Vol. 163, No. 3JOURNAL OF BACTERIOLOGY, Sept. 1985, p. 1180-11850021-9193/85/091180-06$02.00/0Copyright © 1985, American Society for Microbiology

Effect of Calcofluor White and Congo Red on Fungal Cell WallMorphogenesis: In Vivo Activation of Chitin Polymerization

CESAR RONCERO AND ANGEL DURAN*Instituto de Microbiologia Bioquimica, Facultad de Biologia, Universidad de SalamancalConsejo Superior de

Investigaciones Cientificas, Salamanca, Spain

Received 27 March 1985/Accepted 17 June 1985

Calcofluor White (a fluorochrome dye) affected the growth of Geotrichum lactis by causing lysis of cells at thehyphal tips. This effect was prevented by the presence of an osmotic stabilizer. The growth of Saccharomycescerevisiae was also affected, and multicellular aggregates were formed because of incomplete separation ofmother and daughter cells; fluorescence microscopy indicated the presence of abnormally thick septa. Theformation of rudimentary wail material by G. lactis protoplasts was promoted by Calcofluor or Congo red(another dye). The rate of chitin synthesis in protoplasts and growing cells was enhanced by both dyes. Incontrast, both dyes inhibited chitin and j0(1,3)-glucan synthases in isolated cell-free systems. Degradation ratesfor I0(1,3)-glucan or chitin were not affected significantly by the dyes. Wheat germ agglutinin also affectedchitin synthesis in protoplasts.

The fluorescent brightener Calcofluor White M2R New isused commercially to whiten textiles and paper and has beenused as a stain for cell wall materials in fungi, algae, andhigher plants (9, 15, 17, 22). This fluorochrome and anotherdye, Congo red, interact with various polysaccharides (31)but exhibit a particularly high affinity for chitin and cellulose(6, 14, 16). Through hydrogen bonding (with free hydroxylgroups [19] and dipolar interactions [26]) Calcofluor binds tonascent microfibers of cellulose in Acetobacter xylinum (14)and algae within the genus Oocystis (27). As a consequenceof dye interaction the glucan chains are prevented fromcocrystallizing to form microfibrils, and their assembly isthus seriously disrupted (3). Similar results have been de-scribed for chitin microfibril formation in the alga Poteri-ochromonas sp. with both Calcofluor and Congo red (16).Recent work (11, 29) has indicated that the assembly of

chitin microfibrils in yeasts is also altered by Calcofluor orCongo red. Furthermore, the in vitro inhibition of Neuros-pora crassa chitin synthase by Calcofluor has been reported(27).

In this study we investigated the general effects ofCalcofluor and Congo red on fungal wall morphogenesis.The effects of the dyes on the biosyntheses of two structuralpolysaccharides, ,(1,3)-glucan and chitin, during cell growthand protoplast wall generation were particularly explored.The results indicate that Calcofluor inhibits Geotrichumlactis growth but activates the synthesis of wall material byprotoplasts. In the case of Saccharomyces cerevisiae,Calcofluor induces abnormal septa which apparently fail todevelop abscission zones between mother and daughtercells. A similar effect was produced in S. cerevisiae byCongo red (30). The most notable finding in every casestudied was that the rate of chitin polymerization wasspecifically enhanced in vivo by either Calcofluor or Congored. This result confirms previous assumptions about theactivation of chitin synthesis by these dyes in S. cerevisiae(12, 30) and is in concert with observed increases (in thepresence of Calcofluor) of cellulose polymerization in A.xylinum (3). Preliminary results indicated that the lectin

* Corresponding author.

wheat germ agglutinin (WGA) also activates chitin synthesisin G. lactis protoplasts.

MATERIALS AND METHODS

Organisms and growth conditions. The filamentous fungusG. lactis ATCC 48590 was grown in 1% glucose-1% yeastextract (YED; Difco Laboratories, Detroit, Mich.); growthwas quantified as described previously (23). The growth of S.cerevisiae X2180 (ATCC 26109) in YED was monitored bymeasuring the absorbance of cultures in a Coleman Junior IIspectrophotometer at 600 nm.

Chemicals. Calcofluor White M2R New {disodium salt of4,4'-bis[4-anilino-bis(beta-hydroxyethyl) amino-s-triazine-2-ylamino]-2,2'-stilbene disulfonic acid} and the benzidinederivative Congo red were from American Cyanamid Co.,Bound Brook, N.J., and Serva, Heidelberg, Federal Repub-lic of Germany, respectively.UDP-N-acetylglucosamine, UDP-glucose, GTP, N-

acetylglucosamine, digitonin, laminarin, and sorbitol werepurchased from Sigma Chemical Co., St. Louis, Mo.Papulacandin B was a gift from Ciba-Geigy, Basel, Switzer-land. WGA was from Boehringer GmbH, Mannheim, Fed-eral Republic of Germany, and WGA-fluorescein was fromMiles Scientific, Div. of Miles Laboratories, Inc.,Naperville, Ill. UDP-[U-14C]glucose (240 mCi/mmol), UDP-[U-1'4C]N-acetylglucosamine (346 mCi/mmol), and [U-'4C]glucose (295 mCi/mmol) were from Amersham Interna-tional plc, Amersham, United Kingdom.

Chitinase was purified from Streptomyces griseus ATCC23345 as described previously (21). In some experimentschitinase from Serva was used. Zymolyase-5000 was fromKirin Brewery, Tokyo, Japan, and Zymolyase-lOOT wasfrom Miles.

Formation and reversion of protoplasts. S. cerevisiae cellswere converted into protoplasts as described previously (5,11). G. lactis protoplasts were prepared from log-phasemycelia, harvested, washed with distilled water by filtration,and suspended in 30 mM sodium phosphate buffer (pH 6.3)(70 mg [wet weight] per ml) containing 1.2 mg of chitinase(Serva) per ml, 0.75 mg of Zymolyase-5000 per ml, and 1 MMgSO4 as the osmotic stabilizer (final concentrations). Nopretreatment of mycelia was necessary. After 90 min of

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incubation at 30°C with gentle stirring, the conversion ofmycelia into protoplasts was almost complete. Protoplastswere washed twice by centrifugation at 10,000 x g for 5 minand finally suspended in 1 M MgSO4.The reversion of protoplasts from both microorganisms

was carried out in YED liquid medium supplemented with0.8 M sorbitol as the osmotic stabilizer and aureomycin (50,ug/ml) to prevent bacterial contamination. The reversion ofG. lactis protoplasts had an absolute requirement for Mg2"(results not shown).

Fractionation of cell wail polysaccharides. Cell wall mate-rial from cells or protoplasts was extracted twice with 6%NaOH as described previously (23). When revertingprotoplasts or S. cerevisiae cells were used, cells were notbroken before extraction. After alkali extraction, the remain-ing insoluble residues were washed with water until the pHwas neutral, and samples from the pellet were incubated for48 h at 30°C with chitinase or Zymolyase-lOOT. After incu-bation, the residues were centrifuged and washed. Thematerial remaining after digestion with chitinase orZymolyase-lOOT was taken to represent the alkali-insoluble,3(1,3)-glucan or chitin fractions, respectively. Duplicates forevery value were always included in all the experiments.Enzyme preparation. Cell-free extracts from both micro-

organisms were obtained by mechanical breakage with aBraun homogenizer as described previously (25). Chitinsynthase from G. lactis was solubilized by suspending thecrude membranes at 10 mg of protein per ml in Tris-hydrochloride (pH 7.5) containing 5 mM MgSO4 and 1%(wt/vol) digitonin. The suspension was stirred for 45 min atroom temperature and then centrifuged at 50,000 x g for 40min. The supernatant represented the chitin synthase-solubilized enzyme.

G. lactis cells were permeabilized with digitonin (13). A0.1-g/ml (wt weight) cell sample was suspended in 25 mMTris-hydrochloride (pH 7.5)-1% digitonin, incubated for 15min at 30°C, centrifuged at 40,000 x g, and suspended in 1 mlof 25 mM Tris-hydrochloride (pH 7.5-S5 mM MgSO4.Plasma membranes from S. cerevisiae were prepared from

protoplasts reinforced by concanavalin A treatment by apublished procedure (10) recently modified (7). The lysatewas centrifuged at 12,000 x g on a sucrose cushion; themembranes at the interphase were pooled and suspended in50 mM Tris-hydrochloride (pH 7.5)-2 mM MgSO4, centri-fuged as described above, washed once by centrifugation,and finally resuspended in the same buffer.Enzyme assays. P(1,3)-Glucan synthase activity was as-

sayed as described previously (25). Chitin synthase wasalways activated with trypsin (11) to assert the total potentialactivity. The reaction mixture contained all the componentspreviously described (11), but in the case of G. lactisenzyme, the buffer was 0.1 M glycine-NaOH (pH 9.0), asoptimal activity was achieved at this pH (data not shown).Radioactivity from the insoluble 14C-labeled synthesizedproducts [chitin or,B(1,3)-glucan] was determined after fil-tration and washing (18) with a Beckman LS-8100 scintilla-tion spectrometer. Chitinase and 13(1,3)-glucanase activitieswere determined as described previously (21, 24). One unitof enzyme activity was defined as described previously (25).

Other methods. Protein was determined by the Lowrymethod. Total glucose incorporation was monitored bymeasuring the radioactivity in trichloroacetic acid-insolublematerial (23) from cultures supplemented with [U-14C]glucose. Fluorescence was observed with a NikonOptiphot microscope with an epifluorescence attachmentand an HBO 10OW/2 light source. An excitation filter

8

E

0

0

EI-,O.,

6

4

2

2 4 6

Time (hours)FIG. 1. Growth of G. lactis in the presence or absence of

Calcofluor. Symbols: 0, control cultures without 0.8 M sorbitol; *,control cultures with 0.8 M sorbitol; *, Calcofluor-treated (80jig/ml) cultures supplemented with 0.8 M sorbitol; A, Calcofluor-treated (80 ,ig/ml) cultures not supplemented with 0.8 M sorbitol; A,Calcofluor-treated (115 jig/ml) cultures supplemented with 0.8 Msorbitol.

(IF420-485), a dichroic mirror (DM-510), and a barrier filterat 520 to 560 nm were used as recommended by the manu-facturer. Sometimes, to facilitate photography, cells wereimmobilized in agar (6). Samples for electron microscopywere prepared as described previously (4).

RESULTSEffect of Calcofluor on cell growth. A survey of the effect of

Calcofluor was extended to several fungi. Petri dishes con-taining YED solid medium were inoculated, and filter disks(5-mm diameter) dipped into a 2% Calcofluor solution weredeposited on the agar surface. After a growth period, a clearhalo around the disk indicated inhibition of growth. Amongthe fungi tested, Mucor rouxii ATCC 24905, Aspergillusniger ATCC 9642, Penicillium expansum CECT 2287 (Colec-cion Espafiola de Cultivos Tipo), G. lactis ATCC 48590, S.cerevisiae ATCC 26109, Schizosaccharomyces pombeATCC 24843, Candida utilis CECT 1061, Cryptococcusalbidus CECT 1280, and Rhodotorula rubra CECT 1158,only S. pombe was resistant to Calcofluor under the condi-tions used.The addition of Calcofluor (80,ug/ml) to a growing culture

of G. lactis (1 mg [dry weight] per ml) resulted in animmediate arrest of growth (Fig. 1) and a drastic lysis ofmycelia at the hyphal tips. Lower concentrations (15 to 40p,g/ml) caused no significant effect on growth (data notshown). The inhibition of growth could be prevented (Fig.1), to some extent at least, by the presence of an osmoticstabilizer (0.8 M sorbitol) in the culture medium. After freeCalcofluor was removed from a culture exposed to a 5-mintreatment, growth recurred; a delay in this regrowth wasobserved, owing to some lysis.The inhibition of growth depended on the size of the

inoculum; thus, after culturing of a 1-mg (dry weight)/mlinoculum for 6 h in the presence of 65,ug of Calcofluor perml, a 50o growth yield, as compared with the controlculture, was achieved, whereas no growth was observed

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1182 RONCERO AND DURAN

FIG. 2. S. cerevisiae X2180 cells grown without (a) or with (band c) Calcofluor (0.1 mg/ml) and observed in a phase-contrast (aand b) or a fluorescence (c) microscope. Bar, 10 ,um.

with a 0.5-mg (dry weight)/ml inoculum. Calcofluor concen-trations below a threshold value (15 ,ug/ml) had no effect ongrowth. No lytic effect was observed in stationary-phasecultures.The growth of S. cerevisiae was also affected by

Calcofluor. Phase-contrast microscopy revealed the pres-ence of multicellular aggregates, as if the cells were repeat-edly budding without completing the separation betweenmother and daughter cells (Fig. 2a and b). A closer obser-vation revealed the existence of anomalous thick septa,clearly visible under the fluorescence microscope, betweenthe cells (Fig. 2c). Observations in the electron microscopeconfirmed the presence of abnormally thick septa (data notshown) very similar to those induced by Congo red (30). Ina growing culture (107 cells per ml) supplemented with 0.1mg of Calcofluor per ml, the average cell number per cellgroup (total cell number divided by the number of groups,

EE

Q

-

mU.0

C.U0

U

1.5 -

including single cells) varied throughout the treatment, beingmaximal (3.8 cells) at the late log phase and decreasingprogressively to a value similar to that for the control cultureas the treated culture entered the stationary phase (the valuefor the control culture ranged from 1.1 to 1.4 cells).

Reversion of protoplasts. During the initial hours of rever-sion the growth rate (measured as [14C]glucose incorporatedinto trichloroacetic acid-precipitable material) of G. lactisprotoplasts (2 x 107 protoplasts per ml) was considerablyincreased by the presence of Calcofluor (60 to 250 ,ug/ml) orCongo red. The results (Fig. 3) are the averages of duplicatesfrom four different experiments. The reversion patterns ofG. lactis protoplasts were strikingly different, depending onthe presence or absence of the dyes. Both dyes first inducedthe formation of a highly fluorescent, thick, homogeneousshell around almost every protoplast (Fig. 4a). After 3 to 5 hof growth, the protoplasts emitted one to three hyphae in a"germinating-like" fashion (Fig. 4b). In the absence of dyethe protoplasts aggregated in large clumps, and reversionoccurred through aberrant forms from which hyphal tubeseventually emerged (Fig. 4c). In the absence of dye asignificant percentage of protoplasts did not revert.Mycelia from protoplasts reverted in Calcofluor and

proved more resistant to a new addition of dye than myceliagrown in the absence of dye. Material that was 7 h old fromeither reverting protoplasts (with Calcofluor) or germinatingarthrospores (without Calcofluor) was grown with or withoutCalcofluor (125 ,ug/ml). The initial lysis rate (in the presenceof dye) of mycelia from germinating arthrospores was con-siderably higher than that of mycelia from revertingprotoplasts.

0

A

01.0 1

0.5[

AU

QU S

aI

1.5 3.0

Time (hours)

4.5

FIG. 3. Glucose (Glc) incorporation into G. lactis protoplastsregenerating in osmotically stabilized YED medium. Symbols: 0,control culture; *, culture treated with 50 p.g of Congo red per ml;A, culture treated with 60 ,ug of Calcofluor per ml; 0, culture treatedwith 125 p.g of Calcofluor per ml.

FIG. 4. Phase-contrast micrographs of G. lactis protoplasts re-generating in the presence of Calcofluor (125 p.g/ml) (a and b) orWGA (1 mg/ml) not supplemented (d) or supplemented (e) withpolyoxin D (1 mg/ml). (c) Control culture. Bars, 10 ,.m.

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WGA, a plant lectin specific for P(1,4)-linked N-acetylglucosamine residues (1), induced the production ofvery peculiar structures when added at 0.5 to 2 mg/ml toreverting G. lactis protoplasts. A band material which was

somewhat fibrous in appearance and subsequently continuedto be produced polarly surrounded most of the protoplasts(Fig. 4d). These structures seemed to be made of chitinbecause (i) the material showed fluorescence when traceamounts of WGA-fluorescein were added to the revertingprotoplasts; (ii) the material disappeared after chitinasedigestion; (iii) the formation of the material could be drasti-cally reduced in the presence of polyoxin D, a specificinhibitor of chitin synthase (11) (Fig. 4e); and (iv) theproduction of the material was not restrained bypapulacandin B (20 j,g/ml), an inhibitor of P(1,3)-glucansynthase from G. lactis (25).

Effect of Calcofluor or Congo red on cell wall synthesis. (i)Growing cells. As indicated earlier, lysis of G. lactis myceliaby Calcofluor could be prevented by the presence of an

osmotic stabilizer (0.8 M sorbitol). Under these conditions,Calcofluor (80 jig/ml) was added to a growing culture of G.lactis (1 mg [dry weight] per ml), followed 60 min later by theaddition of ['4C]glucose (1 ,uCi/ml). The incorporation oflabel (per milligram [dry weight] of material) into the chitinfraction of Calcofluor-treated mycelia (after 3 h of growth)increased by 52 to 60% of the control culture value in twodifferent experiments; the incorporation into the ,B(1,3)-glucan fraction was not affected (data not shown).

Similar experiments were performed with S. cerevisiae

cultures supplemented with Calcofluor or Congo red (100,ug/ml) (no addition of sorbitol was necessary). In twoindependent experiments for each dye, the incorporation oflabel per cell into the chitin fraction of treated cultures was

at least double the control culture value in every case;similar or slightly lower values than those of the controlculture were found for the P(1,3)-glucan fraction (data notshown).

(ii) Protoplasts. [14C]glucose (5 to 9 p.Ci/ml) was added toG. lactis while protoplasts were growing in a medium with orwithout Calcofluor (250 ,ug/ml). Samples from control andtreated cultures were withdrawn after 1.5 and 3 h of growth,and the incorporation of label into the cell wall fractions wasdetermined as described above. The results indicated spe-cific rates of incorporation into each fraction (counts perminute per fraction/total incorporated counts per minute).Rates of alkali-insoluble ,B(1,3)-glucan and especially chitinsynthesis were dramatically increased (Table 1) byCalcofluor. After 7 h of treatment, the rates for both frac-tions decreased, particularly in the case of chitin, to valuescloser to those for normally growing mycelia (data notshown).The radioactivity remaining in the alkali-insoluble residue

from Calcofluor-treated protoplasts constituted up to 50 to60% of the total incorporated label, whereas in the controlcultures it ranged from 3 to 5%. The metabolism of resting G.lactis protoplasts (stabilized protoplasts suspended in 50 mMsodium phosphate buffer [pH 6.5] supplemented with 1%

glucose) was diverted towards cell wall synthesis byCalcofluor or WGA in such a way that only cell wall materialsurrounding the protoplasts was produced, whereas in thecontrol cultures, middle-sized mycelia were produced.The specific rate of incorporation into the cell wall chitin

fraction of S. cerevisiae protoplasts was also increased byCalcofluor (0.1 mg/ml); the rate of incorporation into the1(1,3)-glucan fraction was almost unaffected (Table 1).In vitro effect of Calcofluor on cell wall synthases. Chitin

TABLE 1. Effect of Calcofluor on the synthesis of cell wallpolysaccharides in reverting protoplasts from G. lactis and S.

cerevisiae'

(cpm incorporated per fraction/total cpm incorporated) x 102b

Organism Polysaccharide Controle treatedcultures culturescultures

A B A B

G. lactis Alkali-insoluble 0.7 0.7 5 13P(1,3)-glucan

Chitin 1.1 1.3 11.7 42

S. cerevisiae Alkali-insoluble 2 9 3.7 8.5,0(1,3)-glucan

Chitin 1.3 4.3 4.6 10.3a For experimental details, see the text.b For G. lactis, A = 1.5 h and B = 3 h. For S. cerevisiae, A = 2.5 h and B =

5 h.

synthase activity [in addition to ,B(1,3)-glucan synthase ac-tivity] from G. lactis was inhibited in vitro by both dyes.Solubilized chitin synthase was also inhibited. The Ki forCalcofluor varied from 0.3 to 0.6 mg/ml (final assay concen-tration). The inhibition kinetics of chitin synthase appearedto be of the "mixed type," so that both the Vmax and the Kmwere altered. The inhibition was independent of the length ofincubation and was irreversible, i.e., the inhibition of par-ticulate synthase after the Calcofluor was washed out bycentrifugation was the same as the inhibition of theunwashed enzyme.

In an attempt to demonstrate the in vitro activation ofchitin synthase we grew G. lactis mycelia in YED supple-mented with sorbitol and Calcofluor (80 ,ug/ml); after 2 h oftreatment, mycelia were harvested, washed by filtration, andpermeabilized as described above. Chitin synthase activityin treated mycelia was always lower than that in controlmycelia. Shorter treatments with variable amounts ofCalcofluor before permeabilization always yielded activitylevels consistently lower than those in the control. Furthertrials consisted of measuring the initial rates of chitin syn-thesis (1 to 2 min) in the presence of Calcofluor (final assayconcentration 0.25 to 100 ,ug/ml). No activation of chitinsynthesis was ever observed, however. Finally, chitin syn-thesis in S. cerevisiae intact plasma membranes was notaffected by Calcofluor (2.5 to 20 jig/ml), and higher concen-trations inhibited the activity.As previously shown for S. cerevisiae (A. Duratn, unpub-

lished results), chitin synthase from G. lactis was neitheractivated nor inhibited in vitro by WGA, even at assayconcentrations as high as 0.5 mg/ml.

In vitro effect of Calcofluor on cell wall hydrolases. Theaddition of Calcofluor to chitinase or ,(1,3)-glucanase assaysresulted in an inhibition of the hydrolysis rates of thecorresponding substrates (Ki, ca. 1 to 2 mg/ml). To evaluatethe possible effect of Calcofluor on the in vivo degradation ofsome cell wall polysaccharides, we synthesized 13(1,3)-glucan and chitin in vitro in the presence or absence ofCalcofluor to enable the dye to be integrated into the nascentpolysaccharide chains; we then submitted them to degrada-tion by specific enzymes (Zymolyase-lOOT or chitinase,respectively). The syntheses of P(1,3)-glucan from S. cere-visiae extracts or chitin from G. lactis extracts were carriedout in the presence of 0.8 or 0.4 mg of Calcofluor per ml,

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0

0

30F

151-.

b-0

E

:0.0)

0

451.

301

15F

C

B

-B 0

0

0 0

-A.30 60 90

Time (minutes)

FIG. 5. Degradation rates of P(1,3)-glucan (A) and chitin (B)synthesized in vitro in the presence (0) or absence (0) ofCalcofluor. The initial amount of polysaccharide to be degraded was

ca. 260 nmol (ca. 300,000 cpm).

respectively. The rates of synthesis were reduced by 50 and60%, respectively, as compared to those of the control.Similar amounts (260 nmol) of each in vitro-synthesizedpolysaccharide were then washed by centrifugation, sus-

pended in the assay buffer, and incubated with Zymolyase-100T (0.9 ,ug) or chitinase (Serva) (3.5 ,ug) in a final volumeof 600 ,ul. The samples were withdrawn at appropriate times,and the radioactivity released into the supernatant was

considered the degraded polysaccharide. The initial rates of,(1,3)-glucan hydrolysis were very similar (Fig. SA),whereas the degradation rate of the Calcofluor-containingchitin was somewhat higher than that of the control (Fig.5B). Identical results were obtained in one additional exper-

iment for each polymer.

DISCUSSIONCalcofluor prevents the in vivo assembly of cellulose

microfibrils by A. xylinum (3) and of chitin by other micro-organisms (12, 16) so that the Calcofluor-induced product isprofoundly different from the native one (14). Because chitinis most probably the main structural polysaccharide in the G.lactis cell wall (23), lysis of growing mycelia by Calcofluormay be caused by abnormal chitin deposition and subse-quent weakening of the wall. This conclusion is favored bytwo facts: (i) lysis of mycelia is prevented by the presence ofan osmotic stabilizer and (ii) no lytic effect is observed underconditions unfavorable for growth.The exposure of S. cerevisiae to Calcofluor induces

multicellular aggregates, as described for Congo red (30).The abnormally thick septa may be the result of a massivedeposition of polymers, mainly chitin. This assumptioncorresponds with the proposed morphogenic role of chitin inthe formation of the yeast primary septum (8). The fact that

S. pombe, whose cells do not contain chitin, is not affectedby Calcofluor would also seem to suggest that chitin is thetarget polysaccharide.

It is feasible to assume that the binding of Calcofluor tonascent ,(1,3)-glucan fibers or chitin fibers or both mayinterfere with the action of the hydrolytic activities presum-ably implicated in cell wall morphogenesis (2). The physio-logical significance of the degradation rates of the polymers(synthesized in vitro in the presence or absence ofCalcofluor) still remains unclear, however.

In addition to the described morphological changes it wasimportant to ascertain whether the action of the dyes couldbe directed towards the synthesis of any specific polysac-charide. Indeed, the results show that Calcofluor increasesthe rate of chitin polymerization in growing G. lactis and S.cerevisiae cells; a similar effect is also induced by Congo redin S. cerevisiae.The overall rate of glucose incorporation into G. lactis

protoplasts is clearly activated by both dyes. A large pro-portion of the incorporated label can be accounted for by thealkali-insoluble wall residue in the treated cultures as com-pared to the control cultures. The specific rates of P(1,3)-glucan and especially chitin synthesis are, indeed, highlyenhanced by Calcofluor. The excess of wall material synthe-sized around the protoplasts may favor the initial reversionsteps in treated cultures and would explain the differencesbetween the reversion patterns of control and treated cul-tures.

Cell wall generation by protoplasts is a morphogenicprocess obviously different from cell wall synthesis in grow-ing mycelia. Nevertheless, the reasons for a higher resist-ance to Calcofluor in protoplasts than in osmotically pro-tected mycelia and for a greater activation by the dyes in thesynthesis of G. lactis protoplasts wall material than in thesynthesis of growing cells are as yet unknown to us.The stimulatory effect of Calcofluor (and Congo red and

WGA) on chitin synthesis is most probably a result of itsdirect binding to the nascent polysaccharide chains as theyare extruded outside the plasma membrane. There are sev-eral lines of evidence to support this assumption: (i) nakedprotoplasts do not bind Calcofluor (or Congo red), as ob-served under fluorescence microscopy; (ii) WGA, whosemolecular weight (36,000 as a dimer) makes penetrationthrough the plasma membrane highly improbable, also ap-parently affects chitin polymerization in vivo; and (iii) sincethe synthesis of chitin from UDP-N-acetylglucosamine byparticulate or solubilized preparations is never activated butrather is inhibited irreversibly by Calcofluor, it follows thatpenetration of the dye into the protoplasts would inhibitchitin synthesis, as in the case of permeabilized mycelia.The effect of WGA on chitin synthesis by G. lactis

protoplasts and the inhibition of fungal growth by the lectinin Trichoderma viride (20) parallel the effect of Calcofluordescribed in this work. It is feasible to assume that WGA andCalcofluor may be acting in vivo in a similar way.The fact that neither Calcofluor, Congo red, nor WGA

activates chitin synthase in vitro under any conditions butactivates it in vivo poses a puzzle. Calcofluor interacts invitro with both internal and external sides of the synthase(chitin synthase is located on the yeast plasma membrane[10]), whereas in vivo only the external side is in contactwith the dye. It could be that the irreversible inhibition ofchitin synthase caused by Calcofluor in vitro (also recentlydescribed in N. crassa [28]) masks its activating effect.The activation of chitin polymerization in vivo by

Calcofluor or Congo red closely resembles the effect de-

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scribed for cellulose synthesis in A. xylinum (3). Accordingto these authors, polymerization and crystallization (forcellulose biogenesis) are tightly coupled, as if the timerequired for crystallization could limit the rate of polymer-ization. Calcofluor, by uncoupling these two processes,would, in this case, activate the synthesis of structurallyabnormal chitin.A precise arrangement of synthesizing sites in relation to

the cell surface, as described for cellulose in Micrasterias sp.(29), may be required for the biogenesis of crystalling chitinfibrils. However, (i) vectorial synthesis of chitin has beenachieved with purified yeast plasma membranes (7), and (ii)the lengths of chitin chains synthesized in vivo or by highlypurified chitin synthase from yeasts are very similar (18). Ananalysis of the X-ray diffraction patterns of the differentlysynthesized chitins and the study of the functioning ofpurified chitin synthase integrated in defined vesiculatedlipid bilayers may shed some light on the problem. Never-theless, it would appear that the in vitro effect of Congo redor Calcofluor on cell wall synthases has no physiologicalrelevance.

ACKNOWLEDGMENTSC.R. acknowledges support from a fellowship granted by the

Ministerio de Educaci6n y Ciencia, Madrid, Spain. This work wassupported by a grant from the CAICYT, Madrid, Spain.We thank R. Black for correcting the manuscript.

LITERATURE CITED1. Allen, A. K., A. Neuberger, and N. Sharon. 1973. The purifica-

tion, composition and specificity of wheat-germ agglutinin.Biochem. J. 131:155-162.

2. Bartnicki-Garcia, S. 1973. Fundamental aspects of hyphal mor-phogenesis, p. 245-267. In J. M. Ashworth and J. E. Smith(ed.), Microbial differentiation. Cambridge University Press,Cambridge.

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