380 · chemical constituents of the organisms are commonly insufficient to permit interpretation of...

PLANT PHYSIOLOGY

Neither the P32-phosphate nor the S35-sulfate weremetabolized by intact seeds until after radicle protru-sion (15 to 18 hours after beginning of imbibition).Punctured seeds, however, metabolized the radioactivephosphate or sulfate within 3 hours after the beginningof imbibition. We concluded that the coats surround-ing the embryo are impermeable to phosphate andsulfate ions. Most of the organic P32 extracted,whether from punctured seeds before radicle protrusionor from unpunctured seeds after protrusion, was foundin phosphorylcholine and phospholipids.

S35 was detected in cysteine, methionine, and nu-merous unidentified compounds, both from puncturedseeds before radicle protrusion and from unpuncturedseeds after radicle protrusion.

Thus within 3 hours after the beginning of im-bibition, punctured seeds could esterify phosphate andreduce sulfate to the level of sulfhydryl.

LITERATURE CITED1. BASSHAM, J. A. and CALVIN, M. The Path of Car-

bon in Photosynthesis. Prentice-Hall, EnglewoodCliffs, New Jersey 1957.

2. BENSON, A. A. and MARUO, B. Plant phospholipids.I. Identification of the phosphatidyl glycerols.Biochim. Biophys. Acta 27: 189-195. 1958.

3. BLOCK, R. R., DURRUM, E. L. and ZWEIG, G. AManual of Paper Chromatography and Paper Elec-trophoresis. Academic Press, New York 1958.

4. CROCKER, W. and BARTON, L. V. Physiology ofSeeds. Chronica Botanica, Waltham 1953.

5. EVENARI, M. The physiological action and biologicalimportance of germination inhibitors. Symposia ofthe Society for Experimental Biology 11: 21-43.1957.

6. EVENARI, M., KLEIN', S., ANCHORI, H. and FEINBRUN,N. The beginning of cell division and cell elonga-tionl in germinating lettuce seed. Bull. Res. Councilof Israel 6D: 33-37. 1957.

7. EVENARI, M. and NEUMANN, G. The germinationof lettuce seed. II. The influence of fruit coat,seed coat and endosperm upon germiniation. Bull.Res. Council of Israel 2: 75-78. 1952.

8. HABER, A. H. and TOLBERT, N. E. Effects of gib-berellic acid, kinetin, and light on the germinationof lettuce seed. In: Photoperiodism and RelatedPhenomena in Plants and Animals, A. P. Withrow,ed. AAAS, Washington, D. C. (In press.)

9. HAGEN, C. E., BORTIIWICK, H. A. and HENDRICKS,S. B. Oxygen consumption of lettuce seed in rela-tion to photocontrol of germination. Bot. Gaz. 115:360-364. 1954.

10. KUNITAKE, G., SALTAIAN, P. and LANG, A. Theproducts of CO2 dark fixation in leaves of long- andshort-day treated Kalanzchot blossfeldiana. PlantPhysiol. 32: 201-203. 1957.

11. LATIEs, G. Respiration and cellular work and theregulation of the respiration rate in plants. SurveyBiol. Prog. 3: 215-299. 1957.

12. MAIZEL, J. V., BENSON, A. A. and TOLBERT, N. E.Identification of phosphoryl choline as an importantconstituent of plant saps. Plant Physiol. 31: 407-408. 1956.

13. POLJAKOFF-MAYBER, A and EVENARI, M. Somefurther investigations on the oxidative systems ofgerminating lettuce seeds. Physiol. Plantarum 11:84-91. 1958.

14. RUNECKLES, V. C. Formation of alkyl phosphatesin wheat leaves. Nature 181: 1470-1471. 1958.

MECHANISMS OF ACTION OF POLYMYXIN 13 ONCHLORELLA AND SCENEDESMUS 1' 2,3

R. A. GALLOWAY AND R. W. KRAUSSDEPARTMENr OF BOTANY, UNIVERSITY OF MARYLAND, COLLEGE PARK, MARYLAND

The investigations of the mechanism of action ofan inhibitor of cell growth or development often yieldfundamental information about the metabolism of thespecies under study. The available data on the bio-chemical constituents of the organisms are commonlyinsufficient to permit interpretation of experimentalresults obtained when the inhibitor is introduced.The examination of the role of polymyxin B, an anti-biotic which shows strikingly different effects on re-lated species, is no exception. Evidence has been ac-cumulating which indicates that polymyxin B acts todisorganize the cell wall which in turn causes dis-

1 Received September 29, 1958.2 This investigation was supported in part by the

Rockefeller Foundation and the Office of Naval Research.3 Contribution no. 2954 Maryland Agricultural Experi-

ment Station, Scientific Article A 718.

ruption of the osmotic equilibrium of the cell (1, 8, 9,10). However, other evidence (2, 11, 17) suggeststhat damage from surface action does not completelyexplain polymyxin inhibition. Osmotic disruptionseems to be but one of several possible effects. War-ren et al (17), studying the sensitivity of polymyxin-treated cells to lysozyme, observed that no correlationcould be demonstrated between the sensitivity of theorganisms to the antibiotic and the lytic response fol-lowing the addition of the enzyme. Galloway andKrauss (2) showed that galactose can protect againstthe inhibition of respiration by polymyxin B in 2species of bacteria.

The present study was undertaken to further ex-amine the role of polymyxin B, especially with regardto the protective effect of galactose on susceptiblealgae, as well as the mechanism of resistance innormally resistant algae. Such an investigation has

380

Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

381GALLOWAY AND KRAUSS--POLYMYXIN B ACTION ON ALGAE

required additional exploration of the metabolism ofboth kinds of organisms.

MATERIALS AND METHODS

ORGANISMS AND MIEDIA: The algae in thesestudies included Chlorella pyrenoidosa Chick, VanNiel's strain, Chlorella vulgaris Beijerinck, Pring-sheim's strain, isolated by Emerson, and Scenedesmullsobliquuts Turp. (Kutz), strain WH-50, isolated byKrauss. The several media differed only with re-spect to the amount of phosphorous and the presenceor absence of tris (hydroxymethyl) aminomethane(Sigma 7-9) buffer. The components common toall media were KNO3 (1.00 g/l), MgSO, - 7H2O(0.25 g/l), and micronutrients in the form of innercomplex salts of ethylenediaminetetra acetic acid(EDTA) as suggested by Thomas and Krauss (15).The micronutrients included iron (5 ppm), manga-nese, calcium, copper, cobalt, and zinc (1 ppm each).The "high-phosphate" medium contained, in additionto the common components, 1.64 X 10-3 M phos-phorous. The "low-phosphate" medium included1.61 phosphorous and Sigma 7-9 (2 g/l).

The "32P medium" contained, in addition to thecommon components, 1 X 10-5 phosphorous and10 MC 32P per Mg of non-radioactive phosphorous.Sigma 7-9 (2 g/l) was also added. The compositionof the "32P-EDTA medlium" was the same, except forthe addition of 6 g/l of EDTA. The "phosphorous-free" medium included Sigma 7-9 (2 g/l). The pHof all media was adjusted to 7.0 with HCl.

MANOMETRY: Manometric experiments involvingthe usual manometric techniques (16), were per-formed in a standard Warburg apparatus at 250 C.The shaking rate was 125 complete cycles per minute.Vessels wv-ere of the 2-side-arm, center-well, venting-plug type. One ml of medium containing a dryweight of cells of about 10 mg was placed in the maincompartment of the vessel. The center well contained0.2 ml of 10 % KOH. One half ml of solution con-

taining various concentrations of polymyxin B was

placedl in 1 of the side arms. The other was usedfor the addition of substrate.

GROWTH: The experimental conditions andequipment have been previously described (2).Briefly, 16-mm culture tubes, containing 5 ml of in-oculated medium, were held at a 150 angle on a recip-rocating shaker. A bank of fluorescent and incan-(lescent lights supplied an incident illumination be-tween 700 and 1000 ft-c at the surface of the shaker.Temperature wvas maintained at 25°C.

PHOSPHOGLUCOSE IsOMfERASE: Phosphoglucoseisomerase was prepare(l from rabbit muscle accordingto the method of Slein (13). The reaction mixtureconsisted of 0.1 ml of crude enzyme preparation whichhad been diluted 1 to 4000 wv-ith distilled water redis-tilled in an all pyrex still and 0.4 ml of buffer solutioncontaining various concentrations of the disodium saltof glucose-6-phosphate. The buffer, 0.06 Sigma

7-9, was adjusted to a pH of 7.0. Incubation was per-formed at 60 C for 35 minutes. Termination of thereaction was accomplished by addition of 3.5 ml of8.3 M HCl. Enzyme activity was assayed by deter-mination of fructose-6-phosphate, using the colori-metric method of Roe (12)-viz.. 1 ml. of 1 % re-sorcinol in 95 % ethanol was addedI and the mixturewas kept at 80° C for 10 minutes; then it was cooledin a water bath at 250 C. The color intensity wasread in a Bauch and Lonmb Spectronic 20 spectropho-tometer at a wave length of 540 mMA.

UPTAKE OF 32P: Algae wvere cultured in 35 mlof either low-phosphate or high-phosphate medium in75 ml test tubes in a 250 C water bath. The cultureswere bubbled with air enriched with 5 % CO.,. Lightwas supplied from 2 sides by fluorescent lamps, eachgroup of which gave an irradiance of approximately1500 ft-c at the surface of the culture tubes. Thecultures were grown to an optical density between 0.75and 1.0 measured at a wave length of 560 mM. Cellsharvested at this time were predominately large, ma-ture light cells. In ordler to obtain predominatelystarved and young dark cells, cultures were placed inthe dark for 24 hours before use. The term "light"cells refers to cells which are at the end of the develop-mental cycle and are nearly ready to reproduce vege-tatively by dividing into (laughter cells. In contrast"dark" cells are young cells whichnmay be formed inthe (lark by division of light cells. They remainsmall and physiologically "young" if kept in the darkwithout an energy source.

From this point on, 1 of 2 procedures was enm-ployeed, depending upon the length of time cells wereto be exposed to 32P. For a treatment of 15 minutesor less, usually the following method was used: Thecells were removed from the nmedium by centrifugationfor 2 minutes at 675 X G in 5-ml conical tubes in anangle-head Sargent centrifuge. Cells were waslhedonce, resuspended in phosphorous-free mediuml, anddispensed into a sintered glass funnel fitted with amillipore filter (lisc (HA plain wrhite, 47 cm in(liameter). Aliquots of 32P-EDTA medium werethen thoroughly mixe(d with the cell suspension in thefunnel. (EDTA was included to prevent the precipi-tation of phosphate salts on the membrane observedin preliminary studies-see Results.) After specifiedperiods of time, the medium was removed by suctionthrough the filter, leaving a tlhin layer of radioactivecells. The filter (with cells) was removed andl fast-enedl with rubber cement to the absorbent pad providedfor eaclh filter by the manufacturer. \Vhen the cellswere dry, their radioactivity- was nmeasured on aTracerlab autoscaler with an end-window Geiger-Mueller tube. Alternatively, if cells were to be incontact witlh 32P for longer than 15 minutes.32P-EDTA medium was usuallv added directly to thecentrifuge tubes. After a given time, the cells werecentrifuged from the medium, resuspended in phos-phorous-free medium, and an aliquot was placed irnthe center of a metal planchet, dried, and assayed asbefore.


PLANT PHYSIOLOGY

SUGAR-PHOSPHATE ESTERs-BIOSYNTHESIS ANDCHROMATOGRAPHY: Algae were cultured in 35 mlof low-phosphate me(lium under the same conditionsas those described above in the studies of 32p uptake.When the optical density of the cultures was between0.75 and 1.0, they were placed in complete darknessfor 24 hours at the end of wlhich time they wereharvested by centrifugation, washedl once in phos-phorous-free medium, and resuspenldedI in 15 to 20 mlof 32P medium to give a calculatedl optical densityof 3.5. After 10 minutes the medliunm vas removed bycentrifugation and the cells were resuspended in phos-phorous-free medium. They were retained 5 mintutesin this medium to allow for equilibration of 32p withthe 31P in the cells. The suspension was again cen-trifuged and the cells resuspended in phosphorous-freeme-lium or in phosphorous-free medium to wvhich hadbeen added 0.05 M concentrations of glucose, galac-tose, or fructose. Aliquots were taken after specifiedperiods, the medium was removed, and the cells wereextracted. Extraction was accomplished by resus-pending the cells in distilled water (1 ml for each mlof 32p medium used) and by placing the cell suspen-sion in a 1000 C water bath. After 1 minute theywere rapidly returned to room temperature in anotherwater bath after which the cells were removed by cen-trifugation. The cell-free extracts were used im-mediately or stored overnight at 10 C.

The extract was applied as a spot 1 3/4 inchesfrom 1 edge of large sheets (18 1/4 in. X 22 1/2 in.)of Whatman no. 3 paper pre-washed in 95 % ethanolglacial acetic acid : and water (2: 1: 1, v/v/v).Usually, several applications of 5 lambda each weremadle, the spot being dried by a stream of air at roomtemperature between applications. Five-lambdaquantities of 0.5 M solutions of each of 4 known com-pounds-glucose- 1 -phosphate, glucose-6-phosphate,galactose-1-phosphate, and galactose-6-phosphate-were applied to the same spot. A partial separationof the extract into its several components was achievedby means of a descending movement in the long di-mension of the paper with the solvent-isobutvricaci(l ammonium hydroxide andl water (57 : 4 : 39,v/v/v)-slightly modified from Kilgour et al (6).A standardl commercial clhromatograplhic chamber wasuse(l for this 1st separation. After about 24 hours(several hours after the front had moved off the end ofthe paper) the papers were removed and dried in acurrent of air at room temperature for 24 hours ormore. An 8 1/4 inch widle strip, which included the4 phosphate esters noted above, was cut across thewidth of each sheet. For development in the 2nd di-mension, the sheets were oriented so that the phos-phorous compounds, located 1 3/4 inches from 1 edgeof the original sheets, were at the bottom of the 8 1/4X 18 1/4 inch sheets which remained. The spotswere further resolved by the use of a 2nd solvent-methanol water (7: 3, v/v), containing 1 % sodiumtetraborate. Movement of the solvent was ascendingand in a perpendicular direction to that of the 1stsolvent. The chambers for the ascending develop-

ment were 2 cylindrical pyrex jars, 18 inches tall and12 inches in diameter, fitted with glass covers whichwvere held in place with weights and sealed to the jarwith silicone grease. The chambers were fitted witll4 glass rods fixed 3/4 inch from the top of the cylinderand 1 112 inches apart. The solvent was placed in thebottom of the chambers and the papers were hungfrom the glass rods so that they were immersed about1/4 inch into the solvent. After about 12 hoursYwvhen the solvent front had nearly reached the topof the papers, they were removed, dried as before,and placed next to x-ray film in Kodak x-ray exposureholdlers. After several days of exposure, dependingon the activity present, the films were removed anddeveloped. The chromatograms were sprayed withHanes and Isherwood's reagent (2), dIried for severalminutes at 850 C, hydrated with steam, and exposedto H,S. With this procedlure the authentic spots ofglucose-i-phosphate and galactose-i-phosphate be-came blue-green, those of glucose-6-phosphate andgalactose-6-phosphate became blue, and the back-ground remained nearly white. (From one-dimen-sional chromatograms made witlh each solvent, theRf values of each of the known compounds had beenestablished, enabling their identification on the two-dimensional chromatograms.) (Table I.) Radio-active spots of phosphate-compounds from the extracts

TABLE IRf VALUES OF AUTHENTIC PHOSPHATE COMPOUNDS

COMPOUND SOLVENT NO. 1* SOLVENT NO. 2**Fructose-1,6-diP 0.22Fructose-6-P 0.24Galactose-6-P 0.31 0.34Glucose-6-P 0.32 0.26Glucose-i-P 0.37 0.37Galactose-i-P 0.37 0.29Ortho phosphate 0.51 0.28

* Isobutyric acid: ammonium hydroxide: water (574 39, v/v/v)

** Methanol: water (7: 3, v/v).

of the cells were of such low concentration that no bluecoloration resulted from their presence. The spotscorresponcling to the 4 known compounds were i(lenti-fied oIn the radioautogram. Semii-quantitative deter-nminations of these compoundls in the extract were ac-complished by assaying the radlioactivity in the corres-pondling spots. They were cut from the chromato-gram and evaluated in a windowless flow counter.

RESULTSPolymyxin B has been shown to have detrimental

effects on the growth and respiration of Chlorellapyrenoidosa, whereas C. vulgaris and Scenedesnilusobliquus are unaffected by far higher concentrations(2). Since previous work (2) has also indicated'that susceptible organisms may become resistant whengrown on galactose, experiments were performed tc,determine if the sugar would function in such a pro-

382


GALLOWAY AND KRAUSS-POLYMYXIN B ACTION ON ALGAEtective role in the case of Chlorella pyrenoidosa. Be-fore proceeding with relatively long-term experimentsinvolving polymyxin B in cultures, however, it seemeddesirable to determine if the antibiotic would be in-activated under the environmental conditions of theculture apparatus. Five-ml aliquots of inorganic-medium containing 5, 10, 20, or 40 ppm of polymyxinB were dispensed into 16-mm test tubes and placed onthe shaker. After 26 days a similar series of media-was again prepared and both sets were inoculatedwith 0.1 ml aliquots of a culture of C. pyrenoidosa,a susceptible organism. The results are shown in-table II. The data indicate that under these condi-

TABLE IIOPTICAL DENSITIES OF CHLORELLA PYRENOIDOSA, GROWN

IN FRESH AND OLD MEDIA CONTAINING VARIOUSCONCENTRATIONS OF POLYvIYXIN B

FRESH MEDIUM 26-DAY-OLD MEDIUM

NsOCU ATION POLYMYXIN B, POLYMYxIN B,(PPM) (PPM)

0 5 10 20 40 0 5 10 20 400 0.07 0.07 0.07 0.07 0.071 0.35 * * * *2 0.77 * * * ** No growth.

0.07 0.07 0.07 0.07 0.070.36 0.31 0.30 0.13 0.090.76 0.72 0.71 0.25 0.09

tions the antibiotic is inactivated with time. In orderto establish more precisely the required time for de-activation, growth measurements were made for alonger period of time in another series of culturesusing freshly prepared media containing polymyxinB (table III). Growth eventually began in the tubescontaining 20 ppm polymyxin (14 days) and stilllater in those with 40 ppm (29 days). Even aftergrowth was visually evident, the rate was alwayssomewhat slower than that in medium with no poly-myxin B. Probably growth commenced prior tocomplete destruction of the antibiotic. The growthrate returned to normal in a few days-a fact wlhich

TABLE IIIEFFECT OF TINME ON THE DECOMPOSITION OF POLYvMYXIN

B, AS INDICATED BY THE GROWTH OF CULTURES OF

CHLORELLA PYRENOIDOSA

DAYS AFTERINOCULATION POLYMYXIN B, PPm

0 5 10 20 40

0 0.10 0.10 0.10 0.10 0.101 0.43 * * * *2 0.78 * * *3 1.05 * * * *4 1.25 0.20 * * *5 ** 0.62 * * *7 ** 1.10 0.20 * *

* No growth.** Measurements discontinued.

would seem to substantiate this conclusion. If growthbegan in the presence of a low concentration of poly-myxin B, less than 5 ppm, it seemed possible thatthese cells had acquired resistance by either geneticor physiological mechanisms. To determine if thiswere the case, a culture which had begun to grow inthe presence of 40 ppm (after 31 days) was washedand used as inoculum in media containing 0, 5, 10,20, and 40 ppm polymyxin B. A comparison of thedata from this type of experiment, table IV, with thatobtained under similar conditions using cells neverexposed to polymyxin B prior to this experiment, tableIII, indicates that resistance was attained althoughnot nearly so much as that reported for Escherichiacoli (14). Further experiments revealed that re-sistance could not be further increased by continualgrowth in low concentrations of polymyxin B.

During these experiments. 2 effects of polymyxinB were observed in addition to its effect on growth:1) cultures became completely bleached in 4 to 6hours in the presence of concentrations sufficient tocause inhibition of growth; and, 2) release of daughtercells from mother cells was inhibited.

TABLE ITNOPTICAL DENSITIES OF POLYIMYXIN-CONDITIONEDCULTURES OF CHLORELLA PYRENOIDOSA GROWNIN MEDIA CONTAINING VARIOUS CON-

CENTRATIONS OF POLYMiYXIN B*

DAYS AFTERINOCULATION POLYMYXIN B, PPMi

0 5 10 20 40

0 0.10 0.10 0.10 0.10 0.101 0.44 0.30 0.26 ** **2 0.80 0.72 0.57 ** **3 1.10 0.92 0.85 ** **4 1.30 1.20 1.15 ** **5 *** *** *** ** **6 *** *** *** ** **7 *** *** *** ** **

* The inoculum was from a culture grown in mediumcontaining 40 ppm of polymyxin B.

** No growth.*** Measurements disconitinued.

The bleaching phenomenon may be explained byassuming that the cells had been killed, subsequentchlorophyll break-down occurring rapidly under therelatively high light intensity of these experiments.Bleaching did not occur in the dark-a fact which alsowvould seem to indicate that it was not a direct effectof the inhibitor. However, bleached cultures wereeventually capable of chlorophyll formation and re-newed growth. Whether the entire population wastemporarily in a resting state, capable of growth ata future time, or whether most cells were killed andthe renewed growth of the culture originated with arelatively few viable cells was a problem worth re-solving. Inasmuch as it proved impossible to dif-ferentiate microscopically between viable and non-viable cells, a culture procedure was emploved.

383


PLANT PHYSIOLOGY'

Aliquots of 0.5-mI of cell suspensions, which had beenexposed for 8 hours to 1, 10, 100, or 1000 ppm ofpolymyxin B, were plated in an inorganic agar medi-um in Petri dishes. The cells which had been in 1ppm of polymyxin B produced 5.75 X 106 coloniesper plate, the aliquot from 10 ppm produced 63, thatfrom 100 ppm produced 1, and that from 1000 ppmproduced none. After 24 hours in contact with poly-myxin B, 0.5-ml aliquots were again removed andplated. Colony production was redlucedI to 1.29 X106 for the 1 ppm level, 3 for the 10 ppm, and nonefor the 100 or 1000 ppm levels. Thus it seems evi-dent that: 1) new growth starts from a few remain-ing survivors and not from renewedl growth of theentire culture; and, 2) the bleached appearance of theculture results from the destruction of chlorophyllafter death of most cells.

The 2nd observation was that abnormally largecells appear in cultures of Chlorella pvrenoidosatreated with polymyxin B. The antibiotic has beenreporte(d to produce thread-like cells of Escherichiacoli (11). In the alga, cell division seemed to pro-gress normally, but subsequent (lisruption of themother cell wall faile(d to take place. In some cases,64 and occasionally 128 (laughter cells were insideof 1 mother cell wall (fig 1). The amount of oldcell wall material in the culture was also abnormallyhighl, another indication that lysing of the cell wall

FIG. 1. A photomicrograph of Chlorella pyrenoidosagrown for 24 hours in an inorganiic medium containing15 ppm of polymyxin B (magnified X 500(0).

10O

5

A - CONTROL (MINUS POLYMYXIN B)O -100 PPM POLYMYXIN B0 -200 PPM POLYMYXIN B /

0.1 0.2 0.3 0.4

I/S (MILLIMOLES)

FIG. 2. A double-reciprocal plot of the effect ofpolymyxin B on the activity of phosphoglucose isomeraseobtained from rabbit muscle. The reaction mixture con-sisted of 0.1 ml of enzyme preparation which had beendiluted 1 to 4000 with double-distilled water, and 0.4 mlof buffer solution, 0.06 M Sigma 7-9, adjusted to a pHof 7.0. Incubation was performed at 60 C for 35 minutes.1/v = inverse of the optical density of the product ofthe reaction, fructose-6-phosphate, assayed according tothe method of Roe. 1/S = inverse of the concentrationsof the substrate, glucose-6-phosphate. Each point is anaverage of 4 determinations.

wras slower than normlal. A similar inhibition of dis-ruption of the cell wall may cause the abnormally longcells of E. coli.

Experiments designe(d to evaluate the effectivenessof galactose against inhibition of growth by polymyxinB were undertaken. Using medium containing0.5 % galactose and polymyxin B concentrations of5, 10, 20, and 40 ppm, it was found that at first nogrowtlh was obtained in the presence of any amountof polymyxin, whether galactose was present or not.However, as the time for sufficient deactivation ofeach higlher concentration of the inhibitor elapsed,growth always began 2 or 3 days earlier in the respec-tive meclium with galactose.

For an organism to be able to utilize galactose inthe presence of an inhibitor which prevents its useof glucose, it was logical to postulate that 1 of severalenzyme systems was being damaged. It was reasone(dthat phosphoglucose isomerase was the most likely

384

l/v l5S

-r'-Ai"c ep


GALLOWAY AND KRAUSS-POLYMYXIN B ACTION ON ALGAE

enzyme to be susceptible. Experiments showed thatpolymyxin B indeed has an inhibitory effect on thisenzyme. In order to determine the type of inhibitionbeing effected the data were plotted on double-recipro-cal axes, after the method of Lineweaver and Burk(7) (fig 2). These data indicate clearly that in-hibition is of the competitive type.

In view of the identification of a probable site ofinhibition in the glycolytic pathway, it became impera-tive to examine the compounds formed during themetabolism of the algae. In order to study differencesin the utilization of carbohydrate substrates in thepresence and absence of polymyxin B, cells were ex-posed to radioactive phosphorous. Phosphorylatedintermediates within the cells were thus made radio-active, and any change in 32p distribution could befollowed by tracer techniques.

Experiments were performed with C. pyrenoidosato determine the conditions under which 32p uptakewould occur. For very short term exposures, cellseparation from the media by means of a milliporefilter seemed most promising. However, it was foundthat when 32p medium was passed through the filter,about 20 % of the total activity was retained by thefilter. Perhaps a precipitate of 32PO4 and magnesiumwas being formed which was too small to be visiblebut too large to pass through the filter. If the 32pwvere placed in distilled water, about 10 % of the ac-tivity still remained in the filter. Furthermore, plac-ing cells in distilled water for even a few minutes haddetrimental effects on the cells, as evidlenced by theirrelease of significant amounts of 32p soon after it hadbeen absorbed. Consequently, a chelating agent,EDTA, was incorporated into the medium. It wasfound that 6 g of Na., * EDTA per liter was sufficientto prevent nmore than 1.5 % 32p retention oIn the filter.

A comparison of the uptake of 32p by maturelight cells and young dark cells is shown in figure 3.It was later observed, by chromatography of cell ex-tracts, that large amounts of metaphosphate areformed in cells grown in medium containing the usualamount of phosphate, referred to in this paper as high-phosphate medium. This polymer not only uselesslyaccounts for a major portion of the activitv, but italso tends to make chromatographic separation ofcompounds containing 32p more difficult. It wasreasoned that storage of this "excess" phosphatemight be eliminated by the use of a medium with avery low phosphate content. Experiments with all3 species of algae indicated that growfth was normaland sufficient when the medium contained 1.61 X10-4 Mt phosphorous. This is in agreement with thework of Kamen and Gest (5). Figure 4 shows thatthe uptake of 32p by cells wvhich had been grown inlow-phosphate medium was more rapi(d andc morenearly complete than that by cells growrn in high-phosphate medium. For this reason, and becauseof the elimination of metaphosphate. as later shownby chromatography, cells were routinely cultured inlow-phosphate medium prior to exposure to 32p.

It was observed that C. pyrenoidlosa cells contain-ing 32P rapidlly released large percentages of it when

LoN CELL 00*o.Loo&mi' CEIiS 6--S

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POLYMYXIN S. PPM

FIG. 3 (top), FIG. 4 (centter). Absorption of 32P inthe dark by Chlorella pyrentoidosa. Figure 3 shows ab-sorption by cells which had been grown in media con-taining 1.64 X 10-3 M phosphorous; Figure 4 showsabsorption by cells which had been grown in niedia con-taining 1.61 X 10--4 M phosphorous. "Light" cells werefrom a lighted culture; "dark" cells were from a culturedarkened for 24 hours prior to exposure to 32p. Themedium in which the algae were exposed contained 10MC 32p per /g of 31p, the latter being at a concentrationof 1 x 10-5 M.

FIG. 5 (bottomii). The percent of 32P retained in cellsof Chlorella pyrenoidosa after 15 minutes in media con-taining various concentrations of polymyxin B.

38.5

0

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0

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0

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PLANT PHYSIOLOGY

placed in medium containing polymyxin B. Theamount of 32p lost varied with concentration of in-hibitor, as shown in figure 5. Inspection of radio-autographs of the material lost from the cells (fig 6)revealed that many compounds were involved.Previous work (2) showed that the rate of respira-tion of glucose by C. pyrenoidosa was temporarilydepressed in the presence of 80 ppm or less of poly-my-xin B. Within 2 hours, however, the rate was

approximately that of untreatedl cells. It is to beexpected that upon resumption of respiration, 32plost from the cells would be resorbed. In order to

cleternline if this were the case, 2 treatnments were

executed. In 1, cells containing 32p were placed inphosphorous-free medium to which various concentra-

tions of polymyxin B, an(d 0.05 M\I glucose had beena(l(le(l. In the 2nd, similar cells were exposed tolight and bubbled with 5 % CO.,-in-air. The amount

of 321P released from the cells in 15 minutes was thesame for all treatments at any given concentration ofpolymyxin B. However, at the endl of 2.5 hours,significant amounts of 32p had been resorbed, espe-

cially at the lower concentrations of polymyxin B.The resistant species, C. vuilgaris and S. obliquiis,

grown on low-phosphate medium and given 24 hoursof darkness were found to absorb between 90 and 95 %of the 32p from 32P-medium in 10 minutes. Cellscontaining 32P, when resuspended in phosphorous-free medium, released only about 1 % of their 32p in

the presence or absence of polymyxin B. Radioauto-grams were prepare(l of the extracts of the 3 speciesof algae after they had been placed in phosphorous-free media containing 0.05 M concentrations of glu-cose, galactose, or fructose for 30 or 90 minutes.Similar ra(lioautograms were made of extracts fromthe 2 resistant species which had been exposedl to 320

Fic. 6 (left). A radioautogram of a filter paper chromatogram showing the 32p radioactivity of compoundsreleased from Chlorella pyrenoidosa after 15 minutes exposure to 320 ppm of polymyxin B. The cells had beenpreviously suspended for 10 minutes in medium containing 1 X 10-5 M 31P and 10 ,_c 32P per jug of 31p. The

origin is at the bottom. The solvent was isobutyric acid :ammonium hydroxide :water (57 :4 :39, v/v/v), 24

hours.

FIG. 7 (top, right), FIG. 8 (bottomtt, right). Radioautograms of two-dimensional paper chromatograms showingthe 32p radioactivity of compounds in water extracts of Scenedesinis obliquus after 30 minutes utilization of glucose(fig 7) and of Chlorella pyrenoidosa. after 30 minutes utilization of fructose (fig 8). The cells had been previouslysuspended for 10 minutes in medium containing 1 X 10-5 M 31p and 10 /c 32p per ,Ag of 31P. The origin, removed

during development in the 2nd dimension, lies off the paper to the lower right. The solvents were: 1st (across-right to left) isobutyric acid ammonium hydroxide water (57 4 39, v/v/v), 24 hours; 2nd (up) methanol

water (7 : 3, v/v) containing 1 % sodium tetraborate, 12 hours.

386

......



TABLE VPERCENTAGE OF THE 32p IN CHLORELLA PYRENOIDOSA

2.5 HOURS AFTER TREATMENT WITH VARIOUSCONCENTRATIONS OF POLYMYXIN B *

% 32p IN THE CELLS **POLYMYXIN B, PPM DARK LIGHT

+ GLUCOSE *** - GLUCOSE

0 96.7 96.920 98.1 97.240 86.0 88.880 48.8 17.0160 38.2 11.0320 19.4 8.2

* Light-grown cells labeled with 32p were treated withthe 5 concentrations of polymyxin B. In 15 minutes allbut 9.8 % of the 39P was lost from the cells regardless ofthe concentration of polvmyxin B.

** This represents the 32P remaining in the cells plusthat resorbed during 2.5 hours.

*** (.05 M.

ppm polymyxin B for 1 hour prior to addition ofsubstrate. Examples of these radioautograms areshown in figures 7 and 8. The amounts of glucose-1-phosphate, glucose-6-phosphate, galactose-i-phos-phate, and galactose-6-phosphate present in the cellsafter 30 minutes of utilization of substrates, as reveal-ed by their radioactivity, are shown in table VI.

DISCUSSION AND CONCLUSIONSIt appears that polymyxin B can injure or destroy

certain organisms in at least 2 ways. It may eitherdisrupt the differential permeability of the cell mem-brane so that cell components are lost, or it can actagainst specific enzymes essential to metabolism. Inregard to inhibition at the enzyme level, it is apparenitthat polymyxin B prevented the formation of fructose-6-phosphate from glucose-6-phosphate by inhibitionof phospho-glucose isomerase. Consequently, re-sistance requires 1st a membrane which withstands

TABLE VICOUNTS OF 32p PER MINUTE IN FOUR HEXOSE-PHOSPHATE ESTERS IN WATER EXTRACTS OF SCENEDESMUS OBLIQUUS,

CHLORELLA VULGARIS AND C. PYRENTOIDOSA AFTER UTILIZING SUBSTRATES FOR 30 MINUTES *

HEXOSE UNTREATED TREATED**SUBSTRATE PHOSPHATE SCENTEDESMUS CHLORELLA CHLORELLA SCENEDESMU S CHLORELLA

ESTER OBLIQUUS VULGARIS PYRENOIDOSA OBLIQUUS VULGARIS

None G-1-P*** (°2$100 151 0 48 426None G~~1~~P*** (25)t (8) (5) (10)Ga-l-Pt ~0 172 175 218 527Ga-i-Pt ° l(8) (26) (22) (13)

G-6-P ~~295 1682 501 711 3010G-6-P 2(75) (80) (74) (73) (74)Ga-6-P 0 90 0 0 107

(4) (3)Glucose G-1-P ~~180 710 0 297 837Glucose G-l-P (24) (6) (7) (9)

Ga-l-P ~~0 2619 362 777 2400Ga-i-P ° 2(22) (29) (18) (26)

G-6-P 3385 8370 887 3354 6128G-6-P ~~(51) (71) (71) (75) (65)

Ga-6-P 188 42 0 0 0(25) (1)Fructose G-1-P ~~127 395 0 192 427Fructose G-l-P (21) (7) (11) (9)

Ga-1-P 0 1578 199 306 1510(28) (32) (18) (32)

G-6-P (466 3564 419 1250 2723G-6-P ~~(79) (63) (68) (71) (59)Ga-6-P 0 82 0 0 0(2)

Galactose G-1-P )194 2780 54 289 837GalactoseG-i-P ~(33) (34) (8) (11) (16)Ga-1-P 0 1188 242 795 1811

(14) (38) (29) (34)G-6-P 268 3861 342 1662 2631

(46) (47) (54) (60) (50)Ga-6-P 119 409 0 0 0Ga-6-P ~~(21) (5)

utilization of substrate.

* Etxtracts were at 100° C for 1 minute.** Treatment consisted of exposure to 320 ppm of polymyxin B for 1 hour prior to*** Glucose-l-phosphate.t Galactose-i-phosphate.t Numbers in parentheses are percentages of total activity of the compounds indicated.

387


PLANT PHYSIOLOGY

attack, and 2nd, a sequence of enzymes capable ofyielding energy to the organisms in the absence ofphosphoglucose isomerase. In the case of the re-sistant species, Chlorella vuilgaris and Scenedesmusobliquus, the evidence indicates that this 2nd require-ment has been met by the utilization of galactose-6-phosphate in the synthesis of fructose-6-phosphate,or intermediates below this in the Embden-Myerhofsequence (fig 9). The "by-way" proposed involvesfirst the mutation of galactose-1-phosphate to galac-tose-6-phosphate under the influence of a phospho-galacto-mutase. The next step in the sequence almostcertainly is the conversion of galactose-6-phosphate bymeans of a phosphogalactose isomerase to tagatose-6-phosphate (4). One of 3 subsequent transforma-tions must occur: 1) tagatose-6-phosphate could beconverted to fructose-6-phosphate by means of aphosphotagatose epimerase; 2) the diphosphate esterof tagatose could be converted to fructose-1,6-diphos-phate under the influence of a diphosphotagatoisomer-ase; or, 3) tagatose-1,6-diphosphate itself could besplit by means of an aldolase to yield the 2 trioseesters. No evidlence yet exists which enables a choiceamong these 3 possibilities of re-entry into the classi-cal glycolytic pathway.

The ability to circumvent a block in the glycolyticpathway in the manner suggested accounts for theprotection affordedl by galactose in the case of certainotherwise susceptible organisms incapable of obtainingenergy from glucose via galactose. This also ac-

H.-OPO, H OPO, HI ,OmHCOH | HWOI HLO

HOCH 0 HOCH 0 -la --- HOCH 0

HC HC HCCH,OH CH,OH CH,OH

GLUCOSE-1- PHOSPHATE GALACTOSE -1- PHOSPHArE GALACrOSE

H. OH H.-OH H.-OHHC0H HC0H HCO;

HOCH ' HOCHO

H 6 H6 HOCHHL01j H6!J HOCHCH,OH CH,OPO,- CH,OPO,'

GLUCOSE CLUCOSE-6-PHOSPHArE CALACrOSE-6- PHOSPHArE

I ICH,OH CH,OH

HC HCHOC HOC;HCOH0° HOCH 0HCJ H0-CH,OPO,' CH,OPO,'

FRuCrOSE-6-PHOSPHArE TAGArOSE -6- PHOSPHAEr

I ~~~~~ICH OPO,' CH90PO,'

HC HC

HCOH> 0 _ __ HOCH0HH

HH

CH,OPO,- GH,OPO.'FRUC rOSE - /, P- DIPHOSPHA rE rAGAroSE - /1 6 -

I 0PHOSPHATE

HVC + CH,OPO,'HCOH C+*OCH,OP0,- CH,OH

6LTCERAL - D/HTDROXY-DEHrDE -3- ACG rOREPHOSPHArf PHOSPHArE

FIG. 9. The sequence of hexose intermediates of analternate pathway in relation to the classical glycolyticscheme.

counts for the syntlhesis of galactose-6-phosphate inresistant organisms. The (lisappearance of galactose-6-phosphate from resistant organisms subsequent toexposure to polymyxin B is interpreted as evidenceof increased utilization of this ester under this condi-tion. Simultaneous increase in galactose- 1 -phos-phate indicates that its mutation (the shift of thephosphate moiety from the 1 to the 6 position) is therate-limiting step in this sequence.

Bacteria which gained protection from inhibitionof polymlyxin B by utilization of galactose (2) are ex-

amples of organisms which meet 1 requirement forresistance, i.e., a membrane not subject to disorgani-zation, but not the other, i.e., a method of obtainingenergy in the absence of phosphoglucose isomerase,except when supplied with galactose. These speciesevi(lently lack at least 1 of the enzyme systemls in-volved in the sequence between glucose-6-phosphateand galactose-6-phosphate.

SUMMARYA studly was ma(le of the effects of the antibiotic,

polymyxin B, on unicellular green algae. The growthandl respiration of C. pvrenzoidosa are inhibited by 5to 10 ppm of polymyxin B, whereas C. vidgaris andScenedesnius obliquus are unaffected. Previous workwitlh susceptible bacteria sholvwed that their rate ofrespiration of galactose remained unchanged upon ex-posure to polymyxin B. Lack of a similar protectiveeffect from glucose gave evidence of a new pathwayof galactose utilization.

Investigation revealed 2 convergent lines of evi-dence which implicate phosphoglucose isomlerase asthe enzyme in glycolysis which is blocked by polynmy-xin B. First, a direct examination of the effect ofthe antibiotic on the extracted enzyme showed an in-hibition of the competitive type. Secondl, galactose-6-phosphate is synthesized by the resistant organisms,Chlorella zvulgaris and Scenedesmii lls obliqtu s.Further transformations of galactose-6-phosphatewould permit re-entry into glycolysis at a point bevondglucose-6-phosphate. A sequence of transformationshas been hypothesized: 1) Galactose becomes plios-phorylated on carbon-6; 2) a shift in the positions ofthe ring results in the formation of a ketose, tagatose-6-phosphate; and, 3) the addition of a 2nd phosphateradical yields tagatose-1,6-diphosphate. Re-entry in-to glycolysis could result fromn: ] ) Conversion oftagatose-6-phosphate to fructose-6-phosphate; 2) con-version of tagatose- 1 ,6-diphosphate to fructose- 1,6-dliphosphate; or, 3) the splitting of tagatose-1,6-di-phosphate into the triose phosphate esters, glyceral(le-lhyde-3-phosphate and (lihydroxyactone phosphate.

Another striking aspect of polymiiyxin B is itsability to disorganize the cellular membrane. Theleakage of phosphorous from cells of the susceptibleChlorella pyrenoidosa is approximately 80 % after 15minutes of exposure to polymyxin B.

388



LITERATURE CITED1. FEW, A. V. and SCHULMAN, J. H. The absorption

of polymvxin E by bacteria and bacterial cellwalls and its bactericidal action. Jour. Gen. Micro-biol. 9: 454-466. 1953.

2. GALLOWAY, R. A. and KRAUSS, R. W. The differen-tial action of chemical agents, especially polymyxinB, on certain algae, bacteria, and fungi. Amer.Jour. Bot. 46: 40-49. 1959.

3. HANES, C. S. and ISHERWOOD), F. A. Separation ofthe phosphoric esters oIn the filter paper chromato-gram. Nature 164: 1107-1112. 1949.

4. HIRST, E. L., HOUGH, L. and JONES, J. K. N. Thestructure of Sterculia setigera gum. Part I. Aninvestigation by the method of paper partitionchromatography of the products of hydrolysis ofthe gum. Jour. Chem. Soc. (London). 1949:3145-3151. 1949.

5. KANIEN, M. and GEST, H. Serendipic aspects ofrecent nutritional research in bacterial photosyn-thesis. In: Phosphorous Metabolism. W. D. Mc-Elroy and B. Glass, eds. Vol. II. Pp. 507-521.Johns Hopkins Press, Baltimore 1952.

6. KILGOUR, G. L., FELTON, S. P. and HUENNEKENS,F. M. Paper chromatography of flavins andflavin nucleotides. Jour. Amer. Chem. Soc. 79:2254-2256. 1957.

7. LINEW\ EAVER, H. and BURK, D. The determinationof enzyme dissociation constants. Jour. Amer.Chem. Soc. 56: 65&-666. 1934.

8. LOWRY, R. J. and SUSSMAN, A. S. Physiology ofthe cell surface of Neurospora ascospores. II. In-terference with dye absorption by polymyxin.

Arch. Biochem. Biophys. 62: 113-124. 1956.9. NEWTON, B. A. The properties and mode of action

of the polymyxins. Bacteriol. Revs. 20: 14-27.1956.

10. NORMAN, A. C. The effect of polymyxin on plantroots. Arch. Biochem. Biophys. 58: 461-477.1955.

11. PRATT, R. anid DUFRENOY, J. Cytochemical mecha-nisms of penicillin action. VIII. Involvement ofribonucleic acid derivatives. Jour. Bacteriol. 57:9-13. 1949.

12. ROE, J. H. A colorimetric miiethod for the determina-tion of fructose in blood and urine. Jour. Biol.Chem. 107: 15-22. 1943.

13. SLEIN, M. W. Phosphohexoisomerases from muscle.In: Methods in Enzymology, S. P. Colowick andN. 0. Kaplan, eds. Vol. I. Pp. 229-306. Aca-demic Press, New York 1955.

14. SZYBALSKI, W. and BRYSON, V. Genetic stuidies onmicrobial cross resistance to toxic agents. I. Crossresistance of Escherichia coli to fifteen antibiotics.Jour. Bacterial. 64: 489-499. 1952.

15. THOMAS, W. H. and KRAUSS, R. WV. Nitrogenmetabolism in Scenedesmus as affected by environ-mental changes. Plant Physiol. 30: 113-122.1955.

16. UMBREIT, W. W., BURRIS, R. H. and STAUFFER, J. F.Manometric Techniques and Tissue Metabolism.Burgess, Miinneapolis 1949.

17. WARREN, G. H., GRAY, J. and YURCHIENCO, J. A.Effect of polymyxin on the lysis of Areisseria catar-rhalis by lysozyme. Jour. Bacteriol. 74: 788-7/93.1957.

ON THE OCCURRENCE OF FREE GALACTURONIC ACID INAPPLES AND TOMATOES 1 2

JOHN H. McCLENDON, C. W. WOODMANSEE AND G. FRED SOMERSDEPART-MENT OF AGRICULTURAL BIOCHEMISTRY AND FOOD TECH., UNIVERSITY OF DELAWARE, NEWARK, DELAWARE

Changes in amiiounts and kinds of pectic sub-stances have long been associated with the ripeningprocess in fruits (12). However, the uronic acidswhich are characteristic constituents of these andmany other plant polysaccharides are not generallyconsidered to occur in free state. Nevertheless, sev-eral reports of their occurrence in sound fruits haveappearedl recently (1, 2, 4, 9, 15, 18, 19). Sincemost of these reports were based on colorimetric as-say methods without characterizing the particularuronic acid concerned, we wish to report here somemore precise indications and a few quantitative dataon changes with ripeness.

AIATERIALS AND METHODSThe fruits used in this study were selected from

the replicate samples prepared for the study of their

Received October 1, 1958.2 Published as Miscellaneous Paper no. 317, with the

approval of the Director of the Delaware AgriculturalExperiment Station.

polysaccharides (20). They were picked as both un-ripe and ripe, with the latter allowed to become over-ripe in the laboratory. Fruits with apparent signsof rotting were discarded.

About 1000 g of the fruit were slicedI into boiling2-propanol (99 %) in an amount sufficient to givea final alcohol concentration of about 70 %. Thefiltered solutions were reduced to a syrup on a steambath and stored in the deep freeze till analyzed.

ISOLATION OF THE FRUIT ACIDS: The acids inthe syrups were freed of neutral sugars and cationsby passing them through a 2-step deionization coltumnin a procedure mainly followving Winkler (18, 19)and M\iills (15). The 1st step was Amberlite IR-120(beads) in the hydrogen form. The 2nd step wasDuolite A-4 (smaller than 40 mesh) in the free baseform. Blanks and standards were run as recommend-ed by Winkler. The anion resin was eluted with20 % formic acid followed by water. Some pigmentappeared in the acid eluates of the unknowns.

PAPER CHROMATOGRAPHY: Uronic acids aremore difficult to separate than the corresponding

389


380 · chemical constituents of the organisms are commonly insufficient to permit interpretation of...

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