Biochem. J. (1974) 144, 503-511Printed in Great Britain

The Biosynthesis of Granulose by Clostridium pasteurianum

By ROBERT L. ROBSON, R. MOYRA ROBSON and J. GARETH MORRISDepartment ofBotany and Microbiology, School ofBiological Sciences,

University College of Wales, Aberystwyth S Y23 3DA, U.K.

(Received 10 July 1974)

"1. Mutant strains of Clostridium pasteurianum were obtained, which are unable tosynthesize granulose (an intracellularly accumulated amylopectin-like ac-polyglucan).2. These mutants lacked either (a) ADP-glucose pyrophosphorylase (EC 2.7.7.27), or (b)granulose synthase (i.e. ADP-glucose-a-1,4-glucan glucosyltransferase, EC 2.4.1.21).3. Although both of these enzymes were constitutively synthesized by the wild-typeorganism, massive deposition of granulose in a sporulating culture coincided with athreefold increase in the specific activity of ADP-glucose pyrophosphorylase. 4. Thesoluble ADP-glucose pyrophosphorylase was partially purified (33-fold). Its ATP-saturation curve was not sigmoidal and its activity was not enhanced by phosphorylatedintermediates of glycolysis, pyruvate, NAD(P)H or pyridoxal 5'-phosphate. ADP atrelatively high concentrations acted as a competitive inhibitor (K, = 19mM). 5. Thedependence of granulose synthase on a suitable polyglucan primer was demonstrated byusing enzyme obtained from a granulose-free mutant strain (lacking ADP-glucose pyro-phosphorylase). 6. Partial purification of granulose synthase from wild-type strains wasfacilitated by its being bound to the native particles of granulose. No activator wasdiscovered, but ADP, AMP and pyridoxal 5'-phosphate were competitive inhibitors,ADP being most effective (K, about 0.2mM). 7. It would appear that the synthesis ofgranulose in Cl. pasteurianum is not subject to the positive, fine control that is a featureof glycogen biosynthesis in most bacteria.

Several species of Clostridium synthesize granulose,an a-polyglucosan whose structure resembles thatof amylopectin (Gavard & Milhaud, 1952; Whyte &Strasdine, 1972), or may be less branched (Laishleyet al., 1974). Electron-microscopic studies haveshown this material to be accumulated intracellularlyin the form oflarge granules (Mackey & Morris, 1971;Rousseau et al., 1971; Krasilnikov et al., 1972;Laishley et al., 1973), whose presence in theseobligately anaerobic bacteria is readily evidenced bytheir intense blue-black staining with I2.

In batch culture in a defined, minimal mediumcontaining only glucose, NH4+ ions, salts andvitamins, sporulation of a wild-type strain of Clos-tridium pasteurianum ATCC 6013 was accompaniedby massive deposition ofgranulose(Mackey& Morris,1971). When it was further found that (a) Clostridiumpasteurianum NCIB 9486, which sporulated poorlyunder the same batch culture conditions, did notaccumulate granulose in such large quantity, and(b) granulose-negative mutant strains were virtuallyunable to sporulate in the usual glucose-rich minimalmedium (Morris & Robson, 1973), it seemed likelythat knowledge of the route and means of regulationof granulose biosynthesis could contribute to ourunderstanding of the metabolic state of the organismat that time when sporulation is initiated,

Vol. 144

In prokaryotes, biosynthesis of the related poly-glucosan, glycogen, proceeds via synthesis andutilization of ADP-glucose, i.e.:

Glucose 1-phosphate+ATP ADP-glucose+PP,(i)

ADP-glucose+ a-1 ,4-glucanADP+ a-1,4-glucosyl glucan (ii)

Reaction (i) is catalysed by ADP-glucose pyrophos-phorylase (i.e. glucose 1-phosphate adenyltransferase,EC 2.7.7.27) and reaction (ii) by glycogen synthase(i.e. ADP-glucose-a-1 ,4-glucan 4-glucosyl transferaseEC 2.4.1.21). The elegant studies of Preiss and co-workers (Preiss, 1969; Preiss et al., 1973) have shownbacterial synthesis of glycogen to be regulated byfine control ofADP-glucosepyrophosphorylase activ-ity. This contrasts with the situation in eukaryoteswhere glycogen is synthesized from UDP-glucose andglycogen synthetase is the locus of regulation.

- It is the chiefpurpose ofthe present paper to reportthat although granulose synthesis in Cl. pasteurianumproceeds via ADP-glucose, we have been unable todetect any allosteric fine control of the ADP-glucosepyrophosphorylase of this organism. Nor is thegranulose synthase subject to sophisticatedregulation.

503

R. L. ROBSON, R. M. ROBSON AND J. G. MORRIS

Part of this work has been reported in a preliminaryform (Robson et al., 1972).

Materials

Organisms

The sporulating strain of Cl. pasteurianum (W-5)A.T.C.C. 6013 was kindly supplied by Mrs. WinifredEgo, University of Hawaii, Honolulu. Cl. pasteur-ianum N.C.I.B. 9486 was obtained from the NationalCollection of Industrial Bacteria, Aberdeen, U.K.

Enzymes

Alkaline phosphatase (EC 3.1.3.1, type Ills fromEscherichia coli), a-amylase (EC 3.2.1.1), deoxyribo-nuclease (EC 3.1.4.5) inorganic pyrophosphatase(EC 3.6.1.1), muramidase (EC 3.2.1.17, grade 1) andribonuclease (EC 3.1.4.22, type X1-A) were obtainedfrom Sigma (London) Chemical Co., Kingston-upon-Thames, Surrey, U.K.

Chemicals

Crystalline bovine serum albumin, N-methyl-N'-nitro-N-nitrosoguanidine, nucleotide sugars andTrizma base were obtained from Sigma (London)Chemical Co. Adenine nucleotides, coenzymes andsugar phosphates were purchased from BoehringerCorp. (London) Ltd., Ealing, London, U.K. DEAE-celluloses were obtained fromWhatman BiochemicalsLtd., Maidstone, Kent, U.K., and Sephadex G-25and G-200 were from Pharmacia Fine Chemicals,Uppsala, Sweden. All radioactive chemicals wereobtained from The Radiochemical Centre, Amer-sham, Bucks., U.K. Amylopectin, dithiothreitol,glycogen and all AnalaR grade reagents were pur-chased from BDH Chemicals Ltd., Poole, Dorset,U.K. Reinforced Clostridial Medium and agar werebought from Oxoid Ltd., London E.C.4, U.K.

Methods

Maintenance andgrowth oforganisms

Washed spore suspensions of Cl. pasteurianum(strain W-5), sub-strains ATCC 6013 and NCIB 9486were prepared as described by Mackey & Morris(1971) and were stored at 2°C. Samples of thesespore suspensions were 'heat-activated' by incubationat 80°C for 10min in sterile, capped test-tubes, beforebeing employed as inocula of suitable volumes ofgrowth medium. The minimal medium contained(per litre), 40g of glucose, 3g of NH4CI, 0.1g ofMgSO4,7H20,0.1 gofNaC,O0.01 gofNaMoO4,2H20,0.01 g of CaCl2, 0.015g of MnSO4,4H20, 0.275g of

ferric sodium sequestrate, 0.121 g of Trizma base,2mg of p-aminobenzoic acid and 0.12mg of biotin.Some 2 drops of aq. 0.05% Methylene Blue wereadded immediately before sterilizing by autoclaving,and after cooling, 1M-potassium phosphate buffer,pH7, was aseptically added to a final concentrationof 0.05M. Freshly prepared medium in an almostcompletely filled 500ml of Ehrlenmeyer flask wasinoculated with heat-activated spores (to give 106spores/ml). The flask was then sealed with a sterile'Suba-Seal' stopper, and with a hypodermic needleattached to a sterile gas filter theatmosphere above theculture was aseptically replaced by 02-free N2+C02(95:5). After overnight incubation at 37°C, thisculture served as an inoculum for a similar 81itreanaerobic culture. For experiments in which thecourse ofgrowth and sporulation were to be measured,a 350ml culture was incubated anaerobically in thefermenter vessel described by O'Brien & Morris(1971).

Mutagenesis

(i) With N-methyl-N'-nitro-N-nitrosoguanidine.Organisms harvested by aseptic centrifugation fromanaerobic cultures in the mid-exponential phase ofgrowth, were resuspended in 25mM-sodium citratebuffer, pH 5.5, to a density of 107cells/ml. The culturesupernatant was transferred to a sterile McCartneybottle and was maintained anaerobically at 37°C.Then 1 ml ofa freshly prepared solution ofN-methyl-N'-nitro-N-nitrosoguanidine (1 mg/ml) in citratebuffer was added to lOml of the cell suspension, andthe mixture was then incubated aerobically for 15minat 371C in a 50ml centrifuge tube. The organismsrecovered by centrifugation at 20000g for 10min at5°C were resuspended in 40ml of the original culturesupematant and allowed to 'grow out' anaerobicallyat 370C.

(ii) With u.v. irradiation. A 10ml sample of anexponentially growing, anaerobic culture (containingabout 107organisms/ml) was exposed to u.v. irradia-tion from a germicidal lamp (30W placed 10cmabove the culture) until 99.9% of the population waskilled. The survivors were allowed to resume anaero-bic growth in the dark at 37°C.

Isolation ofgranulose-negative mutants

Cultures 'grown out' for at least two generationsafter mutagenesis, were plated on glucose minimalmedium solidified with 2% (w/v) agar (Oxoid no. 1).The petri dishes were incubated anaerobically for3 days at 370C [in anaerobic jars containing an02-free atmosphere of H2+N2+CO2 (approx.5:43:2)]. When the opened dishes were then invertedover I2 crystals, the colonies of granulose-negativemutants were identifiable by their being unstained by

1974

504

BIOSYNTHESIS OF GRANULOSE IN CL. PASTEURIANUM

the sublimed I2 vapour. Provided that exposure to I2was not over-prolonged (generally less than 1 min),these mutant strains could be directly subculturedon to anaerobic slopes of agar-solidified minimalmedium.

Preparation of cell extracts

Organisms harvested by centrifugation at 3°C(approx. 100OOg for 10min) were washed andresuspended in0.05M-Tris-HCl buffer, pH8, contain-ing 0.5mM-dithiothreitol. The resultant suspension(about 0.5g wet wt. of organisms/ml) was disruptedby anaerobic passage at 1.4x 108Pa through a chilledFrench pressure cell (Aminco, Silver Spring, Md.,U.S.A.). The process was repeated until microscopicexamination revealed that adequate disruption hadbeen achieved.

Assay methods

Glucose. Glucose was assayed colorimetrically bythe standard 'Glucostat' procedure (Teller, 1956).

Granulose. Granulose was determined as glucoseliberated by acid hydrolysis ofwell-washed organisms.The suspension of organisms in 1 M-H2SO4 washeated at 100°C for 3h and neutralized with KOH.

Growth measurements. (1) Cell counts were donemicroscopically by using an 'improved Neubauer'counting chamber. (2) Culture density was measuredspectrophotometrically at 680nm with a UnicamSP. 600 spectrophotometer. (3) Dry weight deter-minations were done after centrifuging the culturesample at 15000g for 5min and twice washing thecell pellet in water. The washed suspension was thentransferred to a pre-weighed foil container and driedat 105°C to constant weight. (4) Viable counts weredone by the spread-plate technique by using plates ofReinforced Clostridial Agar (Oxoid Ltd.), suitabledilutions being prepared in sterile 'bacterial Ringer'solution (Wilson, 1922). The plates were incubatedanaerobically at 37°C for 3 days before the colonieswere counted.

Protein. Soluble protein was generally measuredby the method of Lowry et al. (1951); a standardcurve was prepared with crystalline bovine serumalbumin (fraction V). Protein in fractions obtainedby column chromatography was assayed by themethod of Warburg & Christian (1941).

Radioactivity. Radioactivity was measured byscintillation counting with a Beckman LS-200Bspectrometer. Aqueous samples (less than 0.5ml)were counted for radioactivity with negligible quench-ing in 10mi amounts of a toluene-Triton X-100-2,5-diphenyloxazole mixture consisting of 4g of2,5-diphenyloxazole (PPO) and 500ml of TritonX-100 in toluene (to a final volume of 1 litre). Driedfilter-paper or anion-exchange-paper discs were

Vol, 144

submerged in 10mI amounts of a toluene-PPOmixture containing 4.8g of PPO/litre of toluene.

Enzyme assays

ADP-glucose pyrophosphorylase. The rate of syn-thesis of ADP-[14C]glucose from ['4C]glucose 1-phosphate and ATP was measured by the methoddescribed by Shen & Preiss (1964). The reactionmixture, which was incubated for 10min at 37°C,contained (in0.2ml): 2umol ofATP, 41umol ofMgCI2,0.172,umol of D-['4C]glucose 1-phosphate (sp. radio-activity 3puCi/4umol), 20jumol of Tris-HCl buffer,pH8.0, 1 unit of inorganic pyrophosphatase andenzyme. The reaction was terminated by heating themixture in a boiling water bath for 30s. Alkalinephosphatase (lOunits) was then added and themixture was incubated for 1 h at 37°C. Duplicate 10l1samples of the phosphatase-treated preparation wereapplied to discs (25mm diam.) of DEAE-cellulose(Whatman DE-81). One disc was thoroughly washedwith water by using a Millipore filtration system(Millipore Corp., Bedford, Mass., U.S.A.) and thenboth discs were dried under an i.r. lamp and countedfor radioactivity.

Granulose synthase. The rate of incorporation ofradioactivity from ADP-[14C]glucose into a-1,4-glucan was measured by the method of Greenberg &Preiss (1965). The reaction mixture, incubated at25°C for 10min, contained (in 0.2ml): 0.05,umol ofADP-[(4C]glucose (0.1 #uCi/pmol), 0.05,ug of amylo-pectin, 5umol of Tris-HCI, pH8, 1 pmol of MgCI2and enzyme. The reaction was stopped by theaddition of 2ml of cold 75% (v/v) methanol con-taining 1 % (w/v) KCI. The mixture was left for10min atO°C and then centrifuged at 3000gfor 10min.The pellet was washed three times by suspension in,and re-centrifugation from, 5ml volumes ofmethanolwith KCI. It was finally resuspended in 50 p1 of 0.1 M-NaOH for assay of its radioactivity.

Enzyme purification

ADP-glucose pyrophosphorylase from wild-typestrains of Cl. pasteurianum. Cultures (81) wereharvested during late exponential growth, by centri-fugation at 100OOg (ra,. 10cm) for 10min at 3C. Theorganisms were twice washed by suspension in, andre-centrifugation from, 25mM-Tris-HCI buffer,pH8. The washed cell pellet (approx. 30g wet wt.)was resuspended under an atmosphere of H2, in5OmM-MgCI2 containing 1 mM-dithiothreitol, to givea suspension containing 0.5g wet wt. of organisms/ml. To this were added 20mg ofmuramidase (approx.4 x I0O units; 1 unit of muramidase will produce aAE450 of 0.001/min at pH6.24 at 25°C in a 2.6mlsuspension of Micrococcus lysodeikticus), 5mg ofdeoxyribonuclease (approx. 7x 103Kunitz units;

505

R. L. ROBSON, R. M. ROBSON AND J. G. MORRIS

1 Kunitz unit will produce a AE260 of 0.001/min perml at pH5.0 at 25°C) and 5mg of ribonuclease(approx. 500Kunitz units). The suspension wasincubated at 25°C for 30min and then centrifugedanaerobically at 20000g (ray. 7.6cm) for 15min at3°C. The clear, brown supernatant was used as thesource ofthe 'crude' ADP-glucose pyrophosphorylaseand could be stored frozen at -30°C under H2 withoutloss of activity. All subsequent operations werecarried out at 0-40C.

Step 1. To 40ml of the cell extract was slowlyadded 10.03g of finely ground solid (NH4)2SO4(low in heavy metals), the mixture being stirredthroughout at 0°C. The final suspension [45%(NH4)2SO4 satn.] was left at 0°C for 30min, and wasthen centrifuged at 20000g for 15min at 3°C. To thesupematant was slowly added a further 3.2g of(NH4)2SO4 (to achieve 55% satn.). A precipitate wasobtained which, after being left at 0°C for 30min, wasrecovered by centrifugation, dissolved in 10ml of25mM-Tris-HCI buffer, pH8, containing 1 mM-dithio-threitol (i.e. Tris-dithiothreitol buffer), and desaltedby passage through a column (250mmx 15mm) ofSephadex G-25 previously equilibrated with Tris-dithiothreitol buffer.

Step 2. A 15ml sample of the Sephadex G-25eluent was applied to a column (185mmx20mm) ofDEAE-cellulose (Whatman DE 23), which hadpreviously been equilibrated with Tris-dithiothreitolbuffer, pH8. Fresh buffer (60ml) was run in, and alinear gradient of increasing concentration of Mg2+ions in Tris-dithiothreitol buffer, pH8 (0-0.4M-MgCI2 in 500mI) was then applied. Fractions (5nml)of the eluent were serially collected, and those whichcontained ADP-glucose pyrophosphorylase activity(usually nos. 33-41, inclusive) were pooled andbrought to 80% satn. with solid (NH4)2SO4. Thepellet obtained by centrifugation at 20000g for 15minwas redissolved in 4ml ofTris-dithiothreitol buffer.

Step 3. The 4ml sample of partially purified enzymefrom Step 2was applied toacolumn(350mm x 25mm)of Sephadex G-200 previously equilibrated withTris-dithiothreitol buffer. This buffer was also usedas the eluent, which was applied at a rate of 30ml/h.Fractions (5ml) were serially collected, and thosecontaining ADP-glucose pyrophosphorylase activity

(usually nos. 16-21, inclusive) were pooled, broughtto 80% satn. with solid (NH4)2SO4 and centrifugedat 20000g for 15min. The pellet was dissolved in 5mlof Tris-dithiothreitol buffer and could be storedanaerobically at -30°C. The complete procedure,which effected a 33-fold purification of the enzyme,is summarized in Table 1.

Granulose synthase from wild-type strains of Cl.pasteurianum. Cultures (81) were harvested duringearly stationary phase and the organisms were twicewashed with Tris-dithiothreitol buffer, pH8. Theresultant 50ml of suspension (containing about 35gwet wt. of cells) was disrupted by passage at 1.4x108Pa through a chilled French pressure cell. Theresultant crude extract could be stored in liquid N2.Approx. 95% of the granulose synthase activity

was associated with the pellet obtained by centri-fugation of this crude cell extract at 100OOg for15min at 3°C. This suggested that partial purificationcould be accomplished by differential centrifugation.

Step 1. Crude cell extract (50mI) equally distri-buted between 2 x 30ml 'Corex' centrifuge tubes wascentrifuged for 30min at 30C at 26000g (ray. 7.5cm).The resulting pellet was horizontally stratified infour distinct bands. The uppermost layer wasremovedby careful agitation and withdrawn with the super-natant fraction. The following (middle) two layerswere carefully resuspended in Tris-dithiothreitolbuffer and the residual (basal) layer containingunbroken organisms was discarded.

Step 2. Further centrifugation (as in Step 1) ofthe suspension derived from the 'middle layers',produced a stratified pellet consisting of three layers.Removal of the uppermost layer (by careful re-suspension in Tris-dithiothreitol buffer) revealed themedial thin white layer which contained the bulk ofthe granulose. This was removed by suspension inTris-dithiothreitol buffer and the remaining basallayer was discarded. The suspensions prepared fromthetwoconserved layerswereseparatelyre-centrifuged(as above) and the granulose-rich, white layers in theresulting pellets were suspended in Tris-dithiothre-itol buffer, pooled, and stored in liquid N2.Attempts were made to solubilize the enzyme by

(a) hydrolysis of the 'carrier' polyglucan by using a-amylase at 10°C, (b) incubation with various solutions

Table 1. Partial purification ofADP-glucose pyrophosphorylase from Cl. pasteurianum

Full details are given in the text. One unit of enzyme activity equals I nmol of ADP-glucose produced/min at 37°C.

FractionCrude extract(NH4)2SO4 precipitateDEAE-cellulose eluateSephadex G-200 effluent

Volume(ml)401545

Activity(units/mI)

2905451635781

Protein(mg/ml)31.024.216.92.54

Specific activity(units/mg ofprotein)

9.3622.5096.80

310.00

Yield

10070.556.433.7

1974

506

BIOSYNTHESIS OF GRANULOSE IN CL. PASTEURIANUM

Table 2. Partialpurification ofgranulose synthasefrom Cl. pasteurianum

Full details of the procedures used are given in the text. One unit of enzyme activity represents 1 nmol of glucoseincorporated/min at 25°C.

FractionCrude extractFirst centrifugationSupematant plus top layer2nd layer3rd layerBottom layer

Subsequent centrifugationsPooled granulose fractions

Volume(ml)50

40111613

5

Activity(units/ml)

266.4

.108.4600.065.5

1137.0

Protein(mg/ml)23.8

22.914.48.31.3

4.9

Specific activity(units/mg ofprotein)

11.2

7.5172.350.4

232.0

Yield

100.0

9.072.16.4

42.7

of salts at high ionic strength (e.g. 2M-LiCI) and (c)brief exposure to mildly acid or alkaline conditions.None ofthese treatments yielded a soluble, granulose-free, enzymically active product.The partial purification procedure used to obtain

the granulose-bound preparation is summarized inTable 2. This material lost its enzymic activity quiterapidly at 37°C, and although addition of bovineserum albumin (Smg/ml) decreased the rate ofdeterioration, kinetic studies were performed at25°C, at which temperature the enzyme proved to beadequately stable.

Results

Deposition ofgranuloseCl. pasteurianum 6013, which undergoes near-

synchronous sporulation in glucose minimal medium(Mackey & Morris, 1971), synthesized little granuloseduring its vegetative growth, but produced massivequantities ofgranulose at the end ofgrowth and onsetof sporulation (Fig. Ia). In contrast, Cl. pasteurianum9486 in the same medium failed to complete theprocess of sporulation despite apparently successfulforespore formation, and accumulated only half ofthe quantity of polyglucan laid down by strain 6013(Fig. lb). Both strains ceased to grow for reasonsother than deprivation of exogenous sources of car-bon and nitrogen since both glucose and NH4+ ionswere present in excess in the final culture super-natants.

A DP-glucose is the precursor ofgranuloseGranulose-negative mutants derived from both

wild-type strains of Cl.pasteurianum (see the Methodssection) were found to lack either ADP-glucosepyrophosphorylase or granulose synthase. Revert-ants able to synthesize granulose had reacquiredthe previously missing enzyme (Table 3). It wastherefore concluded that ADP-glucose is the soleprecursor of granulose in Cl. pasteurianum.

Vol, 144

Synthesis ofthe key enzymes ofgranulose biosynthesis

Both ADP-glucose pyrophosphorylase and granu-lose synthase were present in vegetatively growingcultures of Cl.pasteurianum even during the exponen-tial phase of batch culture when little granulosedeposition was observed. In strain 6013 both enzymeswere present at high specific activity at the onset ofthe stationary phase of the culture, i.e. coincidentwith the period of most dramatic granulose accumu-lation. This. was most marked in the case ofADP-glucose pyrophosphorylase whose specificactivity increased threefold at this time (Fig. 2a).The specific activities of both enzymes were signifi-cantly lower in strain 9486and underwent littlechangeas growth ceased (Fig. 2b).

Properties ofthe ADP-glucosepyrophosphorylase

Nucleotide triphosphate specificity. Crude extractsofwild-type Cl.pasteurianum contained UDP-glucosepyrophosphorylase at approx. 50% of the specificactivity of ADP-glucose pyrophosphorylase. TheUTP-utilizing enzyme was significantly more heat-stable than the ATP-dependent enzyme, and thepartially purified preparation of ADP-glucose pyro-phosphorylase (see the Methods section) displayedlittle activity with UTP, GTP and CTP (Table 4).pH optimum. The partially purified enzyme demon-

strated activity over a broad range of pH values(pH7-10); its optimum pH was about pH8.0.

Effects ofchanges in substrate concentration. Prim-ary plots ofenzyme activity against substrate concen-tration were hyperbolic in the case of both ATP andglucose 1-phosphate; there was no evidence ofsigmoidicity in these curves. Double-reciprocal plots(of 1/v against 1/[S]) were linear, and the apparentKm values for ATP and glucose 1-phosphate were2.5mM and 70AM respectively.

Effects of various metabolites. In all, over 20compounds were tested for their effect on the ADP-glucose pyrophosphorylase activity in (i) crude cell

507

R. L. ROBSON, R. M. ROBSON AND J. G. MORRIS

8.0 ~ t X 200

8.0

o00 eO0

00

El~~~~~~~~~~~~~~~~~~~~~~4C 0

8.0~~~~~~~~~~~u

b7. 0 Zs* 0 2 4 6 8 10 12 14 160~~~~~~~~~~~~

3~~~~~~~~~9.0a

a (b)4) ~~~~~~~~~0

200 lo"0~~~~~~~~~~~~~~'

08.0

100 (

7.0 0

0 2 4 6 8 10 12 14 16

Time (h)Fig. 1. Granulose accumulation by wild-type strains of

Clostridium pasteurianum

Table 3. Granulose-negative mutant strains of Cl. pasteur-ianum

Granulose-negative mutants wereproduced and isolated asdescribed in the Methods section. Revertant strains wereselected as 12-staining colonies obtained on plating theproducts of mutagenesis of cultures of granulose-negative'parent' strains. Ability (+), or inability (-), to synthesizegranulose, was determined both by l2-staining and by assayof the glucose content of acid-hydrolysed washed sus-pensions of the organism (see the Methods section).The total absence (-) or presence (+) in normal specificactivity of the two key enzymes of granulose biosynthesiswas assessed by usingcrude extracts oforganisms harvestedat the late-exponential phase of batch growth (as inFig. 2).

Possession of

Ability tosynthesize

Strain granulose9486

Wild-typeMR 30MR 30 revertantMR31MR 31 revertant

6013Wild-typeMR 505MR 505 revertant

+

ADP-glucose Granulosepyrophos- synthasephorylase

+ + +_ +_+ + +_- +

+

_+_

+

+

++

(a) Cl.pasteurianum 6013. This sporulated well; foresporeswere produced by over 80% of the organisms (at 8h) andthese gave rise to mature refractile spores (at 14h).(b) Cl. pasteurianum 9486, which sporulated very poorly.The organisms were grown anaerobically at 37°C in batchculture in minimal medium containing 4% (w/v) glucose.Towards the end of the exponential phase of growth,cell number (o) and the granulose content of theorganisms (@) were determined at intervals by the pro-cedures described in the Methods section.

extracts and (ii) partially purified preparations.Thesecompounds included thosemetabolites reportedto act as allosteric effectors of the ADP-glucosepyrophosphorylases of other glycogen-synthesizingbacteria (Preiss, 1969). None of the following sub-stances enhanced the activity of the enzyme whensupplied at 3mM final concentration: acetyl CoA,acetyl phosphate, ADP, AMP, butyryl phosphate,fructose 1-phosphate, fructose 6-phosphate, fructose1,6-diphosphate, glucose 6-phosphate, Pi, NAD+,NADH, NADP+, NADPH, phosphoenolpyruvate,6-phosphogluconate, 2-phosphoglycerate, 3-phos-phoglycerate, pyridoxal 5'-phosphate, pyruvate, rib-ose 5-phosphate. Of all the compounds tested, only

ADP was found to inhibit the enzyme; it acted as acompetitive inhibitor of ATP utilization with a K1of 19mM.

Properties of the granulose synthase

Nucleotide sugar specificity. Even in crude cellextracts, UDP-glucose was unable to substitute forADP-glucose as substrate for granulose synthase(when both nucleotide sugars were supplied at a finalconcentration of 0.25mM).

Requirement for a glucan primer. Since the granu-lose synthase obtained from wild-type Cl. pasteur-ianum was invariably bound to particles of granulose(see the Methods section) it was not possible to usesuch a preparation for studies of the requirement ofthe enzyme for a primer. Therefore the granulose-negative mutant strain MR-31 (lacking ADP-glucosepyrophosphorylase) was exploited as a ready sourceof primer-free granulose synthase. The activity ofthis enzyme was measured both in the presence andabsence of amylopectin (2.5mg/ml) by the proceduredescribed in the Methods section. After the reactionhad been stopped, amylopectin was added to thosemixtures from which it had formerly been omitted,so that co-precipitation of any newly synthesized

1974

508

BIOSYNTHESIS OF GRANULOSE IN CL. PASTEURIANUM

9.0 -,

0

8.8

8.6

8.4

(b)

8.2k

8.0k

7.81-

7.6

7.47.2 .,,t

2 3 4 5 6 7 8 9 10

15 00404

0 o

0aa2ce

._

15 X'

N

100.L

_ -S

_ -O

15

0It1 '

Time (h)

Fig. 2. Specific activities of key enzymes of granulosebiosynthesis during growth of wild-type strains of Clos-

tridium pasteurianum

(a) Cl. pasteurianum 6013; (b) Cl. pasteurianum 9486. Theorganisms were grown in batch culture as in Fig. 1. Cellnumber (0) and the specific activities of ADP-glucosepyrophosphorylase (A) and of granulose synthase (A)were measured as described in the Methods section.

Table 4. Nucleotide triphosphate specificity of thle ADP-glucose pyrophosphorylase ofCl. pasteurianum

The ADP-glucose pyrophosphorylase activity of a crudecell extract of Cl. pasteurianum and of a partially purifiedpreparation (see the Methods section) was assayed by theprocedure described in the Methods section. ATP wasreplaced by an equimolar concentration of an alternativenucleotide triphosphate. The resultant activities of theenzyme are expressed as a percentage of that activitydemonstrated with ATP.

Enzyme activity (%)Nucleotidetriphosphate

(1OmM)ATPUTPGTPCTP

Vol. 144

Crudeextract100.047.54.22.1

Partially purifiedpreparation

100.00.62.4

004

30

0 o /-

0 5 10 15 20Time (miin)

Fig. 3. Dependence of activity of granulose synthase onprovision ofapolyglucanprimer

Granulose synthase activity in a crude extract of the ADP-glucose pyrophosphorylase-less (granulose-negative)mutant Cl. pasteurianum MR 31 was assayed in thepresence (0) or absence (o) of amylopectin (2.5mg/ml),by using the radioactive procedure (see the Methodssection) to measure incorporation into granulose of[14C]glucose from ADP-['4C]glucose.

polyglucan could be effected. Negligible synthesis of[I4C]polyglucan from ADP-[14C]glucose was in factachieved in the absence of primer (Fig. 3).pH optimum. The enzyme was maximally active

over a relatively broad range of pH values (pH6.5-8.5).

Effects of changes in substrate concentration. Theprimary plot ofenzyme activity against ADP-glucoseconcentration was hyperbolic (with no trace ofsigmoidicity). The double-reciprocal plot of 1/vversus 1/[S] was linear, and the apparent Km forADP-glucose was about 20uM.

Effects of various metabolites. None of the com-

.pounds tested (above) as possible effectors of ADP-glucose pyrophosphorylase enhanced the activityof granulose synthase when added (at 3mM) tocrude cell extracts or to partially purified preparationsof the enzyme (see the Methods section), nor hadATP or glucose 1-phosphate any effect on the activityof the enzyme. On the other hand ADP, AMP andpyridoxal 5'-phosphate were all competitively inhibi-tory with apparent K1 values of 0.2, 3.5 and 1.2mmrespectively.

Discussion

From the nature of the enzymic lesions in granu-lose-negative mutant strains of Cl. pasteurianum, weconclude unequivocally that the polyglucan is pro-duced from ADP-glucose. The similar bacterialsynthesis of glycogen is regulated by a sophisticated

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510 R. L. ROBSON, R. M. ROBSON AND J. G. MORRIS

combination ofcoarse and fine controls, with produc-tion of the biosynthetic enzymes being subject torepression/derepression and the activity of the ADP-glucose pyrophosphate being modulated by positiveand negative allosteric effectors, whose concen-trations reflect the flow of metabolites through themajor pathway of carbon metabolism, and/or theadenylate energy-charge status of the organism(Preiss, 1969; Preiss et al., 1973; Dawes & Senior,1973).In Cl. pasteurianum 6013 massive deposition of

granulose coincided with a threefold increase in thespecific activity of ADP-glucose pyrophosphorylase.In strain 9486, in which granulose accumulation wasneither so rapid nor so extensive (Fig. 1), this de-repression of synthesis of ADP-glucose pyrophos-phorylase was not observed and granulose synthasewas present at a consistently lower specific activity(Fig. 2).Themost surprisingfindingwas that theADP-glucose pyrophosphorylase of Cl. pasteurianum wasnot activated by metabolites which are positiveallosteric effectors of the analogous enzymes ofbacterial glycogen synthesis. Further, only ADPsignificantly inhibited the enzyme from Cl. pasteur-ianum, and that (a) competitively with glucose 1-phosphate and (b) only at relatively high concen-trations (K, of 19mM compared with a Km of 2.5mMfor ATP and 0.07mM for glucose 1-phosphate).Otto et al. (1972) who reported derepression ofsynthesis ofADP-glucose pyrophosphorylase duringgrowth of Cl. pasteurianum W-5, and who couldsimilarly discover no activator oftheenzyme, reportedit to be inhibited by pyridoxal 5'-phosphate andNADH. We did not observe significant inhibitionof the enzyme by these compounds.The granulose synthase of Cl. pasteurianum

resembles the glycogen synthases of other pro-karyotes (Preiss et al., 1973; Elbein & Mitchell,1973). It effectively used only ADP-glucose asglucosyl donor, its activity was dependent on theprovision of a suitable a-1,4-polyglucan primer,and there was no evidence of complex effector inter-actions or of interconversion of different forms ofthe enzyme (as is the case with regulatory, eukaryoticglycogen synthases; Ryman & Whelan, 1971). Pro-duct inhibition of granulose synthase by ADP(K, = 0.2mM) could be of physiological significancein Cl. pasteurianum, as it has proved to be in glycogensynthesis in other bacteria (Greenberg & Preiss,1965; Elbein & Mitchell, 1973; Dietzler& Strominger,1973). However, the consequences in vivo of thesusceptibility of granulose synthase to inhibition byAMP (K, = 3.5mM) and pyridoxal 5'-phosphate,are less obvious. The intimate association of granu-lose synthase with the product polymer is of interest,since Laishley et al. (1973) have suggested that thenative granules are bounded by a membranous coat.

Since ATP is utilized as a primary substrate, and

ADP is an inhibitor both of granulose synthase and(less effectively) of ADP-glucose pyrophosphorylase,the rate of granulose synthesis in Cl. pasteurianummust reflect the adenylate energy charge (Atkinson &Walton, 1967) and the availability of glucose 1-phosphate. It is tempting to interpret the apparentlack of any more refined control of granulosesynthesis as evidence of the primitive nature of thesystem, in Clostridium. In this light, the ADP-glucose pyrophosphorylase of Cl.pasteurianum wouldseem to be a primordial enzyme, the evolutionaryprogenitor of those forms of the enzyme whosesensitive response to allosteric controls is a feature ofglycogen production in many bacteria.The possible role of branching or other modifying

accessory enzymes which may be concerned withgranulose fabrication, was not investigated in thepresent study. It has been reported that the granuloseof Cl. pasteurianum is an essentially unbranchedglucan with linear sequences of ac[1-+4]-linked glucoseresidues alternating with sequences of a[41-6]-linkedunits (Laishley et al., 1974). Whether this means thatthe granule-bound granulose synthase should consistof at least two distinct transglucosylases (with a[1-4]and a[1->6] specificities respectively) has yet to bedetermined.

This work was supported by a grant from the ScienceResearch Council.

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