JOURNAL OF BACTERIOLOGY, Sept. 1976, p. 1408-1413Copyright © 1976 American Society for Microbiology

Vol. 127, No. 3Printed in U.S.A.

Presence of Polyphosphate of Low Molecular Weight inZygomycetes

SONIA M. C. DIETRICHInstituto de Botdnica, C.P. 4005, Sdo Paulo, Brazil

Received for publication 4 April 1976

Polyphosphate of average chain length corresponding to 10 phosphate unitswas detected in the mycelial extract of zygomycetes. Gel electrophoresis tech-niques commonly used for the separation and characterization of acidic muco-polysaccharides were successfully used for the detection, purification, and char-acterization of the polyphosphate.

Polyphosphates have been known to occur innumerous filamentous fingi and in yeast. Thepolyphosphates obtained from these organismscover a wide range of molecular sizes. Thus,polyphosphates ofup to 10 phosphate units (Pi)have been detected in acid extracts togetherwith material of much higher chain length (fora review, see reference 8).The methods commonly used for the detec-

tion, characterization, and molecular weightdetermination of these compounds involverather elaborate techniques that are neitherrapid nor suitable in small-scale preparations(7, 8).The present paper reports the presence of

polyphosphate of low molecular weight in themycelium of three species of zygomycetes. Fur-thermore, it reports for the first time the appli-cation of microelectrophoretic techniques usedin the analysis of acidic mucopolysaccharidesfor the detection and characterization of poly-phosphates.

MATERIALS AND METHODSOrganisns and culture conditions. Choanephora

cucurbitarum (Berkeley & Ravenel) Thaxter (no.2339), Syncephalis sp. (no. 1995), Mortierella alpinaPeyroud (no. 1928), Mucor javanicus Wehmer (no.1989), Zygorrhynchus exponens Burgeff (no. 1960),and Cunninghamella elegans Lendner (no. 1591)were kindly supplied by the Instituto de Micologia,Universidade Federal de Pernambuco. The follow-ing oomycetes were also utilized: Phytophthora in-festans (Mont.) de Bary (no. 537) from the Se$do deMicologia Fitopatol6gica, Instituto Biol6gico de S&oPaulo; Achlya pseudoradiosa Rogers & Benecke(SPC 30); and Pythium sp. (ATCC 24619 = SPC 35).The numbers in parentheses refer to the suppliers'original collections except for ATCC 24619, whichwas originally from the Instituto de Botanica de S&oPaulo (SPC 35).The cultures of zygomycetes were maintained on

Difco potato dextrose agar slants, and the oomyceteswere maintained on Seymours MSPS-agar medium

(15). The liquid medium used for large-scale cul-tures was Hesseltine's synthetic medium (9) withoutagar for the zygomycetes and Seymour's MSPS (15)for the oomycetes. Actively growing mycelia weretransferred to Erlenmeyer flasks containing up to 40ml of the appropriate medium and were maintainedfor 3 to 4 days at 22°C in the dark. The cultures werethen disrupted with a Waring blender for 30 s, and2.5-ml portions of the suspension were inoculatedinto larger Erlenmeyer flasks containing 100 to 250ml of the same medium. These cultures were grownfor 5 days under the same conditions, harvested byfiltration, and washed three times with cold distilledwater. The cultures were shaken only occasionally.

For the growth in the presence of 3P, the generalprocedure was the same except that the liquid cul-tures (250 ml ofmedium) received an addition of 150Iul (total, 0.25 ,Ci) of carrier-free NaH232PO4 (Insti-tuto de Energia At6mica, University of S&o Paulo)on day 3 of incubation.

Extraction and purification procedures. Thewashed mycelia were homogenized with cold waterin a Virtis 45 homogenizer. In some instances, themycelia were either extracted by maceration withsand in a mortar or subjected to sonic treatmentwith an Insonator model 500 sonic oscillator for 5minutes intermittently, after homogenization,without any significant difference from the previousprocedure. The homogenates were centrifuged at1,000 x g for 30 min, and the supernatants wereprecipitated by the addition of either 2 volumes ofethanol or 5% cetyltrimethylammonium bromide(CETAVLON; BDH Chemicals Ltd., England), inthe proportion of 1.7 ml/100 ml of extract, to whichwas added NaCl up to approximately 0.7%. Afterstanding overnight at 50C, the precipitates wereharvested by centrifugation. The ethanol precipitatewas washed with 95% ethanol and dried. The CE-TAVLON precipitate was resuspended in 2 ml of10% sodium acetate plus 1 drop of ammonium hy-droxide (final pH, approximately 11) and centri-fuged. The supernatant fluid was acidified to pH 5.0with HCI, precipitated with 2 volumes of ethanol at50C, washed with ethanol, and dried. Samples of theextracts were dissolved in water and subjected toagarose gel electrophoresis in gel slides (7.5 by 5 cm)essentially as described by Jaques and co-workers

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(11) for heparin, and by Dietrich and Dietrich (2) forother acidic mucopolysaccharides. Semipreparativeagarose gel electrophoresis (50 mg of crude extract)was performed using a comparatively longer (20 cm)and thicker gel slide (3). After the run, a smalllongitudinal strip of the gel was stained with tolui-dine blue and the remainder was cut transversallyaccording to the location of the spots in the guidestrip. The fractions were then eluted by freeze-thaw-ing according to Dietrich et al. (3).

Purification of polyphosphate was also performedby diethylaminoethyl (DEAE)-cellulose chromatog-raphy using a 32P-labeled extract. A 250-mg amountof the crude extract (containing approximately1,000,000 cpm Of 32p) was diluted in 10 ml of waterand chromatographed in a DEAE-cellulose columneluted by stepwise addition of NaCl of increasingconcentrations. Portions of the fractions were ana-lyzed by scintillation counting, metachromatic reac-tion with toluidine blue, and ultraviolet (UV) ab-sorption at 260 nm. The 32P-labeled fractions devoidof UV-absorbing compounds were precipitated withCETAVLON and obtained as sodium salts, as de-scribed for the crude extract.

Analytical procedures. Total and labile phos-phates were determined by the method of Fiske andSubbaRow (6), pentoses by the method of Mejbaum(13) as modified, proteins by the procedure of Lowryet al. (12), and total carbohydrates by the anthronemethod (16). Molecular weight determinations wereperformed by polyacrylamide gel electrophoresis es-sentially as described by Hilborn and Anastassiadis(10) for the molecular weight determination of acidicmucopolysaccharides, except that gel slabs wereused instead of cylinders. Paper chromatography ofpolyphosphates was performed by using Ebel's no. 1solvent system (4) and the system described byOhashi and Van Wazer (14). The spots were detectedin the paper by the acid molybdate reagent (1).

Synthesis of standard polyphosphate. Sodiumdecaphosphate was prepared according to Felter andco-workers (5). The average molecular weight of thesynthesized compound was determined by polyacryl-amide gel electrophoresis and corresponded to apolyphosphate with an average of 10 to 12 Pi.

RESULTSDetection of polyphosphates by agarose gel

electrophoresis. Figure 1 shows a microelectro-phoresis slide of extracts of nine species of phy-comycetes. The zygomycetes Mortierella al-pina, Mucor javanicus, Zygorrhynchus expo-nens, and Choanephora cucurbitarum showedthe presence of metachromatic substances mi-grating toward the positive electrode. C. cucur-bitarum showed the largest quantity of themetachromatic material. The relative amountsof metachromatic compounds in the extractsapparently did not bear any relationship to theamount of other toluidine blue-staining com-pounds (nucleic acids) extractable under thesame conditions. The electrophoretic migrationof the metachromatic compounds varied from

POLYPHOSPHATE IN ZYGOMYCETES 1409

species to species. Slowly migrating compounds(unknown) were found in Mortierella, Mucor,and (possibly) Zygorrhynchus, whereas fast-moving compounds (polyphosphates) werefound in Mucor, Zygorrhynchus, Choa-nephora, and (possibly) Mortierella. The oomy-cetes Pythium, Achlya, and Phytophthora didnot show the presence of these compounds evenwhen the agarose slides were loaded with fivetimes as much extract.The species that showed metachromatic sub-

stances plus two ofthe species that did not showthem in the previous extraction were grown inthe presence of 32P-labeled monobasic sodiumphosphate. The amounts of phosphate and 32Plabeling obtained are given in Table 1. Themicroelectrophoretic patterns of toluidine blue

r--!+

oly P

NA

-unknown

NOIG I1 2 3 4 5 6 7 S 9 OIIFIG. 1. Agarose gel microelectrophoresis of myce-

lial extracts offungi. (1) Syncephalis sp.; (2) Morti-erella alpina; (3) Mucor javanicus; (4) Zygorrhyn-chus exponens; (5) Cunninghamella elegans; (6)Pythium sp.; (7) Achlya pseudoradiosa; (8) Phy-tophthora infestans; (9) Choanephora cucurbitarum.Extracts were obtained by precipitation with CE-TAVLON. Amounts of extract containing approxi-mately 20 to 30 pg of total phosphate were spotted.

TABLE 1. Total phosphate and 32p incorporation inselected fungi

Species

Syncephalis sp.............Mucorjavanicus ...........Zygorrhynchus exponens ...Achlya pseudoradiosa ......Choanephora cucurbitarum

Total phos-phate (,ug/mg of ex-tract)a23.520.817.630.484.1

32P incorpo-ration (cpm/mg of ex-tract)a

1,2001,000800

1,1004,500

a Extract obtained by precipitation with ethanol.

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1410 DIETRICH

staining and 32P labeling in the extracts areshown in Fig. 2. Only C. cucurbitarum showeda large incorporation of 32P into the metachro-matic compound. The other species showed nei-ther much labeling nor much of the metachro-matic fractions (except for Mucor and Zygor-rhynchus, when the slides were loaded with theequivalent of 20 to 30 ug of total phosphate, asin Fig. 1). Therefore, the extract from Choa-nephora was used for further studies.

Purification and characterization of thepolyphosphate. The polyphosphate from Choa-nephora was purified by DEAE-cellulose chro-matography as shown in Fig. 3. The bulk of thepolyphosphates (metachromatic, UV-nonab-sorbing, 32P-labeled fractions) were eluted atconcentrations of NaCl around 0.5 M (fractions60 to 68). Fractions 62 and 65 were precipitatedwith CETAVLON as described in Materialsand Methods and subjected to agarose gel elec-trophoresis. Each fraction showed one single32P-containing spot and metachromasy. Thequantitative determination of phosphate inthese fractions (Table 2) showed the presence oflabile phosphate which corresponds to the totalphosphate present in that fraction. Ribose de-termination gave negative results. The frac-

tions were also devoid of carbohydrates andproteins (Table 2).

Alternatively, the polyphosphates were puri-fied by semipreparative agarose gel electropho-resis. Fraction 5 of this preparation showed onesingle metachromatic spot with the same elec-trophoretic migration and chemical composi-tion as fraction 62 from DEAE-cellulose (Fig. 4and Table 1).The metachromatic 32P-labeled compound

(fraction 62) was labile to 1 N HCI treatment at100°C for 10 min (Fig. 5), yielding inorganicphosphate, as shown by paper chromatographyof the hydrolysate (Fig. 6) and by colorimetricdeterminations.Molecular weight determination. Paper

chromatography in Ebel's solvent system (Fig.6) indicated that the polyphosphate contained10 or more Pi, since the original polymer didnot move from the origin (4, 18). In the solventdescribed by Ohashi and Van Wazer (14), thecompound migrates with an Rf identical to thatof the 10-Pi standard. The molecular weight,determined by polyacrylamide gel electrophore-sis, gave an average value of 780, thus indicat-ing a polyphosphate with an average chainlength of 10 Pi.

+

IA.

Z.7

IfI\.

-"~-ORIGIN.9 741IA957 4 1 3 9 7 4

FIG. 2. Correlation between 32P labeling and metachromasy in components of mycelial extract of selectedzygomycetes. (A) Toluidine blue staining; (B) Radioautography of agarose gel slide. Species numbers as inFig. 1. Extracts obtained by precipitation with ethanol. About 0.2 mg of extract was spotted.

..I speciesnumber

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POLYPHOSPHATE IN ZYGOMYCETES 1411

0 0.05 M OJ M

74-, _l

3

° 2E0.U

0

02 M 031M 05 M

OD 260nmcpm

I M NoCIcamion

QD.260 nm3

2

7' Fractionnv*

FIG. 3. Fractionation of mycelial extract from Choanephora cucurbitarum by DEAE-cellulose columnchromatography.

TABLE 2. Composition ofpolyphosphate fractionsobtained by column chromatography in DEAE-cellulose and by agarose gel electrophoresisa

Phosphate(,ug) Pen- Glu- Pro-

Fraction tose cose teinLa- Total (Ag) (,g) (/lg)bile

Crude ........... 78.4 84.1 72.0 150.0 623.0No. 62, DEAE ... 18.1 18.5 0 0 0No. 65, DEAE ... 9.9 9.6 0 0 0No. 5, gel ....... 29.5 29.7 0 0

a Determinations correspond to 1 mg for the crude ex-tract, 5 Al out of approximately 100 Al for fraction no. 62, 5,ul out of 50 1,u for fraction no. 65, and 10 ,ul out of 500 Al forfraction no. 5.

DISCUSSIONMetachromatic compounds were detected by

agarose gel electrophoresis in relatively largeamounts in mycelial extracts of the zygomy-cetes Choanephora cucurbitarum, Mucor ja-vanicus, and Zygorrhynchus exponens, andpossibly in Mortierella alpina. The followingproperties of one of these metachromatic com-pounds enable its characterization as a poly-phosphate: (i) highly anionic properties; (ii)

+

u-

_ E.919_l'w

_"_~- ORIGIN

GEL DEAEFr.5 Fr.62

FIG. 4. Comparative electrophoretic migration ofpolyphosphate purified by DEAE-cellulose chroma-tography and by agarose gel electrophoresis.

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1412 DIETRICH

-ORIGIN

A BFIG. 5. Agarose gel electrophoresis of polyphos-

phate before (B) and after (A) acid hydrolysis.

metachromasy; (iii) incorporation of labeledphosphate (32P); (iv) precipitation with CETAV-LON; (v) total lability to 1 N HCl for 10 min at10000 with the production of inorganic phos-phate and loss of metachromasy; (vi) samechromatographic migration as standard poly-phosphate; and (vii) absence of pentose andUV-absorbing compounds. The average molec-ular weight of the polyphosphate from Choa-nephora was 780, which accounts for an averageof 10 Pi. The polyphosphate was extractablewith cold water, suggesting that the compoundis not bound to other molecules in the myceliumof these fungi.The analysis of the compound was performed

by using mainly agarose gel electrophoresis,polyacrylamide gel electrophoresis, and precip-itation with quaternary amines, which arecommon techniques used in the study of acidmucopolysaccharides (2, 3, 11). The agarose gelelectrophoresis permitted the detection ofmeta-chromatic compounds present in minuteamounts in some species (less than 20 to 30 ugof total phosphate) and could be useful in thescreening of these substances in microbial ex-tracts. Furthermore, it permitted rapid purifi-cation of polyphosphate comparable to that ob-tained by DEAE-cellulose chromatography.

CPoy PiCotroll

_PPi

ORIGIN

Pdly Pi +

IN Ha

FIG. 6. Paper chromatography of polyphosphatebefore and after acid hydrolysis. Pi, Inorganic phos-phate; PPi, pyrophosphate (possibly originated dur-ing chromatography). The solvent used was the onedescribed by Ebel (4).

It is noteworthy that none of the oomycetesanalyzed showed this compound even in heav-ily loaded slides. It has been observed that inAchlya pseudoradiosa Rogers & Beneke (oo-

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VOL. 127, 1976

mycetes) there is no detectable polyphosphatein extracts obtained at different stages ofgrowth or development (Dietrich and Matida,unpublished data). Also, there are no data on

the presence of polyphosphates in oomycetes,although polyphosphated glucans have alreadybeen found in this group of fungi (17).

ACKNOWLEDGMENTSThis work was aided by grants from the Fundacao de

Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), andConselho Nacional de Desenvolvimento Cientifico e Tec-nol6gico (CNPq), Brazil.

I wish to express my gratitude to C. P. Dietrich for usefulsuggestions, for help in the preparation of this manuscript,and for the use ofsome ofhis laboratory facilities. Gratitudeis also extended to Hernan Chaimovich, who kindly sup-

plied the radioactive phosphate and gave useful sugges-

tions. The technical help of Margarida K. Shuzuki in grow-ing the fungi is also acknowledged.

LITERATURE CITED1. Burrows, S., F. S. M. Grylls, and J. S. Harrison. 1952.

Paper chromatography of phosphoric esters. Nature(London) 170:800-801.

2. Dietrich, C. P., and S. M. C. Dietrich. 1972. Simplemicro method for the identification of heparin andother acidic mucopolysaccharides from mammaliantissues. Anal. Biochem. 46:209-215.

3. Dietrich, C. P., H. B. Nader, L. R. G. Britto, and M. E.Silva. 1971. Chemical composition of heparitin sul-fate. Fractionation and characterization of four acidicmucopolysaccharides in heparitin sulfate from beeflung tissue. Biochim. Biophys. Acta 237:430-441.

4. Ebel, 3. P. 1954. Separation par chromatographie surpapier des oxyacides du phosphore. Mikrochim. Actap. 679-700.

5. Felter, S., G. Dirheimer, and J. P. Ebel. 1970. Re-cherches sur les polyphosphatases de levure de bou-langerie. Bull. Soc. Chim. Biol. 52:433-446.

6. Fiske, C. H., and Y. SubbaRow. 1925. The colorimetric

POLYPHOSPHATE IN ZYGOMYCETES 1413

determination of phosphorus. J. Biol. Chem. 66:375-400.

7. Harold, F. M. 1960. Accumulation of inorganic poly-phosphate in mutants ofNeurospora crassa. Biochim.Biophys. Acta 45:172-188.

8. Harold, F. M. 1966. Inorganic polyphosphates in biol-ogy: structure, metabolism, and function. Bacteriol.Rev. 30:772-794.

9. Hesseltine, C. W., and R. F. Anderson. 1957. Microbialproduction of carotenoids. I. Zygospores and caroteneproduced by intraspecific and interspecific crosses ofchoanephoraceae, in liquid media. Mycologia 49:449-452.

10. Hilborn, J. C., and P. A. Anastassiadis. 1971. Estima-tion of the molecular weights of acidic mucopolysac-charides by polyacrylamide gel electrophoresis. Anal.Biochem. 39:88-92.

11. Jaques, L. B., R. E. Ballieux, C. P. Dietrich, and L. W.Kavanagh. 1968. A microelectrophoretic method forheparin. Can. J. Physiol. Pharmacol. 46:351-360.

12. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

13. Mejbaum, W. 1939. Uber die Bestimmung kleiner Pen-tosemengen, insbesondere in Derivaten der Aden-ylsaure. Z. Physiol. Chem. 258:117-120.

14. Ohashi, S., and J. R. Van Wazer. 1963. Paper chroma-tography of very long chain polyphosphates. Anal.Chem. 35:1984-1985.

15. Seymour, R. L. 1970. The genus Saprolegnia. NovaHedwigia Z. Krytogamenkd. 19:1-124.

16. Umbreit, W. W., and R. H. Burns. 1964. Chemicalanalysis, p. 205-210. In W. W. Umbreit, R. H. Burris,and J. F. Stauffer (ed.), Manometric techniques, 4thed. Burgess Publishing Co., Minneapolis, Minn.

17. Wang, M. C., and S. Bartnicki-Garcia. 1973. Novelphosphoglucans from the cytoplasm of Phytophthorapalmivora and their selective occurrence in certainlife cycle stages. J. Biol. Chem. 248:4112-4118.

18. Woodis, T. C., Jr., J. R. Trimm, and R. D. Duncan.1973. Separation of polyphosphates by paper chroma-tography with a new solvent. Anal. Chim. Acta65:469-473.

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