JouRNAL OF BACrERIOLOGY, June 1975, p. 1062-1070 Copyright 0 1975 American Society for Microbiology

Vol. 122, No. 3 Printed in U.S.A.

Changes in Glucosamine and Galactosamine Levels During Conidial Germination in Neurospora crassa JOSEPH C. SCHMrr, CLARK M. EDSON,' AND STUART BRODY* Department of Biology, University of California, San Diego, La Jolla, California 92037 Received for publication 23 January 1975

The levels of glucosamine and galactosamine were determined in conidia, germinating conidia, and vegetative mycelia of Neurospora crassa. In the vegetative mycelia about 90% of the amino sugars were shown to be components of the cell wall. The remaining 10% of the amino sugars were tentatively identified as the nucleotide sugars uridine diphospho-2-acetamido-2-deoxy-D-glucose and uridine diphospho-2-acetamido-2-deoxy-D-galactose. Conidia and vegetative mycelia contained about the same levels of glucosamine. During the first 9 h after the initiation of germination, the total glucosamine content had increased 3.1-fold, whereas the residual dry weight of the culture had increased 7.7-fold. This led to a drop in the glucosamine concentration from 100 Amol/g of residual dry weight to 42 ,mol/g. During this time, all of the conidia had germinated and the surface area of the new germ tubes had increased to 10 times that of the conidia. Either germ tubes were initially produced without glucosamine-containing polymers, or these polymers (probably chitin) were deposited only at low densities in the germ tube cell walls. The chitin precursor uridine diphospho-2acetamido-2-deoxy-D-glucose was present at all times during conidial germination. Conida contained very low levels of galactosamine. During germination, galactosamine could not be detected until the culture had reached a cell density of about 0.6 mg of residual dry weight per ml of growth medium. This was observed regardless of the time required to reach this cell density or the fold increase in dry weight. The accumulation of galactosamine-containing polymers does not appear to be necessary for germ tube formation. The levels of soluble galactosamine (uridine diphospho-2-acetamido-2-deoxy-D-galactose) were very low in conidia and increased during germination at the same time that galactosamine appeared in the cellular polymers. In addition, under certain culture conditions, the appearance of galactosamine and the increase in the glucosamine concentration occurred simultaneously. involves a change from an approximately spherical cell to a tubular cell, cell wall structural alterations are expected to occur during germination. The cell wall of N. crassa mycelia is composed of chitin, galactosamine-containing polymers, a partially characterized glucan, protein, and several minor components (3, 8, 10-12, 14, 25, 30). The outer layer of the cell wall of hyphae has been reported to be composed of a glucanprotein-galactosamine complex (19, 20, 26). All of the galactosamine in cell walls of Neurospora mycelia is in this fraction (25). An inner layer of the cell wall is composed of chitin microfibrils embedded in an amorphous glucan (26) or in a protein matrix (20). Most of the glucosamine in Present address: Department of Medical Chemistry, the cell wall is in this chitin fraction (25). As will Kyoto University Faculty of Medicine, Yoshida, Sakyo-ku, be shown in this paper, most of the glucosamine in the mycelia is in the cell wall. Therefore, the Kyoto, 606, Japan.

In the asexual life cycle of Neurospora crassa, there are three basic cellular forms: conidia, vegetative mycelia, and aerial hyphae. Each of these has its own characteristic shape and function. Conidia, or more precisely macroconidia, are vegetative spores produced by budding in long chains from the tips of specialized aerial hyphae (36). During germination, each conidium produces one or two germ tubes that eventually yield the long, tubular, multinucleate cells of the vegetative mycelium. The developmental changes most easily observed in many differentiating systems, and especially in fungi, are changes in cell shape. The major shape-determining element in fungi is its cell wall (4). Since conidial germination I

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glucosamine content of the mycelia is a direct indication of the amount of chitin in the cell wall. Glucosamine has been reported to be present in conidial cell walls (18, 22) at about the same level found in mycelia (18). However, very little of the other amino sugar in Neurospora, galactosamine, was found in either conidia or conidia cell walls (18). The absence of galactosamine in conidia was also observed in our laboratory and led to our initial hypothesis that galactosamine synthesis might be important for the formation of germ tubes during conidial germination. Galactosamine polymers have been found as cell wall components in a number of fungi (3). In some species it is a relatively minor component (2, 6, 8, 19, 38), whereas in others it is the only cell wall carbohydrate (23, 35). Galactosamine was found as a major constitutent of the cell wall of a virus-containing strain of Pennicillium stoloniferum but was a minor component of four virus-free strains of the same organism (5). The only carbohydrate in the cell walls of spores and spherules of the myxomycete Physarum polycephalum were galactosamine polymers (23). Galactosamine polymers have also been found in the bacterial species Neisseria sicca (1). Both Aspergillus parasiticus (12) and N. crassa (31) can excrete high-molecular-weight homopolymers of partially acetylated galactosamine. Galactosamine polymers that are similar to those that are excreted have been extracted from the cell wall of vegetative hyphae from both of these organisms (12, 19, 31). A. nidulans excretes an extracellular heteroglycan composed of galactose and galactosamine (17). In N. crassa the amount of galactosamine in the cell wall has been shown to vary with genetic background (18, 22, 34). Many mutant strains with colonial morphology have either more or less cell wall galactosamine than the wild-type strain (C. Edson and S. Brody, manuscript in preparation). A mutant strain sensitive to osmotic pressure has been shown to have elevated levels of galactosamine (34). A pleiotropic mutant strain of N. crassa, exo-1, which had derepressed levels of the exoenzymes aamylase, glucoamylase, ,3-fructofuranosidase, and trehalase, also contained elevated levels of galactosamine (18). The nucleotide sugar uridine diphospho-2acetamido-2-deoxy-D-glucose (UDP-GlcNAc) has been shown to be the direct precursor of chitin (13, 14, 16), the major glucosaminecontaining polymer in Neurospora (25). Galactosamine is synthesized in bacteria (15) and mammals (9) by epimerization of UDP-GlcNAc

to uridine diphospho-2-acetamido-2-deoxy-D-galactose (UDP-GalNAc). This pathway also occurs in Neurospora (C. Edson and S. Brody, manuscript in preparation). The UDP-GalNAc is thought to be the direct precursor of cell wall galactosamine-containing polymers, but this has not been proven experimentally. The pathway for the formation of chitin and galactosaminecontaining polymers as currently understood is summarized as follows:

Fructose-6-phosphate

-

UDP-GlcNAc - chitin UDP-GlcNAc*-+ UDP-GalNAc -, galactosamine polymers During conidial germination, the cell wall of the germ tube is formed by extension of the existing conidial cell wall (27). Since the cell walls of conidia contained far less galactosamine than those of mycelia (18), it was proposed initially that galactosamine deposition might play a vital developmental role during conidial germination. Therefore, we have determined the changes in the galactosamine content that occurred during conidial germination and subsequent growth. Changes were also observed in glucosamine levels and these are reported. In addition, the amino sugar content of the cell fraction containing the nucleotide sugar precursors (UDP-GlcNAc and UDP-GalNAc) of the cell wall polymers was measured. MATERIALS AND METHODS

Neurospora strain. The wild-type strain RL3-8A (FGSC 2218) of N. crassa was used in these studies and can be obtained from the Fungal Genetics Stock Center, Humboldt State College, Arcata, Calif. Conidial preparations. Conidia were obtained from slant tube cultures containing 6 ml of Vogel minimal medium (37) with 2% glucose and 2% agar. Forty slant cultures were grown in constant light for 7 days at 22 C. Conidia were removed with a sterile loop and suspended in 100 ml of cold, sterile water in a flask containing a magnetic stirring bar. The conidial suspension was vigorously agitated on a magnetic stirrer, and the contaminating fragments of mycelia were removed by filtering through four layers of cheesecloth. The conidial preparation was then centrifuged and washed with cold, sterile water before being used as inoculum. Throughout the preparation of the inoculum, the temperature was maintained at about 5 C. Forty slants of the wild-type strain yielded approximately 1.8 x 1010 conidia. Germination conditions. All conidial germination experiments were carried out in liquid-shake cultures. Either 50 ml of Vogel minimal medium containing 2% glucose in 125-nil flasks or 250 ml of the same medium in 1-liter flasks was employed in most experiments. A "complete" medium containing Vogel salts, 0.1%

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SCHMIT, EDSON, AND BRODY

Casamino Acids, 0.25% yeast extract, 0.4% glycerol, and 0.4% sucrose was used for one experiment. Germination was initiated by adding freshly prepared conidia to each flask giving a final concentration of about 5 x 106 conidia per ml. All cultures were incubated at 22 C on a rotatory shaker at 125 rpm. For germination counts and for determining the average length of the germ tubes, samples were removed at intervals and fixed in 10% formalin. The number of conidia with germ tubes extending at least 50% of the diameter of the conidia was determined. The average germ tube length was measured by using a calibrated ocular micrometer. Harvesting and preparation of alcohol-insoluble and alcohol-soluble fractions. Conidia and germinating conidia were harvested by filtration on membrane filters (type EHWPO4700, Millipore Corp.). Mycelia were harvested on Whatman no. 1 filter paper. The cells were washed quickly with water and plunged into 20 ml of boiling 80% ethanol. The samples were boiled for 10 min, cooled, centrifuged, and filtered through EHWPO4700 Millipore filters. This gave two fractions: (i) the alcohol-insoluble fraction containing the cell wall material as well as protein, deoxyribonucleic acid, ribonucleic acid, etc., and (ii) the alcohol-soluble fraction containing the sugar nucleotides (UDP-GlcNAc and UDP-GalNAc) as well as many other compounds. About 80% of the dry weight of Neurospora was insoluble in boiling 80% ethanol. The alcohol-insoluble material was dried overnight at 90 C and weighed to determine the residual dry weight (RDW). The supernatant (alcoholsoluble fraction) was flash evaporated to dryness on a rotatory evaporator. The samples were suspended in a known volume of water and centrifuged to remove undissolved material. Samples containing large amounts of lipids were extracted with 3 ml of chloroform. Cell wall preparation. Mycelial cell wall was prepared by a modification of the method of Mahadevan and Tatum (25). A 1-g amount of lyophilized mycelia was ground in a Wiley Mill and added to 100 ml of aqueous 1% sodium dodecyl sulfate. The mixture was stirred at 22 C for 3 h followed by 15 h at 4 C. The cell wall material was washed with distilled water six or seven times, lyophilized, and ground to a fine powder in a Wiley Mill. This was followed by treatment with hot 80% ethanol (100 ml/500 mg of cell wall) for 20 min, filtration, and lyophilization. The purity of the isolated cell walls was judged by two criteria. First, less than 2% of the total cellular protein was found associated with the purified cell wall. This was determined by measuring the amino acids released after hydrolysis of the cell wall in 6 N HCl for 24 h at 110 C. In fact, if these amino acids were actually components of the cell wall, there may be no contamination of the purified cell wall by cytoplasmic protein. This possibility was supported by the observation that the amino acid composition of the proteins associated with the cell wall was different from that of the total cellular protein (C. Edson and S. Brody, manuscript in preparation). Second, there was very little phospholipid contamination of cell walls purified by this procedure. When cultures were

J. BACTERIOL.

grown in [14C ]choline, which was incorporated into phosphatidyl-choline, the major phospholipid in the membranes, no radioactivity was found associated with the purified cell walls (B. Hanson and S. Brody, manuscript in preparation). Based on these two criteria, it was concluded that these cell wall preparations were almost completely devoid of contamination with membrane lipids or cellular proteins. Hydrolysis of the cell wall-containing fractions. The lyophilized mycelia, isolated cell wall, and dried, alcohol-insoluble residue were ground to a fine powder with a mortar and pestle, and weighed samples were hydrolyzed in 6 N HCl for 18 h at 90 C in sealed, evacuated hydrolysis tubes. Increasing the temperature to 110 C and the time to 24 h (18, 22) did not increase the yield of amino sugars. After hydrolysis the HCl was removed by repeated flash evaporation. UDP-GlcNAc and UDP-GaINAc levels. Prior to acid hydrolysis, no free glucosamine or galactosamine could be detected in the alcohol-soluble fraction from mycellia by using an amino acid analyzer. Both of these amino sugars were detected in this fraction after hydrolysis in 3 N HCl for 3 h at 110 C. The amino sugar components of the alcohol-soluble fraction from mycelia were characterized and purified by ascending paper chromatography on sheets (46 by 57 cm) of Whatman no. 1 in 1 M ammonium acetate-95% ethanol (30:70; pH 7). After development, the chromatograms were cut into strips. Each strip was eluted with water, and the samples were hydrolyzed in 3 N HCl for 3 h at 110 C. The amino sugar content was then measured by using an amino acid analyzer. When areas of the chromatogram corresponding to the position of standard GlcNAc and GalNAc were eluted and hydrolyzed, no amino sugars were detected. All of the glucosamine and galactosamine released by hydrolysis had the same Rt as UDP-GlcNAc. A good correlation was obtained between the nucleotide content (measured by absorbance at 260 nm) and the total amino sugar content (measured after hydrolysis by using an amino acid analyzer) of the material from this region of the chromatogram. In other control experiments, 90% of the expected amount of glucosamine was recovered after hydrolysis of standard UDP-GlcNAc. It was concluded that UDP-GlcNAc and UDP-GalNAc were the predominant amino sugars in the alcohol-soluble fraction of mycelia. In addition, no free amino sugar could be detected in the alcohol-soluble fraction from conidia prior to hydrolysis. It was concluded that the predominant amino sugars in this fraction from conidia were also nucleotide sugars. In the experiments reported in this paper, the amino sugar content of the alcohol-soluble fraction wab measured after hydrolysis of a portion of the extract in 3 N HCl for 3 h at 110 C. No paper chromatography was used. After hydrolysis, the HCl was removed by repeated flash evaporation. Quantitative measurement of glucosamine and galactosamine. The amino sugar content was determined on a Beckman 120 C amino acid analyzer (29) equipped with an Infotronics integrator. Glucosamine and galactosamine were separated on a 29-cm column filled with 16 cm of Beckman PA35 resin at 55 C by

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CHANGES IN GLUCOSAMINE LEVELS

using pH 5.28 sodium citrate buffer (0.35 M Na+). The amino sugars were detected by their reaction with ninhydrin. Standard solutions of glucosamine and galactosamine were used to calibrate the analyzer. By this procedure, the amino sugar determinations could be completed within 1 h.

RESULTS Cellular localization of amino sugars. To determine the percentage of the total cellular amino sugars associated with the cell wall, the amino sugar content of both unfractionated mycelia and purified cell walls was measured. More than 90% of the total cellular glucosamine and galactosamine co-purified with the cell wall (Table 1). The remainder of the amino sugars could be recovered in the alcohol-soluble fraction as the nucleotide sugars UDP-GlcNAc and UDP-GalNAc. These nucleotide sugars were lost during the preparation of the cell walls. Alcohol fractionation was used as a convenient and rapid technique for separating the cell wall material from the nucleotide sugars. The alcohol-insoluble fraction contained all of the cell wall. Eighty percent of the dry weight of the mycelia was recovered in this fraction. Since only 15% of the dry weight was cell wall (Table 1), many other components of the cell must also be in the alcohol-insoluble fraction. However, more than 90% of the amino sugars in the cell were in the purified cell wall (Table 1). Therefore, the bulk of the amino sugars released from the alcohol-insoluble residue must be associated with the cell wall. Thus, changes that occurred in the amino sugar content of the alcoholinsoluble fraction during germination and growth must be due almost entirely to changes either in the proportion of the alcohol-insoluble fraction that was cell wall or in the percentage TABL 1. Comparison of the amino sugar content of mycelia and purified cell wall8a Amino sugar concn

Sample analyzed

Myceliab Cell Wallc

(pmo/g of lyophilized mycelia)

GIcNAc

GalNAc

62 57

60 58

a Cell walls were prepared by a modified sodium dodecyl sulfate method of Mahadevan and Tatum

(25).

b The total amino sugar content was obtained after acid hydrolysis of lyophilized mycelia from late logphase shake cultures. c About 150 mg of cell wall was obtained from 1 g of lyophilized mycelia. The amino sugars released by acid hydrolysis of this 150 mg of cell wall is given.

1065

of the cell wall that was amino sugar. Since the objective of this research was to determine whether there were any correlations between changes in the amino sugar levels and the appearance of germ tubes, no attempt was made to distinguish between these two possibilities. Amino sugar content of conidia and mycelia. The amino sugar content of both the alcohol-insoluble (cell wall) and the alcoholsoluble fractions (nucleotide sugars) of conidia and mycelia was measured (Table 2). Conidia contained about the same levels of glucosamine in both the alcohol-insoluble and alcohol-soluble fractions as was found in the corresponding fraction from mycelia. About 1.6% of the dry weight of both conidia and mycelia was recovered as glucosamine (RDW = 80% of the dry weight). In contrast, very little galactosamine was detected in either fraction from conidia, but galactosamine was detected in both of these fractions from mycelia (Table 2). Only 0.01% of the dry weight of the conidia was galactosamine, whereas about 0.6% of the mycelia was this amino sugar. Since the alcohol-insoluble fraction contained all of the conidial cell wall, galactosamine polymers could have little, if any, role in maintaining the structural integrity of conidial cell walls. Conidial germination and growth. At 22 C in minimal medium in shake cultures, germ tubes began to appear after 2.5 h (Fig. 1). By 7 h, more than 90% of the conidia had germ tubes. The dry weight of the alcohol-insoluble fraction (RDW) increased exponentially with a doubling time of 3.2 h (Fig. 1). No lag period was observed. This indicated that at least some of the components of this fraction (cell wall, protein, ribonucleic acid, deoxyribonucleic acid, etc.) were being synthesized throughout the early stages of germination before the appearance of germ tubes. Since germ tube cell walls were formed by extension of the existing conidial cell wall (27), the length of the germ tube could be used as an indicator of the presence of newly synthesized cell wall. The average length of the conidium plus its germ tube was measured (Fig. 2). The average initial diameter of the conidia was 6 Mm, with individual conidia varying from 4 to 8 um. By 10 h, the average length of the conidia plus germ tubes was 80 jAm (individuals varied from 14 to 150 MAm). Thus, by 10 h, a considerable amount of new cell wall had been formed. Changes in the glucosamine content of the alcohol-insoluble residue during conidial germination. The glucosamine content of the alcohol-insoluble fraction decreased nearly

1066

SCHMIT, EDSON, AND BRODY

J. BACTERIOL.

TABLE 2. Amino sugar concentration of conidia and mycelia Amino sugar concn

Cell type

Conidia Myceliac

(;&mol/g of RDW)

Alcohol-insoluble fractiona

Alcohol-soluble fractionb

GlcNAc

GalNAc

GlcNAc

GaINAc

110 (62-145) 94d (79-107)

0.2 (0.01-0.5) 35e (20-41)

10.1 (8.2-12.7) 11.0 (10.4, 11.6)

0.5 (0.3-0.6) 3.0 (2.4, 3.6)

a The concentrations given are the average of five determinations, with the lowest and highest concentrations given in the parenthesis. b The average concentrations of the soluble amino sugars were obtained from three determinations for conidia and two for mycelia. c Mycelia was obtained from liquid-shake cultures grown for 24 h at 22 C. d The levels of glucosamine from mycelia are higher in this table than in Table 1 because the data are expressed in terms of micromoles per gram of RDW rather than micromoles per gram of mycelia. Twenty percent of the dry weight was soluble in ethanol. Thus, 94 ,mol/g of RDW is equivalent to 75 ,moVg of mycelia, which is very similar to the levels in Table 1. e The levels of galactosamine obtained in these experiments were lower than those in Table 1. This is not thought to be significant since, as will be presented, the galactosamine content varied considerably depending on the amount of growth.

en

a I 0

80 o I

I

In

60 4 I-

a

a

cromoles per gram of RDW, a decrease was obtained (Fig. 2). Thus, there was an increase in the dry weight of the alcohol-insoluble fraction without a corresponding increase in the glucosamine content. New cell walls were being synthesized during this time, since by 9 h the average length of the conidia plus germ tubes had increased 10-fold (Fig. 2) and the surface area (calculated from

0

O X o tn

*n

20

E

ID Z

2E

cLZ

ar

am

Z

PERIOD OF GROWTH (Hours)

w

I

Z

FIG. 1. RDW and percentage of the conidia with germ tubes. The conidia of the wild-type strain RL38A were germinated in liquid-shake cultures at 22 C. The RDW was the material precipitated by boiling in 80%o ethanol. Conidia were considered germinated if they had germ tubes that were one-half the diameter of a conidium. Symbols: 0, RDW; 0, percentage of the conidia with germ tubes.

threefold during conidial germination (Fig. 2). This decrease was not due to the overall loss of glucosamine, since the glucosamine content per flask did not decrease (Fig. 3). However, the increase in the glucosamine content lagged behind the increase in the residual dry weight (Fig. 3, insert). After 9 h, the RDW of the culture had increased 7.7-fold, but the glucosamine content of the residue had only increased 3.1-fold (Fig. 3, insert). When the glucosamine concentration was expressed in terms of mi-

z

0 n

4)

E

C., Z

-w

4

O

Z

cr

25 20 15 l0 PERIOD OF GROWTH (Hours)

5

FIG. 2. Amino sugar content of the alcohol-insoluble fraction and the average length of conidia plus germ tubes. The amino sugar content of the alcoholinsoluble fraction of samples taken during germination and growth in liquid-shake cultures was determined after acid hydrolysis. The alcohol-insoluble fraction contained all of the cell wall material. The average length of the conidia plus germ tube was determined as described previously. The average diameter of a newly formed germ tube was about 4 gm. Symbols: 0, glucosamine; A, galactosamine; 0, average length of conidia plus germ tubes.

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CHANGES IN GLUCOSAMINE LEVELS

y CO) 4 -J

L -i

zcr

wo

E

Z N

z

Zr

~

5 25 10 20 15 PERIOD OF GROWTH (Hours) FIG. 3. Amino sugar content per flask. The data are from the same experiment shown in Fig. 2. The insert compares the fold increase of glucosamine and RDW. Symbols: 0, glucosamine; A, galactosamine; 0, RDW.

the data in Fig. 2) had increased 11-fold. Since the glucosamine content had only increased 3.1-fold (Fig. 2), the new germ tubes had less than one-third the amount of glucosamine per unit surface than did conidia. In this (Fig. 2) and other experiments, the glucosamine content of the alcohol-insoluble fraction varied considerably depending on when the mycelia were harvested. The lowest concentrations (about 40 Amol/g of RDW) were obtained during germination (Fig. 2), and the highest concentrations (200 umol/g of RDW) were obtained with late stationary-phase cultures after 72 h of growth. In these prolonged growth experiments, the glucosamine content continued to increase after the cultures had reached stationary phase. Appearance of galactosamine in the alcohol-insoluble residue. Very little galactosamine was detected in the alcohol-insoluble fraction until 9 h after the initiation of germination (Fig. 2). By 9 h, essentially all of the conidia had germinated (Fig. 1), and the average germ tube

1067

length was 10 times the original conidial diameter (Fig. 2). The RDW of the culture had increased 7.7-fold (Fig. 3, insert). Germ tubes and, consequently, cell walls, had been formed well before the appearance of detectable levels of galactosamine in the fraction containing the cell wall (Fig. 2). Thus, galactosamine was not a major structural component of the germ tube cell walls. Also, galactosamine polymers were not excreted into the media during conidial germination. No extracellular galactosamine-containing polymers were detected in the media until after 24 h. The isolation procedures of Reissig and Glasgow (31) were employed. After 24.5 h of growth in minimal medium, the concentration of galactosamine in the alcohol-insoluble rQsidue was 20 ,mol/g of RDW (Fig. 2). In other experiments where the mycelia were harvested after 72 h of growth, galactosamine reached concentrations of 40 smollg of RDW. An even higher galactosamine concentration of 90 umoVg of RDW was obtained after 72 h of growth in a "complete" medium. Thus, the concentration of galactosamine in the mycelia was dependent on the culture conditions and the period of growth. Both the appearance of galactosamine and the increase in the glucosamine concentration occurred simultaneously during germination (Fig. 2). This correlation was observed consistently in duplicate experiments. The possibility exists that the synthesis of both glucosamineand galactosamine-containing polymers might share some common controlling element. Changes in the amino sugar content of the

alcohol-soluble fraction during conidial germination. The amino sugar content of the alcohol-soluble fraction was measured during germination (Fig. 4). The only soluble forms of amino sugars in Neurospora were the nucleotide sugars UDP-GlcNAc and UDP-GalNAc. Measuring the amino sugars released by acid hydrolysis of the alcohol-soluble fraction gave a direct assay for the levels of these nucleotide sugars. Glucosamine (UDP-GlcNAc) was detected in the alcohol-soluble fractions at all times during germination (Fig. 4). Therefore, the lag in the synthesis of glucosamine-containing polymers during the first 9 h of germination (Fig. 2) was not due to the absence of the chitin precursor, UDP-GlcNAc. The appearance of galactosamine in the alcohol-soluble fraction (Fig. 4) correlated very well with its appearance in the alcohol-insoluble fraction (Fig. 2). Galactosamine (UDP-GalNAc) was first detected in the alcohol-soluble fraction in the sample taken at 12.5 h.

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SCHM1T, EDSON, AND BRODY

Correlation between galactosamine appearance and culture density. The initial appearance of galactosamine was not dependent on the length of time that the culture had been growing, but rather on the concentration of cells in the culture. In Table 3 are summarized the results from three experiments in which cultures were inoculated with increasing amounts of conidia. The time required for the accumulation of about 6 ismol of galactosamine per g of RDW varied from 24 h with the lowest initial cell density (6 ,ug of conidia per ml) to only 7 h with the highest initial cell density (122 ;g/ml). In the first experiment, there had been a 50-fold increase in RDW, whereas in the third experiment the RDW had only increased fivefold when the galactosamine concentration reached 6 ltmol/g. Thus, the initial appearance of detectable galactosamine levels did not correlate well with the amount of growth (5- to 50-fold increase in RDW) or the length of the growth period (7 to 24 h). However, the appearance of z

o

& -

!z I w

,

b.

- E0 -2

0

25 20 5 10 15 PERIOD OF GROWTH (Hours)

FiG. 4. Amino sugar concentration of the alcoholsoluble fraction. All of the alcohol-soluble glucosamine and galactosamine was in the sugar nucleotides UDP-GlcNAc and UDP-GaINAc. The amino sugar concentrations were determined after acid hydrolysis. Symbols: 0, glucosamine; A, galactosamine.

TABL 3. Period of growth and the culture density required for galactosamine to reach a concentration of 6 gmol/g of RDWb Concn of

Expt

conidial

inoculum" ("g/ml)

1

6

2 3

57 122

Growth (C) temp

Peiod ofo Culture growth density (lag Of (h) --IL

RDW/ml)

23 23 23

24 12.5 7

300 820 600

a The galactosamine concentration was determined in the alcohol-insoluble fraction. bGermination was not affected by varying the amount of the conidial inoculum over this range.

J. BAC-uoL.

galactosamine correlated better with culture density. The culture density varied only from 0.30 to 0.82 mg/ml of culture medium when the galactosamine level reached 6 gmol/g of RDW (Table 3). DISCUSSION Several lines of evidence indicate that chitin is the predominant glucosamine-containing polymer in both conidia and mycelia. In studies with purified cell walls, about 10% of the conidial cell wall (22) and 9.4% of the mycelial cell wall (25) was identified as chitin. After acid hydrolysis of purified cell walls, about 12% of the conidial cell wall (18) and 9% of the mycelial cell wall (Table 1) were recovered as glucosamine. Thus, most of the glucosamine in the conidial and mycelial cell walls was chitin. Since more than 90% of the total glucosamine in the mycelia was in the cell wall (Table 1), the predominant glucosamine-containing polymer in the mycelia was chitin. It is reasonable to assume that chitin is also the predominant glucosamine-containing polymer in germ tubes. During conidial germination, glucosaminecontaining polymers (chitin) were not synthesized initially as fast as the other alcohol-insoluble components of the cell (Fig. 2). Also, when compared to conidia, germ tubes contained less than one-third the amount of glucosamine per unit surface area. Two different explanations can be given for these observations. Chitin could be distributed uniformly but at low levels throughout the new germ tubes, or the germ tubes could be formed initially without chitin and the chitin could be deposited as a "secondary" wall behind the growing tips. It has been suggested that chitin is required for cell wall strength (21). Studies with a temperature-sensitive strain of Aspergillus with defective glucosamine synthesis have shown that this fungus could grow "normally" at the nonpermissive temperature providing the medium was osmotically buffered (21). The hyphae that were produced contained very little chitin and were osmotically fragile. Presumably, chitin played a stiffening or strengthening role in the Aspergillus cell wall. Chitin could play a similar role in Neurospora cell walls. If this is the case, chitin might not be required at high levels until well after germ tubes had been formed and the internal osmotic pressure had begun to increase. In Fusarium culmorum, chitin microfibrils have been reported to be deposited as a secondary wall beneath the "primary" wall behind the hyphal tip in germ tubes (28). Cytological studies with Neurospora have shown that chitin

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CHANGES IN GLUCOSAMINE LEVELS

microfibrils were also located in the inner-most layer of the cell wall next to the plasma membrane (20, 28). Chitin can apparently be deposited in mature Neurospora hyphae, since we have observed that the glucosamine content increased after the cultures had reached stationary phase. Thus, it is possible that chitin synthesis and, consequently, glucosamine accumulation could be delayed until after germ tubes had been formed. Conidial cell walls contained very little galactosamine (Table 2; 18). During germination, galactosamine did not begin to accumulate until well after germ tubes had been formed (Fig. 2). Therefore, galactosamine-containing polymers were not required as a major structural or shape-determining component of the cell wall of either conidia or germ tubes. Also, galactosamine-containing polymers were required, at most, only at very low levels for the formation of hyphae at the growing front of the mycelial mat of surface-grown cultures (32). If galactosamine does play a structural role in cell wall architecture, it must be restricted to cell walls formed after conidial germination and to those in older regions of the mycelial mat of surface cultures. The initiation of galactosamine accumulation was correlated with the concentration of cells in the culture (Table 3). Presumably, this was the result of changes in the composition of the medium. Either something was being removed or added to the medium after a certain amount of growth. The nature of this change in the medium was not determined. The formation of glucosamine-containing polymers in shake cultures may also be dependent to some extent on the culture density since the concentrations of both glucosamine and galactosamine increased simultaneously during germination (Fig. 2). A similar increase in amino sugar levels was observed in shake cultures with chopped mycelia as inoculum (unpublished data). In yeast, chitin synthetase was formed as a zymogen that was activated by proteolytic cleavage (7). Perhaps the formation of chitin and galactosamine polymers in Neurospora also involves zymogen activation. In shake cultures, a specific cell density may be required for the appearance of the proteolytic activities that are necessary for zymogen activation. Galactosamine polymers can account for up to 8% of the dry weight of the mycelial cell wall (Table 1). Since these polymers apparently do not play a major structural role, their physiological function remains unknown. One possibility is that they may be associated with the production of extracellular enzymes (18). Both galac-

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tosamine-containing polymers (31) and extracellular enzymes (18) are excreted into the culture medium during late log phase. Galactosamine and protein are located in the outer, alkali-soluble layer of the mycelial cell wall (26). The positively charged galactosamine-containing polymers (19, 31) could provide binding sites for the attachment of extracellular enzymes. Alternatively, they couldbe accidentally trapped in the cell wall by molecular sieving during excretion as has been reported for extracellular enzymes (33). A mutant strain of N. crassa , exo-1, excretes large amounts of several extracellular enzymes and has elevated levels of galactosamine in its cell walls (18). It would be interesting to know if other Neurospora strains that have elevated galactosamine levels such as the osmotic sensitive strain os-i (34) and the temperature-sensitive colonial strain cot-1 (31) also have increased production of extracellular enzymes. ACKNOWLEDGMENTS The excellent technical assistance of Stan Martins is gratefully acknowledged. This investigation was supported by National Science Foundation grant GB 21227 and by Public Health Service grant GM 19308 from National Institute of General Medical Sciences. One of us (J.C.S.) was supported by Public Health Service postdoctoral fellowship 1-FO2-GM-50,529-02 from the National Institute of General Medical Sciences. LITERATURE CITED 1. Adams, G. A., and A. S. Chaudhari. 1972. Galactosamine polymer isolated from the cell wall of Neisseria sicca.

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