POLYPHOSIHATE ACCUMULATION AND UTILIZATION AS RELATED TOSYNCHRONIZED CELL DIVISION OF CORYNEBACTERIUM

DIPHHTHERIAE'

THEOI)ORE SALL, STUART MUDD, AND ATSUSHI TAKAGI2Department of Mlicrobiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

Received for publication July 3, 1958

In previous studies, Sall et al. (1956) employ-ing resting cells of Corynebacterium diphtheriae,and Mludd et al. (1956, 1958) employing restingcells of Mycobacteria, demonstrated that malateas substrate enhances the accumulation ofpolyphosphate (metaphosphate) in the form ofmetachromatic granules and glucose promotesthe withdcrawal of polyphosphate. Similarly,AIudd et al. (1958), employing p32, showedthat, in growing cultures of Mycobacteriumthamnopheos, malate as substrate or azaserineas inhibitor favors the (leposition of polyphos-phate, whereas glucose as substrate or dinitro-phenol as inhibitor promotes the utilization ofpolyphosphate to form ribonucleic acid.

Various applications of synchronized celldivision to metabolic and cytological studieshave recently been reviewed by Campbell (1957).In the present study, utilizing synchronouslydividing cells of C. diphtheriae, a physiologicaldeposition an(l utilization of polyphosphate hasbeen (lemonstrated in relation to the cycle ofcell division.

MATERIALS AND METHODS

Corynebacteriium diphtheriae mitis A 679 wasused throughout. Stock cultures were kept underoil at 4 C on MIorton-Engley (1945) yeast extracttryptone agar enriched with blood serum. Sub-cultures were preparedl friom the stock culturesby inoculating tubes of Mlorton-Engley bloo10selum broth and incubating for 18 hr at 37 Cin a roller-tube aerator.

Synchronized cell division was obtained in thefollowing manner: 18 hr cultures were harvested,

I This work has been aided by a grant from theUnited States Atomic Energy Commission, AECContract No. AT(30-1)-1342.

2 Fellow of the Theresa F. and Joseph FelsenMemorial Ftund. Present address: Department ofBacteriology, School of Medicine, University ofTottori, Yonago, Tot tori-ken, Japan.

washed 3 times with refrigerated saline solutionand suspend(led in refrigerated saline. The cellsuspension was placed in an ice bath for 45 min.The suspension was then poured into 250 ml ofMIorton-Engley blood serum broth at 37 C andaerated. At 10 min interxvals, 30 ml aliquotswere removed by means of a 50-ml svringeequipped with a 3-way stopcock. One ml wasremoved for total cell count and estimation ofindex of metachromasy. The riemaining 29 mlof cell suspension were anal-zed for the variousphosphorus fractions.

Total cell counts were obtained in the usualmanner employing a Petroff-Hauser countingchamber. One-quarter ml of cell suspension wasdiluted with aqueous solution of 1.5 per centcrystal violet, and mixed vigorously in a smalltest tube. A portion of the diluted cell suspen-sioIn was removed with a capillaiy pipette andwas used to charge the counting chamber. Tenlarge squares were counted, 5 diagonally and 5scattered.

Index of metachromasv w-as determined by theprocedure described by Sall et al. (1956). Theindex of metachromasy is the sum of the productsobtained by multiplying the estimated diameters(4+ = 1,3± =3+ , 2+ = ½,l+ =of the granules by the percentage of cells showingsuch gr'anules.

Phosphorus partitioning was performe(d in amanner previously described (Sall et al., 1956).The acid soluble fraction was determined intoto. The following acid insoluble fractions we?repooled and determined: phospholipid, organic,inorganic, andl 7 miii phosphorus.Thet ultramicromethod of lBerenblum andI Chain, as modified byAMartin and Doty (1956) for the determiniationlof phosphorus was employed. Ogur and Roseni's(1950) method for nucleic acid estimations wasused as an assay confirmatory to the estimationof nucleic acids by fractionation of phosphorus.

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POLYPHOSPHATE IN CELL DIVISION OF C. DIPHTHERIAE

RESULTS

Approximately 80 per cent of the cells dividesynehronously after an incubation period ofabout 20 min. A similar division again tookplace after about 40 min (tables 1 and 3, andfigures la and 8a). The various phosphorus

TABLE 1Relationship of phosphorus fractions to cyclic cell

division; cells initially metachromatic

Time Cell I.M * Total Poly DNA RNA A.S. A.Ins.Count P P P P P P

min X 10-9

0 1.54 119 6.0 2.3 .13 0.84 1.3 0.7810 1.61 63 6.7 1.6 .12 1.7 1.6 1.220 1.65 113 8.7 2.2 .24 1.8 1.7 1.830 3.18 9 5.2 0.31 .16 1.3 1.0 1.440 3.24 62 6.9 1.7 .31 1.6 1.2 2.150 6.09 14 4.2 0.20 .20 1.4 0.66 1.660 6.80 80 5.4 1.2 .29 1.5 0.65 1.7

All phosphorus fractions are expressed asgg X 109 P per cell.

Total cell counts are expressed as cells X 10-9per 29 ml of culture.

* I.M. = Index of metachromasy; Poly P is theacid insoluble polyphosphate P; DNA and RNA =deoxyribonucleic and ribonucleic acid, respec-tively; A.S. is a pool of the total cold trichloraceticacid-soluble phosphorus fractions; A.Ins. is apool of the trichloracetic acid-insoluble fractionsconsisting of phospholipid, inorganic, organic,and 7 min phosphorus.

TABLE 2Ultraviolet absorption determination of the

nucleic acid fractions

Time Total Cell I.M.* DNA RNACount

min X 109 plg Ag

0 1.20 10 3.1 2010 1.32 4 3.0 3220 1.55 42 5.4 4130 2.76 6 3.8 3140 3.09 66 6.6 4250 6.45 8 3.8 2760 7.50 74 5.7 35

The nucleic acid

pgg X 10' per cell.

fractions are expressed as

Total cell counts are expressed as cells X 10-1

per 29 ml of culture.* IbM. = Index of metachromasy; DNA =

deoxyribonucleic acid; RNA = ribonucleic acid.

TABLE 3Relationship of index of nmetachromasy, nucleicacids and polyphosphate to cyclic cell division;

cells initially nonmetachromatic

Time Total Cell J.M.* Poly DNA RNACount P P P

min X 109

0 1.92 6 0.20 .05 0.7810 2.05 3 0.44 .10 1.320 2.12 40 1.2 .24 1.630 3.71 0 0.27 .16 1.240 3.86 81 0.91 .26 1.850 6.82 0 0.26 .16 1.460 7.75 63 0.77 .27 1.4

The nucleic acid and polyphosphate fractionsare expressed as,ug X 109 P per cell.

Total cell counts are expressed as cells X 10-9per 29 ml of culture.

* I.M. = Index of metachromasy; Poly P is theacid insoluble polyphosphate P; DNA and RNA =deoxyribonucleic and ribonucleic acid, respec-tively.

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7

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5

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302

0 10 20 30 40 50 60MINUTES

Figure la. Synchronized cell division of Cory-nebacterium diphtheriae. The culture had pro-gressed through 2 cycles. Cells initially metachro-matic. All P values plotted as,g P per 29 ml ofculture. I.M. = Index of metachromasy; Meta-P = Polyphosphate (metaphosphate); DNA-P =Deoxyribonucleic acid phosphorus.

fractions are expressed as phosphorus or nucleicacid per bacterial cell in tables 1, 2, and 3 andas phosphorus per volume of culture in figuresla and 8a.

Deoxyribonucleic acid phosphorus values percell (tables 1 and 3) were approximately double

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SALL, MUDD, AND TAKAGI

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S4: 6s};*Figures 1-7. Light micrographs of Neisser stained cells which correspond to table 1 and the curve in

figure la. Magnification 1250 X.Figure 1, 0 time cells. Note the many darkly stained metachromatic granules; index of

metachromasy = 119.Figure 2, 10 min cells. Note the fewer metachromatic granules; index of metachromasy = 63.Figure 3, 20 min cells. Note the increase in the number and size of the granules; index of

metachromasy = 113.Figure 4, 30 min cells. Note the absence of the granules; index of metachromasy = 9.Figure 5, 40 min cells. Note the increase in the number of granules; index of metachromasy = 62.Figure 6, 50 min cells. Note the marked decrease in the number and size of the granules; index of

metachromasv = 14.Figure 7, 60 min cells. Note the increase in

metachromasy = 80.

their preceding values at 20 and 40 min andincreased by 45 to 70 per cent at 60 min. Ultra-violet spectrophotometric analysis of the nucleicacids (table 2) indicated similar step-wise in-creases in deoxyribonucleic acid. These data are

in agreement with data from cells of higher forms,which approximately double their chromatincontent prior to cell division. Total, ribonucleicacid, acid soluble, and acid insoluble phosphorusper cell showed a less clear relationship to thecycle of cell division.The acid insoluble polyphosphate (metaphos-

phate) phosphorus values per cell increasedconcomitantly with deoxyribonucleic acid phos-phorus, i.e., at 20, 40, and 60 min, and decreased

the number and size of the granules; index of

markedly during the times of increasing cellnumbers, i. e., at 30 and 50 min. Because of theminute quantities of acid soluble polyphosphateaccurate estimations of this fraction were notfeasible. Fields of cells stained for polyphosphateby the technique of Neisser (Sall et al., 1956)were prepared at intervals corresponding to thepreparations for microchemical analysis. Theindices of metachromasy fluctuated in parallelwith polyphosphate P (tables 1 and 3); thesevalues were strikingly high at 20, 40, and 60min, but low at 30 and 50 min. The ratherdramatic accumulations of polyphosphate gran-

ules at 20, 40, and 60 min are shown in figures1 to 14.

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POLYPHOSPHATE IN CELL DIVISION OF C. DIPHTHERIAE

7-

6

Figure 8a. Synchronized cell division of Cory-nebacterium diphtheriae. The culture had pro-

gressed through 2 cycles. Cells initially non-

metachromatic. All P values plotted as,g P per

29 ml of culture. I.M. = Index of metachromasy;Meta-P = Polyphosphate (metaphosphate);DNA-P = Deoxyribonucleic acid phosphorus.

°- 5 -

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Figures 8-14. Light micrographs of Neisser stained cells which correspond to table 3 and the curvein figure 8a. Magnification 1250 X.

Figure 8, 0 time cells. Index of metachromasy = 6.Figure 9, 10 min cells. Only occasional metachromatic granules; index of metachromasy = 3.Figure 10, 20 min cells. Note the marked increase in the number of metachromatic granules; index of

metachromasy = 40.Figure 11, 30 min cells. Comparatively few granules; index of metachromasy = 0.Figure 12, 40 min cells. Note the large increase in size and number of metachromatic granules; index

of metachromasy = 81.Figure 13, 50 min cells. Note the marked decrease in the size and number of the granules; index of

metachromasy = 0.Figure 14, 60 min cells. Note the increase in the number of granules; index of metachromasy = 63.

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SALL, MUDD, AND TAKAGI

DISCUSSION

The data presented are representative of thesuccessful experiments, about one-half of thetotal number that were performed. The principaldifficulties were involved in obtaining synchro-nized cell division. It is of interest to note thatthe normal generation time of this organismunder the same experimental conditions withthe exception of cold shock, is 22 min, the same

generation time as was observed with synchro-nously dividing cells. However, a period of 45min was necessary for the cold shock treatmentin order to obtain synchronized cell division.This could be accounted for if portions of thepopulation reached the "particular stage of un-

balanced growth" (Campbell, 1957) at differenttimes, resulting in the need for a 45-min shockperiod for at least 80 per cent of the organismsto attain the unbalanced growth stage.

It is characteristic of bacilli of C. diphtheriaeto form sharp angles with each other followingcell division. This characteristic tended to theformation of clumps, which made the total cellcount arduous to obtain. This difficultv was

surmounted, to an extent, by vigorously shakingthe cell suspension with the crystal violet dilutingsolution in a small test tube containing severalsmall glass beads.

Winkler (1953) states that during the earlystages of the log phase Corynebacteria andMycobacteria usuallv have minute metachro-matic granules, which are detectable only with theelectronmicroscope. Towards the end of thelog phase the granules are large enough to beseen in stained preparations with the lightmicroscope, and in the stationary phase thegranules increase in size. This is in agreementwith Kornberg (1957) and Winder and Denneny(1957) who postulate that during periods ofrapid growth, or synthetic processes which re-

quire adenosine triphosphate, the utilization ofpolyphosphate exceeds its formation. This issubstantiated by Mudd et al. (1958) who demon-strated with Mycobacteria that polyphosphatecould be used for nucleic acid synthesis and cellgrowth.

Recently Kornberg et al. (1958) obtained fromE. coli an enzyme which is capable of convertingdeoxyguanosine triphosphate to deoxyguanosineand tripolyphosphate. The implications of thisreaction further support the possibilities thatpolyphosphate can be utilized for nucleic acidsynthesis and cell growth.

The demonstration that polyphosphate maybe stored and utilized in physiological relation-ship to the cycle of cell division is in agreementwith the evidence presented in earlier papers(Sall et al., 1956; Winder and Denneny, 1956;Kornberg et al., 1956; Lieberman, 1956; Muddet al., 1958) that polyphosphate may serve inmicroorganisms as phosphagen. Scott and Chu(1958) have shown that during cell division ofE. coli there is a sharp increase in glucose assim-ilation. It is possible that phosphate may beutilized in the transport mechanisms involved.

SUMMARY

Polyphosphate is found, by cytochemical andmicrochemical procedures, to increase and de-crease in relation to the cyclic division of syn-chronized dividing cells. The peaks of poly-phosphate accumulation are found immediatelybefore the periods of increasing cell numbers;minimal amounts of polyphosphate are foun(dat the ends of the recurrent periods of cell divi-sion. Increments of deoxyribonucleic acid arefound to occur between the periods of cell divi-sion. Accumulation and disappearance of poly-phosphate are, then, physiological events relatedto cell division; these findings support the inter-pretation of polyphosphate as phosphagen.

REFERENCESCAMPBELL, A. 1957 Synchronization of cell

division. Bacteriol. Revs., 21, 263-272.KORNBERG, A., KORNBERG, S. R., AND SIMMS, E.

S. 1956 Metaphosphate synthesis by an en-zyme from Escherichia coli. Biochim. etBiophys. Acta, 20, 215-227.

KORNBERG, S. R. 1957 Adenosine triphosphatesynthesis from polyphosphate by an enzymefrom Escherichia coli. Biochim. et Biophys.Acta, 26, 294-300.

KORNBERG, S. R., LEHMAN, I. R., BESSMAN, M. J.,SIMMs, E. S., AND KORNBERG, A. 1958 En-zymatic cleavage of deoxyguanosiine triphos-phate to deoxyguanosine and trIipolyphos-phate. J. Biol Chem., 233, 159-162.

LIEBERMAN, I. 1956 Inorganic triphosphatesynthesis by muscle adenylate kinase. J.Biol. Chem., 219, 307-317.

AIARTIN, J. B. AND DOTY, D. M. 1956 In lethodsof biochemical analysis, Vol. 3, p. 7. Editedby D. Glick. Interscience Publishers, Inc.,New York.

MORTON, H. E. AND ENGLEY, F. B., JR. 1945The protective action of dysentery bacterio-phage in experimental infections in mice.J. Bacteriol., 49, 245-255.

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MUDD, S., TAKEYA, K., AND HENDERSON, H. J.1956 Electron-scattering granules and re-

ducing sites in Mycobacteria. J. Bacteriol.,72, 767-783.

MUDD, S., YOSHIDA, A., AND KOIKE, M. 1958Polyphosphate as accumulator of phosphorusand energy. J. Bacteriol., 75, 224-235.

OGUR, M. AND ROSEN, G. 1950 The nucleicacids of plant tissues. I. The extraction andestimation of desoxypentose nucleic acid andpentose nucleic acid. Arch. Biochem., 25,262-276.

SALL, T., MUDD, S., AND DAVIS, J. C. 1956 Fac-tors conditioning the accumulation and disap-pearance of metaphosphate in cells of Cory-nebacterium diphtheriae. Arch. Biochem.Biophys., 60, 130-146.

SCOTT, 1). B. McN. AND CHU, E. 1958 Glucoseassimilation by synchronized cultures of E.coli. Bacteriol. Proc., 1958, 96.

WINDER, F. G. AND DENNENY, J. M. 1956 Phos-phorus metabolism of mycobacteria: deter-mination of phosphorus compounds in some

mycobacteria. J. Gen. Microbiol., 15, 1-18.WINDER, F. G. AND DJENNENY, J. M. 1957 The

metabolism of inorganic polyphosphate inMycobacteria. J. Gen. Microbiol., 17, 573-585.

WINKLER, A. 1953 The metachromatic granulesof bacteria. In Symposium, bacterial cytology.Supplement to Rend. ist. super. sanita, pp.

39-43.

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