PATHWAYS OF GLUCOSE CATABOLISM IN BACILLUS SUBTILIS'

MANUEL GOLDMAN2 AND HAROLD J. BLUMENTHALDepartment of Bacteriology, The University of Michigan, Ann Arbor, Michigan

Received for publication 12 April 1963

ABSTRACT

GOLDAIAN, MANUEL (The University ofMichigan, Ann Arbor) AND HAROLD J. BLUMEN-THAL. Pathways of glucose catabolism in Bacillussubtilis. J. Bacteriol. 86:303-311. 1963.-Underaerobic conditions, resting cells of Bacillussubtilis Marburg C4 catabolized 60 to 70% ofglucose by the Embden-Meyerhof pathway andthe remainder by the hexose monophosphatepathway; under anaerobic conditions, the per-centages were 70 to 80 and 20 to 30, respectively.These estimates, based on two different radio-isotopic procedures, were the same whether thecells were grown in a glucose medium contain-ing a complex, organic nitrogen source (C cells)or a simple inorganic nitrogen source (S cells).In C cells, respiration was inhibited by fluoride,whereas S cells were relatively insensitive tothis influence. Factors such as the initial con-centration of inorganic phosphate or glucoseduring growth, and the concentration of inorganicphosphate or even the presence of fluoride duringglucose utilization by resting cells, had no majoreffects on the pathways of glucose catabolism.From an examination of the isotopic distributionin an isolated intermediate, lactic acid, it seemsunlikely that the radioisotopic estimates wereinfluenced by such other factors as participationof an Entner-Doudoroff pathway, extensiverandomization of C, activity into other positionsof the hexose molecule, or extensive CO2 fixation.

If living cells can be altered internally bychanges in external environment, then selectivelyapplied modifications might cause measurablechanges in metabolic pathways. Gary and Bard(1952) reported that the physiological behavior

1 Portion of a thesis presented by the senior au-thor in partial fulfillment of the requirements forthe degree of Doctor of Philosophy, The Univer-sity of Michigan, 1961.

2 Present address: Department of Biology,Wayne State University, Detroit, Mich.

of Bacillus subtilis strain Marburg C4 depends onthe nutritional environment, basing their con-clusions mainly on the effects of inhibitors, rateof adaptation to substrates, and the cellularcontent of certain enzymes. Growth in a Tryp-tone-yeast extract-glucose medium yielded cells(C cells) capable of vigorous respiration andfermentation, whereas growth in an inorganicnitrogen-salts-glucose medium yielded cells (Scells) which exhibited respiratory activity butpractically no fermentative capacity. The twotypes showed quantitative differences in enzymecontent: S cells had limited aldolase activityand contained no detectable glyceraldehyde-3-phosphate dehydrogenase; C cells showed 1.5to 2.0 times the aldolase activity of S cells andalso measurable amounts of nicotinamide adeninedinucleotide-linked glyceraldehyde-3-phosphatedehydrogenase activity. Supposedly, glucose wasoxidized preferentially by the hexose mono-phosphate (HMP) pathway in S cells, accountingfor the fluoride insensitivity of S cells and thefluoride sensitivity of C cells, although both Sand C cells possessed the enzyme of both theEmbden-Meyerhof (EM) and HMP pathways(Gary and Bard, 1952; Gary, Klausmeier, andBard, 1954).We wished to re-examine the quantitative

significance of these findings, using isotopictechniques to estimate the pathways of glucosecatabolism in the two cell types. A preliminaryreport of this work has been published (Goldmanand Blumenthal, 1959). Our results lead us toconclude that glucose is catabolized by the EMand HMP pathways in both S and C cells ofB. subtilis, with the EM pathway predominating.

MATERIALS AND METHODSCultures. Stock cultures of Bacillus subtilis

Marburg C4, obtained from I. C. Gunsalus,University of Illinois, Urbana, N. D. Gary,Wesleyan University, Middletown, Conn., andthe culture collection of the Department ofBacteriology, The University of Michigan, Ann

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Arbor, were prepared by incubation on Trypti-case Soy Agar (BBL) slants in screw-cap tubesfor 48 hr at 37 C followed by storage at 4 C.These cultures were routinely transferredmonthly. As a precaution, cultures were alsostored at -20 C; these were transferred ever.y6 mnonths.

Preparation of cell suspensions. Actively grow-ing cells were prepared by transfer from the4 C culture to tubes containing 10 ml of eitherthe S or C medium of Gary and Bard (1952).The cultures were aerated by bubbling sterileair, at a rate of 10 ml/min, into the mediumthrough a capillary tip placed at the bottom ofthe tube. After incubation for 10 to 12 hr at37 C, 1 ml of the culture was transferred to 200ml of the same medium, in a 250-ml Erlenmeyerflask, and incubated at 37 C for 5 to 7 hr. Sterileair was bubbled through the medium at a rateof 30 ml/min.A more convenient method of limited aeration,

giving results similar to the method just de-scribed, comprised flasks almost filled withmedium. The flasks were shaken on a New Bruns-wick rotary shaker (150 to 200 rev/min). Primaryinocula were obtained by transfer from agarslants to 40 ml of medium in 50-ml Erlenmeyerflasks. Final inocula amounting to 0.5% of themedium volume were placed in 250-ml Erlen-meyer flasks containing 200 ml of medium or inlarger Erlenmeyer flasks containing medium in80% of the flask volume.

Cells were harvested by centrifugation at 4 C,washed once (using one-half the growth volume)with 0.85% NaCl (w/v), and suspended in0.85% NaCl. Turbidity of the cell suspension, asmeasured in a Klett colorimeter using a 420-myfilter, was converted to a dry weight equivalentby means of a previously calibrated curve.

General experimental conditions. After mano-metrically measuring 02 utilization in an airatmosphere, or CO2 production in a 95% N2-5%CO2 atmosphere (Umbreit, Burris, and Stauffer,1957), 0.1 volume of 10 N H2S04 was added tothe cell suspension and the cells were then re-moved by centrifugation. In the anaerobicexperiments, lactic acid was isolated from thesupernatant liquid by continuous ether extrac-tion for 24 hr; in the aerobic experiments, aceticacid was isolated from the supernatant liquid bysteam distillation. The acids were purifiedon a column of silica gel, using a gradient of

chloroform and n-butanol to elute the acids(Bulen, Varner, and Burrell, 1952). The purifiedcompounds were converted to CO2 by combustionwith persulfate (Katz, Abraham, and Baker,1954), and radioactivity was determined asBaCI403. Lactate was decarboxylated with acidpermanganate, using the method described byAbraham and Hassid (1957).

C14 determination. CO2 contained in 2 to 3 NNaOH was converted to BaCO3 with a solutionof 10% BaCI2 and 1% NH4CI. The BaCO3 pre-cipitate was washed twice with, and suspendedin, absolute alcohol. The slurry was evenlydistributed with a capillary pipette over a tared,microporous, porcelain filter disc. After removalof excess ethanol by suction, the sample wasdried under an infrared lamp.

Radioactivity was measured as BaC'403 witha gas-flow, thin-window, Geiger-Miuller tubeand a scaler assembly equipped with an auto-matic sample changer and recorder (Nuclear-Chicago Corp., Des Plaines, Ill.). Determina-tions were made to a standard error of no morethan 3% (about 1,100 counts). Correction wasmade for background; self-absorption was cor-rected to infinite thinness from a previouslyprepared curve relating weight of sample tospecific activity (Schweitzer and Stern, 1950).

Estimation of the pathuways of glucose catabolismi.Pathways of glucose catabolism were estimatedby the method of Blumenthal, Lewis, and Wein-house (1954), which is based on the specificactivity of isolated C14-intermediates, and by theradiorespirometric method of Wang et al. (1956,1958), which is based on the yield of C'4O2 . Forour purposes, the latter method was modifiedoperationally by the simple apparatus shown inFig. 1 and by the use of resting cells instead ofgrowing cells. The flask, containing the cells,buffer, and C'4-labeled glucose (correction factorsused to equalize the amount of C'4 per flask) in afinal volume of 15 ml, was agitated on a rotaryshaker at 200 strokes/min in an incubator (37 C).All flasks were run in duplicate. At hourly in-tervals the center-well tube, which contained0.2 to 0.3 ml of 3 N NaOH, was replaced with onecontaining fresh alkali, and at this time a sampleof the medium was removed for the assay of theglucose. At the end of the experimental period,usually 3 hr, 1 ml of 10 N H?SO4 was added tothe flask; metabolic products were then isolatedand treated as previously described.

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the HMP pathway, even though extracts ofRubber stopper these cells contained enzymes of both the EM

and HMP pathways (Gary et al., 1954).Pathways of glucose catabolism. Using two

different radioisotopic procedures, we made a250ml Erlenmeyer

flaskRESPIRATION FERMENTATION

Rubber band a/150

Incubation medium A, /

V,confainin9,5e La

0 -- 0x 00

FIG. 1. Apparatus for estimation of pathways of 0 20 40 60 80 0 20 40 60 Soglucose catabolism. TIME (MINUTES)

FIG. 2. Effect of fluoride on respiration and fer-Chemical analyses. Glucose was measured mentation of C cells of Bacillus subtilis; 0, no

routinely by a chemical procedure using anthrone fluoride added; 0, 0.02 M NaF. Aerobic Warburg(Seifter et al., 1950), and in certain cases by an flasks containedS0 Amoles of sodium phosphate bufferenzymatic method using glucose oxidase (Worth- (pH 7.0), 6umoles of glucose, 0.2 ml of 20% KOH inington Biochemical Corp.). Barker and Sum- center well, 5 mg (dry weight) of washed cells inmerson's (1941) method was used to determine 0.95% NaCl, water to give a final volutme of 2.2 ml;lactate. air atmosphere. Anaerobic flasks contained bicar-*. . ~~~~~~~~~bonate-CO2 buffer, pH 7.0 (20 Mlmoles of sodium bi-Chemicals. All reagents used were of analytical cbonate) ,6720molesofg ,dr ig of

gradeorothehighst prityavaiablecom-carbonate), 5 jAmoles of glucose, 5 mg (dry weight) ofgrade or of the highest purity available com- washed cells in 0.85% NaCl, water to give a finalmercially. volume of 3.2 ml; 95% N2-5% CO2 atmosphere.

Glucose-i- and -6-C'4 were obtained from theNational Bureau of Standards, Washington,D.C. All other C'4-labeled substrates were ob- RESPIRATION FERMENTATIONtained from Volk Radiochemical Co., Skokie, Ill.

0

RESULTS 300

Respirometric studies. Respiratory and fermen- 2250tative activities of C cells were strongly inhibited S- /by 0.02 M sodium fluoride (Fig. 2), while the .L200 c 1

same concentration of fluoride had little or no = /z/1t50effect on the respiration of S cells (Fig. 3), con- / 0'

firming previously reported results (Gary and 0o 0Bard, 1952). However, the relatively high fermen- , 1 0'tative activity of S cells which we noted was in ° 50 / .0contrast to the results reported by Gary and 0

Bard (1952). The high rate of glucose fermenta- 00 20 40L 60 20 40 60 0tion in S cells from all three strains of Marburg TIME (MINUTES)C4 was strongly inhibited by fluoride (Fig. 3). FIG. 3. Effect of fluoride on respiration and fer-The absence of fluoride inhibition of glucose mentation of S cells of Bacillus subtilis; 0, nooxidation by S cells, and other indirect evidence, fluoride added; ,0.02 M NaF. The protocol was theled Gary and Bard (1952) to conclude that same as that listed for Fig. 2. Identical results wereglucose was catabolized aerobically in S cells obtained with two other strains of B. subtilis Mar-solely by a fluoride-insensitive system, supposedly burg C4.

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TABLE 1. Estimates of the pathways of glucose catabolism in Bacillus subtilis based on the isolation ofC'4-intermediates

RSAa of intermediate C2 or C3 GlucoseCell type formed from Corrected units by utilizedRSA EM by EM

Glucose-U-C"4 Glucose-i-C'4 pathwayb pathwayc

Aerobic (acetate-isolated) dS......0......0.244 0.289 1.18 79 65C ..................... 0.383 0.426 1.11 74 59

Anaerobic (lactate-isolated)S.. ..... 0.866 0.711 0.820 82 70C.. 0.910 0.793 0.872 87 77

a RSA (relative specific activity) equals (specific activity of acetate or lactate carbon atoms)/(spe-cific activity of initial glucose-C'4 carbon atoms).

b Percentage of C2 units by EM (Embden-Meyerhof) = (corrected RSA/1.5) X 100; percentage ofC3 units by EM = corrected RSA X 100.

c Percentage of glucose by EM = (% C2 or C3 units by EM)/200 - (% C2 or C3 units by EM).d Aerobic flasks contained either 650 ,Amoles of potassium phosphate buffer (pH 7.0) or 225 Mumoles

of sodium maleate buffer (pH 7.0), 50,umoles of glucose containing about 50,000 counts/min, 100 mg(dry weight) of resting cells in 0.85% NaCl, 1 ml of 20% KOH in the center well, and water to give atotal volume of 15 ml; air atmosphere.

e Anaerobic flasks contained bicarbonate-CO2 buffer, pH 7.0 (150 ,moles of sodium bicarbonate),50,moles of glucose containing about 50,000 counts/min, 100 mg (dry weight) of resting cells in 0.85%NaCl, and water to give a total volume of 15 ml; 95% N2-5% CO2 atmosphere.

TABLE 2. Estimtates of the pathways of glucosecatabolism in Bacillus subtilis based on C1402

evolutiona

Cell C1402 from C"402 from Total C14 Glucose Glucoseglucose-I- glucose-6- adminis- by bytype C'4 C14 teredb HMPC EMd

counts/min counts/min counts/min % %

S 20,240 7,705 48,000 26 74C 18,350 900 48,000 36 64

a The protocol was identical to that in Table 1except that anaerobic conditions were not tested.

bBased on the amount of glucose utilized fromthe medium.

Per cent glucose by HMP =(C"402 from glucose-i-C'4) -

(C'402 from glucose-6-C'4) X 100.total C14 administered

d Per cent glucose by EM = 100 - per centglucose by HMP.

quantitative estimate of the percentage of glucosecatabolized by the EM and HMP pathways(Tables 1 and 2). Aerobically, the EM pathwayaccounted for 65 to 75% of the glucose used byS cells and 60 to 65% of that used by C cells.Anaerobically, the estimates of glucose catab-olism were similar to those obtained aerobicallyexcept that the participation of the EM pathway

was generally slightly greater. Our estimates ofglucose catabolism in resting cells agree generallywith those reported by Wang et al. (1958) andWang and Krackov (1962), who used growingcells of B. subtilis strain Marburg and a radio-respirometric method.

Attempts to modify pathways of glucose catab-olism. Certain factors were examined for theirpossible effect on the relative extent of glucoseutilization by the HMP pathway in S cells.

1) Effect of fluoride:-Even when S cells usedglucose aerobically in the presence of 0.02 MNaF, little change occurred in the relative degreeof glucose utilization by the HMP pathway(Table 3). Glucose was still catabolized mainlyby the EM pathway.

2) Effect of inorganic phosphate:-Kravitzand Guarino (1958) found that inorganic ortho-phosphate (Pi) apparently influenced the extentof participation of the EM and HMP pathwaysin glucose catabolism by cell-free extracts ofEhrlich mouse ascites cells; relatively high Pilevels favored the EM system, while relativelylow Pi levels favored the HMP pathway. Theorell(1935) showed that Pi inhibited glucose-6-phosphate dehydrogenase. The necessary rolefor Pi in the conversion of 3-phosphoglyceralde-hyde to 1,3-diphosphoglyceric acid is well

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TABLE 3. Effect of fluoride on pathways of glucosecatabolism of Bacillus subtilis, S cells*

NaF C1402 from C'402 from Total C14 Glucose Glucose(0.02 M) glucose-l- glucose-6- adminis- by by(00) C14 C14 tered HMPt EMt

counts/min counts/min counts/min % %O 27,975 13,015 48,000 31 69+ 30,950 13,150 48,000 37 63

* The protocol was the same as that for Table 1for aerobic conditions except for the addition offluoride.

t See the method of calculation in Table 2.

known. Cells grown in S medium containingrelatively low anmounts of Pi might thus utilizethe HMP pathway to a much greater extentthan normally grown cells.

Cells were grown in S medium containing0.001 the normal concentration of Pi, the pHbeing maintained at about 7 by adding sterileNaOH at appropriate intervals. UTnder theseconditions of low Pi, which might favor theactivity of the HMP pathway, glucose was stillutilized predominantly by the EM pathway(Table 4) even though the respiratory activity ofthe cells was only 20% that of normal S cells(Table 5). The addition of Pi, at levels incorpo-rated in normal S medium, to resting cells grownin S medium containing low concentrations of Piresulted in only a 10% increase in utilization ofglucose by the EM pathway (Table 4).

3) Eftect of glucose:-Some workers, usingEhrlich ascites tumor (Racker, 1956) or lactatingmammary gland of the rat (McLean, 1960), foundthat increased conversion of glucose Cl to C02,indicative of greater HMP activity, resultedfrom an increased concentration of glucose in

the medium. Others, however, working withEhrlich ascites tumor (Wenner, Hackney, andMoliterno, 1958) or polymorphonuclear leuko-cytes from guinea pigs (Evans and Karnovsky,1962), found no increased conversion of glucoseCl even over a wide range of glucose concentra-tions. Apparently the effect of the concentrationof glucose in the growth medium on the pathwaysof glucose catabolism in the resulting cell sus-pensions has not been studied. We observed nodifferences in resting, vegetative S cells utilizingglucose at an initial concentration of about0.02%, whether they were first grown in 0.1%or 1% (normal S medium) concentrations ofglucose; the relative amount of glucose catab-olized aerobically by the EM pathway was still60 to 70% in either case.

Possible factors affecting the estimation of

TABLE 5. Effect of orthophosphate concentration in Sgrowth medium on the subsequent respiration of

resting cells in an orthophosphate-freebuffer*

Fraction of orthophosphate Qo relative to controlof regular S growth mediumf 2

0.001 200.01 760.1 1001.0 100

* The protocol was the same as that listed inFig. 2 except that sodium maleate buffer replacedsodium phosphate buffer. The cells were grownin the phosphate-poor S medium as described inthe text.

t The concentration of orthophosphate in theregular S growth medium is 0.03 M.

TABLE 4. Effect of orthophosphate on pathways of glucose catabolism of S cells g-own in 0.001 the normalconcentration of orthophosphate*

Orthophos- Ex*mt C1402 from glucose- C1402 from Total C14 Glucose by Glucose byphate (0.03 m) xpermen -C4 glucose-6-C14 administeredt HMPI EMt

counts/min counts/min counts/min % %0 1 18,530 2,825 45,400 3529 651

2 18,160 7,755 48,000 22f 78f

+ 1 10,700 2,188 38,000 22119 781812 13,050 5,355 47,100 16f 84f

* The protocol was the same as that in Table 1 for aerobic conditions except for the change in thefinal concentration of orthophosphate added to the buffer (maleate).

t Based on the am6unt of glucose utilized from the medium.I See the method of calculation in Table 2.

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pathways of glucose catabolism. The presence ofthe Entner-Doudoroff (ED; 1952) pathway as a

third pathway for glucose catabolism, or exten-sive recycling in the HMP pathway and thepossible randomization of Cl activity into otherpositions of the hexose molecule by the reversi-ble transketolase-transaldolase reactions, wouldaffect the validity of the present calculations.Additionally, any extensive CO2 fixation intothree-carbon intermediates, as reported by Neish(1953) for B. licheniformis (subtilis), might alsoaflect the estimations. The presence of a com-

plete ED system has been observed so far onlyin gram-negative bacteria (Entner and Doudoroff,1952; Gibbs and DeMoss, 1954; Wang et al.,1958; Vardanis and Hochster, 1961; Taylor andJuni, 1961; Jyssum and Joner, 1962). In gram-

positive bacteria, there is only some incompleteevidence for the presence of the enzymes of thissystem in B. cereus (Halvorson and Church,1957; Doi, Halvorson, and Church, 1959).Experiments were performed to examine the

above possibilities, based on the distribution ofC'4 in isolated, labeled three-carbon intermediatesfrom glucose-i-, 6-, or uniformly labeled (U) C14.There was little difference in the percentage ofglucose catabolized by a given pathway, anaero-

bically or aerobically, and lactic acid could beobtained anaerobically without resorting topoisons (Dawes and Holms, 1958) or pools ofunlabeled intermediates (Blumenthal et al.,1954). The rationale for these experiments isgiven in Table 6.

Performance of the experiment anaerobicallycould minimize the question of activity in thecarboxyl group (using glucose-i-C'4), due to an

exchange reaction between the C'402 and thecarboxyl group of lactate, since relatively little

TABLE 6. Rationale for the estimation of pathwaysof glucose catabolism

Pathway Lactate RSA, Distribution of C14 intheory lactate, theory

Glucose-i C14100% EM 1.0 Lactate-3-C14100% HMP 0 One unlabeled

lactate100% ED 1.0 Lactate-i-C14

Glucose-6-C'4100% EM 1.0 Lactate-3-C14100% HMP 2.0 Lactate-3-C14100% ED 1.0 Lactate-S-C'4

TABLE 7. Distribution of C14 in lactate from S cellscatabolizing glucose-C'4 anaerobically*

C'4 in Total C14 in~Lactate from Total C14 in carboxyl carboxyl oflabeled glucose lactate group of lactate

lactate

counts/lmin counts/min %

Glucose-U-C'4 1,752 566 32.3Glucose-i -C14 1,278 41 3.2Glucose-6-C'4 1,599 14 0.9

* The protocol was the same as that in Table 1.Separate samples were used for the determina-tion of total counts and counts in the carboxylgroup of lactate.

C1'402 would be formed and the presence of analkali trap would further reduce the chance ofCO2 fixation. Degradation of the lactate andsubsequent determination of the activity in thecarboxyl group would then indicate the extentof operation of the ED pathway. However, theseresults could not be considered valid if there wasextensive recycling in the HMP pathwav orrandomization of C, activity into other positionsof the hexose molecule prior to entry into theglucose catabolic pathways.

Duplicate 125-ml Warburg flasks, containingglucose-U-, -1-, or -6-C'4 and buffer, were pre-pared with NaOH in the center well and wereflushed with nitrogen gas. Sodium maleate asthe buffer interfered with the isolation andpurification of lactate by silica-gel chromatog-raphy. Of several buffers tested to correct thissituation, 0.05 M imidazole-HCl (pH 7.0) wasfinally chosen. Cells in this buffer had excellentglycolytic activity, and an estimate of the path-ways of glucose catabolism of S cells gave re-sults similar to those previously obtained.About 3% of the total activity from glucose-

i-C'4 appeared in the carboxyl group of theisolated lactate when S cells catabolized glucoseanaerobically (Table 7). These data suggested noextensive operation of the ED pathway, sinceC, activity of glucose would have been retainedin the carboxyl group of a three-carbon inter-mediate such as lactate. CO2 fixation apparentlyplayed only a slight role, since 0.9% of theactivity of glucose-6-C'4 appeared in the carboxylgroup of lactate (Table 7). When this 0.9%correction for CO2 fixation was applied to theglucose-i-C14 data, the possible role of the EDpathway was made even less significant, i.e.,only 2.3% C14 in the carboxyl group; therefore,

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a maximum of about 2% of the glucose could becalculated to be metabolized by the ED pathway.

Randomization of C, activity of glucose-i-C14into other positions of the hexose molecule(especially positions 3 and 4) by reversibletransketolase-transaldolase reactions prior tosubsequent entry into pathways of glucosecatabolism or recycling by the HMP pathway(or both) were likewise considered not to occursignificantly under these conditions. If randomi-zation into positions 3 and 4 of hexose had oc-curred significantly, then subsequent catabolismby the EM pathway (estimated at 80 and 90%by the intermediate and CO2 methods, respec-tively) would have resulted in large amounts ofcarboxyl-labeled lactate.

DISCUSSION

We conclude that, with regard to the relativeextent of participation of the EM and HMPpathways during the aerobic or anaerobic catab-olism of glucose, no major difference exists be-tween cells grown under differing nutritionalconditions (S and C cells). Although methods forestimating the pathways of glucose metabolismusing glucose-C'4 are not entirely adequate (seeKatz and Wood, 1960), the results of our studyshowed good agreement between the estimatesbased on the isolation of C'4-intermediates(Blumenthal et al., 1954) and the yield of C0402(Wang et al., 1958).As did Gary and Bard (1952), we too demon-

strated respiratory insensitivity of S cells tofluoride. Such insensitivity previously had beentaken to mean that only the HMP pathway wasoperative in S cells. But in our aerobic experi-ments the presence of fluoride did not markedlyalter the relative extent to which glucose wascatabolized by S cells through the EM pathway,about 70%.

Nonetheless, there is evidence which does sug-gest some physiological difference(s) between Sand C cells. While S and C cells catabolizedglucose similarly, aerobically and anaerobically,the respiratory and fermentative activities of Ccells and the fermentative activities of S cellswere strongly inhibited by fluoride. Such differ-ences in fluoride sensitivity might be related to(i) differences in permeability to fluoride (Volk,1954), (ii) differences in transport mechanisms(Ramos et al., 1962), or (iii) the presence ofsuitable hydrogen acceptors, such as an amino

acid or nitrate (Hofsten, 1961b), even in thepresence of intracellular fluoride (Hofsten,1961a). We have not determined which, ifany, of these conditions were involved in ourstudy.

Suffice it to say that unless other factors areconsidered, insensitivity of an intact cell to anagent such as fluoride should not necessarilyimply the operation of an alternative pathwayor an innate resistance to the agent by a particu-lar enzyme or set of enzymes.We were unsuccessful, in a limited number of

attempts, in significantly altering the relativeextent of participation of the EM and HMPpathways during the catabolism of glucose.Variation in the concentration of glucose or Piduring growth, or the concentration of Pi orthe presence of fluoride during glucose oxidationby resting cells, failed to cause major changes inthe pathways utilized. Even when the Qo, ofS cells was markedly reduced, as in the case ofcells grown in a medium which was relativelylow in Pi, the percentage of glucose catabolizedby the HMP and EM pathways still did notdiffer greatly from that found in S cells grownat much higher Pi levels. Minor changes in thepathways probably occurred as a result of theenvironmental modifications, but presentmethods are not precise enough to detect suchsubtle changes.

Previous estimates of the pathways of glucosecatabolism in resting-cell suspensions of B.cereus indicated that only 1 to 2% of the glucosewas utilized by the HMP pathway, the EM path-way accounting for the remainder (Blumenthal,1961). In spores derived from such vegetativecells, however, the HMP pathway increased toabout 25% after spore germination and thengradually decreased, reaching the 1 to 2% HMPlevel upon completion of their first cell division(Goldman and Blumenthal, 1960). These resultssuggest that the pathways of glucose catabolismare not the same in the vegetative cells of allBacillus species, and that the degree of utiliza-tion of these alternate pathways in a given speciesis capable of marked change.The participation of the EM pathway in

intact B. cereus spores was confirmed by demon-stration of the necessary EM enzymes in cell-free spore extracts (Goldman and Blumenthal,1961). Previously, several key EM enzymes werereported to be missing from B. cereus spore

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extracts (Halvorson and Church, 1957; Doi et al.,1959). These findings also serve to emphasizethe usefulness of the estimations of the pathwaysof glucose catabolism in intact cells in comple-menting studies with cell-free extracts and viceversa.

ACKNOWLEDGMENTS

This work was supported in part by researchgrants from the U.S. Public Health Service(Al-01443), the Michigan Memorial PhoenixProject, and the National Science Foundation(G-14331).

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