the activity observedin extracts of L-alanine-grown cells. The addi-tion of 10 mM NH4Cl to cultures growing onindividual amino acids or Casamino Acids pro-duced no detectable effect on the levels of ADHactivity. Only minimal specific activities werepresent in extracts of exponential-phase cellsgrown on L-malate or glucose when ammoniumserved as the sole nitrogen source. However, thespecific activity increased significantly in 5-hpostexponential-phase cells cultured on glucoseor malate (Table 5).The increase in ADH activity observed in
postexponential-phase cells cultured on glucosewas investigated further. The specific activity ofADH was measured at several intervals duringgrowth on T-salts-glucose-ammonium medium(Fig. 2). A steady increase in ADH specificactivity occurred during postexponential-phasemetabolism after glucose had been exhaustedfrom the medium. The addition of 20 mML-alanine to a second culture resulted in a rapidincrease in ADH specific activity (Fig. 2). Therewas a 20-fold increase in ADH specific activitywithin less than one cell generation (2 h) afterL-alanine addition. Glucose was totally depletedfrom the culture medium during this period.ADH activity in extracts of cells from the
1.5g
-N1
(QI)
J. BACTERIOL.
-14
12 -
0- - I10 Z
8 d1
6
40
2
0V..
O 2 3 4 5 6 7 8 9HOURS
FIG. 2. Effect of L-alanine on the specific activityof L-alanine dehydrogenase (dashed lines) duringgrowth (solid lines) of B. licheniformis in T-salts-glucose-ammonium medium. L-Alanine (20 mM) wasadded as indicated to one culture (0) and a controlculture (0) received no addition. Culture sampleswere withdrawn just prior to the time of alanineaddition and at several subsequent intervals. ADHspecific activities were determined in crude extractsof cell samples by using standard assay conditions.Glucose concentration (A) in supernatant samplesfrom the control culture was determined.
control culture, to which no L-alanine wasadded, showed only a twofold increase duringthis same period of late vegetative growth.However, a seven- to eightfold increase in spe-cific activity was reached within 5 h aftergrowth was completed in the control culture.This is characteristic of postexponential-phasecells grown on glucose (Table 5).The specific activity of ADH also was mea-
sured at several intervals during growth onT-salts-L-alanine medium (Fig. 3). ADH spe-cific activity increased to maximal value duringvegetative growth and remained at a high levelof activity during postexponential-phase me-tabolism. When 10 mM glucose was added to asecond culture, the characteristic high specificactivity ofADH began to decrease immediately.An approximate 50% decrease in activity oc-cured within one cell generation after the addi-tion of glucose. The specific activity in extractsfrom the control culture (no glucose added)increased approximately twofold during thisperiod. This increase is characteristic of cellsduring growth on L-alanine (Table 5).
DISCUSSIONCrude extracts of B. licheniformis strain A-5
were shown to contain NAD+-dependent ADHactivity. ADH specific activity was minimal
FIG. 3. Effect of glucose on the specific activity ofL-alanine dehydrogenase (dashed lines) duringgrowth (solid lines) of B. licheniformis on T-salts-ala-nine medium. Glucose (10 mM) was added as indi-cated to one culture (0), and a control culture (0)received no addition. Culture sampling and ADHassay were as described in Fig. 2.
during vegetative growth in T-salts-glucose-ammonium medium. The specific activity inthese cells then increased at least sevenfoldduring 5 h of postexponential-phase metabo-lism. During this period of increasing ADHactivity, when glucose was exhausted from themedium, sporulation was shown to occur. Clarket al. (4) have shown that alanine is a majorcomponent of the amino acid pool in B.licheniformis strain A-5 and that alanine ismetabolized rapidly by sporulating cells. Thehigh intracellular concentration of alanine maybe partially responsible for the increase in ADHspecific activity during postexponential-phasemetabolism of cells initially cultured on glu-cose. These observations provide suggestive evi-dence that ADH may serve a prominent role insporulation metabolism in B. licheniformis.However, Freese et al. (5) have shown that amutant strain of B. subtilis lacking ADH activ-ity can sporulate after vegetative growth inglucose medium.
Extracts of vegetative cells cultured on 0.3%Casamino Acids, which included 0.57 mM L-al-anine (12), contained ADH activities two- tothreefold higher than in extracts of cells grownon L-glutamate alone. Moreover, the specificactivity in extracts of these cells declined during
postexponential-phase metabolism to approxi-mately the same levels as seen in extracts ofpostexponential-phase cells cultured on glu-cose, malate, or L-glutamate. Similar interme-diate levels of ADH activity were foundthroughout the growth cycle in extracts of cellsgrown on glutamate.The specific activity of ADH was approxi-
mately 90-fold greater in extracts of vegetativecells when grown on L-alanine than in extractsof cells grown on glucose. Moreover, the addi-tion of L-alanine to cultures of vegetative cells,when glucose had been nearly exhausted fromthe growth medium, resulted in an immediateand rapid increase in ADH specific activity. Onthe other hand, a decrease in ADH specificactivity resulted immediately upon the additionof glucose to vegetative cells growing on L-ala-nine. The specific activity of ADH declinedapproximately 50% within one cell generationafter the addition of glucose, providing strongevidence that ADH is under catabolite repres-sion control in B. licheniformis. This pattern ofcontrol is very similar to the regulation ofenzymes for arginine and proline catabolism inthis organism (8).The results from these experiments demon-
strate that ADH in B. licheniformis may beregulated in a manner considerably differentfrom the NAD+-dependent ADH reported tooccur in B. cereus. McCormick and Halvorson(10) reported that the specific activity of ADHincreased during log-phase growth of B. cereusin a yeast extract medium and then declined tominimal activities during sporulation. Theseauthors (10) also reported that the addition ofL-alanine had no effect on the level of ADHactivity per cell during vegetative growth in thesame medium. Our data generally are in closeraccord with the work of Buono et al. (2). Theseauthors (2) demonstrated NAD+-dependentADH activity in extracts of cells during vegeta-tive growth of B. cereus in glucose-glutamate-glycine-salts medium and noted a sharp in-crease in specific activity during the period offorespore development. 8
Apparent Michaelis constants for substratesof the reactions catalyzed by the partiallypurified enzyme from cells of B. licheniformisare in close agreement with reported values forNAD+-dependent ADH from B. cereus (11, 12)and from B. subtilis (14, 17). Kinetic studiesshowed that the oxidative deamination of L-ala-nine was not inhibited by concentrations ofpyruvate in excess of the Km (0.37 mM) forpyruvate. Concentrations of ammonium ionapproaching the Km for ammonium (12 mM)also did not inhibit the deamination of L-ala-
nine, although concentrations greater thantwice the Km were inhibitory. The reversereaction catalyzed by ADH (reductive amina-tion of pyruvate) was inhibited strongly byconcentrations of L-alanine that are similar tothe intracellular concentration reported ingrowing cells (4).Two kinetic factors provide suggestive evi-
dence of a role for ADH in the catabolism ofL-alanine rather than in biosynthesis: (i) therelatively high Km for ammonium ion (12 mM)and (ii) the sensitivity of reductive amination ofpyruvate to inhibition by alanine. A catabolicrole for ADH is substantiated further by theobservations that ADH activity was increasedat least 20-fold after the addition of L-alanine togrowing cells of B. licheniformis, and that thesynthesis of ADH activity appeared to be understrong catabolite repression control.The pathways for entry of either L-alanine or
L-glutamate into the tricarboxylic acid cycleduring sporulation of B. licheniformis have notbeen elucidated. These two amino acids are themajor components of the combined intracellularand extracellular amino acid pools synthesizedduring vegetative growth of this organism onglucose and both are consumed rapidly duringpostexponential-phase metabolism (4). Buonoet al. (2) have suggested that L-glutamate maynot be oxidized directly via the tricarboxylicacid cycle in B. cereus, but that glutamate-pyruvate-transaminase (GPT) and glutamate-oxalacetate-transaminase (GOT) may convertL-glutamate into other amino acids needed forspore protein synthesis. These authors (2) re-ported that activities of both transaminases(GPT and GOT) and of ADH increase signifi-cantly during sporulation of B. cereus.The absence of a "catabolic" glutamate dehy-
drogenase for the oxidative deamination ofL-glutamate to 2-oxoglutarate (13) and theincrease in ADH specific activity during sporu-lation indicate that a role for ADH in the ca-tabolism of both L-glutamate and L-alanine to2-oxoglutarate and pyruvate may be worthy offurther investigation. One possible ADH-de-pendent mechanism for directing the carbonskeletons of L-glutamate and L-alanine into thetricarqboxylic acid cycle is diagrammed in Fig. 4.We have detected GPT activity (0.02 to 0.03IU/mg of protein) in preliminary experimentswith crude extracts of. L-alanine-grown andsporulating cells. Further studies of the regula-tion of amino acid transaminases may be usefulin determining the pathways for L-alanine andL-glutamate catabolism in sporulating cells ofB. Iicheniformis.
GPT (7)
Glutamate
Pool
Pyruvote..4(-.' 2-0xogluta rate
NH4TAlanine 4 -| Alnine|
/ Pool
FIG. 4. Diagram of one possible mechanism forcatabolism of both L-glutamate and L-alanine insporulating cells of B. licheniformis that is dependenton L-alanine dehydrogenase (ADH) and glutamate-pyruvate transaminase (GPT).
ACKNOWLEDGMENTSWe thank R. W. Bernlohr for supplying us with a culture of
B. licheniformis strain A-5 and for valuable advice during thisinvestigation.
This investigation was supported by Public Health ServiceTraining Grant AI-00382 from the National Institute ofAllergy and Infectious Diseases, and by research grantGB-30916 from the National Science Foundation.
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