Vol. 132, No. 2
JOURNAL OF BACTERIOLOGY, Nov. 1977, p. 691-703 Copyright © 1977 American Society for Microbiology
Printed in U.S.A.
Distribution and Conformation of Crystalline Nigeran in Hyphal Walls of Aspergillus niger and Aspergillus awamori T. F. BOBBITT,l J. H. NORDIN,' M. ROUX,2 J. F. REVOL,2 AND R. H. MARCHESSAULT2 Department of Biochemistry, Uniuersity of Massachusetts, A mherst, Massachusetts 01003,' and Department of Chemistry, University of Montreal, Montreal, Quebec, H3C 3V1 Canada2
Received for publication 28 July 1977
Hyphal walls ofAspergillus awamori containing increased amounts of the aglucan, nigeran, became correspondingly more opaque when viewed in the electron microscope as shadowed preparations. However, increased polymer deposition was not accompanied by any significant change in wall thickness. The nigeran of both A. awamori and Aspergillus niger occurred in situ in a crystalline conformation identical to that of single crystals prepared with pure polysaccharide. Furthermore, this polymer was the dominant crystalline material in the hyphae whether or not they were enriched in nigeran. Enzymic digestion of nigeran in A. niger and A. awamori revealed that the bulk of the polymer was exposed to the cell's exterior. However, a certain fraction was accessible to enzymic attack only after the wall was treated with boiling water. A third portion, detectable only by X-ray diffraction, was associated with other components and could not be extracted, even with prolonged boiling. It was removed by hot, dilute alkali and was associated in the wall with another glucan fraction. Dry heating of A. niger walls altered their susceptibility to enzymic digestion of nigeran in situ. It is proposed that this treatment introduces interstices in the crystal surface that facilitate attack. Nigeran, a hot-water-soluble, linear, alter- has a 21 helical conformation with four glucose nating (1---*3), (1-*4)-a-D-glucan (2, 3) was first residues per helix turn. Using electron diffracisolated by Dox and Neidig (6, 7) from Penicil- tion methods with these crystals, Taylor et al. lium expansum and Aspergillus niger. Reese (21) elucidated the relationship of lattice water and Mandels (16), after surveying a large num- to the polymer's overall crystal structure and ber of fungi, found. that nigeran was synthe- demonstrated that these crystals can exist in sized by only a few species of Aspergillus and either "dry" or "hydrated" forms, depending Penicillium. Both Johnston (12) and Tung and upon the drying history. Nordin (25), studying hypal wall structure, conGold et al. (10) found that the amount of cluded that this glucan is a wall component of nigeran present in Aspergillus hyphae is enA. niger. The latter investigators also noted hanced dramatically by nitrogen deprivation. that nigeran might occupy a buried location Furthermore, they demonstrated that the polywithin the wall, since mycodextranase, an en- saccharide is not utilized as a carbon source zyme specific for nigeran, hydrolyzed the glu- during starvation. We have probed the hyphal wall architecture can to a much greater extent when the wall suspensions were first heated to 100CC. How- of A. awamori and A. niger by a combination ever, an alternative explanation for the results of X-ray diffraction analysis, enzymolysis, and was proposed (25), i.e., nigeran might exist in electron microscopy to gain additional insight some crystalline array that conferred resist- into the structural role played by this polymer. ance to enzymic hydrolysis. Additional confir- The results of these studies form the basis of mation of a wall location came from studies of this communication. Gold et al. (9) in which [3H]glucose was incorMATERIALS AND METHODS porated into nigeran, locating the polymer by Growth of organism. A. awamori Nakazawa QM autoradiography and electron microscopy. 873 (A. luchuensis3 was grown from spores aerobiRecently, Sundararajan et al. (20) developed cally in submerged cultures at room temperature a technique for growing single crystals of this (22 to 23°C) in a complete medium consisting of (per polysaccharide, determined its unit cell dimen- liter of deionized water): sucrose (30.0 g), NH4NOQ sions, and showed by X-ray analysis that it (1.0 g), MgSO4 7H20 (0.3 g), KH2PO4 (1.26 g), yeast 691
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BOBBITT ET AL.
extract (0.01 g: Difco Laboratories, Detroit, Mich.). and peptone (0.01 g; Difco(. Nigeran content of the walls was elevated (10) by aseptically washing 4- to 5-day-old mycelia several times with sterile water and transferring them to sterile incomplete medium (nitrogen deficient) consisting of (per liter of water): glucose (30.0 g). MgSO< 7H.,O (0.3 g). and KH.,PO, (1.26 g). A. niger Van Tieghem NRRL 326 (ATCC 16888) was grown from spores in surface cultures at 22 to 23°C. The growth medium consisted of (per liter of water): sucrose (46.0 g(. tartaric acid (2.7 g), NH4NO8 (0.17 g), K.,CO, (0.47 g), MgCO:, (0.27 g). (NH)H2PO, (0.4 g), FeSO47H.,O (0.047 g). and ZnSO (0.047 g). Before sporulation, mycelial mats were harvested and stored frozen at -20-C for use in preparation of cell walls. In one experiment, the organisms were grown in submerged culture under conditions described above for A. awa mori. Preparation of cell walls and hyphae. Whole hyphae of A. acwamori were collected after 0, 48, and 96 h in nitrogen-free medium, washed several times with water, and freeze-dried. Hyphal walls of both A. niiger and A. aowoarmori were prepared as described by Bardalaye and Nordin (1). Nigeran extraction with boiling water. Freezedried walls or hyphae were extracted in water (5 mg/ml) for 15 min in a boiling-water bath. The suspension was centrifuged immediately while hot f'or 1 min at 1,200 x g; the hot supernatant fluid was withdrawn and passed through a glass-wool filter. The pellet was reextracted twice in an identical manner and freeze-dried when used for X-ray diffraction oi- enzymic digestion studies. Quantitative assays of water-extractable nigeran were conducted on 5 mg of wall specimens. The nigeran precipitates were suspended in water, and the quantity of the substance was determined by the phenolsulfuric acid test (8). Water-extractable values are expressed as a percentage of wall (dry weight). Nigeran extraction with alkali. Samples of A. niger and A. awa"iorn walls were extracted with boiling water as described above. The residues from this treatment were then further extracted by either of two additional procedures. Either they were extracted with cold 0.5 N NaOH at 4'C for 20 h followed by washing to neutrality with distilled water, or, alternatively, they were treated with 1 N NaOH at 100°C for 3 h (1). Wall residues were washed exhaustively with cold water to neutrality and freeze-dried. X-ray diffraction and enzymological experiments were conducted on these preparations. The extracts and washings from cold alkalitreated walls were pooled and neutralized with cold 1 N acetic acid. To this solution was added 4 volumes of cold absolute ethanol. The precipitate that formed was allowed to flocculate overnight at 4-C and then was collected, washed by centrifugation in 75% ethanol, and freeze-dried. Total carbohydrate in each sample was determined by the phenol-sulfuric acid method, and protein was measured by the Lowry method (14). Since glucan and nigeran are liberated together by alkaline extraction (25), the quantity of nigeran in the mixture was measured
J. BACTERIOL.
after complete digestion with mycodextranase (see below) by comparison with the reducing equivalents generated by total enzymic hydrolysis of pure nigeran standards. Total acid hydrolysis of 4 mg of cold alkali-extracted material was carried out under N., in 1 ml of 1 N H.SO in a Teflon-lined screw-cap tube at 110°C for 20 h. After hydrolysis, the sample was neutralized with BaCOj,, concentrated, and chromatographed on Whatman no. 1 paper (descending). The solvent system consisted of pyridine-ethyl acetatewater (2:5:7, vol/vol. upper phase). Sugars were visualized with alkaline AgNO: reagent (22(. Enzymatic hydrolysis of nigeran. Mycodextranase produced and purified according to the method of Tung et al. (26) was used to hydrolyze nigeran enzymatically. Suspensions of A. awaamori walls (3 mg/ml) or whole hyphal material (10 mg/ml) in 1 ml of 0.1 M acetate buffer, pH 4.5, were incubated at 40°C with 0.3 U (26) of mycodextranase. Hydrolysis was measured by the amount of reducing groups liberated (18) based on a glucose standard. Some samples were previously heated for 15 min in a boiling-water bath and cooled prior to enzymolysis. Boiled and unboiled samples of A. niger walls (15 mg/ml) were hydrolyzed under the same conditions described above. In certain experiments, A. niger walls were first incubated for 1 h at 40-C with enzyme to remove all exposed nigeran. Some samples were then lyophilized and heated (dry) at 110WC for 2 h before further enzymatic digestion. Ash content. The ash content of A. awarnori walls was determined by the Microanalysis Laboratorvy of the University of Massachusetts. Electron microscopy. A. awaniori walls containing 5, 12, or 28%7c of water-extractable nigeran (as determined above) were deposited and air dried on collodion-coated copper grids and shadowed with gold-palladium (60:40) at a 20° angle in a Denton DV-502 vacuum evaporator. Hyphal material used for transmission electron microscopy was fixed and stained in a 2% solution of KMnO4 at room temperature for 1 h, dehydrated in a graded acetone series, and embedded in Maraglas resin (19). Sections were cut on a Porter-Blum MT-2B ultramicrotome with glass knives and examined at 60 kV with a Philips 200 electron microscope. X-ray diffraction. X-ray diffraction data were recorded on flat-film cameras. using NaF as calibration for the "film to sample" distance. The cameras could be evacuated while the pattern was being recorded; this was the case when the pattern of dehydrated nigeran was determined while no vacuum was applied when measuring the hydrated nigeran pattern. The X-ray radiation from a copper target tube was suitably collimated through cylindrical pinholes and filtered through Ni foil to yield the CuK,, wavelength. The samples, in the form of flaky powders, were mounted in a special holder, which gave an average thickness of 1 to 2 mm after gentle pressing. The mounted specimen was dried for 12 h at 65WC under vacuum before being transferred to the diffraction camera where evacuation was immediately resumed if the dehydrated form of nigeran was being studied.
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for 96 h were so resistant to the electron beam that it could not penetrate, except at the fragmented ends of the wall (Fig. 3). The appearance of the broken end of the hypha suggests that the material responsible for electron opacity is associated with the external portion of the wall. Removal of the nigeran from such hyphae by boiling, enzymatic hydrolysis, or cold 0.5 N NaOH extraction decreased the opacity (Fig. 4). Whole hyphae (hot-water-extractable nigeran content, 4%/c of wall Idry weightl} were sectioned and observed by transmission electron microscopy. The wall was determined to be 0.25 to 0.30 ,lm thick, composed of a wide nonstaining layer and a narrow deeply stained outer layer (Fig. 5). Similar observations have been recorded by Tsukahara and Yamada (24) with A. n iger hyphae. In contrast, hyphal walls containing 28% hot-water-extractable nigeran had only a wide nonstainable component (Fig. 6). Total wall thickness of cells with these differing amounts of nigeran appeared to be essentially the same. In contrast to hyphae RESULTS with lower nigeran contents, those with 28% Nigeran formation. Surface culture condi- polysaccharide were much more difficult to tions and composition of the A. niger medium section. Glass knives dulled after cutting only were such that, at the time these cells were a few sections. X-ray diffraction of A. awarnori hyphal harvested, the amount of nigeran extractable with boiling water equaled approximately 5 to walls. The results of these experiments are 6%Ic of the wall's dry weight. This value corre- given in Tables 1 and 2. Crystalline chitin and lates with quantities reported previously (25). nigeran could be identified in both the hydrated Whether this organism is grown in submerged and the dry wall samples. The presence of nior surface culture, it accumulates nigeran geran as a distinct crystalline phase shows that it is present in domains where one dimension, when shifted to nitrogen-deficient medium. The walls of A. awamnori (submerged culture) which was studied, is at least 5 to 10 nm thick. contained approximately 4% of their dry weight The X-ray pattern of one wall specimen (samas hot-water-extractable nigeran at the time ple E, Table 2) was recorded by placing wet they were transferred to nitrogen-free medium. wall paste in a glass capillary tube. Since this These low polymer contents in the walls of sample contained only 6% nigeran (based on both organisms reflect relatively high levels of the dry weight of the sample) and the paste nitrogen in their growth medium at the time was 90% water, detection of hydrated crystalcells were harvested. When A. awamori cells line nigeran in this experiment shows the were transferred to the incomplete (nitrogen method's sensitivity and is a testimony to the free) medium, the nigeran content of the cells well-developed crystallinity of the nigeran in increased. After 96 h under these conditions, situ. Interestingly, this preparation also hot-water-extractable nigeran accounted for showed one reflection caused by /-(l1-*3)-glu28% of the wall's dry weight, a sevenfold in- can. X-ray studies of A. niger walls (data not shown) also indicated a crystalline structure crease. Electron microscopy of A. awamori walls. for the polymer in this organism. Finally, fresh, intact hyphae not subjected Shadowed preparations of hyphal walls, at the time of their transfer to incomplete medium, to freeze-drying or other treatments were exreveal a rather typical outer surface (Fig. 1). amined. Reflections, typical of hydrated crysAfter 48 h in nitrogen-free medium, the walls talline nigeran, were obtained. It is remarkable have become more opaque to the electron beam how easily the diffraction rings of the dry (Fig. 2). Hot-water-extractable nigeran content nigeran crystal form were recognizable and of this preparation was approximately 12 to reproducible with respect to relative intensity. 14% of the wall's dry weight. The walls of Although many of the chitin and nigeran remycelia that had been in nitrogen-free medium flections overlap, it was always possible to
Exposure times were usually 6 to 24 h. When the hydrated form of nigeran was being studied, a drop of water was added to the mounted sample. After air drying overnight at room humidity. the conditioned sample was placed in the diffraction apparatus, and the X-ray pattern was recorded without applying a vacuum. A pure preparation of nigeran extracted from A. niger was used as reference material in this study. It yielded typical hydrated and dry lattice diffractions, corresponding to the following unit cells (20, 2-1)-() dry: a, 1.775 nm; b, 0.60 nm; c (fiber axis), 1.462 nm; and (ii) hydrated: a, 1.76 nm; b, 0.735 nm; c (fiber axis), 1.34 nm. The spacings corresponding to those calculated for these unit cells are included in Tables 1 and 2 for comparison with the observations on various experimental samples. Because these cell walls also include a certain percentage of chitin, some reflections of the (a-form of chitin (5) are included in Tables 1 and 2 for comparison and identification. Many of these reflections overlap with those of nigeran, and none corresponds to /3-chitin (4). This finding agrees with Rudall, who reports (17) that fungal chitin is exclusively in the a-crystalline form.
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FIG. 1-4 Shadowed preparations of A. awamori hyphal walls. Bar = 1 ,im. FIG. 1. Photographed at time of transfer of cells to incomplete medium, showing a typical fungal outer surface. The water-extractable nigeran equals 4 to 6% of the wall weight at this time. FIG. 2. Photographed after 48 h in incomplete medium. Water-extractable nigeran equals 12 to 14% of the wall weight. Note the change in outer surface texture when compared to Fig. 1 and the increased opacity to the electron beam. 694
FIG. 3. Photographed after 96 h in incomplete medium. Water-extractable nigeran equals approximately 28% of the dry weight of the hyphal wall. The electron beam is unable to penetrate the hyphal wall. Appearance of broken hyphal ends suggests the increased opacity is associated with the external surface of the wall (arrow). FIG. 4. Cell walls containing 28% water-extractable nigeran, treated with mycodextranase to partially remove nigeran. Note the decrease in opacity of the wall compared with Fig. 3. 695
696
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BOBBITT ET AL.
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FIG. 5 and 6. Thin sections of permanganate-fixed A. awamori hyphal walls. DL, Dark outer layer; W, nonstaining cell wall; P, plasma membrane; and C, cytoplasm. Bar = 0.5 Am. FIG. 5. Photographed at the time of transfer of cells to incomplete medium. The wall is approximately 0.25 gum thick. Two distinct layers, one dark (outermost) and one light, can be obserLed. FIG. 6. Photographed after 96 h in incomplete medium. The total wall thickness is approximately the same as in Fig. 5, but the darker-staining component is absent.
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697
TABLE 1. Interplanar distances for dehydrated cell wall samples of A. awamori Interplanar distance (nm)
Samplesa
Nigeran (calcu- 1.128 lated) 1.13 Nigeran (observed) a-Chitin (calculated) A B C D
0.370
0.345
0.312
0.295
0.248
0.876 0.755 0.529 0.460 0.414 0.371
0.343
0.314
0.292
0.248
0.280
0.248
0.281 0.282 0.301 0.282
0.254 0.255 0.250 0.251
0.887
0.759
0.464
0.878
0.932 0.930 0.930
0.343
0.461
0.942
1.13
0.421
0.753
0.544
0.457
0.752 0.781
0.456 0.459
i|Ij
0.457
0.415 0.413 0.418 0.416
0.373
0.372 0.374 0.376
0.336 0.334 0.343 0.338
0.312
0.315
Samples A-D were freeze-dried after treatments. A, 120-h hyphae, freeze-dried; B, 120-h hyphae, boiled 15 min, freeze-dried; C, 96-h hyphae, heated at 105°C for 15 min; D, 96-h hyphae, treated as in C then extracted 36 h in a Soxhlet with water at the boiling point. a
TABLE 2. Interplanar distances for hydrated cell wall samples of A. awamori Samplesa Nigeran (calculated) Nigeran (observed) a-Chitin (calculated) A B C D E
0.942 0.927 0.967 0.956 0.988 1.295b
Interplanar distances (nm) 0.880 0.736 0.685 0.495 0.477 0.418 0.360 0.339 0.311 0.288 0.257 0.223 0.481 0.421 0.358 0.339 0.309 0.288 0.258 0.222 0.882 0.739 0.608 0.267 0.257 0.421 0.337 0.496 0.750 0.608 0.489 0.737 0.616 0.499 0.744 0.616 0.507 0.505 0.595 0.492
0.420 0.418 0.423 0.356 0.420 0.418
0.338 0.337 0.338 0.343
0.317 0.270 0.257 0.320 0.268 0.257 0.270 0.312 0.275 0.251 0.257
a Samples A through D were freeze-dried after treatments. A, 120-h hyphae, freeze-dried; B, 120-h hyphae, boiled 15 min, freeze-dried; C, 96-h hyphae, heated at 105°C for 15 min; D, 96-h hyphae, treated as in C then extracted 36 h in a Soxhlet with water at the boiling point; E, 0-h hyphae, no treatment. b ( 1,3)-,o-D-Glucan.
state that the latter's diffraction dominated in the cases where dry nigeran crystal form was involved. This dominance resulted from strong reflections at 0.755,' 0.414, and 0.371 nm that do not overlap with chitin diffraction rings. In hydrated form, the relative intensities of various nigeran reflections were less reproducible. Thus, the actual interplanar spacings were the same, but their intensities varied as a function of the various drying treatments. This suggests that internal strains in the polyphase structure of the cell wall hinder insertion of a reproducible and equilibrium water content in the nigeran lattice. With pure nigeran crystals, it has been shown (21) that the hydrate contains 2 mol of water per glucose unit. An important finding in this study is persistence of a nigeran diffraction pattern in the cell walls even after a 36-h extraction with boiling water (Table 1, sample D) and cold 0.5 N NaOH (data not shown). This inaccessible fraction is protected from extraction by its interaction with other wall constituents. Alkaline extraction of hyphal nigeran. Walls fromA. awamori that had been in incomplete medium for 96 h were extracted with boiling water. Nigeran that was removed ac-
counted for 28% of the wall (dry weight). The residue was treated with cold 0.5 N NaOH, as described above. This procedure resulted in diminution of the X-ray diffraction pattern intensity for residual nigeran when compared with an untreated control. However, a more drastic hot-alkali extraction was required to eliminate nigeran completely from the specimens examined. This latter treatment released an additional 11% of wall weight as nigeran. Evidence that some nigeran is in fact released from the wall by cold alkali was obtained by mycodextranase digestion of the neutralized extract. In a similar experiment, digestion of the walls with mycodextranase also reduced the diffraction pattern intensity but did not eliminate it. In all cases, the diffraction pattern of chitin was intensified relative to untreated controls. Cold-alkali extraction of nigeran from these walls also caused release of an a-glucan fraction, as determined by its optical rotation and by paper chromatography of the total acid hydrolysate of the material. This fraction accounted for about 9% of the walls (dry weight). Approximately 8 mg of protein (1% of the weight of the wall) was also released. These
698
BOBBITT ET AL.
J. BACTERIOL.
results strongly suggest that this a-glucan (and of polymer accessible to the enzyme were inpossibly the protein) interacts structurally with creased by boiling (Fig. 8). some portion of the A. awamori wall nigeran Not all of the nigeran in A. niger walls (6% in situ, preventing its removal either by boiling TABLE 3. Distribution of nigeran in A. niger and A. water or mycodextranase. awamori hyphal walls" Boiling-water and hot-alkali extractions of A. niger walls were also conducted. Table 3 Total nigeran removed Total nigeran summarizes nigeran distribution in A. niger by l9 content (mg/100 and A. awamori walls in terms of its susceptimg of wall Hot 1N Boiling Mycobility to removal by boiling water, hot 1 N [dry weight]) NaOH water dextraNaOH, and mycodextranase. nase Mycodextranase-catalyzed release of ni- A. awamori 39 100 72 78 geran from hyphal walls. Susceptibility of A. A. niger 11 100 54 39 awamori walls, containing 28% hot-water-exA. awamori walls were prepared from cells tractable nigeran, to enzymatic hydrolysis was not increased by boiling either walls or whole which were grown in submerged culture and then shifted to nitrogen-deficient medium for 96 h. A. hyphae (Fig. 7). On the other hand, with A. niger walls prepared from cells grown in surniger walls (4 to 6% hot-water-extractable ni- face culture were on complete medium. For details see geran), both the rate of attack and total amount the text. a
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Time (min)
60
70
80
90
FIG. 7. Enzymatic digestion of walls and hyphae of A. awamori containing 28% of wall weight as hotwater-extractable nigeran. Duplicate samples of walls (3 mg) or hyphae (10 mg) were suspended in 1 ml of 0.1 M acetate buffer, pH 4.5. One member of each pair was heated in a boiling-water bath for 15 min and cooled. After preincubation at 400C for 5 min, 0.3 U of mycodextranase was added to each tube, and the reducing equivalents liberated were measured as a function of time.
NIGERAN IN ASPERGILLUS WALLS
VOL. 132, 1977
699
1.050 a)
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.150
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10
20
30
40
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60
70
80
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Time (min) FIG. 8. Enzymic digestion of A. niger walls containing 6% hot-water-extractable nigeran. Walls (15 mg) were suspended in 1 ml of 0.1 M acetate buffer, pH 4.5. Analysis was conducted exactly as described in the legend for Fig. 7.
hot-water extractable) was accessible to hydro- This treatment also caused enzymic hydrolysis lytic attack by mycodextranase. This conclu- of the same total amount of nigeran. In this sion is from data in Fig. 9. Walls were first experiment, the zero-time sample was assayed preincubated with mycodextranase, as de- for reducing power after boiling (but before scribed above, until no more reducing groups addition of enzyme) and found negative. The effect of dry-heat treatment on walls were liberated (usually 1 h). The residue was washed with water, lyophilized, weighed, and after a preincubation with mycodextranase is suspended in two equal portions of buffer. Por- illustrated in Fig. 9B. This experiment was tions of the two suspensions were analyzed for conducted in a manner identical to that shown reducing power before (zero time) and after in Fig. 9A except that after preincubation and lyophilization the walls were subjected to dry addition of mycodextranase (Fig. 9A). A second treatment with enzyme (dashed heat at 110°C for 2 h and then cooled. This type curve) failed to hydrolyze additional polymer. of heating somehow altered the structure (or However, boiling the walls followed by cooling accessibility) of the polysaccharide such that a and addition of fresh enzyme (at 60 min) af- certain portion can now be attacked without boiling (dashed curve). However, the total forded the release of hydrolysis products. The solid curve describes the result when amount of nigeran released by boiling water the suspended wall preparation was simply (arrow, dashed, and solid curves) was identical boiled and cooled and fresh enzyme was added. in both cases.
700
J. BACTERIOL.
BOBBITT ET AL.
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30 40 50 Time (min)
60
70
80
90
FIG. 9. Enzymic digestion of A. niger walls containing 6% hot-water-extractable nigeran. Walls (100 mg) were first digested for 1 h at 40°C with 0.3 U of mycodextranase in 1 ml of 0.1 M acetate buffer, pH 4.5, to remove polysaccharide accessible to the enzyme. The walls were then washed in water to remove the enzyme and digestion products and lyophilized. (A) Duplicate 15-mg samples of predigested walls were submitted to a second digestion with and without boiling. Reducing equivalents liberated were measured as a function of time. In the case of the zero-time sample (solid curve), reducing power was measured after boiling but before addition of enzyme. At 60 min (arrow, dashed curve), the unboiled sample was boiled and cooled, and fresh enzyme was added. (B) Experiment conducted exactly as described in the legend for Fig. 9A except that the walls were dry heated at 110°C for 2 h after lyophilization.
DISCUSSION The experiments described here with A. awamori and A. niger demonstrate that nigeran chains occupy at least three distinct domains or configurations in the hyphal wall and that the polymer's organization in situ is highly crystalline (Tables 1 through 3, Fig. 7 through 9). While it cannot be ruled out that a
certain proportion of noncrystalline nigeran may be present in the wall, all three domains contain the crystalline polymer. The data in Table 3 show that one portion, or fraction, is extractable with boiling water, whereas a second is removed only upon additional treatment with hot, dilute alkali. Also, depending on the organism and growth conditions, different percentages of hot-water-extractable nigeran are
VOL. 132, 1977
accessible to enzymic degradation. X-ray diffraction data (Tables 1 and 2) prove that even prolonged (36 h) boiling of hyphal walls fails to extract a certain portion of the polymer and confirm the results in Table 3. Finally, coupled water extraction and enzymatic studies reveal that not all boiling-water-extractable nigeran is susceptible to digestion in situ by mycodextranase (Fig. 9A). An external location for some of the wall nigeran is suggested by the following observations: boiling of nigeran-enriched A. awamori hyphae or walls does not increase either the rate of subsequent nigeran digestion by mycodextranase or the total amount released (Fig. 7). Therefore, boiling does not "unmask" the nigeran to attack by the enzyme. Since the A. awamori cells used contain massive amounts of polysaccharide accessible to the enzyme, any differences between boiled and unboiled walls in either the rate of attack or in the relative amount of nigeran hydrolyzed are small enough that they are not readily evident in nigeran-enriched walls. Therefore, the bulk of the polymer is located in the external face of the wall. In contrast, A. niger walls, bearing a much lower nigeran content, do show differences in the kinetics of attack by mycodextranase and the total amount liberated, depending on whether the walls are boiled first (Fig. 8). However, this difference in proportion of waterextractable nigeran susceptible to enzymic attack allows the conclusion also that some of the nigeran is exposed to the exterior of the wall. The fraction digested without boiling obviously represents that portion directly exposed to the wall's surface. Beneath this exposed fraction is a second crystalline domain inaccessible to the enzyme but removable with boiling water (Fig. 9A and B). It is these two domains which Tung and Nordin observed in their earlier study (25). Possibly, this more inward-positioned fraction is somehow intercalated with another wall constituent that impedes enzymatic hydrolysis but does not prevent nigeran's release with boiling water. The appearance of broken ends of hyphae in shadowed preparations (Fig. 3) suggests that the opaque component is located on an external surface. This opacity is not the result of salt accumulation from the medium, since the ash content of such walls is less than 0.5%. Shadowed preparations of walls that have first undergone exhaustive digestion with mycodextranase reveal a reduction in wall opacity, confirming that the crystalline nigeran is responsible for this effect. Finally, an observed intensification of chitin diffraction patterns
NIGERAN IN ASPERGILLUS WALLS
701
after renoval of nigeran from wall specimens of both A. niger and A. awamori argues for its external location. X-ray diffraction studies support the conclusion that increased opacity observed in shadowed preparations of nigeran-enriched walls is due to the highly crystalline nature of the nigeran and that crystallinity of the nigeran per se does not confer resistance to enzymic attack. A decrease in intensity of an Xray diffraction pattern after enzymic digestion of wall material argues that crystalline material is being degraded. However, comparative studies of relative rates of attack by mycodextranase on preparations of pure crystalline and noncrystalline nigeran in vitro must await further experimentation. Wessels et al. reported (27) that a-(1( 3)glucan in Schizophyllum commune hyphal walls exists in a crystalline array in situ after hot-water extraction. These authors (27) and Jelsma and Kreger also demonstrated that isolated fungal 3- ( 1-3, /3-(1 -*6)-glucan becomes crystalline after treatment with hot, dilute mineral acid (11). It is suggested that molecular reorganization of polymer molecules into crystallites occurs as a result of acid hydrolysis of some branch points. Troy and Koffler (23) noted chitin as a crystalline wall component of Penicillium chrysogenum, another fungus that produced nigeran (16). However, they did not attempt an exhaustive analysis of these walls for other crystalline materials. Since nigeran is a readily hydrated material, it is not surprising to find that previously undried hyphae exhibit the hydrate diffraction pattern. Reversibility of the diffraction patterns, hydrate dry, is also in keeping with observations on pure nigeran (20, 21). On the basis of the latter studies, however, crystalline reversibility does not necessarily imply morphological reversibility. Observations (20, 21) on nigeran single crystals have shown that dehydration is accompanied by formation of cracks in the thin lamellar crvstals that are from 10 to 20 nm wide. It is unlikely that these would be eliminated by rewetting even though the lattice transforms to the hydrate. Such morphological changes may have important consequences for enzymic biodegradation of cell walls that contain a significant content of crystalline polymers. Whether such alterations are caused by the dry-heat treatment (Fig. 9B) is at present unknown, but experiments attempting to answer this question are being pursued. Gold et al. (9) suggested that walls of A. aculeatus thickened with increased age and when growth ceases. In this study, we found no significant increase in wall thickening asso-
702
BOBBITT ET AL.
ciated with nigeran deposition. These results can be explained by: (i) replacement of wall components by nigeran, (ii) intercalation by nigeran of existing wall material, or (iii) a combination of the two processes. Morton et al. (15) reported that levels of intracellular proteinases of several fungi, including A. niger, increase dramatically within several hours of being placed in a nitrogen-free medium. Nitrogen-starved A. niger and A. awamori cells may transport nitrogen-containing components out of the wall and use them for critical cellular processes. The role that nigeran plays within the fungal wall is unknown. It has been suggested (10) that the nigeran may function in maintaining integrity of the cell surface under adverse environmental conditions. Another possible function is as an overflow product that accumulates when nitrogen metabolism ceases (9). We suggest the possibility that nigeran may be a storage product, utilized during cleistothecium development, but that the enzyme necessary for its breakdown has been lost by mutation. The loss of such an enzyme would explain the findings of Dox (6) and Gold et al. (10) that nigeran is not broken down during starvation. Among species of Aspergillus and Penicillium that Reese and Mandels (16) reported produce nigeran, only one, Penicillium jauanicum, is known to possess a sexual stage in its life cycle. Zonneveld (28) has shown clearly that a correlation exists between glucose utilization by Aspergillus nidulans, formation of a-1,3glucan in the hyphal wall, a-1,3-glucanase activity, breakdown of the hyphal wall, and the production of cleistothecia (sexual stage). if, in A. niger andA. awamori, the ability to produce mycodextranase has been lost, nigeran would accumulate in the wall, and cleistothecia development would be blocked. This possible explanation for nigeran accumulation is under examination in our laboratories. ACKNOWLEDGMENTS This research was supported by grants from the National Science Foundation (BMS 75-22424) (to John H. Nordin and the National Research Council of Canada (to R. H. Marchessault). We wish to thank Stanley Holt of the University of Massachusetts Microbiology Department for use of the electron microscope facilities. LITERATURE CITED 1. Bardalaye, P. C., and J. H. Nordin. 1976. Galactosaminogalactan from cell walls of Aspergillus niger. J.
Bacteriol. 125:655-669. 2. Barker, S. A., E. J. Bourne, D. M. O'Mant, and M. Stacey. 1957. Studies of A. niger. VI. The separation and structures of oligosaccharides from nigeran. J.
J. BACTERIOL. Chem. Soc., p. 2448-2454. 3. Barker, S. A., E. J. Bourne, and M. Stacey. 1953. Studies of Aspergillus niger. I. The structure of the polyglucosan synthesized by Aspergillus niger 152. J. Chem. Soc., p. 3084-3090. 4. Blackwell, J. 1969. Structure of 83-chitin or parallel chain systems of poly-3--(1-4)-N-acetyl-D-glucosamine. Biopolymers 7:281-298. 5. Carlstrom, D. 1957. The crystal structure of a-chitin poly-N-acetyl-D-glucosamine. J. Cell Biol. 3:669-683. 6. Dox, A. W. 1915. Influence of autolysis on the mycodextran content of Aspergillus niger. J. Biol. Chem. 20:83-85. 7. Dox, A. W., and R. E. Neidig. 1914. Mycodextran, a new polysaccharide in Penicillium expansum. J. Biol. Chem. 18:167-175. 8. Dubois, M., K. A. Gilles, J. D. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method of determintion of sugars and related substances. Anal. Chem.
28:350-356. 9. Gold, M. H., S. Larson, I. H. Segel, and C. R. Stocking. 1974. Intracellular localization of nigeran in the wall of Aspergillus aculeatus by autoradiography with the electron microscope. J. Bacteriol. 118:1176-1178. 10. Gold, M. H., D. L. Mitzel, and I. H. Segel. 1973. Regulation of nigeran accumulation by Aspergillus aculeatus. J. Bacteriol. 113:856-862. 11. Jelsma, J., and D. R. Kreger. 1975. Ultrastructural observations on (1-3)-,3-D-glucan from fungal cell walls. Carbohydr. Res. 43:200-203. 12. Johnston, I. R. 1965. The composition of the cell wall of Asperigillus niger. Biochem. J. 96:651-657. 13. Kreger, D. R., and M. Kopecka. 1976. On the nature and formation of the fibrillar nets produced by protoplasts of Saccharomyces cerevisiae in liquid media: an electron microscopic, X-ray diffraction and chemical study. J. Gen. Microbiol. 92:207-220. 14. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 15. Morton, A. G., A. G. F. Dickerson, and D. J. F. England. 1960. Changes in enzyme activity of fungi during nitrogen starvation. J. Exp. Bot. 11:116-128. 16. Reese, E. T., and M. Mandels. 1964. A new a-glucanase mycodextranase. Can. J. Microbiol. 10:103-114. 17. Rudall, K. M. 1969. Chitin and its association with other molecules. J. Polym. Sci. 28:83-102. 18. Somogyi, M. 1945. A new reagent from the determination of sugars. J. Biol. Chem. 160:61-68. 19. Spurlock, B. O., V. C. Kattine, and J. Freeman. 1963. Technical modifications in Maraglas embedding. J. Cell Biol. 17:203-207. 20. Sundararajan, P. R., R. H. Marchessault, G. J. Quigley, and A. Sarko. 1973. Crystalline chain conformation of mycodextran. J. Am. Chem. Soc. 95:2001-2008. 21. Taylor, K. J., H. Chanzy, and R. H. Marchessault. 1975. Electron diffraction for hydrated crystalline biopolymers: nigeran. J. Mol. Biol. 92:165-167. 22. Trevelyn, W. E., D. P. Proctor, and J. S. Harrison. 1950. Detection of sugars in paper chromatograms. Nature (London) 166:444-445. 23. Troy, F. A., and H. Koffler. 1969. The chemistry and molecular architecture of the cell walls of Penicillium chrysogenum. J. Biol. Chem. 244:5563-5576. 24. Tsukahara, T., and M. Yamada. 1965. Cytological structure of Aspergillus niger by electron microscopy. Jpn. J. Microbiol. 9:35-48. 25. Tung, K. K., and J. H. Nordin. 1967. Evidence for a buried location of nigeran in cell wall of Aspergillus niger. Biochem. Biophys. Res. Commun. 28:519-524. 26. Tung, K. K., A. Rosenthal, and J. H. Nordin. 1971. Enzymes that hydrolyze fungal cell wall polysaccha-
VOL. 132, 1977 rides. Purification and properties of mycodextranase an a(1-4) glucanase of Penicillium melinii. J. Biol. Chem. 246:2722-2732. 27. Wessels, J. G. H., D. R. Kreger, R. Marchant, B. A. Regensburg, and 0. M. H. DeVries. 1972. Chemical and morphological characterization of the hyphal
NIGERAN IN ASPERGILLUS WALLS
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wall surface of the Basidiomycete Schizophyllum commune. Biochim. Biophys. Acta 273:346-358. 28. Zonneveld, J. M. 1972. Morphogenesis in Aspergillus nidulans. The significance of a-1,3-glucan of the cell wall and a-1,3-glucanase for cleistothecium development. Biochim. Biophys. Acta 273:174-187.
JOURNAL OF BACTERIOLOGY, Nov. 1977, p. 691-703 Copyright © 1977 American Society for Microbiology
Printed in U.S.A.
Distribution and Conformation of Crystalline Nigeran in Hyphal Walls of Aspergillus niger and Aspergillus awamori T. F. BOBBITT,l J. H. NORDIN,' M. ROUX,2 J. F. REVOL,2 AND R. H. MARCHESSAULT2 Department of Biochemistry, Uniuersity of Massachusetts, A mherst, Massachusetts 01003,' and Department of Chemistry, University of Montreal, Montreal, Quebec, H3C 3V1 Canada2
Received for publication 28 July 1977
Hyphal walls ofAspergillus awamori containing increased amounts of the aglucan, nigeran, became correspondingly more opaque when viewed in the electron microscope as shadowed preparations. However, increased polymer deposition was not accompanied by any significant change in wall thickness. The nigeran of both A. awamori and Aspergillus niger occurred in situ in a crystalline conformation identical to that of single crystals prepared with pure polysaccharide. Furthermore, this polymer was the dominant crystalline material in the hyphae whether or not they were enriched in nigeran. Enzymic digestion of nigeran in A. niger and A. awamori revealed that the bulk of the polymer was exposed to the cell's exterior. However, a certain fraction was accessible to enzymic attack only after the wall was treated with boiling water. A third portion, detectable only by X-ray diffraction, was associated with other components and could not be extracted, even with prolonged boiling. It was removed by hot, dilute alkali and was associated in the wall with another glucan fraction. Dry heating of A. niger walls altered their susceptibility to enzymic digestion of nigeran in situ. It is proposed that this treatment introduces interstices in the crystal surface that facilitate attack. Nigeran, a hot-water-soluble, linear, alter- has a 21 helical conformation with four glucose nating (1---*3), (1-*4)-a-D-glucan (2, 3) was first residues per helix turn. Using electron diffracisolated by Dox and Neidig (6, 7) from Penicil- tion methods with these crystals, Taylor et al. lium expansum and Aspergillus niger. Reese (21) elucidated the relationship of lattice water and Mandels (16), after surveying a large num- to the polymer's overall crystal structure and ber of fungi, found. that nigeran was synthe- demonstrated that these crystals can exist in sized by only a few species of Aspergillus and either "dry" or "hydrated" forms, depending Penicillium. Both Johnston (12) and Tung and upon the drying history. Nordin (25), studying hypal wall structure, conGold et al. (10) found that the amount of cluded that this glucan is a wall component of nigeran present in Aspergillus hyphae is enA. niger. The latter investigators also noted hanced dramatically by nitrogen deprivation. that nigeran might occupy a buried location Furthermore, they demonstrated that the polywithin the wall, since mycodextranase, an en- saccharide is not utilized as a carbon source zyme specific for nigeran, hydrolyzed the glu- during starvation. We have probed the hyphal wall architecture can to a much greater extent when the wall suspensions were first heated to 100CC. How- of A. awamori and A. niger by a combination ever, an alternative explanation for the results of X-ray diffraction analysis, enzymolysis, and was proposed (25), i.e., nigeran might exist in electron microscopy to gain additional insight some crystalline array that conferred resist- into the structural role played by this polymer. ance to enzymic hydrolysis. Additional confir- The results of these studies form the basis of mation of a wall location came from studies of this communication. Gold et al. (9) in which [3H]glucose was incorMATERIALS AND METHODS porated into nigeran, locating the polymer by Growth of organism. A. awamori Nakazawa QM autoradiography and electron microscopy. 873 (A. luchuensis3 was grown from spores aerobiRecently, Sundararajan et al. (20) developed cally in submerged cultures at room temperature a technique for growing single crystals of this (22 to 23°C) in a complete medium consisting of (per polysaccharide, determined its unit cell dimen- liter of deionized water): sucrose (30.0 g), NH4NOQ sions, and showed by X-ray analysis that it (1.0 g), MgSO4 7H20 (0.3 g), KH2PO4 (1.26 g), yeast 691
692
BOBBITT ET AL.
extract (0.01 g: Difco Laboratories, Detroit, Mich.). and peptone (0.01 g; Difco(. Nigeran content of the walls was elevated (10) by aseptically washing 4- to 5-day-old mycelia several times with sterile water and transferring them to sterile incomplete medium (nitrogen deficient) consisting of (per liter of water): glucose (30.0 g). MgSO< 7H.,O (0.3 g). and KH.,PO, (1.26 g). A. niger Van Tieghem NRRL 326 (ATCC 16888) was grown from spores in surface cultures at 22 to 23°C. The growth medium consisted of (per liter of water): sucrose (46.0 g(. tartaric acid (2.7 g), NH4NO8 (0.17 g), K.,CO, (0.47 g), MgCO:, (0.27 g). (NH)H2PO, (0.4 g), FeSO47H.,O (0.047 g). and ZnSO (0.047 g). Before sporulation, mycelial mats were harvested and stored frozen at -20-C for use in preparation of cell walls. In one experiment, the organisms were grown in submerged culture under conditions described above for A. awa mori. Preparation of cell walls and hyphae. Whole hyphae of A. acwamori were collected after 0, 48, and 96 h in nitrogen-free medium, washed several times with water, and freeze-dried. Hyphal walls of both A. niiger and A. aowoarmori were prepared as described by Bardalaye and Nordin (1). Nigeran extraction with boiling water. Freezedried walls or hyphae were extracted in water (5 mg/ml) for 15 min in a boiling-water bath. The suspension was centrifuged immediately while hot f'or 1 min at 1,200 x g; the hot supernatant fluid was withdrawn and passed through a glass-wool filter. The pellet was reextracted twice in an identical manner and freeze-dried when used for X-ray diffraction oi- enzymic digestion studies. Quantitative assays of water-extractable nigeran were conducted on 5 mg of wall specimens. The nigeran precipitates were suspended in water, and the quantity of the substance was determined by the phenolsulfuric acid test (8). Water-extractable values are expressed as a percentage of wall (dry weight). Nigeran extraction with alkali. Samples of A. niger and A. awa"iorn walls were extracted with boiling water as described above. The residues from this treatment were then further extracted by either of two additional procedures. Either they were extracted with cold 0.5 N NaOH at 4'C for 20 h followed by washing to neutrality with distilled water, or, alternatively, they were treated with 1 N NaOH at 100°C for 3 h (1). Wall residues were washed exhaustively with cold water to neutrality and freeze-dried. X-ray diffraction and enzymological experiments were conducted on these preparations. The extracts and washings from cold alkalitreated walls were pooled and neutralized with cold 1 N acetic acid. To this solution was added 4 volumes of cold absolute ethanol. The precipitate that formed was allowed to flocculate overnight at 4-C and then was collected, washed by centrifugation in 75% ethanol, and freeze-dried. Total carbohydrate in each sample was determined by the phenol-sulfuric acid method, and protein was measured by the Lowry method (14). Since glucan and nigeran are liberated together by alkaline extraction (25), the quantity of nigeran in the mixture was measured
J. BACTERIOL.
after complete digestion with mycodextranase (see below) by comparison with the reducing equivalents generated by total enzymic hydrolysis of pure nigeran standards. Total acid hydrolysis of 4 mg of cold alkali-extracted material was carried out under N., in 1 ml of 1 N H.SO in a Teflon-lined screw-cap tube at 110°C for 20 h. After hydrolysis, the sample was neutralized with BaCOj,, concentrated, and chromatographed on Whatman no. 1 paper (descending). The solvent system consisted of pyridine-ethyl acetatewater (2:5:7, vol/vol. upper phase). Sugars were visualized with alkaline AgNO: reagent (22(. Enzymatic hydrolysis of nigeran. Mycodextranase produced and purified according to the method of Tung et al. (26) was used to hydrolyze nigeran enzymatically. Suspensions of A. awaamori walls (3 mg/ml) or whole hyphal material (10 mg/ml) in 1 ml of 0.1 M acetate buffer, pH 4.5, were incubated at 40°C with 0.3 U (26) of mycodextranase. Hydrolysis was measured by the amount of reducing groups liberated (18) based on a glucose standard. Some samples were previously heated for 15 min in a boiling-water bath and cooled prior to enzymolysis. Boiled and unboiled samples of A. niger walls (15 mg/ml) were hydrolyzed under the same conditions described above. In certain experiments, A. niger walls were first incubated for 1 h at 40-C with enzyme to remove all exposed nigeran. Some samples were then lyophilized and heated (dry) at 110WC for 2 h before further enzymatic digestion. Ash content. The ash content of A. awarnori walls was determined by the Microanalysis Laboratorvy of the University of Massachusetts. Electron microscopy. A. awaniori walls containing 5, 12, or 28%7c of water-extractable nigeran (as determined above) were deposited and air dried on collodion-coated copper grids and shadowed with gold-palladium (60:40) at a 20° angle in a Denton DV-502 vacuum evaporator. Hyphal material used for transmission electron microscopy was fixed and stained in a 2% solution of KMnO4 at room temperature for 1 h, dehydrated in a graded acetone series, and embedded in Maraglas resin (19). Sections were cut on a Porter-Blum MT-2B ultramicrotome with glass knives and examined at 60 kV with a Philips 200 electron microscope. X-ray diffraction. X-ray diffraction data were recorded on flat-film cameras. using NaF as calibration for the "film to sample" distance. The cameras could be evacuated while the pattern was being recorded; this was the case when the pattern of dehydrated nigeran was determined while no vacuum was applied when measuring the hydrated nigeran pattern. The X-ray radiation from a copper target tube was suitably collimated through cylindrical pinholes and filtered through Ni foil to yield the CuK,, wavelength. The samples, in the form of flaky powders, were mounted in a special holder, which gave an average thickness of 1 to 2 mm after gentle pressing. The mounted specimen was dried for 12 h at 65WC under vacuum before being transferred to the diffraction camera where evacuation was immediately resumed if the dehydrated form of nigeran was being studied.
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for 96 h were so resistant to the electron beam that it could not penetrate, except at the fragmented ends of the wall (Fig. 3). The appearance of the broken end of the hypha suggests that the material responsible for electron opacity is associated with the external portion of the wall. Removal of the nigeran from such hyphae by boiling, enzymatic hydrolysis, or cold 0.5 N NaOH extraction decreased the opacity (Fig. 4). Whole hyphae (hot-water-extractable nigeran content, 4%/c of wall Idry weightl} were sectioned and observed by transmission electron microscopy. The wall was determined to be 0.25 to 0.30 ,lm thick, composed of a wide nonstaining layer and a narrow deeply stained outer layer (Fig. 5). Similar observations have been recorded by Tsukahara and Yamada (24) with A. n iger hyphae. In contrast, hyphal walls containing 28% hot-water-extractable nigeran had only a wide nonstainable component (Fig. 6). Total wall thickness of cells with these differing amounts of nigeran appeared to be essentially the same. In contrast to hyphae RESULTS with lower nigeran contents, those with 28% Nigeran formation. Surface culture condi- polysaccharide were much more difficult to tions and composition of the A. niger medium section. Glass knives dulled after cutting only were such that, at the time these cells were a few sections. X-ray diffraction of A. awarnori hyphal harvested, the amount of nigeran extractable with boiling water equaled approximately 5 to walls. The results of these experiments are 6%Ic of the wall's dry weight. This value corre- given in Tables 1 and 2. Crystalline chitin and lates with quantities reported previously (25). nigeran could be identified in both the hydrated Whether this organism is grown in submerged and the dry wall samples. The presence of nior surface culture, it accumulates nigeran geran as a distinct crystalline phase shows that it is present in domains where one dimension, when shifted to nitrogen-deficient medium. The walls of A. awamnori (submerged culture) which was studied, is at least 5 to 10 nm thick. contained approximately 4% of their dry weight The X-ray pattern of one wall specimen (samas hot-water-extractable nigeran at the time ple E, Table 2) was recorded by placing wet they were transferred to nitrogen-free medium. wall paste in a glass capillary tube. Since this These low polymer contents in the walls of sample contained only 6% nigeran (based on both organisms reflect relatively high levels of the dry weight of the sample) and the paste nitrogen in their growth medium at the time was 90% water, detection of hydrated crystalcells were harvested. When A. awamori cells line nigeran in this experiment shows the were transferred to the incomplete (nitrogen method's sensitivity and is a testimony to the free) medium, the nigeran content of the cells well-developed crystallinity of the nigeran in increased. After 96 h under these conditions, situ. Interestingly, this preparation also hot-water-extractable nigeran accounted for showed one reflection caused by /-(l1-*3)-glu28% of the wall's dry weight, a sevenfold in- can. X-ray studies of A. niger walls (data not shown) also indicated a crystalline structure crease. Electron microscopy of A. awamori walls. for the polymer in this organism. Finally, fresh, intact hyphae not subjected Shadowed preparations of hyphal walls, at the time of their transfer to incomplete medium, to freeze-drying or other treatments were exreveal a rather typical outer surface (Fig. 1). amined. Reflections, typical of hydrated crysAfter 48 h in nitrogen-free medium, the walls talline nigeran, were obtained. It is remarkable have become more opaque to the electron beam how easily the diffraction rings of the dry (Fig. 2). Hot-water-extractable nigeran content nigeran crystal form were recognizable and of this preparation was approximately 12 to reproducible with respect to relative intensity. 14% of the wall's dry weight. The walls of Although many of the chitin and nigeran remycelia that had been in nitrogen-free medium flections overlap, it was always possible to
Exposure times were usually 6 to 24 h. When the hydrated form of nigeran was being studied, a drop of water was added to the mounted sample. After air drying overnight at room humidity. the conditioned sample was placed in the diffraction apparatus, and the X-ray pattern was recorded without applying a vacuum. A pure preparation of nigeran extracted from A. niger was used as reference material in this study. It yielded typical hydrated and dry lattice diffractions, corresponding to the following unit cells (20, 2-1)-() dry: a, 1.775 nm; b, 0.60 nm; c (fiber axis), 1.462 nm; and (ii) hydrated: a, 1.76 nm; b, 0.735 nm; c (fiber axis), 1.34 nm. The spacings corresponding to those calculated for these unit cells are included in Tables 1 and 2 for comparison with the observations on various experimental samples. Because these cell walls also include a certain percentage of chitin, some reflections of the (a-form of chitin (5) are included in Tables 1 and 2 for comparison and identification. Many of these reflections overlap with those of nigeran, and none corresponds to /3-chitin (4). This finding agrees with Rudall, who reports (17) that fungal chitin is exclusively in the a-crystalline form.
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FIG. 1-4 Shadowed preparations of A. awamori hyphal walls. Bar = 1 ,im. FIG. 1. Photographed at time of transfer of cells to incomplete medium, showing a typical fungal outer surface. The water-extractable nigeran equals 4 to 6% of the wall weight at this time. FIG. 2. Photographed after 48 h in incomplete medium. Water-extractable nigeran equals 12 to 14% of the wall weight. Note the change in outer surface texture when compared to Fig. 1 and the increased opacity to the electron beam. 694
FIG. 3. Photographed after 96 h in incomplete medium. Water-extractable nigeran equals approximately 28% of the dry weight of the hyphal wall. The electron beam is unable to penetrate the hyphal wall. Appearance of broken hyphal ends suggests the increased opacity is associated with the external surface of the wall (arrow). FIG. 4. Cell walls containing 28% water-extractable nigeran, treated with mycodextranase to partially remove nigeran. Note the decrease in opacity of the wall compared with Fig. 3. 695
696
J. BACTERIOL.
BOBBITT ET AL.
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FIG. 5 and 6. Thin sections of permanganate-fixed A. awamori hyphal walls. DL, Dark outer layer; W, nonstaining cell wall; P, plasma membrane; and C, cytoplasm. Bar = 0.5 Am. FIG. 5. Photographed at the time of transfer of cells to incomplete medium. The wall is approximately 0.25 gum thick. Two distinct layers, one dark (outermost) and one light, can be obserLed. FIG. 6. Photographed after 96 h in incomplete medium. The total wall thickness is approximately the same as in Fig. 5, but the darker-staining component is absent.
NIGERAN IN ASPERGILLUS WALLS
VOL. 132, 1977
697
TABLE 1. Interplanar distances for dehydrated cell wall samples of A. awamori Interplanar distance (nm)
Samplesa
Nigeran (calcu- 1.128 lated) 1.13 Nigeran (observed) a-Chitin (calculated) A B C D
0.370
0.345
0.312
0.295
0.248
0.876 0.755 0.529 0.460 0.414 0.371
0.343
0.314
0.292
0.248
0.280
0.248
0.281 0.282 0.301 0.282
0.254 0.255 0.250 0.251
0.887
0.759
0.464
0.878
0.932 0.930 0.930
0.343
0.461
0.942
1.13
0.421
0.753
0.544
0.457
0.752 0.781
0.456 0.459
i|Ij
0.457
0.415 0.413 0.418 0.416
0.373
0.372 0.374 0.376
0.336 0.334 0.343 0.338
0.312
0.315
Samples A-D were freeze-dried after treatments. A, 120-h hyphae, freeze-dried; B, 120-h hyphae, boiled 15 min, freeze-dried; C, 96-h hyphae, heated at 105°C for 15 min; D, 96-h hyphae, treated as in C then extracted 36 h in a Soxhlet with water at the boiling point. a
TABLE 2. Interplanar distances for hydrated cell wall samples of A. awamori Samplesa Nigeran (calculated) Nigeran (observed) a-Chitin (calculated) A B C D E
0.942 0.927 0.967 0.956 0.988 1.295b
Interplanar distances (nm) 0.880 0.736 0.685 0.495 0.477 0.418 0.360 0.339 0.311 0.288 0.257 0.223 0.481 0.421 0.358 0.339 0.309 0.288 0.258 0.222 0.882 0.739 0.608 0.267 0.257 0.421 0.337 0.496 0.750 0.608 0.489 0.737 0.616 0.499 0.744 0.616 0.507 0.505 0.595 0.492
0.420 0.418 0.423 0.356 0.420 0.418
0.338 0.337 0.338 0.343
0.317 0.270 0.257 0.320 0.268 0.257 0.270 0.312 0.275 0.251 0.257
a Samples A through D were freeze-dried after treatments. A, 120-h hyphae, freeze-dried; B, 120-h hyphae, boiled 15 min, freeze-dried; C, 96-h hyphae, heated at 105°C for 15 min; D, 96-h hyphae, treated as in C then extracted 36 h in a Soxhlet with water at the boiling point; E, 0-h hyphae, no treatment. b ( 1,3)-,o-D-Glucan.
state that the latter's diffraction dominated in the cases where dry nigeran crystal form was involved. This dominance resulted from strong reflections at 0.755,' 0.414, and 0.371 nm that do not overlap with chitin diffraction rings. In hydrated form, the relative intensities of various nigeran reflections were less reproducible. Thus, the actual interplanar spacings were the same, but their intensities varied as a function of the various drying treatments. This suggests that internal strains in the polyphase structure of the cell wall hinder insertion of a reproducible and equilibrium water content in the nigeran lattice. With pure nigeran crystals, it has been shown (21) that the hydrate contains 2 mol of water per glucose unit. An important finding in this study is persistence of a nigeran diffraction pattern in the cell walls even after a 36-h extraction with boiling water (Table 1, sample D) and cold 0.5 N NaOH (data not shown). This inaccessible fraction is protected from extraction by its interaction with other wall constituents. Alkaline extraction of hyphal nigeran. Walls fromA. awamori that had been in incomplete medium for 96 h were extracted with boiling water. Nigeran that was removed ac-
counted for 28% of the wall (dry weight). The residue was treated with cold 0.5 N NaOH, as described above. This procedure resulted in diminution of the X-ray diffraction pattern intensity for residual nigeran when compared with an untreated control. However, a more drastic hot-alkali extraction was required to eliminate nigeran completely from the specimens examined. This latter treatment released an additional 11% of wall weight as nigeran. Evidence that some nigeran is in fact released from the wall by cold alkali was obtained by mycodextranase digestion of the neutralized extract. In a similar experiment, digestion of the walls with mycodextranase also reduced the diffraction pattern intensity but did not eliminate it. In all cases, the diffraction pattern of chitin was intensified relative to untreated controls. Cold-alkali extraction of nigeran from these walls also caused release of an a-glucan fraction, as determined by its optical rotation and by paper chromatography of the total acid hydrolysate of the material. This fraction accounted for about 9% of the walls (dry weight). Approximately 8 mg of protein (1% of the weight of the wall) was also released. These
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results strongly suggest that this a-glucan (and of polymer accessible to the enzyme were inpossibly the protein) interacts structurally with creased by boiling (Fig. 8). some portion of the A. awamori wall nigeran Not all of the nigeran in A. niger walls (6% in situ, preventing its removal either by boiling TABLE 3. Distribution of nigeran in A. niger and A. water or mycodextranase. awamori hyphal walls" Boiling-water and hot-alkali extractions of A. niger walls were also conducted. Table 3 Total nigeran removed Total nigeran summarizes nigeran distribution in A. niger by l9 content (mg/100 and A. awamori walls in terms of its susceptimg of wall Hot 1N Boiling Mycobility to removal by boiling water, hot 1 N [dry weight]) NaOH water dextraNaOH, and mycodextranase. nase Mycodextranase-catalyzed release of ni- A. awamori 39 100 72 78 geran from hyphal walls. Susceptibility of A. A. niger 11 100 54 39 awamori walls, containing 28% hot-water-exA. awamori walls were prepared from cells tractable nigeran, to enzymatic hydrolysis was not increased by boiling either walls or whole which were grown in submerged culture and then shifted to nitrogen-deficient medium for 96 h. A. hyphae (Fig. 7). On the other hand, with A. niger walls prepared from cells grown in surniger walls (4 to 6% hot-water-extractable ni- face culture were on complete medium. For details see geran), both the rate of attack and total amount the text. a
l
I
o---o
hyphoe plus enzyme hyphoe boiled plus e
*--
walls plus enzyme
o---o
walls boiled plus en
*-
a)
aI) Ln a) C:
n
5 1.500_ a)
o 1.200
a,.900 E >.600
l0
20
30
40
50
Time (min)
60
70
80
90
FIG. 7. Enzymatic digestion of walls and hyphae of A. awamori containing 28% of wall weight as hotwater-extractable nigeran. Duplicate samples of walls (3 mg) or hyphae (10 mg) were suspended in 1 ml of 0.1 M acetate buffer, pH 4.5. One member of each pair was heated in a boiling-water bath for 15 min and cooled. After preincubation at 400C for 5 min, 0.3 U of mycodextranase was added to each tube, and the reducing equivalents liberated were measured as a function of time.
NIGERAN IN ASPERGILLUS WALLS
VOL. 132, 1977
699
1.050 a)
a)
CI)
0~
a.450 ,
E/ o.300
.150
/,
/ , 0
10
20
30
40
50
60
70
80
90
Time (min) FIG. 8. Enzymic digestion of A. niger walls containing 6% hot-water-extractable nigeran. Walls (15 mg) were suspended in 1 ml of 0.1 M acetate buffer, pH 4.5. Analysis was conducted exactly as described in the legend for Fig. 7.
hot-water extractable) was accessible to hydro- This treatment also caused enzymic hydrolysis lytic attack by mycodextranase. This conclu- of the same total amount of nigeran. In this sion is from data in Fig. 9. Walls were first experiment, the zero-time sample was assayed preincubated with mycodextranase, as de- for reducing power after boiling (but before scribed above, until no more reducing groups addition of enzyme) and found negative. The effect of dry-heat treatment on walls were liberated (usually 1 h). The residue was washed with water, lyophilized, weighed, and after a preincubation with mycodextranase is suspended in two equal portions of buffer. Por- illustrated in Fig. 9B. This experiment was tions of the two suspensions were analyzed for conducted in a manner identical to that shown reducing power before (zero time) and after in Fig. 9A except that after preincubation and lyophilization the walls were subjected to dry addition of mycodextranase (Fig. 9A). A second treatment with enzyme (dashed heat at 110°C for 2 h and then cooled. This type curve) failed to hydrolyze additional polymer. of heating somehow altered the structure (or However, boiling the walls followed by cooling accessibility) of the polysaccharide such that a and addition of fresh enzyme (at 60 min) af- certain portion can now be attacked without boiling (dashed curve). However, the total forded the release of hydrolysis products. The solid curve describes the result when amount of nigeran released by boiling water the suspended wall preparation was simply (arrow, dashed, and solid curves) was identical boiled and cooled and fresh enzyme was added. in both cases.
700
J. BACTERIOL.
BOBBITT ET AL.
(I)
c
.300
-o
*0
5
E: 450 t
^x
0~
o
/
IB
.O
0~~~~~~~~~~~~~~~ .300
.150
0
10
20
30 40 50 Time (min)
60
70
80
90
FIG. 9. Enzymic digestion of A. niger walls containing 6% hot-water-extractable nigeran. Walls (100 mg) were first digested for 1 h at 40°C with 0.3 U of mycodextranase in 1 ml of 0.1 M acetate buffer, pH 4.5, to remove polysaccharide accessible to the enzyme. The walls were then washed in water to remove the enzyme and digestion products and lyophilized. (A) Duplicate 15-mg samples of predigested walls were submitted to a second digestion with and without boiling. Reducing equivalents liberated were measured as a function of time. In the case of the zero-time sample (solid curve), reducing power was measured after boiling but before addition of enzyme. At 60 min (arrow, dashed curve), the unboiled sample was boiled and cooled, and fresh enzyme was added. (B) Experiment conducted exactly as described in the legend for Fig. 9A except that the walls were dry heated at 110°C for 2 h after lyophilization.
DISCUSSION The experiments described here with A. awamori and A. niger demonstrate that nigeran chains occupy at least three distinct domains or configurations in the hyphal wall and that the polymer's organization in situ is highly crystalline (Tables 1 through 3, Fig. 7 through 9). While it cannot be ruled out that a
certain proportion of noncrystalline nigeran may be present in the wall, all three domains contain the crystalline polymer. The data in Table 3 show that one portion, or fraction, is extractable with boiling water, whereas a second is removed only upon additional treatment with hot, dilute alkali. Also, depending on the organism and growth conditions, different percentages of hot-water-extractable nigeran are
VOL. 132, 1977
accessible to enzymic degradation. X-ray diffraction data (Tables 1 and 2) prove that even prolonged (36 h) boiling of hyphal walls fails to extract a certain portion of the polymer and confirm the results in Table 3. Finally, coupled water extraction and enzymatic studies reveal that not all boiling-water-extractable nigeran is susceptible to digestion in situ by mycodextranase (Fig. 9A). An external location for some of the wall nigeran is suggested by the following observations: boiling of nigeran-enriched A. awamori hyphae or walls does not increase either the rate of subsequent nigeran digestion by mycodextranase or the total amount released (Fig. 7). Therefore, boiling does not "unmask" the nigeran to attack by the enzyme. Since the A. awamori cells used contain massive amounts of polysaccharide accessible to the enzyme, any differences between boiled and unboiled walls in either the rate of attack or in the relative amount of nigeran hydrolyzed are small enough that they are not readily evident in nigeran-enriched walls. Therefore, the bulk of the polymer is located in the external face of the wall. In contrast, A. niger walls, bearing a much lower nigeran content, do show differences in the kinetics of attack by mycodextranase and the total amount liberated, depending on whether the walls are boiled first (Fig. 8). However, this difference in proportion of waterextractable nigeran susceptible to enzymic attack allows the conclusion also that some of the nigeran is exposed to the exterior of the wall. The fraction digested without boiling obviously represents that portion directly exposed to the wall's surface. Beneath this exposed fraction is a second crystalline domain inaccessible to the enzyme but removable with boiling water (Fig. 9A and B). It is these two domains which Tung and Nordin observed in their earlier study (25). Possibly, this more inward-positioned fraction is somehow intercalated with another wall constituent that impedes enzymatic hydrolysis but does not prevent nigeran's release with boiling water. The appearance of broken ends of hyphae in shadowed preparations (Fig. 3) suggests that the opaque component is located on an external surface. This opacity is not the result of salt accumulation from the medium, since the ash content of such walls is less than 0.5%. Shadowed preparations of walls that have first undergone exhaustive digestion with mycodextranase reveal a reduction in wall opacity, confirming that the crystalline nigeran is responsible for this effect. Finally, an observed intensification of chitin diffraction patterns
NIGERAN IN ASPERGILLUS WALLS
701
after renoval of nigeran from wall specimens of both A. niger and A. awamori argues for its external location. X-ray diffraction studies support the conclusion that increased opacity observed in shadowed preparations of nigeran-enriched walls is due to the highly crystalline nature of the nigeran and that crystallinity of the nigeran per se does not confer resistance to enzymic attack. A decrease in intensity of an Xray diffraction pattern after enzymic digestion of wall material argues that crystalline material is being degraded. However, comparative studies of relative rates of attack by mycodextranase on preparations of pure crystalline and noncrystalline nigeran in vitro must await further experimentation. Wessels et al. reported (27) that a-(1( 3)glucan in Schizophyllum commune hyphal walls exists in a crystalline array in situ after hot-water extraction. These authors (27) and Jelsma and Kreger also demonstrated that isolated fungal 3- ( 1-3, /3-(1 -*6)-glucan becomes crystalline after treatment with hot, dilute mineral acid (11). It is suggested that molecular reorganization of polymer molecules into crystallites occurs as a result of acid hydrolysis of some branch points. Troy and Koffler (23) noted chitin as a crystalline wall component of Penicillium chrysogenum, another fungus that produced nigeran (16). However, they did not attempt an exhaustive analysis of these walls for other crystalline materials. Since nigeran is a readily hydrated material, it is not surprising to find that previously undried hyphae exhibit the hydrate diffraction pattern. Reversibility of the diffraction patterns, hydrate dry, is also in keeping with observations on pure nigeran (20, 21). On the basis of the latter studies, however, crystalline reversibility does not necessarily imply morphological reversibility. Observations (20, 21) on nigeran single crystals have shown that dehydration is accompanied by formation of cracks in the thin lamellar crvstals that are from 10 to 20 nm wide. It is unlikely that these would be eliminated by rewetting even though the lattice transforms to the hydrate. Such morphological changes may have important consequences for enzymic biodegradation of cell walls that contain a significant content of crystalline polymers. Whether such alterations are caused by the dry-heat treatment (Fig. 9B) is at present unknown, but experiments attempting to answer this question are being pursued. Gold et al. (9) suggested that walls of A. aculeatus thickened with increased age and when growth ceases. In this study, we found no significant increase in wall thickening asso-
702
BOBBITT ET AL.
ciated with nigeran deposition. These results can be explained by: (i) replacement of wall components by nigeran, (ii) intercalation by nigeran of existing wall material, or (iii) a combination of the two processes. Morton et al. (15) reported that levels of intracellular proteinases of several fungi, including A. niger, increase dramatically within several hours of being placed in a nitrogen-free medium. Nitrogen-starved A. niger and A. awamori cells may transport nitrogen-containing components out of the wall and use them for critical cellular processes. The role that nigeran plays within the fungal wall is unknown. It has been suggested (10) that the nigeran may function in maintaining integrity of the cell surface under adverse environmental conditions. Another possible function is as an overflow product that accumulates when nitrogen metabolism ceases (9). We suggest the possibility that nigeran may be a storage product, utilized during cleistothecium development, but that the enzyme necessary for its breakdown has been lost by mutation. The loss of such an enzyme would explain the findings of Dox (6) and Gold et al. (10) that nigeran is not broken down during starvation. Among species of Aspergillus and Penicillium that Reese and Mandels (16) reported produce nigeran, only one, Penicillium jauanicum, is known to possess a sexual stage in its life cycle. Zonneveld (28) has shown clearly that a correlation exists between glucose utilization by Aspergillus nidulans, formation of a-1,3glucan in the hyphal wall, a-1,3-glucanase activity, breakdown of the hyphal wall, and the production of cleistothecia (sexual stage). if, in A. niger andA. awamori, the ability to produce mycodextranase has been lost, nigeran would accumulate in the wall, and cleistothecia development would be blocked. This possible explanation for nigeran accumulation is under examination in our laboratories. ACKNOWLEDGMENTS This research was supported by grants from the National Science Foundation (BMS 75-22424) (to John H. Nordin and the National Research Council of Canada (to R. H. Marchessault). We wish to thank Stanley Holt of the University of Massachusetts Microbiology Department for use of the electron microscope facilities. LITERATURE CITED 1. Bardalaye, P. C., and J. H. Nordin. 1976. Galactosaminogalactan from cell walls of Aspergillus niger. J.
Bacteriol. 125:655-669. 2. Barker, S. A., E. J. Bourne, D. M. O'Mant, and M. Stacey. 1957. Studies of A. niger. VI. The separation and structures of oligosaccharides from nigeran. J.
J. BACTERIOL. Chem. Soc., p. 2448-2454. 3. Barker, S. A., E. J. Bourne, and M. Stacey. 1953. Studies of Aspergillus niger. I. The structure of the polyglucosan synthesized by Aspergillus niger 152. J. Chem. Soc., p. 3084-3090. 4. Blackwell, J. 1969. Structure of 83-chitin or parallel chain systems of poly-3--(1-4)-N-acetyl-D-glucosamine. Biopolymers 7:281-298. 5. Carlstrom, D. 1957. The crystal structure of a-chitin poly-N-acetyl-D-glucosamine. J. Cell Biol. 3:669-683. 6. Dox, A. W. 1915. Influence of autolysis on the mycodextran content of Aspergillus niger. J. Biol. Chem. 20:83-85. 7. Dox, A. W., and R. E. Neidig. 1914. Mycodextran, a new polysaccharide in Penicillium expansum. J. Biol. Chem. 18:167-175. 8. Dubois, M., K. A. Gilles, J. D. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method of determintion of sugars and related substances. Anal. Chem.
28:350-356. 9. Gold, M. H., S. Larson, I. H. Segel, and C. R. Stocking. 1974. Intracellular localization of nigeran in the wall of Aspergillus aculeatus by autoradiography with the electron microscope. J. Bacteriol. 118:1176-1178. 10. Gold, M. H., D. L. Mitzel, and I. H. Segel. 1973. Regulation of nigeran accumulation by Aspergillus aculeatus. J. Bacteriol. 113:856-862. 11. Jelsma, J., and D. R. Kreger. 1975. Ultrastructural observations on (1-3)-,3-D-glucan from fungal cell walls. Carbohydr. Res. 43:200-203. 12. Johnston, I. R. 1965. The composition of the cell wall of Asperigillus niger. Biochem. J. 96:651-657. 13. Kreger, D. R., and M. Kopecka. 1976. On the nature and formation of the fibrillar nets produced by protoplasts of Saccharomyces cerevisiae in liquid media: an electron microscopic, X-ray diffraction and chemical study. J. Gen. Microbiol. 92:207-220. 14. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 15. Morton, A. G., A. G. F. Dickerson, and D. J. F. England. 1960. Changes in enzyme activity of fungi during nitrogen starvation. J. Exp. Bot. 11:116-128. 16. Reese, E. T., and M. Mandels. 1964. A new a-glucanase mycodextranase. Can. J. Microbiol. 10:103-114. 17. Rudall, K. M. 1969. Chitin and its association with other molecules. J. Polym. Sci. 28:83-102. 18. Somogyi, M. 1945. A new reagent from the determination of sugars. J. Biol. Chem. 160:61-68. 19. Spurlock, B. O., V. C. Kattine, and J. Freeman. 1963. Technical modifications in Maraglas embedding. J. Cell Biol. 17:203-207. 20. Sundararajan, P. R., R. H. Marchessault, G. J. Quigley, and A. Sarko. 1973. Crystalline chain conformation of mycodextran. J. Am. Chem. Soc. 95:2001-2008. 21. Taylor, K. J., H. Chanzy, and R. H. Marchessault. 1975. Electron diffraction for hydrated crystalline biopolymers: nigeran. J. Mol. Biol. 92:165-167. 22. Trevelyn, W. E., D. P. Proctor, and J. S. Harrison. 1950. Detection of sugars in paper chromatograms. Nature (London) 166:444-445. 23. Troy, F. A., and H. Koffler. 1969. The chemistry and molecular architecture of the cell walls of Penicillium chrysogenum. J. Biol. Chem. 244:5563-5576. 24. Tsukahara, T., and M. Yamada. 1965. Cytological structure of Aspergillus niger by electron microscopy. Jpn. J. Microbiol. 9:35-48. 25. Tung, K. K., and J. H. Nordin. 1967. Evidence for a buried location of nigeran in cell wall of Aspergillus niger. Biochem. Biophys. Res. Commun. 28:519-524. 26. Tung, K. K., A. Rosenthal, and J. H. Nordin. 1971. Enzymes that hydrolyze fungal cell wall polysaccha-
VOL. 132, 1977 rides. Purification and properties of mycodextranase an a(1-4) glucanase of Penicillium melinii. J. Biol. Chem. 246:2722-2732. 27. Wessels, J. G. H., D. R. Kreger, R. Marchant, B. A. Regensburg, and 0. M. H. DeVries. 1972. Chemical and morphological characterization of the hyphal
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wall surface of the Basidiomycete Schizophyllum commune. Biochim. Biophys. Acta 273:346-358. 28. Zonneveld, J. M. 1972. Morphogenesis in Aspergillus nidulans. The significance of a-1,3-glucan of the cell wall and a-1,3-glucanase for cleistothecium development. Biochim. Biophys. Acta 273:174-187.
