JOURNAL OF BACTERIOLOGY, Dec. 1978, P. 1058-10690021-9193/78/0136-1058$02.00/0Copyright i) 1978 American Society for Microbiology

Vol. 136, No. 3

Printed in U.S.A.

Incorporation of 32Pi into Nucleotides, Polyphosphates, andOther Acid-Soluble Compounds by Myxococcus xanthus

During Myxospore FormationP. Y. MAEBA* AND R. SHIPMAN

Department ofMicrobiology, University ofManitoba, Winnipeg, Manitoba, Canada R3T2N2

Received for publication 11 September 1978

When glycerol was used to induce myxospore formation in Myxococcus xanthusin the presence of 32Pi, the label was incorporated into a variety of acid-solublecompounds. Incorporation into ribonucleotides was approximately fivefold greaterthan in vegetative cells or noninducible mutants grown in glycerol. The label wasalso incorporated into some unknown compounds and material tentatively iden-tified as guanosine tetraphosphate. Marked accumulation into polyphosphates,which were present mainly in culture supernatants, occurred relatively late duringmyxospore formation. The kinetics of accumulation of some of these compoundsand their distribution into acid-soluble cell extracts and culture supernatants aredescribed and compared with those in vegetative cells and noninducible mutants.

The biology of Myxococcus xanthus, a gram-negative, gliding bacterium, has been the subjectof several recent reviews (33, 34). Under starva-tion conditions on solid medium, the cells aggre-gate to form raised mounds or fruiting bodieswithin which the long slender cells undertakemorphogenetic change to form spherical myxo-spores. The asynchronous formation of fruitingbodies is generally complete within 4 to 5 daysat 32°C. The study of events concerned withmyxospore formation has received impetus fromthe discovery that glycerol is able to inducespore formation in liquid medium (9). If themedium is made 0.5 M with glycerol, logarith-mic-phase cells quantitatively and synchro-nously differentiate into myxospores in the ab-sence of fruiting body formation. Cells, morpho-logically indistinguishable from mature myxo-spores, are formed within 2 h after addition ofglycerol (9), although some metabolism contin-ues for an additional 6 h in sporulating cultures(22). During the first 2 h after glycerol addition,numerous metabolic changes take place. Theglyoxalate cycle enzymes, isocitratase and mal-ate synthetase, are induced (33), as are the en-zymes required for formation and incorporationof N-acetylgalactosamine into spore coats (11)and a variety of other enzymes (34). Changes insynthesis ofRNA and DNA also accompany thesporulation process (34).The alterations in biosynthetic events suggest

that regulatory mechanisms must be involved.Since the discovery of guanosine tetraphosphate(6), several laboratories have reported the pres-ence of novel phosphorylated compounds in a

variety of organisms (12, 17, 19, 23, 24). The timeofappearance of these compounds indicates theymay participate in regulatory functions. Origi-nally, this work was intended to look for analo-gous compounds that may appear during myx-ospore formation in M. xanthus. Because of theappearance ofmany compounds, this report rep-resents a survey of the acid-soluble, phosphoryl-ated compounds that appear in cell extracts andsupernatant fractions of M. xanthus culturesinduced to form myxospores in liquid mediumwith glycerol.

MATERIALS AND METHODSOrganism. M. xanthus MD-1 was obtained from

M. Dworkin (University of Minnesota, Minneapolis).A tan isolate, competent in fruiting body formation,was used throughout. Five independently isolatedspontaneous mutants, unable to sporulate in 0.5 Mglycerol, were selected by the method of Burchard andParish (4) and used in certain experiments.

Growth. The growth medium consisted of 1%(wt/vol) Casitone (Difco) containing 10.0 mM MgSO4.The phosphate concentration in this medium, as de-termined by the method of Ames (1), was 0.5 mM.Cells were routinely grown in 40 ml of the medium in250-ml Erlenmeyer flasks, which were incubated at32°C in a gyratory water bath shaker (New BrunswickScientific Co.) set at 160 rpm. When smaller volumeswere required, 5 to 10 ml of the culture was incubatedin 50-ml Erlenmeyer flasks under the same conditionsexcept the flasks were tilted at a 300 angle.

For myxospore formation, logarithmic-phase cellswere centrifuged at 12,000 x g for 10 min at 4°C. Thecell pellet was dispersed to the same density in freshmedium and allowed to resume growth at 32°C withshaking. After 60 min, sterile glycerol (6.8 M) was

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PHOSPHORYLATED COMPOUNDS IN M. XANTHUS 1059

added to a concentration of 0.5 M, and incubation wascontinued at 32°C with shaking. For vegetative cellcontrols, sterile water was added instead of glycerol.Cell and spore counts of cultures, diluted with distilledwater, were made microscopically with a Petroff-Hausser bacterial counting chamber (Hausser Scien-tific Co.).

Labeling and extraction. Orthophosphate, 32Pi(New England Nuclear Corp.), was added to culturesat a concentration of 250 to 500,uCi/ml, 30 min beforeaddition of glycerol (or water). Samples (0.5 to 1.0 ml)were removed from the cultures at various times andtransferred to ice-cold centrifuge tubes, then centri-fuged at 12,000 x g for 10 min. The supernatant werefrozen at -75°C and lyophilized, and the resultingviscous residue was made to 1/5 the sample volumewith 0.1 M formic acid (pH 3.0) and analyzed. Identicalresults were obtained if the residue was dissolved in1.0 M formic acid. The cell pellet was washed with ice-cold medium containing 0.5 M glycerol, extracted with1/5 volume of ice-cold 2.0 M formic acid (pH 30) for30 min, then centrifuged to remove cell debris beforeanalysis (6).

For pulse-labeling, centrifuged cells were allowed togrow for 60 min in fresh medium as described above.After addition of glycerol to 0.5M to induce myxosporeformation, 1.0-ml amounts were placed in 15.0-mlround-bottom centrifuge tubes, and incubation, withshaking, was continued at 32°C. As judged by micro-scopic examination, the time course of myxosporeformation was the same as in larger culture. At speci-fied times 250 ,uCi of 32Pi was added to the culture, and,after 30 min of incubation, the culture was cooled inice, then separated into cells and supernatants, whichwere extracted as above.Chromatography. Commercially available thin-

layer chromatography plates were washed with dis-tilled water before use. Five- to 10-pl amounts of theextracted samples were spotted on poly(ethylene)-imine-cellulose thin-layer plates (20 by 20 cm; PEIplates; Brinkmann Instruments, Inc.) for chromatog-raphy in two dimensions according to the method ofCashel et al. (7). The first-dimension buffer was 3.3 Mammonium formate and 0.68 M boric acid adjusted topH 7.0 with NH40H. The second-dimension bufferwas 1.5 M KH2PO4 (pH 3.65). In some cases, culturesupematant extracts were run in one dimension withthe phosphate buffer on long (20 by 40 cm) PEI-cellulose thin-layer plates (5). When smaller plates (20by 20 cm) were used for one-dimensional chromatog-raphy, two cycles of chromatography with the samebuffer were employed. In these cases, the plates werewashed with distilled water and methanol betweencycles. After chromatography, the thin-layer plateswere exposed to Kodak RP-1 X-ray film (EastmanKodak Co.) for 24 to 48 h and developed. Regions onthe chromatogram corresponding to the radioactiveareas detected on the film were cut into 1-cm2 areasand counted in a Beckman LS 230 scintillationcounter. The scintillation cocktail consisted of 15.2 gof 2,5-diphenyloxazole and 0.35 g of 1,4-bis-[2-(5-phen-yloxazolyl)]-benzene per gallon of toluene.

In some cases, 32Pi was purified by adsorption to acolumn of A-25 diethylaminoethyl-Sephadex (Phar-macia) equilibrated in 0.01 M triethylammonium bi-

carbonate (pH 8.0) and eluted with a 200-ml lineargradient of 0.01 to 0.5 M triethylammonium bicarbon-ate. The radioactivity was monitored by Cerenkovcounting of aliquots of 1.0-ml fractions. The first peakfractions with radioactivity were pooled, evaporatedto dryness under vacuum, and brought to the originalvolume in distilled water. The preparation was made1.0 N with concentrated HCI, placed in a boiling waterbath for 15 min, then neutralized with 10 N NaOH.ATP was measured by the firefly luciferase assay

method (29). Cells were collected on 25-mm-diametermembrane filters (0.45-,um; Millipore Corp.) and ex-tracted in 4.0 ml of 0.05 M tris(hydroxymethyl)amino-methane-hydrochloride (pH 7.7) at 100°C for 8 min asdescribed by Strehler and Totter (29). Measurementswere made on a model 2000 ATP Photometer (JRB,Inc.). Charcoal adsorption of 0.5-ml amounts of 32p_labeled extracts was carried out according to themethod described by Griffin et al. (13). Material thatremained unadsorbed or was eluted from charcoal wasconcentrated under vacuum in a Evapomix (Buchler)before analysis by one-dimensional chromatography.

Standard nucleotides used for markers and enzymeswere obtained from Sigma Chemical Co. The "unu-sual" nucleotides guanosine 5'-triphosphate-3'-diphos-phate and guanosine 5'-diphosphate-3'-diphosphatewere purchased from ICN Pharmaceuticals.

RESULTS

Figure 1 (left) shows autoradiograms aftertwo-dimensional chromatography of cell and su-pematant extracts obtained from 32P-labeledcultures 4 h after glycerol induction. By thistime, all phosphorylated compounds that can bedetected under these conditions have appeared.The faint spot immediately to the right of GTPwas likely dGTP (7) and was not counted. Thoseappearing just below GTP were not always ob-served and were not counted. The schematicdrawing in Fig. 1 (right) gives the lettering sys-tem used to identify the compounds studied.Since volumes for cell and supematant extractswere the same, as well as sample volumes placedon chromatograms, the intensity of labeling ofthe compounds on different chromatogramscould be compared. On this basis, they weredivided into three groups. One group of com-pounds labeled more intensely in cell extractsthan in supematant fractions and are indicatedby open symbols in the schematic drawing inFig. 1. These include GTP, ATP, and GDP. Thelatter two could not be resolved in this systemand were counted as one spot, ATP-GDP. Thesecond group of compounds were found in bothfractions but labeled much more intensely insupernatant extracts. These are designated bythe solid symbols in the schematic drawing andinclude those marked X, A, B, C, D, E, and 0(Fig. 1). The final group, shown by diagonalmarkings in the schematic drawing, were found

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1060 MAEBA AND SHIPMAN

CELLS

4..A',

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SUPER NATE_ i_

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FIG. 1. Autoradiograms after two-dimensional chromatography of cell extracts (top, left) and supernatantextracts (bottom, left) from 32P-labeled cultures (400 ,uCi/ml). Cultures were induced with 0.5 M glycerol for 4h before extraction (text). The schematic drawing (right) is a composite of the autoradiograms and shows thelettering system used to identify the spots that appeared. G4P, Guanosine tetraphosphate. Arrows indicate the

direction of chromatography and the buffer system employed.

only in cell extracts and are compounds 2 and 3and a compound tentatively identified as gua-nosine tetraphosphate.The accumulation of 32Pi into GTP and ATP-

GDP in induced and control cultures to whichwater, instead of glycerol, was added is summa-rized in Fig. 2. Although radioactivities remainedconstant in control cells, there was increasedaccumulation of label into nucleotides of sporu-

lating cultures. Accumulation into cellular nu-

cleotides began 30 min after addition of glycerol,whereas that into supernatant nucleotides began2 h after induction, suggesting that intracellularnucleotides reach a certain level before excretioninto the culture medium. The accumulation was

complete by 3 h, the time at which virtually all

(>95%) of the cells appeared, morphologically,as myxospores. The accumulation represented a

fivefold increase, approximately, in labeling overthat in vegetative cultures. The ratio of label insupernatant to cellular nucleotides was always

higher for GTP than ATP-GDP. The labeling ofUTP and CTP followed a similar pattern,though not as marked (Shipman and Maeba,unpublished data), indicating glycerol inductionhas a pronounced effect on general nucleotidemetabolism. It should be noted that in controlcultures the radioactivity in nucleotides, calcu-lated on a cell basis, remained constant fromzero time (Fig. 1). This indicates that the 30-minprelabeling time before glycerol addition was

sufficient for equilibration of the label and thataccumulation was not a result of increasing spe-cific activities in phosphate pools. Accumulationof 32Pi into nucleotides was basically the same

regardless of prelabeling periods up to 4 h.The amount of ATP present in cultures was

measured by the firefly luciferase method as

described by Strehler and Totter (29). A twofoldincrease in ATP levels was observed in inducedcultures, but the levels remained constant incontrols (Fig. 3). The smaller increase as com-

A

G4P

B

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PHOSPHORYLATED COMPOUNDS IN M. XANTHUS 1061

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FIG. 2. Accumulation of 32Pi (250 t,Ci/ml) intoATP-GDP and GTP by glycerol-induced cultures(closed symbols) and control, vegetative cultures(open symbols). Glycerol or water was added at zerotime, 30 min after addition of 32Pi, and cell extracts(0, 0) and culture supernatant extracts (U, 5) wereanalyzed (text). The counts shown are relative to themaximum amount incorporated into cell extracts byinduced cultures. For ATP-GDP the maximum was57,000 cpm/108 cells, and for GTP it was 30,000cpm/l08 cells.

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1 2 3 4 5 6HOURS POST- GLYCEROL

FIG. 3. ATP levels in glycerol-induced cultures(0) and control vegetative cultures (0). Samplestaken at times indicated were extracted in boilingwater, and ATP content was measured by the fireflyluciferase assay system described in the text.

pared with 32P-labeled cultures (Fig. 2) may haveresulted from differences in extraction proce-dures (Materials and Methods). It may also in-dicate that ATP formed during glycerol induc-tion proceeded by de novo and/or salvage path-ways whereby more than one phosphate mole-cule was incorporated. At any rate, the accu-mulation of label into ATP, -and presumablyother nucleotides, was due, in part, to increasingpool sizes. To determine whether increased ratesof synthesis of nucleotides were responsible for

accumulation, pulse-label studies were carriedout. The amount of 32p; incorporated into intra-cellular GTP in 30-min pulses increased five- toeightfold during myxospore fornation as com-pared to vegetative cells (Fig. 4). The amountincorporated by control vegetative cultures isshown by the first vertical bar in Fig. 4. In-creased incorporation began 30 min after induc-tion and continued at maximal rates for 2 hbefore decreasing. The fact that significantamounts of GTP were synthesized at 4 h post-induction, when accumulation has ceased andmay even be decreasing (Fig. 2), indicates turn-over may be taking place. Similar results wereobtained when incorporation into ATP-GDPwas measured (not shown).Although the above results do not give de-

tailed information regarding nucleotide metab-olism during myxospore formation, they stronglysupport the contention that nucleotides accu-mulate during glycerol-induced myxospore for-mation. As discussed later, this accumulationmay be important in explaining the appearanceof the phosphorylated compounds, A, B, C, D,E, and 0, that appear predominantly in theculture supernatant fluids during myxospore for-mation. The appearance of these compounds insupernatant extracts was analyzed by one-di-mensional chromatography. Spots G4P, 2, and3 were not found in supernatants (Fig. 1) andtherefore did not interfere with the analysis.Figure 5 shows the time course of appearance oflabeled supernatant compounds during myxo-spore formation. After 30-min labeling of vege-tatie cells (Fig. 5, zero time track), extracellularATP and GTP, spot A, spot 0, and traces of

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FIG. 4. Pulse-labeling ofcell extract GTP with 3"Pi(250 ,uCi/ml) during myxospore formation. Glycerol-induced cultures (1.0 ml) were incubated for 30-minintervals with 32Pi at times indicated, then centri-fuged, extracted, and analyzed (see the text). The firstvertical bar represents incorporation by vegetativecells that were pulsed I h after transfer to freshmedium.

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U$t * * ¢ GDPATP

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FIG. 5. One-dimensional chromatography of supernatant extracts from glycerol-induced cultures. 32P-labeled cultures (250 ,uCi/ml) were induced at zero time, and samples were removed at times indicated. A 5-t,l sample of the extract was run on PEI-cellulose thin-layer plates (20 by 40 cm). Only the lower 20 cm of thechromatogram is shown.

compounds B and C were seen. After glycerolinduction, the intensity of labeling increased inall compounds with the exception of spot A,which appeared to decrease to a constant level.Extracellular nucleotides began accumulatingbetween 1 and 2 h postinduction, whereas labelinto the series of spots B to 0 did not accumulateuntil 2 to 4 h after induction. Spot X, just above

GTP, did not appear until 6 h after glyceroladdition.The accumulation of label into spot 0 was

followed in sporulating and vegetative cultures.During myxospore formation, there was a 50-fold increase in labeling of this material in su-pernatant extracts (Fig. 6A), reaching a plateau4 h after induction. In contrast, 32Pi did not

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HOURSFIG. 6. Accumulation of 32Pi into spot 0 in glycerol-induced cultures (closed symbols) and control vegetative

cultures (open symbols). (A) Accumulation in supernatant extracts from cultures engaged in myxosporeformation. (B) Accumulation in cell extracts of sporulating cultures (-) and vegetative cultures (a) and insupernatant extracts of vegetative cultures (0). Glycerol or water was added at zero time, 30 min afteraddition of 32Pi (250 ,uCi/ml).

accumulate into intracellular compound 0, andthe low levels present decreased still furtherbetween 3 and 4 h postinduction (Fig. 6B), sug-gesting the material was excreted from sporulat-ing cells. In control cultures, compound 0 accu-mulated slowly in both cell and supematantextracts, but the total amount labeled after 6 hwas 20-fold smaller than in sporulating cultures(Fig. 6B). Accumulation of 32Pi into spot C isshown in Fig. 7. The incorporation patterns intospots D and E were almost identical to that intoC and are not shown. Accumulation into super-natant and cell extracts began 2 h after glyceroladdition and continued into 7 h postinduction.Most, but not all, of compound C accumulatedin supernatant extracts. In vegetative culturesupernatant extracts, there were detectableamounts of C at zero time which remained con-stant, so that the amount per cell decreased withtime (Fig. 7), suggesting cells did not synthesizethis material. It was not detected in cell extractsfrom labeled vegetative cultures. Although theradioactivity accumulating in spot B wassmaller, the pattern of accumulation was thesame as that into C (Fig. 7).The amount of label incorporated in 30-min

pulses into the supernatant compounds wasmeasured and is shown in Fig. 8 for some ofthese compounds. In all cases, maximum incor-poration occurred between 1 and 3 h after in-duction. The increase in rate of incorporationduring myxospore formation can be judged bycomparison with that incorporated by controlcultures as represented by the first bar in eachof the histograms (Fig. 8). The data indicatedthat the increased accumulation noted in Fig. 6

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HOURSFIG. 7. Accumulation of 32P, (250 ,uCi/ml) into spot

C in cell extracts (0) and supernatant extracts (0)from glycerol-induced cultures and into supernatantextracts (O) ofcontrol vegetative cultures. Also shownis the labeling of spot B (A) in supernatant extractsfrom sporulating cultures. Conditions for labeling aredescribed in the legend to Fig. 6.

and 7 was due in part to increased rates ofsynthesis. On occasion, anomalous pulse-label-ing was observed, as shown for spot C in Fig. 8,where incorporation rates were much higherthan expected although the amount of the in-

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FIG. 8. Pulse-labeling of spot 0, spot D, and spotC in culture supernatant extracts during myxosporeformation. Glycerol-induced cultures (1.0 ml) wereincubated for 30-min intervals with 250 ,uCi of 32Pi atindicated times, and, after centrifugation, the super-natant was extracted and analyzed (see the text). Thefirst bar in each of the histograms represents incor-poration into control vegetative culture supernatantsthat were pulsed 1 h after transfer to fresh medium.The incorporation is given relative to the maximumincorporated into each spot, which was 18,600cpm/108 cells for spot 0, 400 cpm/108 cells for spot D,and 1,680 cpm/108 cells for spot C.

crease was not. This was noted especially forsupernatant compounds with greater chromato-graphic mobilities, i.e., spots A, B, and C. Theresults appeared as artifacts due to high back-ground levels of the compound. Subsequently itwas found that background levels were due tocontaminants present in commercial 32Pi.

Figure 9 shows an autoradiogram after chro-matography of 32Pi, taken directly from a com-

mercial preparation, as compared to that of la-beled supernatant extracts from cultures 8 hafter induction. Altough the bulk of the radio-activity in the commercial stock migrated as Pi,contaminants with the same mobilities as spotsA, B, C, D, and 0 were clearly visible. Thepresence of the contaminants explains theanomalous result observed in pulse-label exper-iments (Fig. 8). Also, the appearance of spots A,B, C, and 0 at zero time in accumulation exper-iments (Fig. 5 and 7) was likely due to thesecontaminants. Since contaminants were likely tobe inorganic polyphosphates (3), then the ma-terial that accumulated in culture fluids duringmyxospore formation was probably inorganicpolyphosphates.To show that sporulating cells did not selec-

tively concentrate and release contaminantspresent in the label, accumulation of these com-

pounds was measured with 32Pi purified as de-scribed above. The purity can be judged bycomparing the Pi channels in Fig. 9 and 10. Alsoshown in Fig. 10 is the accumulation of the samecompounds when purified 32Pi was used for la-beling. The amounts accumulated were basicallythe same as in previous experiments except thezero time counts were lower for spots A and 0

ATP

GTP'

A

B

C

DE0

FIG. 9. Comparison of 32P-labeled supernatant ex-

tract from glycerol-induced cultures (left) and 10 JACiof 32Pi taken directly from a commercial preparation(right) by one-dimensional chromatography on PEI-cellulose thin-layer plates. The supernatant sampleis the same as in Fig. 5. Both chromatograms were

run under identical conditions, though not at thesame time.

(not shown). Portions of the chromatogram cor-

responding to positions of traces of contaminantsin purified 32Pi were cut out and counted. From

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PHOSPHORYLATED COMPOUNDS IN M. XANTHUS 1065

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FIG. 10. One-dimensional chromatography of supernatant extracts from glycerol-induced cultures labeledwith 32Pi (200 MCi/ml) purified as described in the text. Approximately 10 tiCi of the purified label waschromatographed in the "Pi" channel. Samples were removed at indicated times, and the supernatant, afterextraction, was chromatographed with 1.65Mphosphate buffer (pH 3.65).

the counts it was estimated that if contaminantswere responsible for the appearance of excretedmaterial, their levels in the medium would haveto be 5- to 10-fold greater to produce the resultsin Fig. 10.

Further confirmation that the material accu-mulating in the medium was polyphosphatecame from charcoal adsorption studies. Acid-washed charcoal was mixed with 32P-labeled su-pernatant extracts from cells induced for 4 haccording to the procedure described by Griffinet al. (13). After adsorption, only GTP, ATP,and GDP could be detected in eluates (channel2, Fig. 11). The material that did not adsorb to

charcoal (channel 3, Fig. 11) was identified aspolyphosphates. When the unadsorbed materialwas incubated with inorganic pyrophosphatase,spots A and B were degraded (channel 4, Fig.11), indicating these may be pyrophosphate andpossibly tripolyphosphate, respectively. Treat-ment with alkaline phosphatase, organic pyro-phosphatase, ribonuclease A, spleen and snakephosphodiesterase, and various combinations ofthese enzymes did not alter the mobilities of theunadsorbed compounds.The labeling of nucleotides and other phos-

phorylated compounds was analyzed in five in-dependently isolated mutants of M. xanthus

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1066 MAEBA AND SHIPMAN

GDP

ATP

GTP

A

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BCDE0

FIG. 11. One-dimensional chromatography of 32P-labeled supernatant extracts fractionated by charcoal(see the text). Extracts were from cultures labeled for 4 h after glycerol induction. Channel 1 shows extractsbefore charcoal adsorption. Channel 2 shows the material eluted from charcoal, after adsorption, bywater-ethanol-NH3 (65:35:3). The remaining channels show the material that did not adsorb to charcoalbefore (3) and after (4) incubation with 100 pg of inorganic pyrophosphatase per ml for 30 min at 30°C.

that were unable to form myxospores in 0.5 Mglycerol. The pattern of labeling after additionof glycerol was the same as that of vegetativecells in the absence of glycerol. The radioactivi-ties of intracellular compounds remained con-stant (not shown), and those of some extracel-lular compounds are shown in Fig. 12. The ra-dioactivity of GTP remained constant, and thatof spot 0 increased as in vegetative cells (see

above). The levels of polyphosphates A, B, C,and D declined (Fig. 12); this was probably dueto the presence of contaminating compounds instock 32Pi which were degraded and/or dilutedas the cells grew. At any rate, the increasedlabeling of phosphorylated compounds observedduring glycerol-induced sporulation was not dueto glycerol per se, but accompanied the forma-tion of myxospores.

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HOURS POST-GLYCEROLFIG. 12. Accumulation of "2Pi into culture supernatant extracts by a glycerol-noninducible mutant growing

in 0.5M glycerol and 250 ,uCi of 32p, per ml.

Guanosine tetraphosphate, tentatively identi-fied by co-chromatography with standards, wasdetected only in cell extracts of sporulating cul-tures (Fig. 1). The maximum accumulation be-tween 2 and 3 h after induction (Fig. 13) corre-lates fairly well with the time at which reductionin net RNA synthesis takes place (2, 22). Gua-nosine pentaphosphate was not detected underthese conditions; it should appear between spotsB and C in Fig. 1. The appearance of radioactiv-ity in three unidentified spots, 2, 3, and X (Fig.1), increased after induction (not shown). Theidentification of these compounds is in progress.

DISCUSSIONDuring myxospore formation the amount of

32Pi incorporated into nucleotides was approxi-mately fivefold greater than in vegetative cells.Although the ratio of intracellular/extracellularamounts varied, a significant portion was ex-creted into the medium of sporulating cultures.The effect was not restricted to nucleoside tri-phosphates, since in cases where ATP and GDPwere resolved (as in Fig. 5) increased labelinginto both compounds was observed. The accu-mulation could not be explained by RNA deg-radation that takes place during induction, sincethe turnover rate maintains RNA at a constantlevel (2, 20, 22). Nor could the effects be attrib-uted to osmotic effects, since mutants that werenot induced by glycerol failed to accumulatenucleotides, as well as other phosphorylatedcompounds, in the presence of glycerol. The fact

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FIG. 13. Incorporation of 32P, (250 ACi/ml) intoguanosine tetraphosphate in cell extracts ofglycerol-induced cultures. Glycerol was added at zero time,30 min after addition of the label, and cell extractswere prepared at indicated times (see the text).

that prelabeling periods up to 4 h yielded essen-tially the same results indicates accumulationwas not a result of increasing specific activitiesof phosphate pools. Furthermore, direct assaysof ATP content of cells (Fig. 3) and pulse-label

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1068 MAEBA AND SHIPMAN

experiments (Fig. 4) indicated that the pool sizesand synthesis of nucleotides increased duringglycerol-induced myxospore formation.The period of maximum accumulation of nu-

cleotides was one of active synthesis of spore-specific enzymes and spore material (9, 11, 30,33) and may represent a time of increased pro-duction of nucleotides for energy and formationof spore-specific RNA. However, the markedaccumulation of both intra- and extracellularnucleotides indicated an overproduction of thesecompounds for which there is no obvious expla-nation. Perhaps they represent nucleotide re-serves in myxospores that may also be requiredfor germination. In Bacillus species, increaseswere noted in nucleotide pools during sporula-tion (8, 28).

Their accumulation predominantly in culturesupernatant and the kinetics of accumulationand synthesis indicated that spots 0, B, C, D,and E were related compounds that were likelypolyphosphates of varying chain lengths (Re-sults). Recently, Ludwig et al. (18) showed thatpolyphosphates could be identified by the chro-matographic procedure of Cashel (5) as used inthis study. Comparison of the data shows thatspots A, B, and C were likely pyrophosphate andtri- and tetrapolyphosphates, respectively. Theother compounds, D, E, and 0, are probablypolyphosphates with greater chain lengths.Since spot 0 did not migrate in this system itwas not possible to determine whether it wascomprised of one or more compounds. Someunpublished data from this laboratory indicatethat spot 0 is heterogeneous. If these spotsrepresent a family of polyphosphates, then spotO would be the largest and may be the parentmolecule from which the others were formed. Itreached its peak level within 3 h after glyceroladdition (Fig. 6), whereas the others showedgreatest accumulation between 3 and 4 h andcontinued to increase (Fig. 7). Also, spot 0 ac-cumulated in vegetative cells and showed thegreatest increase during myxospore formation.The presence of contaminants in stock 32P1, pre-sumably polyphosphate (3), could not accountfor their appearance in the medium of sporulat-ing cells (Results). Although the polyphosphatesaccumulated in much greater quantities in themedium, their presence in small amounts in cellextracts indicated they were formed within thecytoplasm. During vegetative growth in 10 mMphosphate, polyphosphates were deposited asgranules within cells (32). This is in contrast tothe extracellular location of the polyphosphatespecies formed during myxospore formation;nevertheless, it does demonstrate these orga-nisms have the capacity to synthesize these pol-ymers.

J. BACTERIOL.

The accumulation of polyphosphate has beenstudied in other procaryotes. In Nitrosomonas,polyphosphate accumulated when generatedATP was not efficiently used to promote anincrease in cell mass (31). In Aerobacter, poly-phosphate accumulated whenever nucleic acidsynthesis ceased due to nutritional deficiency,regardless of its nature (14). During myxosporeformation, net RNA synthesis is reduced afterglycerol addition (2, 22), at a time when nucleo-tides are accumulating (Results). It is probablethat under these conditions a diversion of ATPpools to polyphosphate takes place. The time atwhich polyphosphate synthesis was observed in-dicates polyphosphates do not play a role ininitiating myxospore formation. That guanosinetetraphosphate accumulated between 120 and180 min (Fig. 13) suggests it may play a role inmediating the rates of RNA synthesis.

It seems unlikely that extracellular polyphos-phates could serve as energy or phosphate re-serves (27). This may be a mechanism by whichinternal phosphate levels are lowered to facili-tate myxospore formation. It is known that theformation of spores in Myxococcus is inhibitedby orthophosphate (2); also, their accumulationmay be related to the observation that a germi-nation factor that could be replaced by Pi wasproduced by germinating myxospores (22). Per-haps the polyphosphates, which were synthe-sized late during sporulation, accumulate and,with the onset of germination, are mobilized,e.g., by a polyphosphatase, to an active formthat stimulates germination. Since the organismnormally grows on solid medium where diffusionof polyphosphates may be limited, this schememay not be far-fetched. Recent reports implicatepolyphosphates in various regulatory functions(15, 18).The identity of spots 2, 3, and X remains un-

known. It does not appear likely that compoundX plays a role in initiating myxospore formationsince it appears fairly late in the process. Furtherwork in progress may determine the role ofcompounds 2 and 3, which accumulate from theonset of spore formation. It is interesting thatspot 2 has chromatographic properties similar toHPN III reported by Rhaese et al. in sporulatingBacillus (25). The reported structure is ppZpUp,where Z is a monosaccharide (26). A role for thismolecule in sporulation has not been deter-mined.

ACKNOWLEDGMENTThis work was supported by an operating grant from the

National Research Council of Canada.

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