JOURNAL OF BACTERIOLOGY, Nov. 1968, p. 1586-1594Copyright @ 1968 American Society for Microbiology

Control of Dimorphism in Mucor by Hexoses:Inhibition of Hyphal Morphogenesis

S. BARTNICKI-GARCIADepartment of Plant Pathology, University of California, Riverside, California 92502

Received for publication 12 August 1968

In anaerobic cultures of Mucor rouxii, morphogenesis was strongly dependent onhexose concentration as well as pCO2. At low levels of hexose or C02, or both,hyphal development occurred; at high levels, the fungus developed as yeast cells.Other dimorphic strains of Mucor responded similarly to hexose and CO2 but dif-ferred in their relative sensitivity to these agents. Glucose was the most effectivehexose in eliciting yeast development of M. rouxii; fructose and mannose were next;and galactose was last. The fungus may be grown into shapes covering its entiredimorphic spectrum simply by manipulating the hexose concentration of the me-dium. Thus, at 0.01% glucose, hyphae were exceedingly long and narrow; at highersugar concentrations, the hyphae became progressively shorter and wider; finally,at about 8% glucose, almost all cells and their progeny were isodiametric (sphericalbudding cells). Such yeast development occurred without a manifested requirementfor exogenous CO2. The stimulation of yeast development by hexose is not an arti-fact due to increased production of metabolic CO2 (hyphae or yeast cells releasedmetabolic CO2 at similar rates). Presumably, the effect was caused by some otherhexose catabolite which interfered with hyphal morphogenesis (apical growth); de-prived of its polarity, the fungus grew into spherical yeastlike shapes. Although10% glucose inhibited the development of hyphae from germinating spores, it didnot prevent the elongation of preformed hyphae. This suggests that hexose inhibitshyphal morphogenesis not by blocking the operation of the enzyme complex re-sponsible for apical growth but by preventing its initiation; such inhibition may beregarded as a repression of hyphal morphogenesis.

Various species of Mucor have the capacity,like other dimorphic fungi, to develop vegeta-tively as either molds or yeasts. As molds, theyform a typical mycelium of branched coenocytichyphae; as yeasts, they grow as individual roundcells and multiply by budding. The investigationsof Pasteur, Reess, Brefeld, and others (1, 10; J.Beauvarie, Thesis, p. 131-158. Univ. of Lyon,France, 1900) revealed that the capacity of Mucorspp. to adopt either one of these two strikinglydifferent growth patterns was governed by theatmosphere of incubation.Mucor rouxii (IM-80), a strain which we have

studied in detail, has developed exclusively in theyeast form when grown under anaerobic atmos-pheres containing at least 30% CO2 (3). In theabsence of CO2, aerobically or anaerobically,development was typically mycelial. No othernutritional factors were previously reported toaffect the dimorphism of this strain significantly(4). The data presented in this paper support theconclusion that the atmosphere of incubation is a

primary determinant of vegetative morphogenesisin Mucor, but with the modification that oxygenand CO2 are not the only important factors. Theconcentration and type of hexose in the mediumare also a main factor in dimorphism. The oldand sometimes disputed notion that "sugars"favor the formation of yeast cells in mucors (1,10, 12) has now been specifically characterized asa seemingly indispensable requirement for aminimal hexose concentration.

MATERIALS AND METHODS

Mucor rouxii strain IM-80 was the main organismtested. Other strains of Mucor examined were kindlydonated by C. W. Hesseltine (Northern UtilizationResearch and Development Division, Peoria, Ill). Allcultivations were performed in media containingyeast extract, peptone, and a hexose as C source. Thebasic culture medium (YPG) consisted of 0.3% Difcoyeast extract, 1.0% Difco peptone, and 2% glucose.The initial pH was 4.5. Hexose solutions were auto-claved separately from the yeast extract-peptone and

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then aseptically combined. For solid cultures, plasticpetri dishes were used containing 6 ml of liquid me-dium with 2.5% agar. The agar was autoclaved withthe hexose solution and then mixed while hot withsterile yeast extract-peptone.

Cultures were inoculated with a washed spore sus-pension harvested from a YPG agar culture that hadbeen aerobically incubated for 3 to 7 days. Liquidmedia were inoculated to a final concentration of 104spores per ml. The surface of agar plates was evenlyinoculated with 103 spores.

All cultures were incubated at 28 C under strictanaerobic conditions. Three different gases were usedto provide anaerobic atmospheres: (i) prepurifiednitrogen, (ii) a mixture of 30% CO2 plus 70% pre-purified nitrogen obtained from the Air ReductionPacific Co., Vernon, Calif., and (iii) carbon dioxide(Coleman Instrument Grade; Matheson Co., Inc.,Newark, Calif.). In some experiments, the prepurifiednitrogen was consecutively bubbled through solutionsof alkaline pyrogallol and chromous sulfate to furtherreduce any traces of oxygen present. This added pre-caution, however, was unnecessary; cellular mor-phology did not change significantly as a result of thisscrubbing.The atmosphere of incubation in liquid cultures was

regulated by two methods: (i) By continuously flush-ing the gas over 25 ml of liquid medium contained ina 125-ml Erlenmeyer flask, placed on a reciprocatingwater bath shaker. This is essentially the methoddescribed previously (3), except that the rubberstoppers were replaced by ground-glass connectors.(ii) By bubbling the gas through 20 ml of liquid me-dium contained in a culture tube with ground-glassgassing connectors (Fig. 1; tubes A or B). The tubeswere immersed in a water bath. In either method, thegas-flow rate was adjusted to approximately 40 to 50cm3/min, except during the first hour when the ratewas ten times higher to remove dissolved oxygen asrapidly as possible.

Petri dish cultures were incubated in a desiccator.The atmosphere of incubation was adjusted by evacu-

FIG. 1. Apparatus for cultivation under controlledatmospheres (bubbling method). A and B are culturetubes (50 ml). C is a sampling tube usually immersed inwater or, when needed, into a C02-trapping solutioncontained in a liquid scintillation vial.

ating the desiccator with a mechanical pump and fill-ing it with the desired gas. This process was repeatedfive to six times to eliminate oxygen as thoroughly aspossible.

Shifts in glucose concentration were performed,avoiding any exposure of the cells to air, by connectingtwo culture tubes in series (Fig. 1; tubes A and B).Tube A contained 10 ml of the initial culture solutionand tube B 10 ml of YPG medium, with twice thedesired final sugar concentration. Tube A was inoc-ulated, connected to tube B, and incubated for theinitial period under prepurified nitrogen. At shiftingtime, tube A was simply rotated 1800 on its ball andsocket joints, allowing its entire contents to flow intotube B.

Morphological development was followed period-ically by removing samples of 2 to 3 ml through thegas outlets (Fig. 1, C). The cells were sedimented bycentrifugation and examined in the light microscopewithout staining. A photographic record of mor-phological development was made with a Polaroidcamera. Since essentially 100% of the spores germi-nated and since the vast majority of them developedmore or less in synchrony, it was possible, by observ-ing overall changes in the population, to make con-clusions about the effect of the environment on asingle cell.The influence of various glucose concentrations on

fermentation rate was tested radioisotopically withunifonnly labeled "4C-D-glucose; in all cases, the finalspecific activity of glucose was 1.6 X 107 disintegra-tions per min per g. Cells were grown under nitrogen,and the 14CO2 released was swept and trapped directlyinto 3 ml of 2-aminoethanol + 2-methoxyethanol(1:2; v/v) contained in a liquid scintillation vial(Fig. 1, D). Every hour, the vial was replaced with afresh one; 15 ml of counting solution (6) was added,and the radioactivity was measured in a Packard Tri-Carb spectrometer by use of an internal standard todetermine total activity.

RESULTS

Control of dimorphism by glucose. The twomodes of vegetative development of M. rouxii,hyphae, or yeast cells may be obtained under thesame atmosphere of incubation simply by usingdifferent hexose concentrations in the medium.This can be clearly demonstrated in a dividedpetri dish incubated anaerobically under 30%CO2 for 36 hr (Fig. 2). On that half containing alow concentration of glucose (0.05% or less),the fungus developed exclusively as a thin mat ofmycelium. On the other half, where the initialglucose concentration was 5% or higher, develop-ment was entirely as discrete yeast colonies. Onpetri plates containing intermediate glucose con-centrations, from 0.05% to 2%, morphologyvaried progressively from hyphal to yeast forms.In liquid cultures, the effect of glucose on vegeta-tive morphogenesis was similar, although theeffective concentrations were different. Thus under

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an atmosphere of 30% Co2, spores germinatedinto long hyphal tubes at a glucose concentra-tion of 0.01%, but they produced multipolar bud-ding yeast cells exclusively at or above 0.1% glu-cose (Fig. 3 and 4). This control of dimorphism ofM. rouxii by glucose was only effective underanaerobic conditions. In aerobic cultures, liquidor solid, there was no evidence of yeast develop-

ment in media containing from 0 to 20% glucose;only mycelial development occurred and, further-more, the diameter of young hyphae was notsignificantly affected by the level of hexose.

In cultures under N2, the diameter and thelength ofM. rouxii hyphae were markedly affectedby glucose concentration. This was clearly ob-served in spores germinated and grown for 12 hr

FiG. 2. Control ofdimorphism by glucose concentration. Agar plate under 30% CO2- (left) Mycelium on 0.05%glucose; (right) yeast colonies on 5% glucose. The plate was incubated for 30 hr. (left) Spores were inoculatedin a single M-shaped streak; (right) spores were deposited on the full width of the Y area.

4~~~~~~~~~~~~~~~~~~~~~A

Fio. 3, 4. Control of dimorphism by glucose concentration. Liquid cultures under 30% CO2. (left) Hyphae in0.01% glucose; (right) yeast cells in 1% glucose. Incubation time was 24 hr. Magnification marker = 100 Am.

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in liquid YPG medium (Fig. 5-8). At the lowestglucose concentration tested (0.01 %O), hyphaewere long and narrow (approximately 6 ,m indiameter); as the sugar concentration increased,the cells became progressively shorter and wider(Fig. 6, 7), until a point was reached (ca. 8%glucose) at which essentially all of the sporesgave rise to perfectly isodiametric cells, i.e.,spherical buds. These daughter buds abscissedand later multiplied again by budding off newspherical cells, giving rise to a typical yeast cul-ture equivalent to that obtained at 2% glucoseunder a high pCO2 (3). Evidently, yeast develop-ment of M. rouxii occurs in the absence of exog-enous CO2 if the glucose concentration of YPGmedium is about 8% or higher. There was noevidence that 10% glucose was detrimental toanaerobic growth; in fact, spores in 10% glucosestarted budding about 1 hr before spores in 1%glucose began emitting germ tubes.

Effect of other hexoses on dimorphism. Fruc-tose, mannose, and galactose, in decreasing orderof efficacy, also stimulate yeast development ofM. rouxii, but proportionately higher concentra-tions of these sugars are required to elicit thesame degree of yeast development obtained withglucose.The different intensity in morphological re-

sponse to the various hexoses was clearly mani-fested on agar plates containing 0.5% hexose.These plates were streaked with a dilute sporesuspension of M. rouxii and incubated anaero-bically under 30% CO2 for 2 days. With glucose,the fungus developed as large yeast colonies sur-rounded only by small, thin halos of mycelium;with fructose or mannose, the yeast colonies weresmaller, and the halos of mycelial growth weredenser and larger; with galactose, only mycelialcolonies developed.

Influence of glucose concentration on CO2 pro-duction. The possibility that the stimulation ofyeastlike development by high levels of glucosewas caused indirectly through an increased pro-duction of metabolic CO2 was next examined anddiscounted. The rates of liberation of CO2 weremeasured radioisotopically in liquid cultures con-taining 0.01%, 0.1%, 1.0%, and 10% glucose.The liquid medium was inoculated with sporesand bubbled with a stream of prepurified nitrogen.In all cultures, CO2 evolution increased exponen-tially during the first 10 hr of incubation (Fig. 9).Only at the lowest glucose concentration (0.01 %)was the rate of CO2 evolution limited by glucoseavailability. From 0.1 to 10% glucose, carbondioxide was released at closely similar rates dur-ing the initial 10 hr of incubation. This periodwas sufficiently long for the establishment ofwell-defined morphological development pat-terns. It may be concluded that hyphae growing

FIG. 5-8. Control of dimorphism by glucose concen-tration. Liquid cultures under nitrogen: Fig. 5, 0.01%;Fig. 6, 0.1%; Fig. 7, 1.0%; Fig. 8, 10%. Incubationtime 12 hr. Magnification marker = 100 um.

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106.

105-

1042

ci

1-

1-

E- 2

2

2 6 10 14 18 22 26 30T i m e (hours)

FIG. 9. Production of CO2 as a function of initialglucose concentration.

in 0.1% glucose released as much metabolic CO2as the yeast cells growing in 10% glucose.

Dimorphic colonies. Yeast colonies of M. rouxiihave a variable tendency to develop a halo ofhyphal growth in the latter states of development.The tendency of a colony to become dimorphicis largely determined by the initial cultivationconditions. In general, if the conditions arestrongly favorable for yeast development (e.g.,2% glucose under 100% CO2), the colony re-mains yeastlike with few or no peripheral hyphaedeveloping at the end of the growth phase. Atless favorable concentrations (e.g., 0.5% glucoseunder 100% CO2), the initial development maybe entirely in the yeast form, but within 1 day thefungus may shift to hyphal morphogenesis (Fig.10). This morphogentic shift is probably causedby the depletion of hexose down to a level un-suitable for yeast development.Commitment to hyphal morphogenesis. When a

suspension of growing, slender hyphae of M.rouxii, obtained by incubating spores in liquidYPG with 0.01% glucose (under nitrogen for 8to 12 hr), was shifted to YPG medium with aglucose concentration favorable for yeast develop-ment (10%), the hyphae neither ceased theirapical growth nor shifted into yeastlike budding,but continued to elongate (Fig. 11-14). Thesepreformed hyphae were seemingly committed to

their initial pattern of morphogenesis; the onlynoticeable response, which occurred about 1 to2 hr after the transfer to high glucose, was aremarkable increase in the diameter of the newlyformed hyphal segments. The older and muchnarrower hyphal segment, which had grown in0.01% glucose, remained essentially unaltered(Fig. 11-14). The commitment of the originalhyphae to apical growth was observed to last forover 12 hr after the shift to a higher glucose con-centration. Simultaneously, about 4 to 6 hr afterthe shift, a few budding yeast cells began to ap-pear. These cells were probably derived fromround cells, present at the time of the shift, whichhad not been committed to hyphal morphogenesis(Fig. 11-14, arrows). It is, perhaps, highly signifi-cant that while these budding cells appeared andmultiplied, the original hyphae were activelyelongating, thus showing that the two morpho-genetic patterns may proceed under the sameenvironmental conditions provided that a priorcommitment to hyphal morphogenesis had beenestablished in part of the population.Hexose control of dimorphism in Mucor spp.

The morphological response of 11 strains ofMucor spp. to glucose and CO2 was assessed onYPG agar plates adjusted to two levels of glucose,0.01% and 2%, and incubated under two differ-ent partial pressures of CO2, 30% and 100%, allunder anaerobic conditions.Although only a small number of strains were

examined, a wide spectrum of morphologicalresponses was noted (Table 1). Only 5 of the 11strains tested manifested a potential for dimorphicdevelopment. Generally, the nondimorphic rep-resentatives grew poorly or failed to grow en-tirely under anaerobic conditions; wheneveranaerobic growth occurred, it was always my-celial.

In all dimorphic strains of Mucor, the morpho-logical phenotype was determined by the levels of

FIG. 10. Dimorphic colony of M. rouxii. Agar platewith 0.05% glucose incubated under 100% C02 for 72hr. Magnification marker = 100 ,um.

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*@ '~~~~~~~~~~~~~~~~~~~~~~.:Z : : :^ ~~~~~~~~~~~~.

: ..: .e~~~~~~~~~~~~~~~~......:.: 6: .~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.:.''.''1.',X."Sp1''t'',"iXX~........

FIG. 11-14. Commitment to hyphal morphogenesis.Hyphae grownfor 18 hr under nitrogen in 0.01% glucose

both glucose and CO2. All strains were ostensiblyyeastlike when cultivated in the presence of 2%glucose, under an atmosphere of 100%7 C02; atlower concentrations of glucose or CO2, hyphaldevelopment was obtained. The tendency todevelop hyphae or yeast cells in response to hexoseand CO2 varied with the strain. M. subtilissimusNRRL 1909 exhibited the highest propensity todevelop yeast cells (Table 1). For instance, in0.01 % glucose, a concentration at which otherstrains grew exclusively in mycelial fashion, M.subtilissimus 1909 developed yeast forms.The stimulation of yeast development by CO2

may not be readily evident unless the hexose con-centration is sufficiently low. For example, M.subtilissimus 1909 was previously believed (3)from tests made with 2% glucose to have adimorphism independent of CO2; however, bycultivation at a much lower glucose concentration(0.01 %O), a partial stimulation of yeast develop-ment by CO2 can be demonstrated (Table 1).

Haidle and Storck (5), working with M. rouxiiNRRL 1894, concluded that CO2 played nospecific role in the maintenance of yeast growth.However, this strain of M. rouxii responds toCO2 much in the same way as M. rouxii IM-80did (Table 1). In our laboratory, M. rouxii 1894developed exclusively in the mycelial form whengrown anaerobically in YPG (2% glucose) underprepurified nitrogen, provided that a small in-oculum (e.g., 104 spores/ml) was used. Under thesecultivation conditions, yeast development ensuedonly if a high pCO2 was also present. In view ofthe large inoculum employed by Haidle andStorck (5), it is possible that the yeast develop-ment obtained in the absence ofCO2 correspondedlargely to "swollen" spores rather than buddingyeast cells. This was the case with heavily in-oculated cultures of M. rouxii IM-80 (4).

DISCUSSION

Hexoses and C02 in dimorphism of Mucor. BothCO2 and hexoses are primary determinants ofyeast development in Mucor spp., and theireffects are seemingly complementary; at lowhexose concentration a high pCO2 is needed forpure yeast development and vice versa. The needfor CO2, however, is not manifested if the level ofhexose in the medium is high enough, e.g., 8 to10% glucose for M. rouxii IM-80 or <2% glu-cose for M. subtilissimus NRRL 1909 (3).Although hexoses were previously recognized

as an indispensable requirement for anaerobicgrowth of M. rouxii (4), a direct influence on

were transferred to 10%,0 glucose and observed at I hr(Fig. 11); 2.7 hr (Fig. 12); 8 hr (Fig. 13); and 12 hr(Fig. 14). The morphology at the time of the transferwas similar to Fig. 11. Magnification marker = 100 ,um.

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TABLE 1. Morphological response of diverse species and strains of Mucor to different levels ofglucose and CO2 in anaerobic cultivationa

100% C02 30% C02Organisms __

2% Glucose 0.01% Glucose 2% Glucose 0.01% Glucose

Dimorphic strainsbM. subtilissimus NRRL 1909.............. Y Y Y Y + MM. rouxii NRRL 1894.................... Y M Y + M MM. racemosus NRRL-1427c................ Y M Y + M MM. rouxii IM 80.......................... Y M Y+M MM. racemosus NRRL 1428................ Y NO Y + M M

Nondimorphic strainsM. hiemalis NRRL 2461.................. NO NO M MM. mucedo NRRL 1424................... NO NO M MM. subtilissimus NRRL 1743c.............. M NO M MActinomucor elegans NRRL 3104...... M NO M MM. pusillus NRRL 1426................... NO NO NO NOM. rammanianus NRRL1296.NO NO NO NO

a On YPG agar plates incubated for 48 hr. Y, yeast development; M, mycelium; NO, no growth.bDimorphic strains are listed in decreasing order of yeastlike tendency.c These two strains were inadvertently interchanged in a previous communication (3).

mold-yeast dimorphism was then discounted onthe basis of tests made in cultures containingfrom 0.1% to 10% glucose incubated undereither N2 or CO2. In all cases, the presence of ahigh pCO2 was the only recognized condition foryeast morphogensis. The additional need for aminimal glucose concentration was overlookedsince, as now shown, such need is manifested atconcentrations below 0.1 % glucose. In a previousinvestigation, incubation of M. rouxii with 10%glucose, under N2, resulted in the formation ofnumerous spherical cells but, contrary to presentresults, these were mainly arthrospore chains, andonly few were budding yeast cells (4). Perhaps,changes in the genotype or experimental condi-tions, or both, may account for the present greatersensitivity of the fungus to the yeast-promotingaction of hexoses.

It is important to note that the concentrationsof hexose or CO2 effective in controlling morpho-genesis of M. rouxii IM-80 pertain only to testsmade in media containing 0.3% yeast extractand 1% peptone. The effective concentrations ofhexose or CO2 are modified by other factors pres-ent in the complex medium. These substanceshave been tentatively and partly characterized asheavy metals and unsaturated fatty acids (un-published data). Thus, it appears that the tendencyof a given strain of Mucor to undergo eitherhyphal or yeast morphogenesis depends on a com-plex interplay of environmental factors, 02, CO2,hexoses, and others. An earlier conclusion that"'according to the strain (of Mucor), C02 may or

may not be necessary to induce yeastlike develop-ment" (3), should be amended to read: "accord-ing to the concentrations of other nutrients in themedium, exogenous CO2 may or may not b-necessary for yeastlike development of Mucor."

Although the stimulation of yeast developmentby CO2 has been amply confirmed, the fact thatyeast cells could be grown in the absence of carbondioxide (high glucose media) raises questionsconcerning the function and indispensability ofCO2 in the biochemical processes ultimatelyresponsible for the budding of spherical cells ofM. rouxii. Because of the continuous productionof metabolic CO2 during growth, tests in thecomplete absence of this gas are not possible.However, a comparison of the rates of CO2evolution in media with either low or high glucoseconcentration demonstrated no significant dif-ference between hyphae and yeast cells. Hence, itis unlikely that hexoses promoted yeast growthby an increased production or accumulation ofCO2; more likely, the morphogenetic action wascaused by another hexose metabolite. The obser-vation that, even in the presence of a high pCO2(Fig. 2, Table 1), yeast development did notnecessarily ensue unless a sufficiently high levelof glucose or other hexose was also present fur-ther supports the contention that hexoses arebona fide stimulants of yeast development. Thequantitatively different response of M. rouxii tothe various hexoses could be conveniently ascribedto the rapidity with which each hexose was ab-sorbed or metabolized.

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Although the experimental evidence ruled outan exogenous accumulation of CO2 or an in-creased rate of production as the manner bywhich a hexose elicits yeast development, it didnot exclude the possibility that metabolic CO2participated jointly with the hexoses in the bio-chemical processes leading to yeast budding. Con-ceivably, a sufficiently high level of glucose mightlower the requirement for exogenous CO2 to thepoint at which metabolically generated CO2would be sufficient to satisfy the requirements foryeast development. There is no reason to abandon.an earlier speculation (4) that CO2 operates bycausing the formation of an internal metabolite"Y", ultimately responsible for yeast formation.Several possible routes for the participation of ahexose in this scheme are depicted in Fig. 15,including the possibility that hexoses and CO2may be acting separately through unrelated path-ways but both eventually leading to the same final.action.

Hexoses, oxygen, and dimorphism. The stimula-tion of yeast development by hexose, initially re-ported for anaerobic forms of M. rouxii IM-80(S. Bartnicki-Garcia, Bacteriol. Proc. p. 123,1967), also extends to the aerobic yeastlike cells,of M. rouxii NRRL 1894 grown by Terenzi andStorck (14). These aerobic yeastlike forms ob-tained in the presence of phenethyl alcohol (5), atthe expense of severe growth inhibition, require aminimal glucose concentration of 2 to 5% (14).A case of mold-yeast dimorphism that normally

occurs under aerobic conditions, that of Candidaalbicans, is seemingly also under hexose control.Nickerson and Mankowski (11) showed thatglucose prevents filamentation and favors thedevelopment of yeast cells in this organism. How-ever, in C. albicans (11), unlike M. rouxii (unpub-lished data), the effect of glucose can be replacedby adding sulfhydryl compounds to the medium.Presumably, the morphological control by hexosein these two fungi may be mediated by some-what different mechanisms.

Repression of hyphal morphogenesis. The pres-ence or absence of apical growth has been pro-posed as the key event determining whether Mucordevelops into hyphae or into yeast cells, respec-tively (1, 2). In this context, a yeast-promoting-agent, such as hexose or C02, or both, or its

CO2 r - fixation producthexose metabolite N y

HEXOSE J--...Phexose metabolite.erFIG. 15. Hypothetical schemes for the participation

.of hexoses and CO2 in the formation of Y morphogen,the presumed internal effector ofyeast development.

internal effe ctor "Y," may be more accuratelydescribed as an inhibitor ofhyphal morphogenesis(apical growth) rather than as an inducer of yeastdevelopment (3). There are two levels, broadlyspeaking, at which this inhibitor may be acting.It could block the function of the enzymaticmechanism responsible for apical growth or,alternatively, it could prevent its formation. Thelatter case may be considered as one akin toenzyme repression. The results obtained by trans-ferring growing hyphae to glucose concentrationsfavorable for yeast development indicated thatthe hexose does not readily stop the operation ofapical growth in preexisting hyphae; conse-quently, the inhibitory action of glucose on hyphaldevelopment of Mucor is more likely by prevent-ing the initiation or formation of centers of apicalmorphogenesis. This action could result from arepression of some component(s) of the enzymaticcomplex responsible for localized cell wall syn-thesis. Catabolite repression (7) has been pro-posed to interpret the control by nutrients ofsporogenesis in bacteria (13) and yeast (8). Like-wise, the morphogenetic effect of hexoses on M.rouxii may operate via catabolite repression. Thispossibility is consistent with the observation(unpublished data) that the concentration ofradiolabeled catabolites (cold 5% perchloric acidsoluble fraction) is two to three times greater incells incubated with 10% than with 1% "4C-D-glucose. The repressor hypothesis seems especiallyattractive to interpret the formation of dimorphiccolonies of Mucor (Fig. 10). This dual behaviormay be regarded as a morphological version ofMonod's diauxic growth (9), with its correspond-ing and consecutive phases of repression and in-duction. The initial yeast phase develops whenglucose concentration is high enough to represshyphal morphogenesis; subsequently, as glucosebecomes depleted, a derepression or induction ofhyphal morphogenesis may take place.

ACKNOWLEDGMENTS

This investigation was supported by Public HealthService research grant AI-05540 from the NationalInstitute of Allergy and Infectious Diseases.The skillful and dedicated assistance of E. Reyes

and the critical advice of W. L. Belser are gratefullyacknowledged.

LITERATuRE CITED1. Bartnicki-Garcia, S. 1963. Symposium on the bio-

chemical bases of morphogenesis in fungi. III.Mold-yeast dimorphism of Mucor. Bacteriol.Rev. 27:293-304.

2. Bartnicki-Garcia, S., N. Nelson, and E. Cota-Robles. 1968. A novel apical corpuscle in thehyphae of Mucor rouxii. J. Bacteriol. 95:2399-2402.

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3. Bartnicki-Garcia, S., and W. J. Nickerson. 1962.Induction of yeastlike development in Mucorby carbon dioxide. J. Bacteriol. 84:829-840.

4. Bartnicki-Garcia, S., and W. J. Nickerson. 1962.Nutrition, growth, and morphogenesis ofMucor rouxii. J. Bacteriol. 84:841-858.

5. Haidle, C. W., and R. Storck. 1966. Control ofdimorphism in Mucor rouxii. J. Bacteriol. 92:1236-1244.

6. Jeffay, H., and J. Alvarez. 1961. Liquid scintilla-tion counting of carbon-14. Anal. Chem. 33:612-615.

7. Magasanik, B. 1961. Catabolite repression. ColdSpring Harbor Symp. Quant. Biol. 26:249-256.

8. Miller, J. J., and 0. Hoffman-Ostenhoff. 1964.Spore formation and germination in Sac-charomyces. Z. Allgem. Mikrobiol. 4:273-294.

9. Monod, J. 1947. The phenomenon of enzymaticadaptation. Growth 11:223-289.

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10. Nadson, G., and G. Philippov. 1925. Une nouvellemucorinee. Mucor guilliermondi. n. sp. et sesformes levures. Rev. Gen. Botan. 37:450-461.

11. Nickerson, W. J., and Z. Mankowski. 1953.Role of nutrition in the maintenance of theyeast-shape in Candida. Am. J. Botany 40:584-592.

12. Ritter, G. E. 1913. Die giftige und formativeWirkung der Saueren auf die Mucoraceen undihre Beziehung zur Mucorhefebildung. Jahrb.Wiss. Botan. 52:351-403.

13. Schaeffer, P., J. Millet, and J. P. Aubert. 1965.Catabolic repression of bacterial sporulation.Proc. Natl. Acad. Sci. U.S. 54:704-711.

14. Terenzi, H. F., and R. Storck. 1968. Stimulationby phenethyl alcohol of aerobic fermentation inMucor rouxii. Biochem. Biophys. Res. Com-mun. 30:447-452.

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