JOURNAL OF BACTERIOLOGY, June 1972, p. 1041-1049Copyright 0 1972 American Society for Microbiology

Vol. 110, No. 3Printed in U.SA.

Regulation of Exocellular Proteases inNeurospora crassa: Induction and Repression of

Enzyme SynthesisH. DRUCKER

Biology Department, Battelle, Pacific Northwest Laboratories, Richland, Washington 99352

Received for publication 27 January 1972

Neurospora crassa strain 74A grown on Vogel's medium containing bovineserum albumin (BSA) as principal carbon source secretes proteolytic enzymeswhich appear in the culture filtrate. Low concentrations of sucrose (0.1%) are

necessary for growth from conidia, as conidia will not germinate on BSA alone.Onee growth is initiated, however, protease production begins and at 5 to 6 hrgrowth and enzyme production are parallel. Higher concentrations of sucrose

(0.5-2%) repress protease synthesis. Other metabolizable materials (sugars,amino acids, peptide mixtures) also repress protease synthesis. Some sugarswill not sustain growth but allow germination and full induction of protease inthe presence of protein. A material found in culture fluids of cells during in-duction of protease synthesis when added to repressed cultures causes a five-fold increase in the amount of protease production, although this is still ap-

proximately half that of normally induced cells. This material appears to beproduced by induced cells in as little as 2 hr of culture, which is before detect-able levels of protease can be found. It is heat-stable, of low molecular weight,and is not a simple product of protein digestion by the N. crassa proteases.

Bacteria, fungi, and streptomycetes are ca-pable of producing large quantities of exocel-lular proteases under varying conditions ofgrowth. In a few cases, as with Pseudomonasana Arthrobacter, exocellular proteolytic ac-tivity would appear to be constitutive (3, 10,11) and not dependent on the presence of pro-tein during growth. In the case of Strepto-myces griseus and S. fradiae, both capable ofsecreting a potent mixture of exopeptidasesand endopeptidases into their milieu (12, 13), acomplex medium is required for growth andenzyme production, and thus the question ofregulation of these enzymes remains open.This is also the case for Aspergillus species (1).There has been one report of an inducible ker-atinase in the literature (14), but the existenceof this phenomenon has been questioned (12).

In Bacillus species, exocellular proteaseshave been implicated in germination (18) andsporulation (5, 9), but there have been no re-ports of induction of enzyme during log-phasegrowth.Many microorganisms are capable of de-

grading proteinaceous substrates in nature, butno thorough examination has been made of theregulation of the exocellular proteases required

for this activity. In this communication, wewill describe a system employing Neurosporacrassa, where the organism grows on a proteinas principal carbon source in synthetic me-dium within 24 hr and where the biosynthesisof exocellular protease is regulated.

MATERIALS AND METHODSGrowth of organism. N. crassa, strain 74A, was

grown on Vogel's minimal salts medium (23) withthe carbon sources described in Table 1. The me-dium was inoculated with conidia as described byTurner and Matchett (20), and the cells were grownon a rotary shaker at 30 C for varying time periods.Experiments were done on cells in early phase ofgrowth (0-6 hr after inoculation) and during logphase of growth (6-16 hr after inoculation). No ex-periments were done with mycelial mats.

Preparation of substrate. Two grams of Ham-mersten quality casein (Nutritional Biochemicals)was suspended in 60 ml of distilled water, and thesuspension was dissolved by raising the pH of thesolution to 12.0 with 1 N NaOH. The pH was thenlowered to 8.0 with 1 N HCl, and the solution wasmade 2% (w/v) in casein and 0.01 M in tris(hydroxy-methyl)aminomethane (Tris)-hydrochloride buffer,pH 8.0.Assay of proteolytic activity. Exocellular pro-

tease was assayed by a modification of the method of1041

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TABLE 1. Composition of media for induction andrepression of exocellular protease synthesis in

Neurospora crassa

Medium Composition

Inducing Vogel's salts, 1% BSA,a and0.1% sucrose

Repressing Vogel's salts, 1% BSA,G and2% sucrose

a Bovine serum albumin (BSA) was Pentex Frac-tion V.

McDonald and Chen (7). Cells were removed by fil-tration through Whatman no. 1 paper, and from 0.5to 1 ml of culture filtrate was added to a tube con-taining 0.1 ml of 1 M Tris, pH 8.0, and distilledwater to make this solution 0.01 M in Tris, 1 ml finalvolume. A blank and an assay tube were prepared inthis fashion, and 2 ml of precipitating agent (8% tri-chloroacetic acid) was added to the blank prior tothe addition of substrate. The substrate (2% casein)was then added to both the blank and assay tube,and incubation was carried out at 37 C for 30 min.At the end of this time, 2 ml of precipitating agentwas added to the samples containing active en-zyme, and the tubes were incubated for an addi-tional 30 min at 37 C to allow complete precipitationof undigested casein and precipitable partial-diges-tion products. The precipitate was removed by fil-tration through Whatman no. 1 paper, and 1 ml offiltrate was removed for assay. A 5-ml amount ofbiuret reagent (consisting of 100 ml of 2% Na2CO3,10 ml of 10% NaOH, 1 ml of 2.7% sodium potassiumtartrate, and 1 ml of 1% CuSO4) was added to thissample. After 10 min at room temperature, 0.5 ml ofFolin-Ciocalteau reagent diluted 1:1 with water wasadded, the tubes were immediately shaken, andcolor was allowed to develop for 1 hr. The tubes wereread at 700 nm against the blank of culture filtrate.One unit of activity (PU) is defined as that amountof enzyme which releases the color-equivalent of onemicrogram of tyrosine in one minute (2). All assayswere performed in duplicate, and data points repre-sent an average of the two determinations.

Crude extracts of N. crassa were made by grindinglyophilized cells (20), and 0.25 to 0.5 ml of extractwere assayed for protease activity as describedabove.The colorimetric assay described could not be

used directly when cells were grown on tryptone orCasamino Acids, as these materials would lead tovery high blank values for filtrate. Prior to assay ofculture fluid of cells grown on 1% tryptone or Casa-mino Acids, 5 g of dry Sephadex G-25 was added to100 ml of culture fluid, and the Sephadex was re-moved by centrifugation. This was repeated, and theresulting material, free from high concentration ofinterfering substances, was assayed.

Sephadex chromatography. To characterize theprotease-inducing factor described in the text, 5 mlof culture filtrate of N. crassa grown 8 hr on the in-ducing medium was placed on a column (3 by 35 cm)of Sephadex G-25. The void volume of the column

had been determined with 5 ml of blue Dextran 2000(Pharmacia), and two fractions were taken for assayof inducing ability. The first fraction was materialappearing in the void volume, and the second frac-tion consisted of material from the included volumeof the gel (from end of void volume to twice voidvolume).

RESULTSWhen conidia of Neurospora are inoculated

into the inducing medium, exocellular pro-teases are found in the medium within 1 hr ofearly log growth, and appearance of enzymethen parallels log growth (Fig. 1A) up to latelog phase. The enzyme produced would appearto be an exocellular enzyme, as levels of intra-cellular protease in terms of units per milli-gram of cells remain constant throughout theinduction period, whereas specific activity ofexocellular enzyme rises (Fig. 1B). In addition,growth on inducing medium does not changethe intracellular amounts of protease as com-pared with growth on 2% sucrose alone, a me-dium which does not induce exocellular pro-tease, suggesting that induction is not affectedby increases in intracellular protease and sub-sequent leakage. Since the growth of the orga-nism on this medium is approximately halfthat of cells grown on sucrose as carbon source(see Table 3), bovine serum albumin (BSA)would appear to be a relatively poor carbonsource. Low concentrations of sucrose are re-quired in the inducing medium since germina-tion of conidia will not occur on BSA alone.The specific activity of intracellular and

exocellular protease was not measured before 6hr of culture for the following reasons: (i) fulllog-phase growth of cells does not occur until 8hr of culture (21), (ii) the low concentrations ofmaterial found in the interval 0 to 6 hr (Fig. 1),and (iii) the problem of events occurring ingermination of conidia in the interval 0 to 4 hr.A preliminary analysis of the number and

types of protease that might be present in cul-ture filtrate of cells grown on inducing me-dium was performed by examination of the pHstability profile and the effect of ethylenedi-aminetetraacetic acid disodium salt (EDTA)on proteolytic activity. Culture filtrate fromcells grown 8 hr on inducing medium con-taining 0.691 PU/ml of culture filtrate was di-luted 1: 1 with 0.1 M buffers of pH 3.0 to 10.2.The samples were incubated for 60 min at 37C, and 1 ml of the sample was then assayed forproteolytic activity. The pH stability profile(Fig. 2) appears to show three overlappingpeaks, one with maximum at acid pH (pH 4.6),one at neutral pH (6-7) and one at alkaline pH

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VOL. 110, 1972 REGULATION OF NEUROSPORA PROTEASES

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FIG. 1. (A) Growth and protease production of N. crassa 74A on the protease-inducing medium of 0.1%sucrose-1% BSA. Conidia were inoculated into 1,500 ml of medium, and cells were grown as described in thetext. At the intervals shown, 50-ml samples were removed from the culture. Cells were collected by filtration,lyophilized and weighed, and culture filtrate was assayed for proteolytic activity. (0) Cell concentration, mil-ligrams (dry weight) per milliliter of media. (x) Proteolytic activity in protease units per milliliter of culturefiltrate. (B) Specific activity of intracellular and exocellular proteases produced by N. crassa 74A grown on

inducing medium. Cells from 50 ml of culture were collected by filtration, lyophilized, and extracted as de-scribed in text. Culture filtrate was assayed for exocellular proteolytic activity. (0) Exocellular enzyme spe-

cific activity in protease units per milligram of cells (dry weight). (x) Intracellular enzyme specific activityin units per milligram of cells (dry weight).

concentrations of EDTA did not change the0.4 degree of inhibition. Neutral proteases are in-0 X-X_X /^ \^ hibited by EDTA as they depend upon diva-

0.3 lent metals (usually zinc) for their activity (19).The culture filtrate contains a considerable

E0.2 _ / \ / 0 amount of salts and a relatively small amountof enzyme, which may explain the high con-

centration of chelator required for inhibition as0.1 compared to the amount required for purified

enzyme (19).llExocellular enzyme is not formed by activa-

30 4.0 5.0 6.0 7.0 0.0 9.0 10.00tion of inactive materials since protein syn-

pH thesis is involved in enzyme production. If cy-FIG. 2. pH Stability profile of induced exocellular cloheximide, an inhibitor of protein synthesis

protease from Neurospora crassa 74A. Culture fil- (17) is added to a culture of N. crassa 2 hrtrate containing 0.691 PU/mI of proteolytic activity after the first appearance of exocellular pro-

from cells grown 8 hr on inducing medium was di- tease, no further exocellular enzyme is secreted

luted 1:1 with: (0) 0.1 M citrate buffer, (x) 0.1 M nto the d (F 3)

phosphate buffer, (A) 0.1 M borate buffer for 60 min Wn Nemedium ais3grat 37 C. One milliliter of the sample was then as- When Neurospora iS grown on a wide varietysayed for proteolytic activity as described in text. of carbon sources, such as carbohydrates, Casa-

mino Acids, or tryptone, protease activity isnot found in the culture fluid (Table 3),

(pH 9.0). The profile would suggest that more implying that the exocellular protease ob-than one enzyme capable of acting on casein served when Neurospora grows on BSA is an

exists in the culture filtrate (see below). induced activity, with the protein BSA servingCulture filtrate from cells grown 8 hr on as inducer. Since Casamino Acids or peptide

inducing medium was assayed for proteolytic mixtures do not induce protease synthesis, theactivity in 0.1 M Tris, pH 8.0, containing con- process of induction of protease activity mustcentrations of EDTA of from 10-5 to 0.1 M. require high-molecular-weight polypeptides.EDTA at a concentration of 10-2 M inhibited Easily metabolizable carbon sources such as

proteolytic activity by 33% (Table 2). Higher sucrose or glucose, when present at substrate

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/xINTRACELLULAR ENZYNE

EXOCELLULAR- ENZYME

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TABLE 2. Effect of EDTA on the proteolytic activityof cell-free filtrates from Neurospora crassa grown

on inducing mediuma

Proteolytic PUb control/Concentration of activity PU EDTAEDTA(M) ~(PU/ml)b added

Control (0) 0.90910-5 1.024 1.1310-4 0.967 1.0610-3 0.839 0.9210-2 0.591 0.6510- 0.587 0.65

aOne milliliter of culture filtrate from cells of N.crassa, strain 74A, was added to 0.2 ml 0.5 M Tris,pH 8.0, containing the desired concentration of eth-ylenediaminetetraacetic acid (EDTA). The samplewas then assayed for proteolytic activity as de-scribed in Materials and Methods.

h One unit of activity (PU) is defined as thatamount of enzyme which releases the color-equiva-lent of one microgram of tyrosine in one minute (2).

levels (2%) in the inducing medium, repressprotease biosynthesis as much as 86%, whereasgrowth was stimulated by 20% (Table 3). Fruc-tose, maltose, and cellobiose, when provided assole carbon and energy source, supportedgrowth, although to a lesser extent than su-crose. The addition of BSA to medium con-taining these three sugars resulted in a 26, 135,and 138% increase (for fructose, maltose, andcellobiose, respectively) in cell mass over thatfound in medium without BSA. In addition,these cells produced twice as much exocellularprotease per unit of cell mass as cells grown onsucrose or glucose plus BSA. This would sug-gest that the increased growth observed inthese media upon addition of BSA is causedby utilization of the peptides and amino acidsreleased by the exocellular proteases. A largegroup of sugars (lactose, mannitol, melibiose,ribose, fucose, arabinose, arabitol) were incap-able in themselves of sustaining growth ofNeurospora but apparently would allow germi-nation in the presence of protein substrate, fol-lowed by induction of proteolytic activity.Galactose and sorbose were the only sugarsthat did not support growth or germination,and thus would not allow protease induction inthe presence of inducer.The kinetics of protease production in re-

pressing medium (Fig. 4) are complex. Pro-tease activity appears at the same period(early log-phase growth) as in growth on in-ducing medium but plateaus almost immedi-ately. Sucrose may act as a repressor by pre-venting synthesis of some proteases or by dras-tically lowering the rate of synthesis. These

data do not allow a clear choice between thesepossibilities. Repression by sucrose is a func-tion of the concentration of the sugar (Fig. 5),with 50% repression occurring at 0.5% sucrose

and maximal repression occurring between 0.7to 1% sucrose. As the cells deplete sucrosefrom the medium containing the protein in-ducer, they synthesize protease, implying dere-pression of the protease genes. There wouldappear to be some repression of protease at a

sucrose concentration as low as 0.1%, judgingfrom the 1- to 2-hr lag of protease synthesisbehind growth in the inducing medium (Fig.1A). The kinetics of growth on sucrose aloneare almost identical to the kinetics of growthon the repressing medium (Table 3 and Fig. 4),but no exocellular protease is produced.

If sucrose functions by preventing the syn-

thesis of one protease, the product of whichinduces the synthesis of another, then additionof such a product to a culture of Neurosporagrowing on repressing medium should cause an

increase in protease levels. To test this possi-bility, cells were grown in inducing mediumfor 8 hr, and 4 ml of culture filtrate was re-

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GROWTH TINE IN HRS

FIG. 3. The effect of cycloheximide on inductionof protease from Neurospora crassa 74A. Conidia ofN. crassa, strain 74A, were inoculated into two flaskseach containing 250 ml of inducing medium. Bothflasks had a Klett reading of 66 with a blue filter onthe Klett Summerson colorimeter at 0 time ofgrowth. The cells were incubated at 30 C, and sam-

ples of cells were removed for turbidimetric assay ofgrowth. Cells were removed by filtration, and 1-mlsamples of culture filtrate were assayed for exocel-lular protease. When the Klett reading had doubledin both flasks (128 Klett units at 8 hr of growth) cy-cloheximide was added to a final concentration of 4tig/ml in flask 1. The control flask received no addi-tions. Exocellular protease from both flasks was thenassayed for 12.5 hr. (0) Control flask, no cyclohexi-mide added; (x) cycloheximide added at 8 hr ofgrowth.

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REGULATION OF NEUROSPORA PROTEASES

TABLE 3. The effect of carbon source on protease productiona

Growthc (mg/ml) Protease production Specific activity(PU/mi)Carbon source' oto

-BSA +1% BSA -BSA +1% BSA +1% BSA %Controiculture culture culture culture cells (PU/mg) production

Sucrose 3.48 4.14 0 1.202 0.291 14.9Glucose 3.52 4.20 0 1.152 0.274 14.0D-(-)-Fructose 3.11 3.91 0 1.998 0.511 26.2D-(+)-Lactose 0 0.412 0 1.164 2.825 145.0D-(-)-Galactose 0 0 0 0 0 0D-(-)-Maltose 1.694 3.98 0 1.713 0.431 22.1D-( -)-Mannitol 0.062 1.35 0 2.168 1.606 82.3L-(-)-Sorbose 0.028 0.040 0 0 0 0D-(+)-Cellobiose 1.75 4.18 0 2.175 0.520 26.7a-Melibiose 0.20 0.364 0 1.509 4.147 213.0D-Ribose 0.090 0.868 0 2.035 2.344 120.2L-Fucose 0.034 0.044 0 0.064 1.465 75.1D-Xylose 0.880 1.600 0 0.388 0.242 12.4D-( -)-Arabinose 0.024 0.450 0 0.562 1.250 64.1L-Arabinose 0.224 1.126 0 2.003 1.779 91.2L-Arabitol 0.056 0.636 0 1.426 2.242 115.0D-(+)-Arabitol 0.022 0.544 0 1.168 2.147 110.1Glycerol 0.222 1.292 0 2.566 1.986 102.0Casamino Acids 4.0 4.2 0 0 0 0Tryptone 4.1 4.3 0 0 0 0ControlInducing medium 2.06 4.017 1.950 100.0

a BSA, Bovine serum albumin. One unit of activity (PU) is defined as that amount of enzyme which re-leases the color-equivalent of one microgram of tyrosine in one minute (2).

b All media are Vogel's salts plus 2% carbon source and 1% BSA where indicated.c All parameters were measured at 12 hr of cjture.

moved. This was added to repressing medium,which was then inoculated with Neurosporaconidia, and growth and protease levels weredetermined. The added material appeared tostimulate protease production (Fig.. 6 andTable 4). Protease levels under these condi-tions were about fivefold higher than the levelnormally found in repressing medium andwere half that found in inducing medium. Inaddition, protease appeared before log growth(2-4 hr), whereas under both induced or re-pressed conditions protease appears after thecells are fully in log growth.The protease-inducing material, which will

be referred to as "factor" in this communica-tion, is distinct in heat stability from the en-zymes produced during induction (Table 5)and is 100% active after treatment for 30 minat 100 C.

Fractionation of culture filtrate on a columnof Sephadex G-25 showed that low-molecular-weight material, void of all proteolytic activ-ity, possessed the ability to relax sucrose re-pression, whereas high-molecular-weight mate-rial containing active protease could not (Fig.

7). The increase in protease found at 10 to 14hr of growth in the presence of the high-mo-lecular-weight fraction and the somewhatlarger rate of production for exocellular pro-tease in the 4 to 8 hr interval may be due tocontamination of the high-molecular-weightfraction by the low-molecular-weight fraction,as the control with no additions showed noevidence for relaxation of sucrose repression.

Factor does not appear to be a directproduct of the action of N. crassa proteases onBSA. The following experiments suggestedthis. BSA (2%) was incubated for periods of 10min to 8 hr with 0.2 to 0.4 PU of Neurosporaprotease per ml which had been stripped offactor by Sephadex G-25 filtration. This is anamount of protease equivalent to that found inthe first 2 hr of protease biosynthesis. In onecase, 8 ml of this material was added to 100 mlof repressing medium which was then inocu-lated with conidia, and the culture was allowedto grow for 16 hr. No increase in protease be-yond the amount normally found in repressedmedium was observed. In the second case, 50ml of this partially digested material was

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FIG. 4. Growth and protease production of N.crassa strain 74A on the repressing medium of 2%osucrose-1% BSA. Protocol and figure legend areidentical to Fig. 1.

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PERCENT SUCROSE (WEIGHT/WEIGHT)FIG. 5. Effect of sucrose concentration on pro-

tease biosynthesis in N. crassa strain 74A. Fifty mil-liliters of media containing Vogel's salts, 1% BSA,and sucrose in per cent (w/w) as shown were inocu-lated with conidia of N. crassa, strain 74A, and theculture was grown for 16 hr. Samples of culture fil-trate were removed and assayed for protease activityin the caseinolytic assay (see text). Maximal induc-tion (100% induced protease activity) occurs at 0.1%sucrose with protease activity corresponding to 2.17PU/ml.

added to a medium containing Vogel's saltsplus 4% sucrose, resulting in a medium ofidentical composition to repressing medium interms of sugar and protein concentration. This

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FIG. 6. Effect of factor on protease production ofN. crassa strain 74A grown in repressing medium. A100-ml amount of inducing medium was inoculatedwith conidia of N. crassa strain 74A, and the culturewas allowed to grow for 8 hr. At the end of this time,12 ml of culture filtrate was removed and added to1,500 ml of repressing medium which was then inoc-ulated with conidia of N. crassa strain 74A. At theintervals shown, 50-ml samples were removed fromthe culture, lyophilized and weighed, and the culturefiltrate was assayed for proteolytic activity. A con-trol of cells grown in repressing medium was in-cluded in the experiment, and results for proteaseproduction and growth were similar to those de-picted in Fig. 4. (0) Cell concentration (dry weight),mg/ml media. (x) Proteolytic activity in PU/mI cul-ture filtrate.

TABLE 4. Effect of factor on protease production ofNeurospora crassa growing in repressing mediuma

Repress-InuigRepress- ing

Determinations mediumc ing mediummedium" plus

factorb

Growth (mg/ml) ........ 0.5 1.03 1.0Protease (PU/ml) ....... 1.0 0.180 0.815Specific activity of pro-

tease (PU/mg cells) ... 2.0 0.175 0.815

,'One unit of activity (PU) is defined as that amount ofenzyme which releases the color-equivalent of one micro-gram of tyrosine in one minute (2).

" All parameters were measured after 8 hr of culture in thedesignated medium.

medium was inoculated, cultured for 16 hr,and again no relaxation of sucrose repressionwas found.To determine the kinetics of factor accumu-

lation during the induction of exocellular pro-teases, cells were grown on inducing medium,and at 2-hr intervals for a total of 12 hr 4-mlamounts of culture filtrate were removed.These samples were separately added to 100ml of repressing medium, which was then inoc-

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TABLE 5. Heat stability of factor and proteolyticenzymes produced by Neurospora crassa strain 74A

% Enzyme activitya % Factor activity"Temp of remaining after 30 remaining after 30incubation min at designated min at designated

temp temp

0 100 10030 95 8040 78 8850 42 9060 6 85100 0 100

a Proteolytic enzyme activity is from an 8-hr cul-ture of N. crassa grown on inducing medium. 0 Cincubated enzyme had 2.6 units of activity (see Ma-terials and Methods) per ml in this experiment andwas taken as 100% active enzyme.

b Samples (10 ml) of 8-hr culture filtrate, assayedas per footnote a, were heated at the indicated tem-perature for 30 min and then added to 200 ml of re-pressing medium. The medium was inoculated withconidia of N. crassa, and the culture was grown for 8hr. Samples of culture filtrate were assayed for pro-tease activity and corrected for amount of enzymeproduced in normal repressed condition by sub-tracting units of enzyme produced in a control cul-ture in repressing medium containing no factor;added enzyme activity from factor was also sub-tracted. One-hundred per cent activity correspondedto 0.143 units/ml in the culture containing 0 C incu-bated factors.

ulated with conidia and allowed to grow for 12hr. Samples from the repressing media weretaken hourly, and proteolytic activity wasmeasured. When the time-of-sampling factorproduced in the inducing medium was plottedas a function of rate of protease production inunits per milliliter per hour in the repressingmedium (Fig. 8), factor concentration appearedto increase in the time interval 2 to 6 hr, plateauat 8 hr, and decrease in the interval 8 to 12 hr.When the same experiment was performed

with cells growing in repressing medium as thesource of factor, no increased rate of proteasebiosynthesis was observed, implying thatunder repressed conditions the material is ei-ther not synthesized or not secreted.As cyclic 3'5'-adenosine monophosphate

(AMP) has been found to relax catabolite re-pression in a number of bacterial systems (15),2 x 10-3 M cyclic AMP was added to a cultureof Neurospora growing in repressing mediumin place of factor. There was no effect on pro-tease production over a control with no addi-tion. Low levels (0.1%) of Casamino Acids,tryptone, and peptone, did not substitute forfactor in the relaxation of sucrose repression.

DISCUSSIONN. crassa is able to synthesize exocellular

proteolytic enzymes when grown on a mediumcontaining a protein as principal carbonsource. These enzymes are not constitutive, as

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GROWTH TIME (HRS)FIG. 7. Effect of Sephadex G-25 fractionation on

activity of factor produced by N. crassa strain 74Agrown on inducing medium. N. crassa was grown 8hr in 100 ml of inducing medium, and 5 ml of cul-ture filtrate was removed. This was placed on acolumn (3 by 35 cm) of Sephadex G-25 previouslycalibrated for void volume with 5 ml of blue dextran.High-molecular-weight material, containing all theprotease activity in the sample, was eluted in thesame volume as blue dextran. Low-molecular-weightfractions, possessing no proteolytic activity, wereeluted in two times void volume. A 10-ml amount ofeither fraction was added to 100 ml of repressingmedium which was then inoculated with conidia ofN. crassa strain 74A. Samples of culture filtrate weretaken at the intervals shown and assayed for proteo-lytic activity. A control flask of cells grown in in-ducing medium and a control of cells grown in re-pressing medium (no additions) provided data onprotease production similar to those depicted in Fig.JA and 4, respectively. (0) Proteolytic activity inPU/ml of filtrate from culture to which high-molec-ular-weight fraction was added; (x) proteolytic ac-tivity in PU/ml of filtrate from culture to which low-molecular-weight fraction was added.

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FIG. 8. Time course of factor production by N.crassa strain 74A-growing on the inducing medium.Conidia of N. crassa 74A were inoculated into 100 mlof inducing medium and cultured. At the time inter-vals shown, 4-mI samples were removed. These sam-ples were then added to separate flasks containing100 ml of repressing medium inoculated with conidiaof N. crassa which were then cultured for 12 hr. Acontrol culture received no factor. At hourly inter-vals during the log phase of growth and proteaseproduction, samples of culture filtrate were removed,assay for proteolytic activity was performed, andfrom these data rates of protease production in unitsper milliliter per hour of growth were determined.

they are not produced when cells grow on non-

proteinaceous substrates (carbohydrates,amino acids, peptide mixtures). As proteinsynthesis is involved in the production of theseenzymes, it would appear that an inductioninvolving a macromolecular inducer (protein)occurs in these cells. This observation is of in-terest since the proteinaceous substrate maynot' be capable of entering the cell intact (16).Yet the cell is capable, in some fashion, of rec-ognizing the presence of protein in the me-

dium and of producing the enzymes essentialfor its degradation and subsequent utilization.The amount of enzyme produced in a protein-containing medium appears to be regulated bythe composition of the medium with respect toother metabolites. That is, easily metaboliz-able compounds such as sucrose or glucose re-press the levels of protease made, as do aminoacids or peptides. Sugars that are poorly me-tabolized appear, in some cases, to allow ger-mination of conidia under the conditions usedin this study, with induction of protease thenoccurring in amounts identical to the amountsproduced in normal induction. Since Neuro-spora will not grow on a medium containing

the protein BSA alone, but requires a trace ofsugar (0.1% in the case of sucrose), it wouldappear that protein carbon sources are notcapable of entering conidia and inducing ger-mination. Given the general impermeability ofcells and spores to macromolecules, this resultwould be expected.More than one type of exocellular protease

appears to be produced during induction.Analysis of the pH stability profile of N. crassaexocellular enzyme produced during log-phasegrowth implies that at least three enzymes aresynthesized, as there are three peaks with re-spect to stability. One peak at pH 4.6 is sim-ilar to that of acid protease (22), the second issimilar to that of neutral protease (6), and thethird is similar to that of alkaline protease (4).In addition, the pH stability profile of crudeexocellular protease fractions from S. fradiae(12) is similar to that reported here for N.crassa enzyme. The observation that EDTAinhibits N. crassa exocellular protease by 33%also implies the existence of a neutral proteasein the exocellular filtrate (19). On the basis ofthe available data, the exact number of en-zymes produced during induction cannot bedetermined, but the evidence strongly suggeststhat more than one enzyme is made, and per-haps three.The material referred to as factor in this

study is produced during growth on a mediumcontaining a protein as principal carbonsource. It is not produced on repressing me-dium. The compound would appear to be oflow molecular weight and would appear to beproduced during the stationary phase ofgrowth on a protein. Factor appears to be ca-pable of partially relaxing sucrose repression.If levels of protease in the presence of sucroseare controlled by catabolite repression (8), thenfactor may be acting to increase the rate ofprotease biosynthesis and not as a specificinducer of proteolytic enzyme. Cyclic 3', 5'-AMP has been found to relax catabolite re-pression in bacteria (15) but does not appear toaffect protease synthesis under repressed con-ditions in this system.

If sucrose were to act as a specific repressorof one or more of the induced proteases, factormight relax repression by acting as an inducerof these enzymes. There is more than one pro-tease produced during induction, but the time-course of synthesis of individual enzymesduring induction and repression has not beendetermined, and thus a model of this kindcannot be substantiated at this time.

Resolution of the role of factor in proteaseinduction and in relaxation of sucrose repres-

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REGULATION OF NEUROSPORA PROTEASES

sion will be dependent upon chemical identifi-cation of factor, an analysis of the kinetics ofprotease induction in terms of individual en-zymes, and resolution of the biosyntheticprocess for factor. Studies now in progress mayshed some light on these problems.

ACKNOWLEDGMENTS

We would like to thank Louise Neil for her excellenttechnical assistance, Jan Turner for his advice and sugges-tions throughout this work, and W. R. Wiley for his help andencouragement throughout this study.

This research was supported by the Battelle Institute andby contract AT(46-1)-1830 from the U.S. Atomic EnergyCommission.

ADDENDUMA recent report has demonstrated that the derma-

tophytic fungus Microsporum canis will produceprotease when grown on a medium where the solecarbon and nitrogen source is casein, but not onCasamino Acids. The existence of more than oneproteolytic activity was inferred, and the process ofproduction was considered to involve secretionrather than autolysis (J. O'Sullivan, and G. E. Ma-thison, J. Gen. Microbiol. 68:319-326, 1971).

LITERATURE CITED1. Bergkvist, R. 1963. The proteolytic enzymes of Asper-

gillus oryzae. H. Properties of the proteolytic en-zymes. Acta Chem. Scand. 17:1541-1551.

2. Hagihara, B., H. Matsubara, M. Nakai, and K. Oku-nuki. 1958. Crystalline bacterial proteinase. I. Prepa-ration of crystalline proteinase of Bac. subtilis. J.Biochem (Tokyo) 45:185-194.

3. Hofsten, B. V., and C. Tjeder. 1965. An extracellularproteolytic enzyme from a strain of Arthrobacter. I.Formation of the enzyme and isolation of mutantstrains without proteolytic activity. Biochim. Biophys.Acta 110:576-584.

4. Ito, M., and M. Sugiura. 1968. Studies on Aspergillusproteinase. I. Purification, crystallization, and someproperties of alkaline proteinase from A. melleus.Yakugaku Zasshi 88:1576-1582.

5. Levisohn, S., and A. I. Aronson. 1967. Regulation of ex-tracellular protease production in Bacillus cereus. J.Bacteriol. 93:1023-1030.

6. McConn, J. D., D. Tsuru, and K. T. Yasunobu. 1964.Bacillus subtilis neutral proteinase. I. A zinc enzymeof high specific activity. J. Biol. Chem. 239:3706-

3715.7. McDonald, C. E., and L. L. Chen. 1965. The Lowry

modification of Folin reagent for determination ofproteinase activity. Anal. Biochem. 10:175-177.

8. Magasanik, B. 1961. Catabolite repression. Cold SpringHarbor Symp. Quant. Biol. 26:249-256.

9. Mandelstam, J., and W. M. Waites. 1968. Sporulation inBacillus subtilis. The role of exoprotease. Biochem. J.109:793-801.

10. Morihara, K. 1964. Production of elastase and pro-teinase by Pseudomonas aeruginosa. J. Bacteriol. 88:745-757.

11. Morihara, K. 1965. Production of proteinase on noncar-bohydrate carbon sources by Pseudomonas aeruginosa.Appl. Microbiol. 13:793-797.

12. Morihara, K., T. Oka, and H. Tsuzuki. 1967. Multipleproteolytic enzymes of Streptomyces fradiae. Produc-tion, isolation, and preliminary characterization.Biochim. Biophys. Acta 139:382-397.

13. Narahashi, Y., K. Shibuya, and M. Yanagita. 1968.Studies on proteolytic enzymes (pronase) of Strepto-myces griseus K-1. II. Separation of exo- and endo-peptidases of pronase. J. Biochem. (Tokyo). 64:427-437.

14. Noval, J. J., and W. J. Nickerson. 1959. Decompositionof native keratin by Streptomyces fradiae. J. Bac-teriol. 77:251-263.

15. Pastan, I., and R. Perlman. 1970. Cyclic adenosine mon-ophosphate in bacteria. Science 169:339-344.

16. Rogers, H. J. 1961. The dissimilation of high molecularweight substances, p. 257-318. In I. C. Gunsalus andR. Y. Stanier (ed.), The bacteria, vol. 5. AcademicPress Inc., London.

17. Schneider, R. P., and W. R. Wiley. 1971. Transcriptionand degradation of messenger ribonucleic acid for aglucose transport system in Neurospora. J. Biol.Chem. 246:4784-4789.

18. Sierra, G. 1967. Germination of bacterial endosporeswith subtilopeptidases. Can. J. Microbiol. 13:489-501.

19. Tsuru, D., J. E. McConn, and K. T. Yasunobu. 1964. B.subtilis neutral protease. A zinc enzyme of high ac-tivity. Biochem. Biophys. Res. Commun. 15:367-371.

20. Turner, J. R., and W. H. Matchett. 1968. Alteration oftryptophan-mediated regulation in Neurospora crassaby indoleglycerol phosphate. J. Bacteriol. 95:1608-1614.

21. Turner, J. R., W. A. Sorsoli, and W. H. Matchett. 1970.Induction of kynureninase in Neurospora. J. Bacteriol.103:364-369.

22. Uchino, F., Y. Kurono, and S. Doi. 1967. Purificationand some properties of crystalline acid protease fromAcrocylindrium sp. Agr. Biol. Chem. 31:428-434.

23. Vogel, H. J. 1964. Distribution of lysine pathwaysamong fingi: evolutionary implications. Amer. Natur.98:435-446.

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