proceeded m),(r buffer plus inducer, 10-3 m). the washed-cell suspensions were adjusted to an...

DE NOVO SYNTHESIS OF a-AM\IYLASE BY BACILLUS STEAROTHERMOPHILUS1N. E. WELKER2 AND L. LEON CAMPBELL2

Department of Microbiology, School of Medicine, Western Reserve University. Cleveland, Ohio, andDepartment of Microbiology, University of Illinois, Urbana, Illinois

Received for publication 29 June 1963

ABSTRACT

WELKER, N. E. (Western Reserve University,Cleveland, Ohio and University of Illinois, Ur-bana), AND L. LEON CAMPBELL. De novosynthesis of a-amylase by Bacillus stearother-mophilus. J. Bacteriol. 86:1202-1210. 1963.-ThepH optimum for the synthesis of a-amylaseby washed-cell suspensions was 6.7. a-Amylasesynthesis began soon after the addition of the in-ducer (maltose, methyl-3-D-maltoside, or phenyl-a-i)-glucoside, at 10-3 M), proceeded at a linearrate for 60 min, and then leveled off. Cell suspen-sions without inducer produced small amounts ofa-amylase. The addition of glucose (2 X 10-3M), sucrose (10-3 M), or glycerol (4 X 10-3 M) towashed-cell suspensions failed to stimulate theproduction of a-amylase. Nitrogen starvation ofwashed cells for 60 min with fructose as a carbonsource or by induction with pure maltose showedthat the ability to produce a-amylase was lost.Examination of the amino acid pool at this timeshowed a general depletion of amino acids andthe complete disappearance of tyrosine, phenyl-alanine, proline, and valine. Replenishment ofthe amino acid pool with casein hydrolysate(0.5%) restored the ability of the cells to producea-amylase. Chloramphenicol and 8-azaguaninewere shown to inhibit a-amylase synthesis. In-hibition was observed immediately upon theaddition of chloramphenicol to cell suspensionspreinduced for varying periods of time. Actino-mycin D and mitomycin C also inhibited a-amylase synthesis when added to induced washed-cell suspensions. The amino acid analogues,norvaline, norleucine, and ethionine, inhibiteda-amylase formation by 72, 53, and 38%, re-spectively. p-Fluorophenylalanine inhibited the

I Part of the dissertation of Neil E. Welker,presented to the Graduate Faculty of WesternReserve University in partial fulfillment of re-quirements for the Ph.D. degree.

2 Present address: Department of Microbiology,University of Illinois, Urbana.

synthesis of active a-amylase by 92% and theincorporation of proline-C'4 into a-amylase andcellular proteins by 95 and 74%0,, respectively.

The kinetics of induction of a-amylase bygratuitous and nongratuitous inducers in growingcultures of Bacillus stearothermophilus and theisolation and characterization of the naturalinducers (maltodextrins) were described in pre-vious papers (Welker and Campbell, 1963a, b, c).The present paper presents evidence for the denovo synthesis of a-amylase by washed-cell sus-pensions of this organism.

MIATERIALS AND MEIETHODSPreparation of washed-cell suspensions. The

growth media and preparation of cells were thesame as those described for growth experiments(Welker and Campbell, 1963a) except that thecells were washed two times with and suspendedin 10 ml of a sterile salt buffer solution of thefollowing composition: K2HPO4, 2.5 g; KH2PO4,1.0 g; NaCl, 1.0 g; and CaCI2 H20, 5 mg perliter of distilled water. The pH was adjusted to6.7. This buffer is referred to as R buffer.

Cells used for nitrogen starvation experiments,examination of amino acid pools, and proline-C'4incorporation experiments were also prepared inthis manner.

Induction of a-amylase by washed-cell suspen-sions. Induction of a-amylase by washed-cellsuspensions was carried out in 250-ml nephelom-eter flasks containing 30 ml of induction medium(R buffer plus inducer, 10-3 M). The washed-cellsuspensions were adjusted to an absorbancy of0.5 to 0.66 at 525 m,u and incubated at 55 C in arotary water-bath shaker at a speed of 133 rev/min. a-Amylase activity was measured as de-scribed by WVelker and Campbell (1963a).

Nitrogen starvation and replenishment of aminoacid pools. Nitrogen starvation was accomplishedin two ways: by the addition of fructose as acarbon source or by addition of an inducer ofa-amylase. The washed cells were placed in a

1202

on April 5, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

DE NOVO SYNTHESIS OF a-AMYLASE

500-ml Erlenmeyer flask containing 150 ml ofR buffer supplemented with either fructose(2.0%) or inducer (10-3 %i). The flasks were in-cubated at 55 C in a rotary water-bath shaker.At 30-min intervals, 5.0-ml samples were re-moved, centrifuged, and washed two times withR buffer. The washed cells were suspended in250-ml flasks containing 30 ml of inductionmedium. The induction flasks were incubated at55 C in the water-bath shaker, and the forma-tion of a-amylase was followed.

Replenishment of nitrogen-starved cells wasaccomplished by removing 5.0-ml samples fromstarvation flasks, washing the cells two timeswith R buffer, and suspending the cells in 250-mlflasks containing 20 ml of the appropriate re-l)ienishment medium (R buffer supplementedwN-ith either 0.5%0 casein hydrolysate or 1.0%1,NH,CI). After 20 min, the cells were centrifuged,washed two times with R buffer, and placed ininduction flasks.

Isolation and quantitation of amino acid pools.Amino acid pools were obtained by the procedureof Gale (1947). Cells to be treated for the isola-tion of amino acid pools were washed two timeswith deionized water and then brought to a finalvolume of 20 ml with water. A 0.1-ml samplewas removed, taken to dryness in a vacuumdesiccator, and weighed.The cells were heated in a boiling-water bath

for 20 min. The cell debris was removed by cen-trifugation, and the supernatant liquid was de-canted. The pellet was washed with water (10to 20 ml), centrifuged, and the supernatantliquid decanted. The supernatant fluids werecombined.The combined supernatant liquid containing

the amino acids was passed through a column,2.0 cm in diameter, packed to a height of 5.5cm with Dowex 5OW (8X, 200 to 400 mesh) inthe H+ form. The column was washed thoroughlywith water, and the amino acids were eluted with20 ml of 1.5 N NH40H. The amino acid mixturewas taken to dryness in a vacuum desiccatorover NaOH pellets. The amino acids were dis-solved in 10 ml of sodium citrate buffer (pH 2.2),centrifuged to remove any precipitate, and storedat -14 C until analyzed on the Beckman/Spincoamino acid analyzer.

Incorporation of labeled DL-proline into cellularprotein and a-amylase. The procedure used formeasuring the incorporation of DL-proline-C'4

into cellular proteins was that of Neidhardt andMagasanik (1960). Samples of the culture (5 to10 ml) to be analyzed for proline-C'4 incorpora-tion were removed and centrifuged, and the super-natant liquid was decanted. The cells were sus-pended in 5.0 ml of 5%7 trichloroacetic acid andstored at 4 C for 12 hr.The cells were removed by centrifugation, and

the supernatant fluid was decanted. The pelletwas washed once with 5.0 ml of 500 trichloro-acetic acid, suspended in exactly 5.0 ml of 5%trichloroacetic acid, and heated in a boiling-waterbath for 30 min. The mixture was cooled, cen-trifuged, and the supernatant liquid decanted.The pellet was dissolved in 0.1 ml of 1.0 N NaOH,diluted to 10 ml with distilled water, and assayedfor protein by the method of Lowry et al. (1951).Samples (0.1 ml) for radioactivity measurementswere placed on aluminum planchets, mixed withfour to five drops of methanol, and dried underan infrared lamp. 'T'he planchets were countedin a Nuclear-Chicago end-window gas-flow Geigercounter (model D-47) operating in the propor-tional range.

For determination of proline-C14 incorporationinto a-amylase, the enzyme was removed fromthe culture fluid by the technique of Thayer(1953). The a-amylase was adsorbed on a 2:1soluble starch-Celite no. 535 (Johns-Manville,New York, N.Y.) mixture. The adsorbent waspacked in a column, 2.5 cm in diameter, to aheight of 1.5 cm. The adsorbent was washed with100 ml of 0.1 M sodium acetate buffer (pH 4.6).The enzyme was eluted by pulling through thecolumn, under a vacuum, 5 to 8 ml of an a-amylase digest of a 1% soluble-starch solution.The samples containing 95% of the enzymaticactivity were pooled, 0.1 ml was placed on analuminum planchet, and the radioactivity meas-ured.

Synthesis of methyl-13-D-maltoside. The proce-dure used for the synthesis of acetylbromomal-tose was that of Fischer and Fischer (1910).Pfanstiehl maltose (cp, 10 g) was mixed withacetylbromide (20 ml) in a 1-liter beaker im-mersed in an ice bath. After 10 to 15 min, thereaction was complete, and 800 ml of ice waterwere poured into the reaction beaker. The beakerwas placed at 4 C for 12 hr. rhe white powderymaterial formed was filtered on a Biuchner filterand washed four times with 800-ml portions ofice water. The acetylbromomaltose was dried in

VOL. 86, 1963 1203


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

WELKER AND CAMPBELL

TABLE 1. Effect of pH on the formation of a-amylase by Bacillus stearothermophilus*

pH (t-Amylase Percentage ofpH for(untsmi) maximal activity

4.5 0 05.0 5 65.5 16 186.0 57 656.3 68 786.5 72 816.7 87 1007.0 26 297.5 20 238.0 5 68.5 2 3

* Washed cells were suspended in R buffer atthe pH indicated with pure maltose (10-3 M) asthe inducer. After 60 min of incubation at 55 C ina water-bath shaker, the cells were removed bycentrifugation and the supernatant liquid was as-sayed for a-amylase activity under optimal con-ditions as described by Welker and Campbell(1963a).

a vacuum desiccator over NaOH and stored at-14 C.The bromo derivative (20 to 50 g) was dis-

solved in 400 ml of absolute methanol (Schoch,Wilson, and Hudson, 1942). The mixture wasdried by adding Na2SO4, filtered, and thenshaken with Ag2CO3 (20 g). The mixture washeated under reflux for 1 hr, treated with char-coal (Darco-60), filtered, and concentrated underreduced pressure. A white powder of heptaacetyl-methyl-maltoside formed and was reprecipitatedfrom ethanol. The reprecipitated product wasdried in vacuo over NaOH.

Deacetylation of the heptaacetyl-methyl-maltoside was accomplished by dissolving thecompound (20 g) in absolute methanol (20 to 30ml) and saturating with ammonia at 0 C. Themixture was placed at -14 C for 12 hr, filtered,and concentrated in vacuo to a syrup. The syrupwas dissolved in 10 ml of water and further puri-fied by large-scale ascending paper chromatog-raphy (Welker and Campbell, 1963a).The methyl-3-D-maltoside had a specific rota-

tion of [a]'4 = +82.4°, which agrees with thevalue reported by Pazur, Marsh, and Ando(1959). The methyl-13-D-maltoside was chromato-graphically pure. After three ascents in a solventcontaining n-butanol-ethanol-water (4:1:1 by

volume), the synthesized compound had an RFvalue identical with that of known methyl-j3-Dmaltoside.

Chemicals. DL-Proline-C"4 (carboxyl labeled)was obtained from Calbiochem. 8-Azaguaninewas purchased from Nutritional BiochemicalsCorp., Cleveland, Ohio. p-Fluorophenylalaninewas obtained from Sigma Chemical Co., St. Louis,Mo. L-Norvaline, 5-methyl-DL-tryptophan, L-norleucine, and D-ethionine were purchased fronmMann Research Laboratories, Inc., New York,N.Y.Mitomycin C, actinomycin D, chlorampheni-

col, and phenyl-a-D-glucoside were obtained fromSol Spiegelman, Department of MicrobiologyUniversity of Illinois.

RESULTS

Optimal conditions for a-amylase synthesis. Theeffect of pH on the synthesis of a-amylase isshown in Table 1. The pH optimum for the in-duced biosynthesis of a-amylase by washed-cellsuspensions was 6.7.The kinetics of a-amylase formation with pure

maltose as the inducer are shown in Fig. 1. a-Amylase synthesis began soon after the additionof the inducer, proceeded at a linear rate for 60min, and then leveled off.When the gratuitous inducers methyl-/3-D-

maltoside or phenyl-a-D-glucoside were used, thesame kinetics of a-amylase formation were ob-served (Fig. 1) as when pure maltose was used asthe inducer. Under these conditions, the saturat-ing concentration of inducer (pure maltose,phenyl-a-D-glucoside, or methyl-3-D-maltoside)was 10-s M. Control cells without inducer pro-duced 5.6 units per ml of ca-amylase after 150 minof incubation. The addition of glucose (2 X 1O3M) sucrose (10-s M), or glycerol (4 X 10-3 M) towashed-cell suspensions did not stimulate theformation of a-amylase.

Effect of nitrogen starvation on ca-amylase syn-thesis and amino acid pool. The depletion of theamino acid pool was accomplished by shaking cellsuspensions with 2.0% fructose for 2 hr. Table 2shows that the ability to synthesize a-amylasewas lost after 60 min of nitrogen starvation. Re-plenishment of the amino acid pools with 0.5%casein hydrolysate at this time restored 90% ofthe enzyme-forming ability. After 120 min of ni-trogen starvation, only 60% of the enzyme-form-ing ability was restored. Replenishment with

1204 J. BACTERIOI,.


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


E._

LI 40-cn

60 90TIME (MIN.)

FIG. 1. Induction of a-amylase in washed-cellsuspensions of Bacillus stearothermophilus by puremaltose, phenyl-ce-D-glucoside, and methyl-,6-D-maltoside. Cells were suspended in 100 ml of Rbuffer. Inducers were added at zero time. Maltose(10-3 M), 0; phenyl-e-D-glucoside (10-i M), A;methyl-,8-D-maltoside (10-3 M), E3.

NH4Cl (1.0%) did not restore a-amylase synthe-sis.The amino acid pool was examined for its amino

acid content after 60 and 150 min of starvation.These pools were compared with the nonstarvedamino acid pool (Table 3). Examination of Table3 reveals that the amino acid pool was signifi-cantly depleted after 60 min of starvation or in-duction. Phenylalanine, tyrosine, proline, andvaline disappeared from the pool. Starvation for150 min depleted the amino acid pool to levelswhere free amino acids could not be detected bythe methods used. Depletion of the pools wasmore complete in the case of induction with malt-ose than with starvation with fructose.

Effect of chloramphenicol and 8-azaguanine on a-amylase synthesis. Figure 2 shows that the addi-tion of chloramphenicol or 8-azaguanine to puremaltose-induced washed-cell suspensions immedi-

ately inhibited the synthesis of oa-amylase. Theresults of the addition of chloramphenicol at vari-ous times after addition of the inducer is shown inFig. 3. It may be seen that the addition of chlor-amphenicol to induced washed-cell suspensionscompletely inhibited further synthesis of enzymeregardless of the time of addition.

Effect of amino acid analogues on ao-amylase syn-thesis. Figure 4 shows that 5-methyl-DL-trypto-phan (5-MT) did not inhibit a-amylase synthesis,

TABLE 2. Effect of nitrogen starvation onthe ability of Bacillus stearothermo-

philus to form ai-amylase*

Starvation Medi-t a-Amylase Per centtime (units/ml) inhibitionmin

0 Buffer 520 NH4Cl 500 Complete 49

30 Buffer 20 39.030 NH4Cl 2530 Complete 45


90 Buffer 0 100.090 NH4Cl 0 -90 Complete 38


* Cells were suspended in a 500-ml flask con-taining 50 ml of R buffer plus 2.0o fructose andplaced on a rotary shaker at 55 C. At the de-scribed times, three samples of 5.0 ml each wereremoved and washed two times with R buffer.One sample was placed in an induction flask con-taining 30 ml of induction medium (R buffer plusmaltose, 103 M); the other two samples wereplaced in 20 ml of the appropriate replenishmentmedium (0.5% casein hydrolysate or 1.0% NH4Cl).After 20 min, cells were centrifuged, washed twotimes with R buffer, and placed in inductionflasks. Induction was carried out for 60 min.

t Complete = cells which have been replenishedby exposure to 0.5% casein hydrolysate. NH4Cl =cells which have been replenished by exposure to1.0% NH4Cl. Buffer = cells receiving no nitrogenreplenishment.

VOL . 86, 1963 1205


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

WELKER AND CAMPBELL

while ethionine, norleucine, norvaline, and p-fluorophenylalanine inhibited active a-amylaseformation by 38, 53, 72, and 80%, respectively.

Effect of p-fluorophenylalanine on the incorpora-tion of DL-proline-C'4 into a-amylase and cellularproteins. The data in Table 4 show that p-fluoro-phenylalanine inhibited the synthesis of activea-amylase by 92% and the incorporation of pro-line-C14 into a-amylase and cellular protein by 95and 74%, respectively.

Effect of mitomycin C and actinomycin D on a-amylase synthesis. Figure 5 shows that both acti-

TABLE 3. Amino acid pools of starved andnonstarved cells of Bacillus stearo-

thermophilus*

Depleted poolsNondepleted Fructose Maltose

Amino acid pool starva- induc-(0 min) tiont tion4(60 min) (60 min)

Tyrosine ......... 1.20 0 0Phenylalanine .... 3.20 0 0Lysine ........... 3.07 1.8 0.093Histidine ......... 0.23 0.15 0.075Arginine ......... 1.30 0.44 0.023Aspartic acid ..... 2.50 1.80 0.095Threonine ........ 2.00 0.19 0.01Serine ............ 2.50 0.64 0.035Glutamic acid.... 2.80 1.30 0.07Proline ........... 0.87 0 0Glycine .......... 2.15 1.20 0.065Alanine ..... 4.10 0.57 0.03Valine .......... 0.50 0 0Methionine ....... 0.30 0.15 0.023Isoleucine ........ 2.23 0.15 0.0075Leucine .......... 1.80 0.34 0.018Tryptophan ..... 0 0 0

* Cells were suspended in 20 ml of deionizedwater and heated in a boiling-water bath for 20min. The cell debris was removed by centrifuga-tion, and the supernatant fluid was decanted. Thepellet was washed once with deionized water andcentrifuged. The supernatant fluids were com-bined and taken to dryness. The amino acid con-tent of the pools was determined in the Beckman/Spinco amino acid analyzer. Results are expressedas micromoles of amino acid per 100 mg (dryweight) of cells.

t Cells were suspended in 150 ml of R buffer(pH 6.7) containing 2.0% fructose.

t Cells were suspended in 150 ml of R buffer(pH 6.7) containing 103 M pure maltose.

-

z

w

ct-j

'I

0 lTIME, HOURS

2

FIG. 2. Chloramphenicol and 8-azaguanine in-hibition of a-amylase synthesis by washed-cell sus-pensions. Cells were suspended in 100 ml of R buffercontaining pure maltose (9 X 104 M) as an inducer.Chloramphenicol (100 ug/ml) and 8-azaguanine(0.6 iumole/ml) were added 45 min after addition ofthe inducer. 8-Azaguanine, r3; chloramphenicol,0; control, +.

nomycin D and mitomycin C inhibit the synthesisof active ca-amylase.

DIscUSSioNThe kinetics of a-amylase formation in washed-

cell suspensions were shown to be linear over a60-min period and then leveled off. The levelingoff of a-amylase synthesis presumably resultsfrom the depletion of the amino acid pool. Starva-tion or induction of cells resulted in a general de-pletion of amino acids and the complete disap-pearance of phenylalanine, tryosine, proline, andvaline from the pool. Replenishment of the aminoacid pool with casein hydrolysate restored theability of the cells to synthesize a-amylase. Simi-lar results were found by Eisenstadt and Klein(1961) where starvation of Pseudomonas saccharo-philia resulted in the inability of cells to syn-thesize a-amylase. The cessation of a-amylasesynthesis, in this system, was due to thedisappearance of aspartate, glutamic acid, and

1206 J. BACTERIOL.


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


one unknown compound from the amino pool.Halvorson and Spiegelman (1953) showed thatinduction of maltozymase in Saccharomyces cere-visiae resulted in a marked decrease in cell com-ponents of the amino acid pool. Although theexperiments with starved cells do not definitelyestablish the de novo synthesis of a-amylase, theydo support this hypothesis.

100

80

60 _ t - -

40

20

20

0lf I0 1 2

TIME, HOURS

FIG. 3. Inhibition of a-amylase synthesis bychloramphenicol added at various times after induc-tion. Cells suspended in 50 ml of R buffer containingpure maltose (9 X 104 m) as an inducer. Chloram-phenicol (100 ,ug/ml) was added as follows: at timeof inducer, +; 15 min, [0; 0 min, A; control, 0.

E :w

-J

0

0 0.5 1.0TIME, HOURS

FIG. 4. Effect of amino acid analogues on a-amylase synthesis. Cells suspended in 15 ml of Rbuffer containing pure maltose (10-3 M) as inducer.The amino acid analogues (10-3 M) were added atzero time. Control, +; 5-MT, (0; ethionine, 0;norleucine, A; norvaline, *; p-fluorophenylalanine,A.

Mandelstam (1957) demonstrated that the rateof protein turnover in nongrowing cells of Escher-ichia coli is sufficient to permit a differential rateof synthesis of ,B-galactosidase identical to thatobserved in rapidly growing cells. If protein turn-over occurs in the cell suspensions of B. stearother-mophilus, it is not sufficient to support the synthe-

TABLE 4. Effect of p-fluorophenylalanine on the incorporation of DL-proline-C4 into cellularproteins and the a-amylase of Bacillus stearothermophilus*

Control p-FluorophenylalanineTime Cellular A a-Amylase Cellular a-Amylase

proteint mt activityt proteint Amt activityt

min units/ml units/ml5 167 8.4 27 39 3.6 315 300 10.8 59 211 4.0 330 750 32.0 69 296 4.8 445 860 57.0 98 249 5.2 560 1290 68.4 145 328 5.6 7

* Cells were suspended in 38 ml of R buffer containing pure maltose (10-3 M) as an inducer and pro-line-C'4 (2.1 X 104 counts per min per ml). p-Fluorophenylalanine (10-3 M) was added to one flask atzero time.

t Activity expressed as counts per minute per 10 ml.

VOL. 86, 1963 1207


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

1208 WELKER AND CAMPBELL

0 20 40 60TIME (MIN.)

FIG. 5. Actinomycin D and mitomycin C in-hibition of a-amylase synthesis by washed-cell sus-pensions. Cells were suspended in 100 ml of R buffercontaining pure maltose (10-3 M) as an inducer.Actinomycin D (10.0 ug/ml) and mitomycin C (0.5,ug/ml) were added 15 min after addition of the in-ducer. Actinomycin D, A; mitomycin C, 0; control,

sis of a-amylase or to prevent the net loss ofphenylalanine, tyrosine, proline, or valine fromthe pool during enzyme induction.The immediate inhibition of a-amylase forma-

tion by chloramphenicol, irrespective of the timeof addition to washed-cell suspensions, supportsthe proposal that the a-amylase of B. stearother-mophilus is formed de novo. If an a-amylase pre-cursor had accumulated in uninduced cells, a lagin chloramphenicol inhibition, upon induction ofthe cells, would be expected. No such lag was ob-served. Chloramphenicol has been shown toinhibit a-amylase formation by washed-cell sus-pensions of P. saccharophilia (Eisenstadt andKlein, 1961) and by various strains of B. subtilis(Nomura et al., 1958; Coleman and Elliott, 1962;Oishi, Takahashi, and Maruo, 1962).The inhibition of enzyme synthesis by amino

J. BACTERIOL.

acid analogues has been reported by several work-ers [see the reviews of Gale (1962) and Richmond(1962)]. Munier and Cohen (1956, 1959) anidCohen and Munier (1959) have shown that, insome cases, p-fluorophenylalanine does not in-hibit protein synthesis but gives rise to proteinscontaining p-fluorophenylalanine which are de-void of enzyme activity. The incorporation ofamino acid analogues into proteins can also resultin the formation of altered structures with re-duced enzyme activity. Yoshida (1960) hasshown that p-fluorophenylalanine replaced 9% ofthe phenylalanine in B. subtilis ca-amylase, result-ing in a loss of 20 to 30% of its activity. Nomuraet al. (1956, 1958) reported that a-amylase syn-thesis by B. subtilis was not affected by p-fluoro-phenylalanine; Coleman and Elliott (1962)showed, with a different strain of B. subtilis, thatthis analogue inhibited a-amylase synthesis by44 %. The inhibition of active a-amylase synthesisby amino acid analogues in our system might bedue to the synthesis of a structurally altered pro-tein with reduced activity or one devoid of en-zyme activity. However, in the case of p-fluoro-phenylalanine, this seems unlikely in view of therapidity with which this analogue inhibits activea-amylase synthesis and the concomitant inhibi-tion of proline-C14 incorporation into a-amylase.The failure of 5-methyl-tryptophan to inhibita-amylase formation is consistent with the factthat the a-amylase produced by this strain of B.stearothermophilus does not contain tryptophan(Campbell and Manning, 1961).8-Azaguanine has been shown to inhibit the

synthesis of constitutive and inducible enzymes(Creaser, 1956; Richmond, 1959; Chantrenne andDevreux, 1960). Fukumoto, Yamamoto, andTsuru (1957) and Nomura and Hosoda (1958)have demonstrated that the synthesis of a-amyl-ase by B. subtilis is inhibited by 8-azaguanine.Our data show that this analogue inhibits a-am-ylase formation by B. stearothermophilus.Although 8-azaguanine can replace as much as40% of the guanine in ribonucleic acid [(see re-view of Davis and Feingold (1962)], the mecha-nism by which it inhibits protein synthesis is notknown.Mitomycin C and actinomycin D were also

found to inhibit a-amylase formation by B. stearo-thermophilus. The mechanism of their inhibitionis not known. Actinomycin D combines withdeoxyribonucleic acid (Kawamoto and Imanishi,

-j

U)

z

U)

-Jllcn


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


1961) and thereby prevents the formation of"messenger" ribonucleic acid. Mitomycin C ap-pears to cause the degradation of deoxyribonu-cleic acid (Kersten, 1962). The net effect of bothof these polypeptide antibiotics is to stop ribonu-cleic acid synthesis [see Davis and Feingold(1962)].

It should be mentioned that in every casewhere a compound was found to inhibit a-amyl-ase synthesis the same level of inhibitor had noeffect on the a-amylase activity of the enzymeproduced in the control experiments.The combined results presented in this paper

lead us to conclude that induction of resting-cellsuspensions of B. stearothermophilus results in thede novo synthesis of a-amylase from pre-existing,low molecular weight compounds in the cells.

ACKNOWLEDGMENTS

N. E. Welker was a predoctoral trainee (2E-75;2G-510) of the National Institutes of Health, U.S.Public Health Service, during the tenure of thiswork. These studies were aided by contracts (NR108-330) between the Office of Naval Research,Biochemistry Branch, Department of the Navy,and Western Reserve University, and betweenthe Office of Naval Research and the Universityof Illinois.

LITERATURE CITED

CAMPBELL, L. L., AND G. B. MANNING. 1961. Ther-mostable a-amylase of Bacillus stearothermo-philus. III. Amino acid composition. J. Biol.Chem. 236:2962-2965.

CHANTRENNE, H., AND S. DEVREUX. 1960. Res-turation de la synth6sea d'enzymes apresinhibition par l'azaguanine. Biochim. Biophys.Acta 41:239-245.

COHEN, G. N., AND R. MUNIER. 1959. Effets desanalogues structuraux d'aminoacides sur lacroissance, la synthese de proteines et la syn-th6se d'enzymes chez Escherichia coli. Bio-chim. Biophys. Acta 31:347-356.

COLEMAN, G., AND W. H. ELLIOTT. 1962. Studieson a-amylase formation by Bacillus subtilis.Biochem. J. 83:256-263.

CREASER, E. H. 1956. The assimilation of aminoacids by bacteria. 22. The effect of 8-azagua-nine upon enzyme formation in Staphylococcusaureus. Biochem. J. 64:539-545.

DAVIS, B. D., AND D. S. FEINGOLD. 1962. Anti-microbial agents: mechanism of action anduse in metabolic studies, p. 343-397. In I. C.Gunsalus and R. Y. Stanier [ed.], The bac-teria, vol. 4. Academic Press, Inc., New York.

EISENSTADT, J. M., AND H. P. KLEIN. 1961. Evi-dence for the de novo synthesis of the alpha-amylase of Pseudomonas saccharophilia. J.Bacteriol. 82:798-807.

FISCHER, E., AND H. FISCHER. 1910. Uber einigeDerivate des Mulchzuckers und der Maltoseund uber zwei neve Glucoside. Chem. Ber.43:2521-2536.

FUKUMOTO, J., T. YAMAMOTO, AND D. TSURU.1957. Effects of carbon sources and base ana-logues of nucleic acid on the formation ofbacterial amylase. Nature 180:438-439.

GALE, E. F. 1947. The assimilation of amino acidsby bacteria. 1. The passage of certain aminoacids across the cell wall and their concentra-tion in the internal environment of Strepto-coccus faecalis. J. Gen. Microbiol. 1:53-76.

GALE, E. F. 1962. The synthesis of proteins andnucleic acids, p. 471-576. In I. C. Gunsalusand R. Y. Stanier [ed.], The bacteria, vol. 3.Academic Press, Inc., New York.

HALVORSON, H. O., AND S. SPIEGELMAN. 1953. Netutilization of free amino acids during the in-duced synthesis of maltozymase in yeast. JBacteriol. 66:601-608.

KAWAMOTO, J., AND M. IMANISHI. 1961. Mecha-nism of action of actinomycin, with specialreference to its interaction with deoxyribo-nucleic acid. Biken's J. 4:13-24.

KERSTEN, H. 1962. Zur Werkungsweise von Mito-mycin C. I. Einfluss von Mitomycin C auf denDesoxyribonucleinsaure in ruhenden Bak-terien. Hoppe-Seylers Z. Physiol. Chem.329:31-39.

LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR,AND R. J. RANDALL. 1951. Protein measure-ment with the Folin phenol reagent. J. Biol.Chem. 193:265-275.

MANDELSTAM, J. 1957. Turnover of protein instarved bacteria and its relationship to theinduced synthesis of enzyme. Nature 179:1179-1181.

MUNIER, R., AND G. N. COHEN. 1956. Incorpora-tion d'analogues structuraux d'aminoacidesdans les prot6ines bacteriennes. Biochim.Biophys. Acta 21:592-593.

MUNIER, R., AND G. N. COHEN. 1959. Incorpora-tion d'analogues structuraux d'aminoacidesdans les prot6ines bacteriennes au cours deleur synth6se in vivo. Biochim. Biophys.Acta 31:378-391.

NEIDHARDT, F. C., AND B. MAGASANIK. 1960. Stud-ies on the role of ribonucleic acid in the growthof bacteria. Biochim. Biophys. Acta 42:99-116.

NOMURA, M., AND J. HOSODA. 1958. Studies onamylase formation by Bacillus subtilis. IV.The relationship between nucleic acid syn-

1209VOL. 86, 1963


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

WELKER AND CAMPBELL

thesis and amylase formation. J. Biochem.(Tokyo) 45:123-131.

NOMURA, M., J. HOSODA, B. MARUO, AND S. AKA-BORI. 1956. Studies on amylase formation byBacillus subtilis. II. Effect of amino acidanalogues on amylase formation by Bacillussubtilis: an apparent competition betweenamylase formation and normal cellular pro-tein synthesis. J. Biochem. (Tokyo) 43:841-850.

NOMURA, M., J. HOSODA, H. YOSHIKAWA, ANDS. NISHIMURA. 1958. Studies on the mechanismof amylase formation by Bacillus subtilis.Proc. Intern. Symp. Enzyme Chem. TokyoKyoto, 1957, p. 359-366.

OISHI, M., H. TAKAHASHI, AND B. MARUO. 1962.The synthesis of a-amylase by a cell-free sys-tem from Bacillus subtilis. Biochem. Biophys.Res. Commun. 8:342-347.

PAZUR, J. H., J. M. MARSH, AND T. ANDO. 1959.The synthesis and the enzymolysis of methylglycosides of malto-oligosaccharides. J. Am.Chem. Soc. 81:2170-2172.

RICHMOND, M. H. 1959. Effect of inhibitors onlytic enzyme synthesis by Bacillus subtilis R.Biochim. Biophys. Acta 34:325-340.

RICHMOND, M. H. 1962. The effect of amino acidanalogues on growth and protein synthesis inmicroorganisms. Bacteriol. Rev. 26:398-420.

SCHOCH, T. J., E. J. WILSON, AND C. S. HUDSON.1942. The stability of 03-methylmaltoside to-ward hot alkali. J. Am. Chem. Soc. 64:2871-2872.

THAYER, P. S. 1953. The amylases of Pseudomonassaccharophila. J. Bacteriol. 66:656-663.

WELKER, N. E., AND L. L. CAMPBELL. 1963a. Ef-fect of carbon sources on formation of a-amyl-ase by Bacillus stearothermophilus. J. Bac-teriol. 86:681-86.

WELKER, N. E., AND L. L. CAMPBELL. 1963b. In-duction of a-amylase of Bacillus stearothermo-philus by maltodextrins. J. Bacteriol. 86:687-691.

WELKER, N. E., AND L. L. CAMPBELL. 1963c. In-duced biosynthesis of a-amylase by growingcultures of Bacillus stearothermophilus. J.Bacteriol. 86:1196-1201.

YOSHIDA, A. 1960. Studies on the mechanism ofprotein synthesis; incorporation of p-fluoro-phenylalanine into a-amylase of Bacillussubtilis. Biochim. Biophys. Acta 41:98-103.

1210 J. BACTERI OL.


http://jb.asm.org/

Dow

nloaded from
http://jb.asm.org/

proceeded m),(r buffer plus inducer, 10-3 m). the washed-cell suspensions were adjusted to an...

Documents