JOURNAL OF BACTERIOLOGY, Aug. 1970, p. 439-446Copyright 0 1970 American Society for Microbiology

Vol. 103, No. 2Printed in U.S.A.

Macroconidial Germination in Microsporum gypseumt

T. J. LEIGHTON,2 J. J. STOCK, AND R. A. KELLN

Department of Microbiology, The University of British Columbia, Vancouver, British Columbia, Canada

Received for publication 18 May 1970

Biochemical events which occur during macroconidial germination have beenstudied in the dermatophyte Microsporum gypseum. The specific activity levels ofvarious metabolic enzymes have been assayed during germination time periods. Theaccumulated levels of several of these enzymes, as a function of exogenous carbo-hydrate source, have been investigated. M. gypseum was found to possess a consti-tutive glyoxalate shunt, a constitutive glucokinase, a fructose phosphoenolpyruvatetransferase, and a mannitol phosphoenolpyruvate transferase. The integration ofendogenous reserve utilization during germination is discussed. The purificationand properties of an alkaline phosphatase and its possible relationship to sporula-tion and spore germination also are described.

There have been many studies of the biochemis-try of fungal conidial germination. The data ac-cumulated from much of this work are availablein several excellent reviews (5, 8, 26). Most of theorganisms examined thus far preserve the vegeta-tive nuclear-cytoplasmic ratio in the conidium. Inthe dermatophyte Microsporum gypseum, thevegetative nuclear-cytoplasmic ratio of two nucleiper hyphal septated unit is not preserved duringmacroconidial development (Leighton and Stock,in preparation). The macroconidium of M. gyp-seum may contain from one to eight nuclei perseptated spore compartment. Nuclear distribu-tion analyses indicate that greater than 70% ofthe total nuclear population is distributed abovethe two nuclei per spore compartment level. Thestructural complexity of the macroconidium andits unusual nuclear distribution properties suggestthat the analysis of macroconidial germination inM. gypseum could yield further informationconcerning fungal cell differentiation processes.This preliminary investigation describes severalbiochemical aspects of spore germination in M.gypseum.

MATERIALS AND METHODS

The organism, sporulation, and germination condi-tions were as previously described (14, 15).

Sporulation and spore germination in the presence ofmannitol, fructose, and acetate. Mannitol (1%, w/v),fructose (1%, w/v), or sodium acetate (3%, w/v) wassubstituted for glucose (1%, w/v) where indicated.

IA preliminary report covering part of this work was pre-sented at the 70th Annual Meeting of the American Society forMicrobiology, Boston, Mass., 26 April-i May 1970.

2 Present address: Department of Biochemistry and Biophysics,University of California, Davis, Calif. 95616.

Sporulation and spore germination were always car-ried out by using the same carbon source. That is, iffructose was the carbon source used for spore ger-mination studies, these spores were harvested from asporulation medium containing fructose as the singleadded carbohydrate.

Preparation of cell-free extracts. Sonically disruptedspore and mycelial extracts were prepared as pre-viously described (15). Extracts were also obtainedby the liquid nitrogen method of Bleyman and Woese(2). Debris was removed from the liquid nitrogengrindate by centrifugation at 10,000 X g for 10 minat 4 C. In all cases, cell-free extracts were bufferedin 0.2 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, pH 7.4.

Analytical determinations. Protein was estimated bythe method of Lowry et al. (18). Crystalline bovineserum albumin was used as the standard. Totalcarbohydrate was determined by the anthrone reac-tion (19), with glucose used as the standard. In-organic phosphate was quantitated by the method ofChen et al. (4). Ribonucleic acid (RNA) was meas-ured by the orcinol reaction (22), with yeast RNAused as a standard.

Carbohydrate extraction and identification. The80% alcohol and sulfuric acid-soluble extracts wereprepared as described by Lingappa and Sussman(16). Alcohol-soluble extracts were desalted by pas-sage through a mixed bed of Dowex-1-x-8 (Cl-) andDowex-50W-x-4 (H+). Immediately after extractionand desalting, carbohydrates in the alcohol-solublepool were separated by descending paper chromatog-raphy by use of a pyridine-ethyl acetate-water (50:120:40, v/v) solvent system. Sugars were detected bydeveloping the chromatograms with silver nitrate (27)or benzidine (10) reagents. Unknown spots wereidentified by co-chromatography with authenticstandards.Enzyme assays. Enzyme activities were determined

by following the changes in optical density of the

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reaction mixtures at 25 C with an automatic recordingspectrophotometer (model 2400; Gilford InstrumentLaboratories, Oberlin, Ohio). Absorbance changeswere measured with reference to reaction mixturesminus substrates. Measurements of reaction rateswere made under conditions in which the initialmeasured rate was linear with respect to time. Spe-cific activities are reported as the change in opticaldensity units per milligram of cell-free extract proteinper minute. The following enzyme activities weremeasured: alkaline phosphatase, glucose-6-PO4 de-hydrogenase, aldolase, glyceraldehyde-3-PO4 dehy-drogenase, enolase, fumarase, and nicotinamideadenine dinucleotide phosphate (NADP)-linkedisocitric dehydrogenase (as described in BiochimicaCatalogue, Boehringer Mannheim Corp., New York,N.Y.); mannitol-l-PO4 phosphatase (25); NADP-linked mannitol dehydrogenase (1); nicotinamideadenine dinucleotide (NAD)-linked mannitol-l-PO4dehydrogenase (25); fructokinase (24); mannitolkinase (24); glucokinase (24); transaldolase (6);transketolase (6); isocitrate lyase (23); glucosephosphoenolpyruvate (PEP) transferase (9); fructosePEP transferase (9), and mannitol PEP transferase(9).

Alkaline phosphatase purification. All steps of theprocedure were carried out at 4 C unless otherwisestated. Alkaline phosphatase assays were carried outon all samples at each step of the procedure. Finelyground, solid ammonium sulfate was used to producethe various saturation levels.

Step 1. The cell-free extract of M. gypseum my-celia was centrifuged at 110,000 X g in a Spincomodel L ultracentrifuge for 2 hr. The supernatantfluid was removed and stored in an ice bath. Thepellets were suspended to original volume in 1.0 MTris-hydrochloride buffer (pH 8.0) containing 10-4M MgCl2 prior to determination of alkaline phos-phatase activity.

Step 2. With 100 ml of the cell-free extract super-natant fraction, 50% saturation was achieved by theaddition of 25 g of ammonium sulfate with constantstirring on an NaCl ice bath. The mixture was allowedto stand for 10 min and then centrifuged at 13,000 X gfor 15 min. The supernatant fluid was brought to 60%saturation by the addition of 7 g of ammonium sulfatein the previously described manner. After standingfor 10 min, the suspension was centrifuged as before,and the pellet was discarded. The supernatant fractionwas brought to 65% saturation through the furtheraddition of 3.5 g of ammonium sulfate and allowedto stand for 10 min. The suspension then was centri-fuged as before. The pellet was retained and held at0 C. The supernatant liquid was then brought to 75%saturation by the further addition of 7 g of ammoniumsulfate and again allowed to stand for 10 min. Thesuspension was centrifuged as before, and the pelletwas retained and stored at 0 C. The supernatant fluidwas brought to 80% saturation by the addition of3.5 g of ammonium sulfate. The suspension wasallowed to stand for 10 min and then was centrifugedas before. The pellet was retained, and the superna-tant fraction was discarded. The pellets from the 65 to80% saturations were dissolved and pooled in 1 M

Tris-hydrochloride buffer (pH 8.0) containing 10-4M MgCl2 to a final volume of 15 ml. The solution wasdialyzed for 24 hr against two successive 2-litervolumes of 0.02 M Tris-hydrochloride buffer (pH 8.0)containing 10-4 M MgC12.

Step 3. Diethylaminoethyl ether (DEAE) cellulosewas equilibrated with 0.02 M Tris-hydrochloride buffer(pH 8.0) containing 10- m M9gC2, and then packedto a height of 10 cm above a piece of finely meshednylon in an 11-mm diameter glass column. A 7.5-ml volume of the dialyzed enzyme preparation frompurification step 2 was applied to the top of thecolumn. After the enzyme solution had entered thecolumn, the phosphatase was eluted from the columnby using 300 ml of Tris-hydrochloride buffer (pH8.0), containing 10- M MgCI2, in a linear gradientfrom 0.02 to 0.25 M. Five-milliliter fractions were ob-tained by using a Warner-Chilcott fraction collector(Warner-Chilcott Inc., Richmond, Calif.). The ab-sorbance of the fractions was read at 280 nm with aGilford model 2400 spectrophotometer. Those frac-tions containing the major portiori of enzymaticactivity were pooled and concentrated to a 10-ml finalvolume with a Diaflo apparatus, by using a membraneof 1,000 molecular weight retention. The pooled,concentrated enzyme preparation was then dialyzedfor 24 hr against two successive 2-liter volumes of0.02 M Tris-hydrochloride buffer (pH 8.0) containing10-4 M MgCI2.Step 4. Column chromatography was carried out

on this enzyme preparation in the same manner asdescribed in Step 3, except that the total volume ofbuffer used in the gradient was reduced to 200 ml.The absorbance of fractions was read at 280 nm andthose containing the major portion of enzymatic ac-tivity were pooled and concentrated to a final volumeof 10 ml as before. The final enzyme preparation wasfrozen and stored at -70 C as 1 -ml volumes untilrequired.

Disc gel electrophoresis. Disc gel electrophoresis(pH 9.3) was carried out by the procedure for theCanalco model 6 system (Canalco, Rockville, Md.).

Activity of alkaline phosphatase on various phos-phorylated compounds. The concentration of all phos-phorylated compounds employed was 8.5 X 10-2 M.Reaction mixtures contained 50 ,uliters of purifiedenzyme preparation, 6.0 ml of 1 M Tris-hydrochloridebuffer (pH 8.0), 5.0 ml of distilled water, and 1.0 mlof substrate. Control reaction mixtures contained allconstituents except the enzyme preparation. Allassays were carried out at 37 C, allowing 10 min ofincubation for each of the various substrate reactionmixtures and control mixtures to equilibrate to tem-perature. Samples of 2.0 ml were taken from both con-trol and reaction mixtures at set time intervals andadded to 1.0 ml of cold 1 N H2SO4. The amount of in-organic phosphate in the reaction and control sampleswas determined by the procedure of Chen et al. (4).The background level of inorganic phosphate in thecontrol mixtures was subtracted from that of the reac-tion mixtures for each substrate. Rates of hydrolysisby the alkaline phosphatase were calculated and com-pared for each substrate.

Chemicals. All substrates for enzyme assays were

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purchased from Boehringer Mannheim Corp., withthe exception of fumaric acid, isocitric acid, and D-mannitol, which were purchased from Calbiochem(Los Angeles, Calif.). Semi-carbazide was purchasedfrom Fisher Scientific Co., Fairlawn, N.J. All ana-lytical standards and carbohydrates were obtainedfrom Calbiochem. Mannitol-1-PO4 was the generousgift of L. Klugns0yr (Physiological Institute, Uni-versity of Bergen, Bergen, Norway). Reagents fordisc gel electrophoresis were obtained from Canalco,Rockville, Md. Tris, N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES), and N-2-hydroxy-ethylpiperazine-N'-2-ethane sulfonic acid (HEPES)were purchased from Calbiochem.

RESULTSMacroconidial utilization of alcohol- and acid-

soluble carbohydrates (saline-germination system).Utilization of endogenous carbohydrate reservesis essential for the germination of conidia in manyfungi. Since M. gypseum macroconidia are capa-ble of germinating in the absence of exogenousnutrients (14), we thought it was possible thatendogenous carbohydrate reserves could providepart of the energy necessary for this germinationprocess.Chromatographic analysis of the zero-time 80%

alcohol-soluble pool indicated the presence ofglucose, fructose, mannitol, trehalose, and inositol.The mannitol spot was no longer detectable at 3hr after initiation of spore germination. Trehaloseand inositol spots were no longer detectable after8 hr. Figure 1 illustrates the time course utiliza-tion of alcohol- and acid-soluble carbohydratefractions during germination. By 8 hr, nearly allthe available alcohol-soluble pool had beenutilized. No further germ-tube elongation was ob-served after this time.Enzyme specific activity changes during periods

of spore germination (glucose-germination sys-tem). It was our experience that a 0.2 M Tris-hydrochloride buffer, pH 7.4, was superior to 0.2M phosphate buffer, pH 7.4, for the preparationof cell-free extracts. Phosphate at 0.2 M appea;edto be an inhibitor to several of the enzymes as-sayed. Neither HEPES nor TES at 0.2 M, p1i 7.4,gave appreciably higher specific activities whencompared with Tris-hydrochloride buffer. Experi-mental variance in single enzyme specific activi-ties between subsequent experiments was nevergreater than 10%. The enzyme assays cited werefound to produce optimal reaction rates in thissystem (unpublished data).Table 1 depicts the time course of the 12 meta-

bolic enzymes assayed. Each enzyme also hasbeen assigned an activity ratio which is the zero-time specific activity divided by the 24-hr specificactivity. The observed pattern does not appear tobe an artefact of cell disruption. Spores disrupted

1 2 3 4 5 * 7 aHOURS

FIG. 1. Utilization ofalcohol anid acid-soluble carbo-hydrate fractions during macroconidial germination.Symbols: 0, alcohol-soluble fraction; 0, acid-solublefraction.

by sonic oscillations or liquid nitrogen gave iden-tical activity ratio level profiles.

Mannitol metabolism in germinating macro-conidia (mannitol, fructose, or glucose-germinat-ing system). Sporulation on the various carbo-hydrates was completed by 7 days, and the har-vested spores from each carbohydrate source ex-hibited similar germination time periods. That is,spores harvested from the homologous carbohy-drate source germinated to the 100% level by 8hr in all cases (unpublished data). Macroconidia ofM. gypseum contained an NAD-linked mannitol-1-PO4 dehydrogenase, a mannitol-l-PO4 phospha-tase, and an NADP-linked mannitol dehydro-genase.

Table 2 lists the specific activities of glucosePEP transferase, fructose PEP transferase, andmannitol PEP transferase after 24 hr of germina-tion in the presence of glucose, fructose, or man-nitol.Table 3 lists the specific activities of glucoki-

nase, fructokinase, and mannitol kinase after 24hr of germination in the presence of glucose, fruc-tose, or mannitol.The above data suggested the presence of a

"constitutive" glucokinase and that mannitolPEP transferase and fructose PEP transferaseaccumulated to higher specific activities whentheir respective substrates were added exoge-nously. It is of interest that both the fructose and

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TABLE 1. Metabolic enzyme levels during spore germination

Specific activity atEnzyme ratiob

O Hra 6 Hr 12 Hr 24 Hr

Fumarase ..................................... 0.100 0.072 0.077 0.030 3.33Isocitrate lyase... 0.050 0.034 0.021 0.013 3.85Glyceraldehyde-3-phosphate dehydrogenase. 0.247 0.136 0.124 0.084 2.94Transaldolase ................................. 1.55 1.01 0.90 0.42 3.69Transketolase.................................. 0.248 0.103 0.068 0.068 3.65Enolase...................................... 0.310 0.126 0.082 0.084 3.71Aldolase ...................................... 1.05 0.908 0.718 0.500 2.10Mannitol-l -phosphate dehydrogenase ..... ..... 1 .04 0.673 0.416 0.259 4.02Isocitrate dehydrogenase ........ .............. 0.155 0.142 0.158 0.138 1.12Glucose-6-phosphate dehydrogenase ..... ...... 0.557 0.426 0.408 0.250 2.23Mannitol dehydrogenase .....................0..620 0.668 0.670 0.555 1.10Alkaline phosphatase......................... 1.12 1.27 1.03 0.233 4.81

a Specific activity = A optical density per minute per milligram of cell-free extract protein.b Activity ratio = zero-time specific activity/24-hr specific activity.

TABLE 2. Hexose phosphoenolpyruvate transferaseactivities after 24-hr spore germination

Carbohydrate source forsporulation and spore germination

EnzymeGlucose Mannitol Fructosespecific specific specific

activitya activity activity

Glucose phosphoenol-pyruvate transfer-ase.................0.022 0.026 0.028

Fructose phospho-enolpyruvate trans-ferase ........... 0.026 0.023 0.047

Mannitol phospho-enolpyruvate trans-ferase ........... 0.026 0.065 0.067

Specific activity = A optical density per min-ute per milligram of cell-free extract protein.

mannitol PEP enzymes accumulated coordinatelyin the presence of fructose.

Table 4 depicts the specific activity changes ofmannitol dehydrogenase, mannitol-1-P04 phos-phatase, and mannitol-1-PO4 dehydrogenaseafter 24 hr of germination in the presence of glu-cose, mannitol, or fructose. Identical enzyme ac-cumulation data were obtained when the sporeswere allowed to germinate for longer time peri-ods, i.e., up to 120 hr.

Evidence for the occurrence of a constitutiveglyoxylate shunt (acetate or glucose-germinationsystem). Table 5 lists the changes in specific ac-tivities of glyoxylate and tricarboxylic acid cycleenzymes during macroconidial germination. It isapparent that M. gypseum possesses a constitu-tive glyoxylate shunt. It is also evident that zer-

TABLE 3. Hexose kinase activities after 24-hrspore germination

Carbohydrate source forsporulation and spore germination

EnzymeGlucose Mannitol Fructosespecific specific specificactivitya activity activity

Glucokinase... 0.180 0.176 0.195Fructokinase.0....... 0.010 0.010 0.010Mannitol kinase....... 0.012 0.011 0.011

a Specific activity = A optical density per min-ute per milligram of cell-free extract protein.

mination in acetate resulted in increased levels ofisocitrate lyase and fumarase and a decrease in thelevel of isocitrate dehydrogenase.

Purification and properties of an alkaline phos-phatase. Alkaline phosphatase-type activities areknown to be associated with the terminal steps insporulation of fungi and related organisms (7, 17,20). Since an alkaline phosphatase appeared toaccumulate maximally during terminal sporula-tion of M. gypseum (unpublished data), we wereinterested in investigating the function of thisenzyme and its behavior during spore germina-tion.Table 6 lists the recovery and purification of

alkaline phosphatase throughout the isolationprocedure. The purified enzyme was found to behomogeneous with respect to alkaline phospha-tase activity as judged by disc gel electrophoresis.The enzyme has an apparent affinity constant of

6.3 X 10-4 (moles of p-nitrophenyl phosphate/liter) at 37 C, pH 8.0 (Fig. 2). It appears that theenzvme is inhibited bv orthoDhosphate (Fig. 3).

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TABLE 4. Activities of mannitol metabolismenzymes after 24-hr germination

Carbohydrate source forsporulation and spore germination

EnzymeGlucose Mannitol Fructosespecific specific specificactivity activity activity

Mannitol dehydrogen-ase5............0.5400.995 1.07

Mannitol-1 -phosphatedehydrogenase .......0.300 0.320 0.568

Mannitol-1 -phosphatephosphataseb........ 9.5 16.0 27.0

Specific activity = A optical density per min-ute per milligram of cell-free extract protein.

I Specific activity = micromoles of inorganicphosphate released per 0.18 mg of cell-free extractprotein per 75 min at 37 C.

TABLE 5. Glyoxalate and tricarboxylic acid cycleenzyme activities during macroconidial

germination

Carbohydrate source forsporulation and spore

germinationEnzyme

Acetate Glucosespecific specificactivitya activity

Isocitrate lyase............ 0.022 0.012Isocitrate dehydrogenase. 0.061 0.128Fumarase. .048 0.024

a Specific activity = A optical density per min-ute per milligram of cell-free extract protein.

The rate of phosphorylytic activity of the enzymeagainst several products of intermediary metabo-lism, as measured by release of orthophosphate,is summarized in Table 7. The enzyme has highactivity against fructose-6-PO4 and glucose-6-PO4(5). Since both of these compounds are important

3.

00

10 a

1.W"3M 2u3"M 3xU3MA 4Xa 3M

I (p-NT1OtINYL PHOsPATE)FIG. 2. Measurement of the apparent affinity con-

stant for alkaline phosphatase with p-nitrophenyl phos-phate (PNPP). Conditions: 0.5 M Tris-hydrochloridebuffer (pH 8.0) at 37 C; v = micromoles of PNPPhydrolyzed per minute per milligram ofprotein.

100

40

20

4x103M 1.2pi0-2M 2x0-2MKH2PQ CONCENTRATION

FIG. 3. Inhibition by orthophosphate of the activityof alkaline phosphatase against p-nitrophenyl phos-phate. Conditions: 0.5 M Tris-hydrochloride buffer(pH 8.0) at 37 C; p-nitrophenyl phosphate concen-tration at 0.6 mg/ml.

TABLE 6. Purification of Microsporum gypseum alkaline phosphatase

Total Poen Enzyme Yed PrfctoFraction enzyme Pmg/rmi units/mg Y(Id (Purfication

unitsP of protein

Cell-free extract 92 5.1 18 100 1Supernatant from 110,000 X g centrifugation 92 1.0 92 100 565 to 80% saturation with (NH4)2SO4 80 0.24 320 87 18Pooled fractions from the final DEAE-cellulose 22 0.0052 4,230 24 240columnb

a One unit of enzyme activity was defined as the quantity of enzyme that catalyzes the release of 1JAmole of p-nitrophenol in 1 min under the conditions specified.

b Values corrected for the fact that only one half of the total volume from the 65 to 80% ammoniumsulfate saturation fraction was applied to the first diethylaminoethyl (DEAE)-cellulose column.

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TABLE 7. Substrate specificity of alkalinephosphatase

RelativeSubstrate reaction

velocity

p-Nitrophenyl phosphate............. 1.0Fructose-6-phosphate ................ 1.7Glucose-6-phosphate ................. 1.5Ribose-5-phosphate.................. 1.4Mannitol-1 -phosphate.1.......... .. . 1.2a-Glycerophosphate. 1.2j3-Glycerophosphate. 1.23-Phosphoglyceric acid. 0.8Fructose-I -6-diphosphate ......... 1.0Bis-p-nitrophenyl phosphate. 0

substrates for intermediary metabolism and re-serve synthesis, these data suggested that thealkaline phosphatase may be active in the shut-ting off of intermediary metabolism and the syn-thesis of carbohydrate reserves during the termi-nal periods of sporulation.RNA to cell-free extract protein ratios during

spore germination (glucose-germination system).Table 8 lists the RNA to cell-free extract proteinratios in zero time and 24-hr spores. It is apparentthat RNA is not concentrated in the spore andevidently is synthesized to a greater extent thancell-free extract protein during spore germina-tion.

DISCUSSION

Since there has been only one previous studyof macroconidial germination in M. gypseum (1),this preliminary investigation was undertaken toascertain what basic processes occurred duringspore germination, and hopefully to suggest pos-sible germination-specific proteins which might beexamined subsequently in further detail.The composition of the alcohol-soluble pool

was very similar to that observed in Aspergillus(11, 12) and Neurospora (12, 16). The ordereddisappearance of mannitol and subsequentlytrehalose and inositol was very similar to theAspergillus system. Horikoshi et al. (11) haveshown that mannitol is a repressor of trehalaseand must be utilized prior to any trehalose degra-dation.The utilization pattern of the alcohol-soluble

pool was found to be very similar to that reportedfor Neurospora (16). The biphasic nature of theutilization curve is probably the result of mannitolutilization prior to the degradation of trehaloseand inositol. It appears that M. gypseum isunique in its ability to utilize a considerable por-

TABLE 8. Relative concentrations of sporeribonucleic acid and protein in zero-time

and 24-hr cell-free extracts

Time RNA Protein Ratio

hr pg/mi pg/mi

0 396 7,200 0.05524 910 6,500 0.140

tion of the acid-soluble pool during spore germi-nation.The pattern of enzyme specific activity changes

observed during spore germination was some-what unusual. All enzymatic activities eitherremained constant or decreased to some extentduring the germination and hyphal outgrowthtime period. This may be explained most easilyby the fact that the vegetative nuclear-cytoplasmicratio was not preserved in the macroconidium.Upon germination, the concentrated spore nu-clei migrate into the growing germ tube and re-establish the lower mycelial nuclear cytoplasmicratio (Leighton and Stock, in preparation). Sincegermination enzymes of necessity would have tobe synthesized prior to and during germ-tubeelongation, i.e., "concentrated" prior to rapidgerm-tube outgrowth, there could be a dispro-portionate amount of non-enzymatic proteinssynthesized during the outgrowth time period.This would give rise to an apparent decrease inenzyme specific activities during spore germina-tion. In this type of a system, enzymes whichmaintain a constant specific activity during germi-nation may in fact represent a considerableamount of enzyme accumulation.

It has been suggested by other workers thatmannitol dehydrogenase and isocitrate dehydro-genase are involved in fungal spore germination(3, 8, 11; J. C. Galbraith and J. E. Smith, Proc.Soc. Gen. Microbiol, p. 12, 1969). It is interestingthat these two enzymes have a low activity ratioin the M. gypseum germination system. It ispossible that these enzymes are involved ingermination processes. Further de novo synthesisstudies are presently in progress to clarify thisobservation.

Investigations involving other fungi and relatedorganisms have suggested that alkaline phospha-tase (7, 17, 20) and isocitrate lyase (3, 8; Gal-braith and Smith, Proc. Soc. Gen. Microbiol.1969:xii) are present at high levels during fungalsporulation. In addition, it has been shown thatM. gypseum alkaline phosphatase is producedmaximally during periods of terminal differentia-tion (Leighton and Stock, in preparation).

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The specificity data obtained for this enzymehave suggested that it may function in the shut-off of intermediary metabolism and carbohydratereserve synthesis. We also knew that the releaseof inorganic phosphatase is an essential feature ofmacroconidial germination in M. gypseum(Leighton, Page, and Stock, in preparation). Thisphosphate could be derived easily from the actionof alkaline phosphatase on fructose-6-PO4 andglucose-6-PO4. Since the enzyme is inhibited byhigh concentrations of orthophosphate, it mayregulate its own shut-off by a type of end-productinhibition. This enzyme is also being studied forde novo synthesis during germination time peri-ods since its high activity ratio suggested it maynot accumulate during spore germination.Many fungi possess a constitutive glyoxylate

shunt (5), and M. gypseum appears to be no ex-ception. When spores of this organism weregerminated in acetate, isocitrate lyase accumu-lated to a higher level than in glucose-germinatedspores. In the presence of acetate, the isocitricdehydrogenase level decreases. This may be ex-plained most readily by the observation of Osakiand Shiio (21), which indicates that isocitratedehydrogenase is repressed by glyoxylate cycleproducts. Hence, an increase in glyoxylate cycleactivity would be expected to result in a decreasedlevel of isocitrate dehydrogenase activity. Thefact that fumarase levels were high during acetategrowth may be a result of increased succinatelevels and apparently a lack of inhibition offumarase by glyoxylate cycle products.

It is not surprising that M. gypseum macro-conidia do not contain a high ratio of RNA tocell-free extract protein. The terminal steps ofsporulation occur under what are essentiallystarvation conditions (William Page, unpublisheddata). This type of environment does not favor ahigh ratio of RNA to cell-free extract protein.

Fungal conidia cannot survive temperature ex-tremes (26). In the case of M. gypseum, themacroconidia are capable of surviving tempera-tures of 50 to 55 C for only short periods of time(14). However, macroconidia are capable ofsurviving for several months in the range 4 to 25C. The ability of the macroconidium to survivedoes not seem to reside in its possessing a pro-tective physical environment, as is the case withfungal ascospores (26). We suggest that the in-creased nuclear-cytoplasmic ratio in the macro-conidium results in a large number of copies ofessential information in the spore. This selectiveconcentration of nuclei increases the number ofavailable targets and concomitantly the numberof potentially lethal events that the spore is capa-ble of surviving.

ACKNOWLEDGMENT

We thank William Page and Louise A. Avent for excellenttechnical assistance and Audrey F. Gronlund for helpful advice.

This investigation was supported by a grant (MT-757) fromthe Medical Research Council, Ottawa, Canada.

LITERATURE CITED

1. Barash, I., M. L. Conway, and D. H. Howard. 1967. Carboncatabolism and synthesis of macromolecules during sporegermination of Microsporum gypseum. J. Bacteriol. 93:656-662.

2. Bleyman, M., and C. Woese. 1969. Ribosomal ribonucleicacid maturation during bacterial spore germination. J.Bacteriol. 97:27-31.

3. Caltrider, P. G., S. Ramachandran, and D. Gottlieb. 1963.Metabolism during germination and function of glyoxalateenzymes in uredospores of rust fungi. Phytopathology53:86-92.

4. Chen, P., T. Toribara, and H. Warner. 1956. Microdetermina-tion of phosphorus. Anal. Chem. 28:1756-1758.

5. Cochrane, V. W. 1966. In M. F. Madelin (ed.), The fungusspore, p. 203. Butterworth & Co., London.

6. De Vries, W., Sj. J. Gerbrandy, and A. H. Stouthamer. 1967.Carbohydrate metabolism in Bifidobacterium bifidum. Bio-chim. Biophys. Acta. 136:415-425.

7. Gezelius, K., and B. E. Wright. 1965. Alkaline phosphatasein Dictyostelium discoideum. J. Gen. Microbiol. 38:309-327.

8. Gottlieb, D. 1966. In M. F. Madelin. (ed), The fungus spore,p. 227-228. Butterworth & Co., London.

9. Hanson, T. E., and R. L. Anderson. 1968. Phosphoenol-pyruvate dependent formation of D-fructose-l-phosphateby a four-component phosphotransferase system. Proc.Nat. Acad. Sci. U.S.A. 61:269-276.

10. Harrocks, R. H. 1949. Paper partition chromatography of re-ducing sugars with benzidine as a spraying agent. Nature(London) 164: 444.

11. Horikoshi, K., S. lida, and Y. lkeda. 1965. Mannitol andmannitol dehydrogenases in conidia of Aspergillus oryzae.J. Bacteriol. 89:326-330.

12. Ikawa, M., P. T. Borowski, and A. Chakravarti. 1968. Cho-line and inositol distribution in algae and fungi. Appl.Microbiol. 16:620-623.

13. Lee, W. H. 1967. Carbon balance of a mannitol fermentationand the biosynthetic pathway. Appl. Microbiol. 15:1206-1210.

14. Leighton, T. J., and J. J. Stock. 1969. Heat-induced macro-conidia germination in Microsporum gypseum. Appl. Mi-crobiol. 17:473-475.

15. Leighton, T. J., and J. J. Stock. 1970. Biochemical changesduring fungal sporulation and spore germination. I. Phenylmethyl sulfonyl fluoride inhibition of macroconidial ger-mination in Microsporum gypseum. J. Bacteriol. 101:931-940.

16. Lingappa, B. T., and A. S. Sussman. 1959. Endogenous sub-strates of dormant, activated and germinating ascosporesof Neurospora tetrasperma. Plant Physiol. 34:466-472.

17. Loomis, W. F., Jr. 1969. Developmental regulation of alkalinephosphatase in Dictyostelium discoideum. J. Bacteriol. 100:417-422.

18. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Ran-dall. 1951. Protein measurement with the Folin phenol re-agent. J. Biol. Chem. 193:265-275.

19. Morris, D. L. 1948. Quantitative determination of carbo-hydrates with Dreywood's anthrone reagent. Science 107:254-255.

20. Nagasald, S. 1968. Cytological and physiological studies onphosphatases in developing cultures of Aspergillus niger.J. Gen. Appl. Microbiol. 14:263-277.

445VOL. 103, 1970

on April 14, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

LEIGHTON, STOCK, AND KELLN

21. Ozaki, H., and Shiio. 1968. Regulation of the TCA and

glyoxalate cycles in Brevibacterium flavum. 1. Inhibition ofisocitrate lyase and isocitrate dehydrogenase by organicacids related to the TCA and glyoxalate cycles. J. Biochem.64:355-363.

22. Schneider, W. C. 1945. Phosphorus compounds in animaltissues. J. Biol. Chem. 161:293-303.

23. Sjogren, R. E., and A. H. Romano. 1967. Evidence for mul-tiple forms of isocitrate lyase in Neurospora crassa. J. Bac-teriol. 93:1638-1643.

24. Slein, M. W., G. T. Cori, and C. F. Cori. 1950. A compara-tive study of hexokinase from yeast and animal tissues. J.Biol. Chem. 186:763-780.

25. Strandberg, G. W. 1969. D-Mannitol metabolism by Asper-gillus candidus. J. Bacteriol. 97:1305-1309.

26. Sussman, A. S., and H. 0. Halvorson. 1966. Spores, theirdormancy and germination. Harper and Row, New York.

27. Trevelyan, W. E., D. P. Procter, and J. S. Harrison. 1950.Detection of sugars on paper chromatograms. Nature(London) 166:444-445.

446 J. BACTERIOL.

on April 14, 2021 by guest

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Dow

nloaded from