Vol. 141, No. 2JOURNAL OF BACTERIOLOGY, Feb. 1980, p. 946-9550021-9193/80/02-0946/10$02.00/0

Subcellular Distribution of Marker Enzymes in Cells of aMinute Fungus, Fusidium sp. 100-3

DEAN HANDLEYt AND B. K. GHOSH*CMDNJ-Rutgers Medical School, Department of Physiology and Biophysics, Piscataway, New Jersey 08854

An electron microscope cytochemical technique was used to determine thesubcellular distribution of marker enzymes in Fusidium sp. 100-3 cells. Nucleosidediphosphatase was found in the nuclear envelope and intracytoplasmic membranesegment. Thiamine pyrophosphatase was found to be associated with the meso-somes. Cytochrome c (oxidase) activity was found only in the mitochondrialcristae. Strong alkaline phosphatase activity was present in the vacuole; inaddition, the enzyme activity was discretely dispersed throughout the cytoplasmwithout any association with any membrane material. The overall characteristicsof the cell ultrastructure and subcellular enzyme distribution of Fusidium sp. 100-3 cells compare fairly well with those of a fungal cell. But there are considerabledifferences from the characteristics of higher eucaryotic cells. Detailed data onthe marker enzymes distribution in a variety of fungal cells are not available.Therefore, it is not possible to conclude whether the marker enzyme distributionof Fusidium sp. 100-3 cells is unique or is typical of any fungal organism. Detailedstudies of cell ultrastructure of and marker enzyme distribution in minute fungalcells and their comparison to the ultrastructure ofand marker enzyme distributionin other fungal organisms may be helpful in understanding the phylogenetic andontogenic development of subcellular organelles.

Recently we reported the isolation of a minutefungus, Fusidium sp. 100-3 (9). This organismhas the size of a bacteria, but contains bothmitochondria and nucleus. The nuclear and mi-tochondrial membranes show extensive associa-tion with the plasma membrane; comparableassociations are also seen between mitochondrialand nuclear membranes. Frequently, thesemembrane-membrane associations are so tightthat the membranes appear to be fused. Thecells contain no endoplasmic reticulum, butsmall segments of membrane material are dis-persed throughout the cytoplasm.

Subcellular differentiation and organelle bio-genesis are based on the classical dogma thatincreases in cell size demand organelle formationfor increased cellular efficiency (11, 22). Ourpresent study is aimed at examining the subcel-lular organization of the cells of this organismbecause one expects from the small size of thecells that there will be miniimal subcellular dif-ferentiation among the cells.The specific function ofan organelle emanates

from a unique enzyme content referred to as amarker enzyme. It is possible that if a subcellularorganelle has not developed in a cell, the char-acteristic enzyme(s) of the missing organelle will

t Present address: Department of Physiology, College ofPhysicians and Surgeons, Columbia Medical School, NewYork, NY 10032.

be found in a different subcellular location.Therefore, the study of the distribution ofmarker enzymes can be used to identify subcel-lular organelles based on their specific function.

In this paper we present data correlating thesubcellular organization of Fusidium sp. 100-3with the distribution of marker enzymes. Themarker enzymes used in the study are as fol-lows: (i) nucleoside diphosphatase (NDPase), amarker for endoplasmic reticulum; (ii) thiaminepyrophosphatase (TPPase), a marker for Golgibodies; (iii) cytochrome c, a marker for the outerand inner mitochondrial membranes; (iv) alka-line phosphatase (APase), a marker for the per-iplasm and the plasma membrane. Finally, theresults on enzyme distribution are compared tothe structure of the organelles.

MATERIALS AND METHODSOrganism growth. Fusidium sp. 100-3 was grown

in Bennett liquid medium containing beef extract,yeast extract, and cerelose (8). The culture was shakenfor 28 to 32 h at 280C in a rotary shaker. These cells,at the exponential phase of growth, are commonlysingle and rod-shaped (9) and were used in all theexperiments.

Fixation to study cell ultrastructure. The cellswere harvested and washed by centrifugation (4,000x g, 10 min) in a GTP nonionic buffer (PM) containing1 mM GTP, 1 mM MgSO4, 2 mM ethylene glycol-bis(,B-aminoethyl ether)-N,N-tetraacetic acid, and 100

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mM piperazine-N,N'-bis(2-ethanesulfonic acid) at pH6.9 (10). The washed cells were fixed in 5% glutaral-dehyde diluted in PM buffer. After fixation for 3 h atroom temperature, the cells were washed in 0.1 Msodium-cacodylate buffer (pH 7.4) and subsequentlytransferred into 1% OS04 in the same buffer for 18 h inthe dark at room temperature. Finally, the fixed cellswere dehydrated in a standard ethanol series andembedded in Spurr resin (21).

Cytochemistry. (i) NDPase, TPPase, and cyto-chrome c (oxidase). The cells were washed in TMbuffer (i.e., 0.24 M Tris-maleate, pH 7.25) for NDPaseand TPPase localization; for cytochrome c localization,the cells were washed in PM buffer. Equal volumes ofcell suspension and 2% ion agar (dissolved in TM or

PM buffer for respective samples) were thoroughlymixed at 37 to 400C. This mixture was layered on a

glass slide; after a few minutes of hardening at 40C,the agar layer was cut into small (1-mm cube) blocks.The blocks, for determination ofNDPase and TPPase,were fixed for 60 min at 40C in a freshly preparedmixture of 3% glutaraldehyde and 1% H202 in TMbuffer (7). The blocks for the determination of cyto-chrome c were'fixed in 2.25% glutaraldehyde in PMbuffer for 80 min at 40C. The aldehyde-fixed blockswere rinsed several times in the appropriate buffer(TM or PM) and were incubated at 370C for 60 to 90min in the specific incubation medium for the differentenzymes. (i) For NDPase activity, IDP-lead medium(pH 7.25) consisted of 5.0 mg of Na-IDP, 0.3 ml of 1%Pb(No3)2, 0.5 ml of 0.025 M Mg9l2, and 1.0 ml of TMbuffer, and 0.7 ml of distilled water to make the volumeup to 2.5 ml. (ii) For TPPase activity, TPP-lead me-

dium (pH 7.25) contained 5.0 mg of thiamine pyro-phosphate (chloride), 0.2 mM ATP, 0.5 ml of 0.025 MMgCl2, and 1.0 ml of TM buffer. ATP was added toenhance the staining of TPPase activity (7). (iii) Forcytochrome c, diaminobenzidine medium at pH 6.0consisted of 20 mg of 3,3'-diaminobenzidine (DAB) or3,3'-diaminobenzidine tetrahydrochloride (DAB-HCl),8.9 ml of 0.05 M acetate buffer (pH 6.0), 1 ml of 0.05M MnCl2, and 0.1 ml of freshly prepared 0.1% H202; 1mg of cytochrome c per ml was added to enhancecytochemical staining (15-17).

After incubation in a specific cytochemical reactionmixture, the blocks were washed in PM or TM bufferfollowed by 0.1 M Veronal acetate buffer at pH 7.1.The washed blocks were fixed in 1% OS04 in Veronalacetate buffer for 18 h at room temperature in thedark. The fixed blocks were dehydrated and embeddedas described above.The control samples were incubated without spe-

cific substrates, i.e., Na-IDP for NDPase; thiaminepyrophosphate chloride for TPPase, and DAB-HClfor cytochrome c (oxidase). If a specific inhibitor wasavailable, cytochemical reaction was performed afterinhibition of enzyme activity by inhibitor treatment ofthe cells, e.g., NDPase activity was inhibited by 0.01M uranyl nitrate (16), and cytochrome c (oxidase)activity was inhibited by 0.01 M KCN (17).

(ii) APase. It is known from our previous studiesthat glutaraldehyde fixation inactivates APase activityof bacterial cells (6). The size of Fusidium sp. 100-3cells is comparable to that of bacterial cells; therefore,the effect of glutaraldehyde fixation on the APase

activity of Fusidium sp. 100-3 was examined. The cellsfrom the growth medium were washed in 0.01 M Tris-acetate-hydrochloride buffer (pH 8.5) (TA) and sus-pended in the same buffer (6); the final concentrationof cells in this stock suspension was 25mg (wet weight)per ml. One-milliliter portions from the stock suspen-sion were treated at 40C for 30 min with differentconcentrations of glutaraldehyde (i.e., 0.1, 0.5, 1, 2, 3,and 5%). After fixation the cell suspensions werewashed free of aldehyde and finally suspended in 1 mlof TA buffer; APase contents of these suspensionswere determined immediately.The distribution of APase was also examined in the

cytosol and the particulate fractions. One-milliliterportions of the stock suspension were ruptured at 40Cby intermittent ultrasonic vibrations in a Bronson 200cell disrupter. Microscopic examination showed thatthe majority of the cells were disrupted after 2 min oftreatment. The ruptured cell suspension was centri-fuged for 30 min at 80,000 x g in an SW-27 rotor(Beckman L2065B preparative ultracentrifuge); theresulting particulate and soluble materials were sepa-rated. The particulate material was suspended in 1 mlof TA buffer; enzyme activity was determined in theclear supernatant and in the particulate fraction.APase assay. All the samples having 1-ml volumes

were mixed with 3.9 ml of 1.0 M Tris-hydrochloridebuffer (pH 8.4) which contained 2 mM MgCl2 and 100ml of 10 mg of p-nitrophenyl phosphate (PNPP) perml. The mixture was incubated at 370C; after 30 minthe reaction was stopped with the addition of 1 ml of2.0 N NaOH into each sample. All the samples werecentrifuged at 20,000 x g for 20 min, and the color ofthe resulting clear supernatant was measured at 410nm in a Beckman model 24 spectrophotometer. APaseactivity was expressed as nmoles ofp-nitrophenol pro-duced per 25 mg (wet weight) of cell.The subcellular distribution of APase activity was

determined by a cytochemical method previously de-scribed (6). One-milliliter portions of stock cell suspen-sion were fixed in 5% glutaraldehyde in TA buffer at40C for 30 min. The fixed cells were washed free ofglutaraldehyde. The following incubation mixture wasprepared with the fixed cells: 0.5 ml of fixed cellsuspension, 39.5 mM sodium-potassium tartrate, 3mMlead nitrate, and 100 pl of 100-mg/ml PNPP. Thismixture was incubated at 37 C for 30 min with occa-sional shaking. After incubation the cells in the incu-bation medium were washed with Veronal acetatebuffer by centrifugation. The washed cells were fixedin 1% 0804, dehydrated, and embedded as describedabove. The control samples were prepared by (i) notincluding any PNPP substrate in the cytochemicalreaction mixture and (ii) inhibiting the enzyme activityof the cells with 2 mM EDTA treatment (5) beforeincubation in the cytochemical reaction mixture.Thin sections (60.0- to 80.0-nm thickness) for each

preparation were stained with lead citrate (18) andviewed in a JEOL 100 C transmission electron micro-scope at 60 or 80 kV. Frequently, unstained sectionswere also examined.

RESULTSSubcellular organization. Gross subcellular

organization of Fusidium sp. 100-3 has been

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described earlier (9). We fixed the cells followingan improved fixation technique using a GTPnonionic buffer system. This procedure is re-ported to be superior in preserving the details ofthe membrane structure microtubules, ribo-somes, and other fine fibrillar components (10).

Clarity of the ultrastructure is remarkable(Fig. 1 to 3a and b) when compared to samplesprepared following conventional techniques asdemonstrated in our earlier paper (9). Exami-nation of a large number of sections did notshow any microtubular or fibrillar structures inthe cells. Individual ribosomes were distinct andevenly dispersed throughout the cytoplasm; thenucleus, mitochondria, vacuole, and mesosomesdid not contain any ribosomes. The averagediameter of the ribosomes was 24 nm; this valueagrees well with the 80S ribosomes of eucaryoticcells (20). The preservation of mitochondria wasexcellent; they contained amorphous intramito-chondrial material (Fig. 1 and 2). Frequently,one profile of a mitochondrion was seen to coverthe entire length of the cell; such a mitochon-drion usually had many lobes which were inter-connected by narrow stalks (Fig. 1, arrowhead).The evidence that multiple profiles of a mito-chondrion (Fig. 2) in one section is the part of asingle interconnected body has been presentedin our previous publication (9). There were alarge number of cristae usually oriented parallelto the longitudinal axis of the mitochondria; thelumen of the cristae was about 25 nm and waselectron lucid.The pleomorphic nucleus contained amor-

phous nucleoplasm and a clearly defined nucleo-lus (Fig. 1 to 3). Chromatin material is notclearly visible in Fig. 1 to 3. It is known thatchromatin material can be demonstrated in elec-tron micrographs only after condensation, whichfrequently depends upon specific salt concentra-tions in the suspending medium. It has beenshown in our earlier publication that in the cellsof fusidia, chromatin is condensed in high-mo-larity phosphate buffer (9). In this specific con-dition of incubation, chromatin material can bedemonstrated to be bound to the nuclear mem-brane. The latter consists of two layers of unitmembrane with an intervening 12- to 15-nm gap(i.e., perinuclear space), which is much narrowerthan the nuclei of plant or animal cells. Thenuclear and plasma membranes were veryclosely apposed; the gap between these mem-branes was much less than 5 nm (Fig. 1 to 3). Inmany regions two membranes of the nuclearenvelope were so closely apposed that they ap-peared to be fused; hence, no perinuclear spacecan be demonstrated (Fig. 3). Although from thedata presented in this paper it is not possible to

establish that the two membranes of the nuclearenvelope are fused, a detailed study has beenmade with freeze-fracture and thin-sectioningtechniques (D. Handley and B. K. Ghosh, Abstr.Annu. Meet. Am. Soc. Microbiol. 1976, J14, p.135; unpublished data). These data clearlyshowed that the two membranes of the nuclearenvelope of fusidia are largely fused. The nuclearpores were very few and irregularly spaced alongthe nuclear membrane (Fig. 1 and 2).Short segments of membrane were dispersed

in the cytoplasm (Fig. 1 and 2). Apparently,these intracytoplasmic membrane segments areequivalent to endoplasmic reticulum. In thinsections of cells fixed as described previously (9),these membrane segments appeared as sheets ofmembrane without any luminal space. Althoughin GTP nonionic buffer a narrow luminal space(5 to 10 nm) can be demonstrated, there wasneither any terminal swelling nor any ribosomeassociated with the membrane surface. It hasbeen discussed earlier (4) that endoplasmic re-ticulum having these characteristics are foundin some fungi, whereas others have typical en-doplasmic reticulum as found in plant or animalcells. Vacuoles are always empty in the cellsfixed by the procedure described before (9), butthe cells fixed in GTP nonionic buffer mostlycontained amorphous dense material and somemultilamellar membrane whorls. The vacuoleswere delimited by distinct membranes.The mesosomes can be observed in cells fixed

by both techniques (Fig. 2 and 3a). These aremultilamellar bodies frequently enclosing cyto-plasmic material. The structure of these bodiesdiffer appreciably from fungal lomasomes, whichare a collection of vesicles or tubules surroundedby a delimiting membrane and located in theperiplasm, occasionally sequestered in the spacebetween the cell wall and the plasma membrane.It has been suggested that all these polymorphicmembranous bodies can be considered underone general group (4). It seems unlikely thatthese bodies are artifacts of fixation becauseboth the lomasomes and the mesosomes havebeen demonstrated in frozen, etched specimens(3, 4) of fungal cells.Cytochemistry. (i) NDPase. Glutaralde-

hyde-hydrogen peroxide fixation preserved boththe ultrastructure and enzyme activity sufficientfor cytochemical localization. The sites ofNDPase activity were seen as electron-densedeposits (Fig. 4 and 5, arrowheads) associatedwith the nuclear envelope and the intracyto-plasmic membrane segments. The vacuolarmembrane also showed discrete electron-densepatches; the intravacuolar contents were freefrom any comparable deposit. The reaction

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FIG. 1 to 3. Fusidium sp. 100-3 cells fixed in glutaraldehyde-GTP nonionic buffer followed by OS04 fixativein Veronal acetate buffer. The cells contain a nucleus (N), one or manyprofiles ofmitochondria (M), a vacuole(19, intracytoplasmic membrane segments (S), nuclearpore complexes (NPC), and mesosomes (Me); note thatconsiderableportions ofthe nuclear envelope are tightly associated with theplasma membrane. Multilamellarwhorls ofmesosome can be seen in Fig. 3b. Bars, 0.5 pim except for that in Fig. 3b.

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product ofNDPase activity cannot be located inthe mitochondrial matrix, the nucleoplasm, orthe plasma membrane. However, some regionsof the plasma or mitochondrial membrane,which are in apposition with the nuclear enve-lope, show electron-dense deposits (Fig. 5, ar-rowheads). The control cells were prepared intwo ways. (i) The cells were incubated withoutIDP substrate; (ii) the cells were treated withuranyl nitrate to inhibit NDPase activity. Thesections from these cells showed no electron-dense deposits.

(ii) TPPase. The TPPase activity appearedas highly electron-dense patches of membranewithout any formation of discrete deposits as-sociated with the membrane (as seen forNDPase activity). The staining caused byTPPase activity is substantially intensified bythe addition of ATP to the cytochemical reac-tion mixture at neutral pH (7). Therefore, in allthe experiments, the ATP intensificationmethod was used. TPPase activity was primarilylocated in the mesosomes (Fig. 6 to 8). Theactivity was distributed throughout the mem-brane material (Fig. 6) as well as throughout theintramesosomal contents. The nuclear mem-brane associated with the mesosomal membranehad TPPase activity, but the rest of the nuclearmembrane was free from any such activity (Fig.8). These sections were not stained with leadcitrate; therefore, the contrast of the nuclearenvelope is poor. But the arrows show thatbeyond the point where nuclear envelopes de-flect away from the mesosome membrane, nostaining of the nuclear envelope can be seen.Portions of the vacuolar membrane and the in-tracytoplasmic membrane segments also showedenzyme activity. Besides these specific locations,TPPase activity was absent from any other sub-cellular locations, including the plasma mem-brane. The control cells were prepared by incu-bation in the absence ofthiamine pyrophosphatechloride substrate. No characteristic reactionwas seen in these cells.Localization of cytochrome c. Two differ-

ent substrates, i.e., DAB and DAB-HCI, wereused in the incubation mixture for the cytochem-ical reaction of cytochrome c (oxidase). The useofDAB failed to give any specific staining of the

mitochondria (Fig. 9); arrowheads in Fig. 9 in-dicate that the DAB reaction product appearsas small patches of electron density in the mi-tochondrial membrane, but a similar reactionwas also seen in the vacuolar membrane. Incontrast, the specific staining with DAB-HCI isclear in Fig. 10 and 11; the cristae of mitochon-dria had intense electron density, but the outermitochondrial membrane showed no such elec-tron density. No other membrane materialshown had comparable electron density. It ap-pears from this notable difference in the stainingbetween DAB- and DAB-HCl-treated cells thatplasma or mitochondrial membranes or both areimpermeable to DAB but are permeable to itschloride form. The control cells were preparedin two ways. (i) The cells were incubated in theabsence of DAB-HCI substrate; (ii) the cyto-chrome oxidase activity was inhibited by KCNtreatment. In these control cells mitochondrialcristae did not show any specific staining.APase. We observed in our preliminary stud-

ies that the whole cell and the culture mediumhave negligible amounts ofAPase activity. How-ever, the enzyme activity of whole cells wasreadily detected by biochemical assay after pre-fixation with glutaraldehyde. Furthermore, afterultrasonic disruption of the whole cells, the en-zyme activity could be detected. The rupturedcellular material was fractionated by differentialcentrifugation into cytosol and'particulate ma-terial; the former contained 92% and the lattercontained 8.0% of the total APase activity of theunfractionated, ruptured whole cell material.The addition of differing concentrations of glu-taraldehyde to the ruptured cells substantiallyinactivated the enzyme activity. The APase ac-tivity of the intact whole cells, on the otherhand, increased with the treatment of increasingconcentrations of glutaraldehyde. But very highconcentrations of aldehyde treatment (e.g.,-10%), caused almost total inactivation of theAPase activity of the unruptured whole cells.These results suggest that APase activity is pos-sibly cryptic in Fusidium sp. 100-3 cells. Alde-hyde treatment may have caused a loss of selec-tive permeability of the plasma membrane and,therefore, PNPP became available to the crypticAPase activity. In other words, the APase activ-

FIG. 4 and 5. NDPase activity is seen as discretely distributed electron-dense deposits. These deposits werebound mainly to the region of the nuclear envelope associated with the plasma or mitochondrial membrane(arrowheads). Frequently, the deposits were also bound to the intracytoplasmic membrane segments (S) orvacuole membrane (V). M, Mitochondria; N, nucleus. Bars, 0.5 pun.

FIG. 6 to 8. Subcellular distribution of TPPase activity. In Fig. 6 note a large and a small mesosome (Me)having high electron density; in Fig. 7, the arrowhead and the enlarged inset show that electron density isuniform throughout the membrane and the amorphous intramesosomal material of the mesosome; in Fig. 8,the arrowheads and the inset show electron density of the mesosomal membrane associated with nuclearenvelope; some vacuole membrane (19 and intracytoplasmic membrane segments (S) show electron density. N,Nucleus. Bars, 0.5 um except for those in insets.

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ity demonstrated by cytochemical procedure inthe aldehyde-treated cells represents theamount of residual intracellular enzyme whichescaped the denaturing effect of the aldehyde. Itappears from several experiments that 5% glu-teraldehyde is the optimum concentration whichdestroys the permeability barrier to PNPP butdid not appreciably affect the APase activity. Itshould be noted that the concentration optimumof aldehyde only holds good for the specific cellconcentration (25 mg [wet weight] per ml) usedin our experiments.The loss of selective permeability ofFusidium

sp. 100-3 cells after aldehyde treatment was fur-ther confirmed by comparing the uptake of animpermeable sugar (C'4-labeled mannitol) be-fore and after aldehyde treatment. The dataclearly show that mannitol was taken up poorlybefore aldehyde fixation, but after fixation thissugar diffused into cells in appreciable amounts(details of the transport will be published else-where).The cytochemical reaction product of APase

appeared as 10-nm, electron-dense deposits ran-domly distributed throughout the cytoplasm,nucleoplasm, and mitochondrial matrix (Fig. 12and 13, arrowheads). The vacuolar membraneshowed large amounts of electron-dense depos-its. The intense reaction of the APase activitywas also noted at the region of bud formation inFig. 13 (bf). No electron-dense deposits werefound to be associated with the plasma mem-brane, nuclear membrane, or mitochondrialmembrane. The APase activity was demon-strated in cells grown in media containing highamounts of inorganic phosphate. Therefore, thedistribution of the APase activity of these cellsrepresents the constitutive enzyme activity ofthe cells. The control cells were prepared in twoways. (i) The cells were incubated in the absenceof PNPP substrate; (ii) the cells were inhibitedby 10-2 M EDTA treatment. These control cellswere free from any electron-dense deposits char-acteristic for the APase activity.

DISCUSSIONThe results presented in this paper show that

the subcellular morphology of Fusidium sp. 100-

3 cells is grossly similar to that of fungal cells.Although there are exceptions (e.g., vesiclescommonly found at the region of bud formationor at the hyphal tip were not present, extensiveportions of the nuclear envelope were intimatelyassociated with the plasma membrane), similarclose opposition was also observed between mi-tochondrial membrane and the nuclear envelopeor plasma membrane: nuclear pores were veryfew; perinuclear space was almost absent; therewere many mesosomes; above all, the organelleswere very small relative to those in a fungal cell.The distribution of marker enzymes chosen in

this paper has been studied extensively in fullydeveloped eucaryotic cells (e.g., mammaliancells [14, 15]). Therefore, our data could not becompared with those of fungal cells. In animalcells, cytochemical localization of NDPase andTPPase depends critically on the pH of theincubation mixture; at pH 7.0 NDPase activityhas been localized in the nuclear membrane andthe endoplasmic reticulum; at the same pHTPPase activity has been found solely in Golgibodies. In cells of fusidia, NDPase was locatedin the nuclear membrane and in the intracyto-plasmic membrane segments, but TPPase activ-ity was found only in the mesosomes.The NDPase distribution data can be readily

interpreted as partly comparable to the distri-bution in animal cells because the enzyme ispresent in the nuclear membrane. The presenceof NDPase activity in the intracytoplasmicmembrane segments suggests that the mem-brane material is equivalent to the endoplasmicreticulum of animal or plant cells. But it shouldbe noted that the ultrastructure of the intracy-toplasmic membrane segments differs substan-tially from the animal cell endoplasmic reticu-lum. It has been discussed earlier (4) that thereis a lack of systematic comparative data on theultrastructure of the endoplasmic reticulum infungi. It is possible that the endoplasmic retic-ulum in some fungi, including fusidia, is unusualif compared to that of animal or plant cells. It ispossible that although this membrane materialhas differentiated from the nuclear membrane,its differentiation is incomplete. In other words,the intracytoplasmic membrane segments are

FIG. 9. Cytochrome c staining with DAB. A large mitochondrion (M) with cristae can be seen; besidessmall electron-dense deposits (arrowheads), no specific staining is seen. N, Nucleus; V, vacuole; s, intracyto-plasmic membrane segments. Bars, 0.5 ,um.

FIG. 10 and 11. Cytochrome c staining with DAB-HCl. In Fig. 10 one mitochondrion (M) and in Fig. 11many profiles of mitochondria (M) clearly show intensely stained cristae. N, Nucleus; V, vacuole. Bars, 0.5,un.

FIG. 12 and 13. APase activity is seen as 10- to 15-nm deposits (arrowheads) randomly distributedthroughout the cytoplasm without having any specific association with the membrane. Vacuoles (V7 containdense aggregates of deposits. Bud membrane (bt) shows uniform electron d.ensity. N, Nucleus; M, mitochon-drion. Bars, 0.5 p,m.

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the precursors of the endoplasmic reticulum. Ithas been shown that in fungi the nuclear mem-brane, endoplasmic reticulum, and Golgi mem-branes constitute part of the general endomem-brane system; the hypothesis is that the differ-entiation ofendomembranes proceeds vectorallyfrom the nuclear envelope to the endoplasmicreticulum to the Golgi membrane (12, 13).

Following the same line of argument, absenceof Golgi membranes and the presence ofTPPaseactivity in the mesosome can be considered tosuggest that the mesosomes in fusidium maypossess some characteristics of Golgi. However,the Golgi body is such a complex subcellularorganelle that the presence of only one enzymecannot be used to identify that organelle. It hasbeen discussed earlier that typical Golgi bodieshave been demonstrated in many fungal orga-nisms (4); whereas, in others, Golgi bodies maybe typical or they are present in an uncharacter-istically simple form. Hence, the presence orabsence of Golgi bodies represents another ex-ample of diversity of fungal subcellular organi-zation. But it is clear that the mesosome mem-brane, in spite of being structurally continuousto the plasma membrane, contains an enzymeactivity (TPPase) which is not present in theplasma membrane. Therefore, it is possible thatin this organism the mesosome membrane hasdifferentiated from the plasma membrane andpossesses a unique characteristic absent in theplasma membrane.DAB-HCI has been extensively used for the

localization of specific oxidation reduction en-zymes (15). The conditions used in the experi-ments reported here are optimal for the cyto-chemical staining of mitochondria. Althoughthere is considerable controversy whether theDAB-HCI method localizes cytochrome c or cy-tochrome c oxidase, the pH 6.0 incubation me-dium used in our experiments is certainly knownto be specific for the localization of cytochromec (17, 19). In animal cells, both the outer and thecristae membrane are stained after incubation inpH 6.0 medium. In contrast, the mitochondriaof Fusidium sp. 100-3 incubated in the samemedium showed staining of cristae only. There-fore, the outer mitochondrial membrane of thecells of fusidia differ from the mitochondrialmembrane of animal cells. It is not possible todetermine the cause of this difference, but it isclear that the outer mitochondrial membrane ofanimal cells and the cells of Fusidium differappreciably in their function because of the ab-sence of cytochrome c. Further investigation ofthe enzyme composition of the mitochondrialmembrane subfractions will be very useful.APase has been localized in fungal, bacterial,

and animal cells. This enzyme shows considera-ble nonspecificity of subcellular distribution inanimals cells. In fungal cells this enzyme ismainly located in vacuoles which have beenconsidered the lysosomal equivalent of animalcells (1). In gram-positive bacteria, APase isassociated with the inside surface of the plasmamembrane (6); in gram-negative bacteria, it islocated in the periplasm (2). In Fusidium sp.100-3, the APase was located mainly in the vac-uoles although some activity could also be notedfree in the cytoplasm. Therefore, this distribu-tion of APase is characteristic of fungal orga-nisms.With all the facts taken together, we can con-

clude that the subcellular organization of Fusi-dium sp. 100-3 cells is, in general, comparable tothat of fungal cells; but Fusidium sp. 100-3 cellsdiffer considerably from the higher eucaryoticcells. It is not possible to comment on whetherthese characteristics are unique for Fusidiumsp. 100-3 or constitute the general characteristicsof any fungal cell unless comparable data fromother fungal organisms is available.

If one considers the size of this organism, it isevident that in spite of having the size of abacterium, this organism possesses a basic eu-caryotic cytology. This raises a fundamentalquestion: what constitutes the critical selectionpressure for the origin of subcellular organelles?The most commonly accepted view is that anincrease in cell size requires compartmentaliza-tion of cell function (11, 12) and this compart-mentalization is achieved during phylogeneticdevelopment of eucaryotic cells through de novogenesis of subcellular organelles and throughpossible incorporation of procaryotic celLs fromthe extracellular environment into evolving eu-caryotic cells. Therefore, the existence of anorganism having the size of a bacterium butcontaining a full complement of eucaryotic or-ganelles suggests that either organelle formationis unrelated to the increase of cell size or thisorganism, or any other related ones, is a rareexample of a degenerated form of a previouslyexisting, fully developed eucaryotic cell. How-ever, this appears unlikely because irrespectiveof cell size, subcellular morphology of fungalcells varies widely. Hence, fungal cells may dem-onstrate differing phylogenetic developmentalstages of subcellular organelles.

In conclusion, we suggest that a minute funguscan be easily neglected because of its size. Butstudies of the subcellular morphology of minutefungi may help us in understanding the phylo-genetic development of subcellular organelles,which, in turn, will be useful in understandingthe ontogenic development of organelles.

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ACKNOWLEDGMENTS

This investigation is supported by financial assistance fromgrant PCM 77-25718 from the National Science Foundation.D.H. acknowledges a graduate fellowship from the Depart-ment of Physiology and Biophysics, CMDNJ-Rutgers MedicalSchool.We thank M. P. Lechevalier and H. Lechevalier for help

during the investigation, S. C. Holt for helpful comments, andM. Smolensky for help in preparing the manuscript.

LITERATURE CITED

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