Molecular architecture of the hyphal wall in the water mold, Achlya ambisexualis Raper

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Title:
Molecular architecture of the hyphal wall in the water mold, Achlya ambisexualis Raper
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Water mold
Achlya ambisexualis
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xi, 163 leaves : ill. ; 28 cm.
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Reiskind, Julia Barth, 1941-
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Saprolegniaceae   ( lcsh )
Molds (Fungi)   ( lcsh )
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non-fiction   ( marcgt )

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Thesis:
Thesis--University of Florida.
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Includes bibliographical references (leaves 150-162).
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by Julia Barth Reiskind.
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Typescript.
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Vita.

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University of Florida
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MOLECULAR ARCHITECTURE OF THE HYPHAL WALL IN THE
WATER MOLD, ACHLYA AMBISEXUALIS RAPER


















BY

JULIA BARTH REISKIND


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY




UNIVERSITY OF FLORIDA


1980


































I would like to dedicate this dissertation to the memory of

John R. Raper, who initiated physiological studies of Achlya by his

pioneering research on the hormonal aspects of mating, and who,

years later, stimulated my interest in fungal physiology and

genetics.














ACKNOWLEDGEMENTS


I would like to thank Drs. Mildred M. Griffith, Chesley B. Hall,

Thomas E. Humphreys, and Paul H. Smith for serving as members of my

supervisory committee. I appreciate their availability and advice during

the course of this research.

I would also like to thank Drs. R. Michael Roberts and S. M.

Mahaboob Basha for the use of their facilities for the gas chromatographic

studies and for their advice on this aspect of the research. Appreciation

also goes to Dr. Arnold S. Bleiweis and Mr. Steven F. Hurst for their

assistance and facilities in the amino acid analyses, to Drs. Henry C.

Aldrich and Gregory W. Erdos for their help and equipment in the ultra-

structural studies, to Dr. Christine E. Carty for the techniques of

lipid extraction and analyses, and to Drs. Stephen G. Zam and Francis C.

Davis for their consultation throughout this study. Thanks also go to

Mr. Charles K. Cottingham for the preparation of cellulase from Achlya

czbisexualis, to Dr. Michael LaBarbera for the use of the polarizing

light microscope, to Dr. Lewis Berner for aid in microphotography, and

to Dr. Jerome M. Aronson who did the x-ray diffraction analyses.

I would like especially to thank the chairman of my supervisory

committee, Dr. J. Thomas Mullins, for his support and advice throughout

this research. This study could not have been completed, much less

started, without his initial definition of the problem and subsequent

assistance.


iii










Final thanks go to my husband, Jon, for his time, patience and

understanding during the long course of this task. I especially

appreciate his willingness to help in child care and other household

duties. I also thank my children, Alix and Michael, for their patience

during this study.















TABLE OF CONTENTS


ACKNOWLEDGEMENTS. . . ... ..... iii

LIST OF TABLES. . . ... ....... vii

LIST OF FIGURES . . ... ....... viii

ABSTRACT. . . ... . x

INTRODUCTION. . . ... . 1

LITERATURE REVIEW . . ... 3

The Organism, Achyla ambisexualis Raper . 3
Higher Plant Cell Walls . . 5
Fungal Walls. . . ... 9
Fungal Walls, Chemical Structure. . ... 12
Fungal Walls, Physical Structure. . ... 24
Fungal Walls, Morphology. . . ... 25
Fungal Walls, Growth. . .... 28

MATERIALS AND METHODS . . ... ..... 30

Organism and Culturing Techniques . ... 30
Hyphal Wall Isolation and Purification. . ... 30
Chemical Fractionation of the Wall. . ... 31
Chemical Analyses of Wall Constituents. . ... 33
Hydrolysis of Buffer-Water Washed Walls by
A. ambisexualis Cellulase. . ... 41
Ultrastructural Studies . . ... 42

RESULTS . . ... . 45

Criteria for Wall Purity. . . ... 45
Chemical Fractionation of the Wall. . ... 45
Chemical Analyses of Wall Constituents. . ... 50
Hydrolysis of Buffer-Water Washed Walls by
A. ambisexualis Cellulase. . ... 78
Ultrastructural Studies . . ... 78

DISCUSSION. . . ... . 100

The Preparation of Wall Samples . .. 100
Chemical Fractionation of the Wall. . ... 100
Chemical Analyses of Wall Constituents. . ... 107










Hydrolysis of Buffer-Water Washed Walls by
A. ambisexualis Cellulase . 111
Ultrastructural Studies . . .. 112

CONCLUSION . . . 120

APPENDICES . . . 122

A TECHNIQUES . . ... 123

Buffer-Water Washing of Isolated Walls . ... 123
Chitin Isolation . . ... .. .123
Cellulose I Isolation . .. ... 124
Preparation of Acid Swollen Cellulose . ... 125
Enzyme Purification . . .. 125
Enzymatic Hydrolysis of Laminarin . .. 126
Hydrolysis of the Unfractionated Wall with H2S04 127
Description of Analyses Used for the Detection of
Neutral Sugars . . ... .. 127
Solubility Analysis of the Hexosamine Component of the Wall 134
Uronic Acid Analysis. . . .. 135
Lipid Extraction and Analysis . .. 135
Phosphorus Analysis . . .. 136
Ultrastructural Studies Thin Section . ... 136

B RECIPES. .. . ... 137

Growth Media for A. ambisexuais. . 137
Cadoxen Reagent . . 137
Schweitzer's Reagent. . . ... 138
Anthrone Reagent. . . .. 138
Glucostat Test . . . 139
Cellulase Viscometric Assay . .. 139
DMAB Assay . . . 140
Folin Test . .. . 141
BioRad Protein Assay. . . ... 141
Lipase Assay. . . .. 141
Carbazole Test. . . .. 142
Fiske-Subbarow Assay. . . ... 143

C PERIPHERAL STUDIES. . . 144

Dry Weight Determination of Washed Mycelium .. 144
GLC Analyses of H2S04 Hydrolysates of Unfractionated Walls. 144
Congo Red Stain of the Wall and its Fractions .. 144
Wall Width as Measured from Thin Section Micrographs 146
Observations of Enzymatically Treated Material by
Phase Microscopy . . .. 146
Observations of Hyphal Branching by Polarizing Light
Microscopy . . 149

REFERENCES. . . . 150

BIOGRAPHICAL SKETCH . .... .. 163














LIST OF TABLES


Table

1 Conditions of enzyme hydrolysis . .... 35

2 Carbohydrate fractions of A. ambisexualis wall. ... 49

3 The separation of enzyme hydrolysates of wall fractions
of A. anbisexualis by paper chromatography. ... 51

4 The separation of acid hydrolysates of wall fractions of
A. amnbisexualis by paper chromatography ... 52

5 Periodate consumption and format liberation of
A. ambisexualis wall fractions. . ... 66

6 Periodate consumption and format liberation of
known polysaccharides. . .. 67

7 X-ray diffraction analysis of Schweitzer's and cadoxen
reagent-soluble fractions of A. cabisexualis wall .... 68

8 Analysis of solubility of glucosamine from
unfractionated walls of A. ambisexualis ... 72

9 Amino acid profile of the total wall of A. ambisexuaZis
after chemical or buffer-water cleaning ... 73

10 Comparison of amino acid profiles of samples of the
total wall of A. ambisexualis during various stages
of chemical cleaning. . . ... 75

11 Chemical constituents of the buffer-water washed
walls of A. ambisexuaZis. .. . ... 79

12 Microfibrillar diameter of various preparations from
A. ambisexuaZis . .... ..... 99


vii














LIST OF FIGURES


Figure

1 Phase contrast photographs of cleaned walls ... 47

2 Decrease in total protein as a measure of wall purity 48

3 GLC of the TMS derivatives of the monosaccharides released
by hydrolysis of the wall fractions or the total wall by
unpurified A. niger cellulase . ... 55

4 GLC of the TMS derivatives of the mono- and disaccharides
released by acid hydrolysis of wall fractions and total
wall. . . ... .... 58

5 GLC of the TMS derivatives of the mono- and disaccharides
released by hydrolysis of the wall fractions or the total
wall by laminarinase. . . ... 61

6 GLC of the TMS derivatives of the monosaccharides released
by hydrolysis of the total wall with laminarinase and
unpurified A. niger cellulase . .... .64

7 X-ray diffraction patterns of cellulose II isolated from
A. ambisexualis walls . .... 70

8 Comparison of amino acid profiles of samples of the total
wall of A. ambisexualis during various stages of chemical
cleaning. . . ... ..... 77

9 Increase in reducing sugars of isolated wall after
hydrolysis by A. ambisexualis cellulase (uncorrected
data) . . ... . 81

10 Increase in reducing sugars of isolated wall after
hydrolysis by A. ambisexualis cellulase ... 83

11 Surface replicas of isolated walls treated chemically 85

12 Surface replicas of wall fractions. . ... 88

13 Surface replicas of live hyphae after chemical treatment. 90

14 Surface replicas of live hyphae after single enzyme
treatment . . ... ..... 93


viii










15 Surface replicas of live hyphae after sequential
enzyme treatment. . . ... ... 95

16 Surface replicas of live hyphae after sequential
enzyme treatment. . . ... ... 97

17 Molecular model of the carbohydrate portion of the
hyphal wall . . ... .... 106

18 Scheme for explaining the apparent increase in micro-
fibrillar width as a result of enzymatic or chemical
treatment . . ... ...... 117

19 Diagrammatical representation of the hyphal wall based
on ultrastructural evidence . ... 119

20 Periodate and iodate oxidation blanks . .. 131

21 Periodate consumption of the wall fractions and the
total wall. . . ... ..... 132

22 Formate liberation of the wall fractions and the total
wall. . . ... .... 133

23 GLC of the TMS derivatives of the monosaccharides
released by H2SO4 hydrolysis of the unfractionated
wall. . . ... .... .145

24 Apical and subapical sections of an A. ambisexualis
hypha . . ... . 148











Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy


MOLECULAR ARCHITECTURE OF THE HYPHAL WALL IN THE
WATER MOLD, ACHLYA AMBISEXUALIS RAPER

By

Julia Barth Reiskind

June 1980

Chairman: J. Thomas Mullins
Major Department: Botany


In order to elucidate the molecular architecture of the hyphal wall

in Achlya cabisexualis Raper both chemical and morphological analyses

were done. Isolated cleaned walls were fractionated chemically. Acid

and enzyme hydrolysates of the resulting polymers or of the unfractionated

wall were analyzed for their neutral sugar content and for their pattern

of linkage and branching. Glucose was the only monosaccharide found, but

three disaccharides were detected, laminaribiose, gentiobiose, and cello-

biose, indicating the presence of $1,3; 1,6; and $1,4 linkages. Close

to 40% of the wall was found to consist of acid-soluble glucans of B1,3

linkages with single B1,6 linked glucose units every fifth monomer. A

much lower percentage (7%) of the wall was soluble in alkali following

the acid treatment. The structure of this fraction was determined to be

a linear polymer of B1,3 and B1,4 linkages with occasional B1,6 side

chains. About 20% of the wall was solubilized by known cellulose solvents

and was considered to be cellulose II based on x-ray diffraction analysis.

Nearly 6% carbohydrate remained after these treatments. This insoluble

residuum was found to have a linkage pattern similar to the alkali-soluble









fraction. Almost 3% of the wall was found to be glucosamine, most of

which was in an insoluble form. After additional analysis it was

concluded that this component was a weakly acetylated chitin. A 10%

protein component was found in the wall, and amino acid analysis revealed

the total spectrum of amino acids including hydroxyproline. Very small

amounts of uronic acids and phosphorus were found, but virtually no

lipid was detected.

Ultrastructural analyses of carbon-platinum surface replicas of

hyphae treated either chemically or enzymatically, of isolated walls

treated chemically, and of various wall fractions were performed. Both

laminarinase, laminarinase plus protease, and acid plus alkali treatments

removed the acid- and alkali-soluble glucans and revealed the underlying

microfibrils of cellulose. The addition of cellulase to the laminarinase

plus protease mixture resulted in virtual dissolution of the hyphae.

Cadoxen, following acid and alkali treatments, resulted in almost total

removal of the microfibrillar pattern. Observations of the surfaces of

the various wall fractions indicated that the acid-soluble phase was

amorphous, the alkali-soluble and insoluble residuum both faintly micro-

fibrillar, and the cellulose II strongly microfibrillar. The cellulose I

and chitin fractions were both uniformly microfibrillar. Morphologically,

the hyphal wall of A. ambisexualis is similar to that of other Phycomycetes.

Basically, the wall consists of an outer amorphous portion of 61,3 and

01,6 glucans which covers an inner microfibrillar component.

From these studies two models of the wall were designed. One is

a molecular model which attempts to describe the molecular architecture

of the wall. The other model is a diagram of the various wall components

based upon both chemical and ultrastructural studies.














INTRODUCTION


Hyphal walls in fungi, as in other organisms, provide an invalu-

able function. In addition to protecting the protoplast from environ-

mental damage, they also, by their rigid nature, aid the organism in

maintenance of its characteristic morphology. Although rigid, walls are

also pliant allowing the necessary morphological changes which occur with

growth. They also play a role in cellular recognition between different

organisms, as in host-parasite interactions and immune responses. Walls

are considered to be an integral part of the living system (Preston 1979),

perhaps as a single macromolecular entity (Keegstra et al. 1973). A

number of studies have been made in an attempt to elucidate the molecular

structure of the cell walls of various organisms. The common pattern

which emerges is that of a rigid fibrillar structure embedded in and

covered by an amorphous matrix. Pores, perhaps proteinaceous in nature

(Wrathall and Tatum 1973), are thought to exist in the wall and to allow

the passage of macromolecules (Farkas 1979).

In this study, the hyphal wall of the water mold AchZya ambisexualis

Raper was analyzed both chemically and ultrastructurally, and a model of

its molecular structure is proposed. Previous research on this organism

has indicated a correlation between lateral branching and increased

production and secretion of the enzyme cellulase (Thomas and Mullins

1969). It has been theorized that localized hydrolytic action by this

enzyme "softens" or restructures the wall allowing branch initiation to

occur (Thomas and Mullins 1969). A logical step in the study of the role









of this enzyme in branching and growth is an in-depth analysis of the

"substrate," i.e., the hyphal wall.

One question which this study attempted to answer is "What are the

bonds of interest in the hyphal wall?" What aspect or aspects of the

wall are responsible for its integrity? Early observations that lysis

of A. ambisexualis hyphae occurred within two hours following the

application of a cellulase led to the suggestion that the structural

integrity of the hyphal wall resides directly in the cellulosic

component or indirectly between the cellulosic and another component

(Mullins 1979). This is in sharp contrast to Preston (1974a) in his

analyses of higher plant walls, who states that the matrix portion of

the wall is the key to its integrity. Another feeling, however, is that

both components are necessary for the integrity of fungal walls (Bartnicki-

Garcia and Lippman 1967; Sietsma et al. 1968, 1969; Hunsley and Burnett

1970; Tokunaga and Bartnicki-Garcia 1971). Whatever the case, the two

functions of the wall, maintenance of hyphal morphology and plasticity

to allow growth, must be borne in mind in the consideration of its

molecular structure. One further point is the observation of Hunsley

and Burnett (1970) of a more loosely arranged fibrillar structure at

growing hyphal apices than in more distal or nongrowing regions. As

branching occurs subapically, the conformation of the wall must be

changed in order for this to occur and it may be here that cellulase or

related hydrolytic enzymes play a key role.














LITERATURE REVIEW


The Organism, AchZya ambisexualis Raper


Achlya ambisexualis Raper belongs to the Class Phycomycetes,

Series Biflagellate, Order Saprolengniales, Family Saprolegniaceae

(Alexopoulos 1952). An alternate classification divides the Phycomycetes

into two subclasses, one of which is the Oomycetes which have differen-

tiated gametangia and to which Achlya belongs (Alexopoulos 1952). One

of the distinguishing characteristics of the Saprolegniales is the

possession of cellulosic cell walls, a situation not commonly found in

the fungi. Fungi of the genus AchZya inhabit fresh water and generally

form colonies around pieces of decaying plant and animal material. AchZya

ambisexualis is filamentous and is made up of coenocytic hyphae surrounded

by a rigid wall. A mass of hyphae is termed a mycelium. Septa, complete

plates in this fungus, are formed only at the base of the reproductive

structures or are sometimes found in aging mycelia. Vegetative growth

occurs at the hyphal apices, also the area of sporangial formation.

Sporangial spores are motile and biflagellate, one flagellum of the

whiplash type and the other of the tinsel type. Sexual reproduction is

by gametangial contact in which there is transfer of male gametes produced

in an antheridiumtofemale gametes or oospheres produced in a oogonium

via fertilization tubes (Alexopoulos 1952).

Sexual reproduction is initiated and sequentially controlled by a

series of diffusible hormones. Raper (1939a, b; 1940) first described

3









this hormonal mechanism in Achyla. The sequence is described as follows:

(1) vegetative female hyphae secrete hormone A into the growth medium;

(2) this hormone is taken up by the male hyphae and the result is the

production of numerous lateral branches, termed antheridial hyphae; (3)

the induced male now secretes hormone B, (4) which is taken up by the

female and causes the induction of oogonial initials; (5) two additional

hormones, C and D, were postulated to be involved in the attraction and

appression of the antheridia to and onto the oogonium; (6) this is followed

by septal delimitation at the antheridial tip and at the base of the

oogonium to form the functional sexual organs. These two cells are the

site of meiosis and gametogenesis. Fertilization takes place with the

formation and inward growth of fertilization tubes from the appressed

antheridia into the oogonium. The species name, A. ambisexualis, was

chosen by Raper (1939a) to emphasize the presence of a wide range of

sexual capacities among the various isolates, i.e., pure female or pure

male, or either male and female depending upon its mating partner.

Hormone A was chemically characterized by McMorris and Barksdale

(1967) and renamed antheridiol. Several structures were proposed

(Arsenault et aZ. 1968) and two isomers of one of the proposed structures

were synthesized, one of which resembled natural antheridiol in its

physical and biological properties (Edwards et al. 1969). Hormone B

was similarly chemically characterized and renamed oogoniol by McMorris

et al. (1975). Both antheridiol and oogoniol are steroids and are the

best characterized sex hormones of this structure found in the plant

kingdom.

Strain E 87, a pure male, was used in this research. The response

by this strain to antheridiol has been examined biochemically. Both









antheridial branching and an increase in the secretion of cellulase into

the wall and its subsequent release into the medium have been found as a

result of antheridiol treatment. The role of cellulase has been postulated

to cause localized wall softening allowing antheridial branching to occur

(Thomas and Mullins 1969). Observations of lateral wall thinning at the

sites of branch initiation gave support to the wall softening theory

(Mullins and Ellis 1974). RNA and protein synthesis have been found to

be required for both branching and cellulase production (Kane et al.

1973; Horowitz and Russell1974; Timberlake 1976).


Higher Plant Cell Walls


Although the principal subject of this dissertation is the study of

a fungal wall, some discussion of higher plant cell wall structure and

growth is relevant. Albersheim and co-workers (Albersheim et al. 1973;

Bauer et al. 1973; Keegstra et al. 1973; Talmadge et al. 1973; Albersheim

1974) have done the most recent and major work in this area and this has

generated considerable interest (Monro et al. 1974, 1976). In terms of

carbohydrate polymers, the higher plant cell wall is more complex than

that found in fungi. The technical approach, however, for studying both

the chemical and physical aspects of walls is similar.

In general, more detailed wall analyses have been done on primary

walls rather than secondary. Primary walls are less differentiated and

occur in plant cells which are still growing. The basic cell wall

structure of a number of dicotyledonous plants has been analyzed and

found to be basically similar (Albersheim et al. 1973). Where differences

in structure occur, they are usually in the linkage, number, and types

of attached residues which act as side chains. Such differences, for









example in the hemicellulose or pectic portion of the wall, result in

changes in physical properties and thus biological function (Aspinall

1973). Pectic substances and hemicelluloses together with cellulose

form the bulk of the primary cell wall of higher plants. The carbohydrate

polymers of the plant cell wall are: (1) cellulose; (2) hemicellulose

(xylans and glucamannans); (3) pectic substances [galacturonans, arabinans,

galactans and/or arabinogalactans, and rhamnogalacturonans (Talmadge et

al. 1973)]; (4) glycoproteins (Aspinall 1973).

Most of these studies employed cultured cells and involved isolating

and cleaning the cell walls, followed by fractionation either by the use

of purified hydrolytic enzymes, or alkali, urea, and mild acid (Albersheim

et al. 1973; Bauer et al. 1973; Talmadge et al. 1973; Albersheim 1974).

Monosaccharides, fractionated polysaccharides, and total wall poly-

saccharides were analyzed for their monomeric structure, type of glycosidic

linkage, and anomeric configuration by a combination of gas chromatography-

mass spectrometry methylation analysis.

Analyses of an endopolygalacturonase digest indicated that the

pectic polysaccharide portion consists of a rhamnogalacturonan main

chain with side chains of arabinans and galactans. The galactan has been

postulated to serve as a bridge to the hemicellulosic portions of the

wall (Talmadge et al. 1973). The hemicellulose portion of the wall is

basically a xyloglucan polymer. This component consists of two fragments,

one, a seven-unit sugar, and the other, a nine-unit one. In addition to

xylose and glucose, small smounts of galactose and fucose are present in

the larger fragment (Bauer et al. 1973; Albersheim 1974). It has been

speculated that covalent linkages exist between the pectic polysaccharides

and the hemicellulosic portion of the wall, while non-covalent linkages










link the cellulosic and hemicellulosic wall components (Bauer et al.

1973). The amount of xyloglucan is sufficient to cover, via the formation

of hydrogen bonds, all of the cellulose fibrils (Bauer et al. 1973).

Lamport and Northcote (1960) reported the existence of a specific

protein occurring in plant walls which contains an imino acid, hydroxy-

proline, which is usually found only in trace amounts in cytoplasmic

protein. It was suggested that this protein might be responsible for

cross linking various wall components and that wall extension might be

caused by the enzymatic reduction of disulphide bridges (Lamport 1965)

or at least by the liability of certain covalent linkages in this glyco-

protein (Lamport 1970). Structural studies of hydroxyproline-rich

glycopeptides have indicated a polypeptide backbone with oligoarabinose

side chains (Lamport 1967, 1969). Subsequently a hydroxyproline-rich

glycopeptide was isolated which contained galactose bound by the hydroxyl

group of serine, and a tentative structure was devised consisting of a

serine with an attached galactose and four hydroxyprolines each with

four arabinose molecules (Lamport 1973). There are many questions concern-

ing cell wall proteins; for example,what sugar is covalently bound, is

there more than one structural protein in the wall, and to which

component of the wall is the protein attached (Preston 1979)?

The only molecular model which will be considered in detail is that

designed by Keegstra et al. (1973) based on the chemical analyses of

sycamore cell walls. Briefly, the matrix of the cell wall includes the

pectic substances, the proteinaceous component, and the hemicellulosic

materials while the cellulosic portion makes up the microfibrillar region.

Covalent cross linkages are postulated to hold the matrix together, while

hydrogen bonds are responsible for cementing the cellulose portion and










for binding the cellulosic molecules to the xyloglucan component of the

matrix. The hydrogen bonding is so extensive that it is considered to

have strength comparable to the covalent linkages. In the model the

glucose moiety of the hemicellulose component lies parallel to the axis

of the cellulose fiber and is bonded by hydrogen bonds. Arabinogalactan

chains lying perpendicular to the cellulose fibrils bind the hemicellulose

to the pectic substances by glycosidic linkages and may also play a role

in binding the hydroxyproline-rich protein (Albersheim et al. 1973).

Taking all the bonds into account a rigid matrix is formed (Albersheim

1974).

Cell elongation, as envisioned in the model, occurs by the ability

of the cellulose molecules to slide past each other, suggesting that

certain bonds are labile. It is postulated that for nonenzymatic

creep-the slow yielding of the cell wall under constant stress

thought to be responsible for cell growth (Preston 1974a)-to occur, only

four consecutive hydrogen bonds need to be broken and it is thought that

these bonds exist between the xyloglucan chains and the cellulose

microfibrils. The rate of creep can be enhanced by lowering the pH or

raising the temperature. Thus auxin may act because of its ability to

stimulate growth via the activation of a hydrogen ion pump (Keegstra

et al. 1973). An alternative suggestion (Albersheim 1974) is that bond

breakage and reformation is mediated enzymatically with the involvement

of a hydrolase and a synthetase.

This model has been criticized on several points (Preston 1979;

Monro et al. 1976). In addition to criticisms of the techniques used,

Preston (1979) felt the binding of xyloglucan and cellulose is unlikely

because of the highly branched nature of the hemicellulose. Stronger










criticism of the model comes from the work of Monro et al. (1974, 1976).

The model, or working hypothesis, devised by these researchers differs

from Albersheim's in several respects. A small amount of hemicellulose

(30% or less) is thought to serve as a covalent bridge between the

protein and the microfibrils. In addition, a fraction of the wall

protein itself or in conjunction with a polysaccharide is felt to be

covalently bound to the microfibrils. More hydrogen bonds are implied

in this model, especially in the matrix. Monro et al. (1974, 1976)

have suggested that the bonds controlling creep should be at right

angles to the direction of elongation, which is not the case in the

Albersheim model. The Albersheim model is an explanation for cell

expansion rather than for cell elongation (Monro 1976). Longitudinal

growth in the Monro model occurs in the hydrogen bonded matrix region

by the shearing of these bonds and is independent of microfibrillar

orientation. These authors state also that studies of the roles of

synthetic and hydrolytic enzymes must be done in order to reach a

better understanding of what occurs in cell enlargement.



Fungal Walls


In general fungal walls appear to be simpler in structure than

those of higher plants, at least simpler in the types of sugar monomers

present. Hyphal walls are described as complex microfibrillar systems,

the microfibrils embedded in a matrix, generally made up of glucans,

mannans, and galactans (Northcote 1963; Aronson 1965; Rosenberger 1976).

The microfibrillar component is usually chitinous, but in a few cases is

cellulosic. A commonly used analogy of the combination of the matrix










and microfibrils is that of reinforced concrete (Rosenberger 1976).

Fungal walls consist generally of 60 to 90% polysaccharide; other

components are uronic acids, protein, lipids, melanin (in some cases),

polyphosphates, and inorganic ions. Carbohydrate-protein complexes

are formed by ester, o-glycosidic, and glucosamine linkages (Sturgeon

1974). A few detailed studies on yeasts and dermatophytes have been made

on wall glycoproteins and peptido-polysaccharides (Gander 1974). In

general these compounds function as enzymes or recognition sites in

mating type or host-pathogen relationships (Gander 1974) and do not

appear to have a role in wall structure.

Ultrastructural studies depict the wall as existing in basically

two layers. The outer layer is the matrix and the inner, nearest the

plasma membrane, is the microfibrillar. The change in layers in the

wall is gradual rather than abrupt (Bartnicki-Garcia 1973). In some

structures there is a third layer, melanin, which lies outside the matrix.

Basic wall form seems to be similar in the various taxonomic groupings

of fungi, even though the chemical composition differs (Bartnicki-Garcia

1973).

A correlation exists between the chemical structure of the wall

and the major taxonomic groups of fungi (Bartnicki-Garcia 1968). Eight

wall categories were created and the various taxonomic groups were

placed in the appropriate one. Members of the first two categories

contain cellulose as the microfibrillar portion, but one has glycogen

as the matrix and the other glucan. The Acrasiales belong to the

former group, while the Oomycetes belong to the latter. Organisms

of the third category have both cellulose and chitin microfibrils









and are represented by members of the Hyphochytridiomycetes. The

fourth is known as the chitosan-chitin category and includes the

Zygomycetes. The fifth and by far the largest group is the chitin-

glucan one which includes the Chytridiomycetes, the Ascomycetes, the

Basidiomycetes, and the Deuteromycetes. Most yeasts belong in the sixth

category, the mannan-glucan one. Yeasts with carotenoid pigments are

placed in the seventh category, the mannan-chitin one. The last

category consists of polygalactosamine-galactan, and are represented by

the Trichomycetes.

In terms of the various polymers found in fungal walls, a distri-

bution pattern can be made (Rosenberger 1976). R-glucans with B1,3 and

81,6 linkages are found in most groups except the Mucorales, while

S-glucans, al,3 linked,are limited to the Ascomycetes and the Basidiomycetes.

Cellulose is found in a few Phycomycetes, while chitin is more universal.

Chitosan and a polysaccharide of galactosamine are found in the

Mucorales, and the Ascomycetes and Hyphomycetes respectively. Poly-

uronides are known in the Mucorales. Protein, or at least the common

amino acids, is found universally. Hydroxyproline is reported in those

which have cellulose in their walls. A more recent study has noted the

presence of hydroxyproline in the basidiomycete TremeZla (Cameron and

Taylor 1976), where chitin occurs.

By altering the metabolism of the cell wall constituents, a

fungus can change its morphology (Dow and Rubery 1977). Such alterations

may involve changing from a mycelial to a yeast form or the reverse, or

changing to a reproductive, survival, or invasive mode. Studies with

Mucor rouxii indicated that there are higher quantities of protein and

mannose in the cell walls of yeast forms as opposed to mycelial










(Bartnicki-Garcia and Nickerson 1962; Bartnicki-Garcia 1968; Dow and

Rubery 1977). Additional differences include the presence of weakly

acidic polysaccharides in yeast walls and strongly acidic ones in

mycelial walls (Dow and Rubery 1977). Quantitative differences in the

chemical composition have been noted in the walls of the different

structures, such as sporangial and hyphal, within a single organism

(Bartnicki-Garcia and Reyes 1964; Bartnicki-Garcia 1968; Cole et al.

1979; Mendoza et al. 1979).


Fungal Walls, Chemical Structure

Before describing the chemical structure of fungal walls certain

inherent shortcomings of studies of this nature will be discussed. The

first problem can be stated simply by the questions, "what is a clean

wall?" and "what is the method used for determining cleanness?" Walls

cleaned with hot alkali appeared pure microscopically, and chemical

analyses revealed that many covalently bound amino acids were released

(Cameron and Taylor 1976). Are these components part of the wall

structure? Loosely bound wall constituents may protrude into periplasmic

space; are these inherent structural compounds? An isolated wall is

quite a different thing from a wall which is part of a living system

and this must also be borne in mind (Crook and Johnston 1962; Cameron

and Taylor 1976). Enzyme degradation is a commonly used method for

studying wall composition but there are drawbacks to this technique, such

as the use of impure enzymes and rearrangements in wall architecture

caused by partial digestion (Farkas 1979). The products of chemical

degradation must also be viewed with reservation due to the liability

of certain constituents (Talmadge et al. 1973). Bearing these thoughts









in mind, it becomes clear that the results of an analysis of wall

composition must be regarded with circumspection (Cameron and Taylor

1976). This does not mean that tentative wall models cannot be drawn,

but that they must be considered in the light of the above restrictions.

Basidiomycete Walls

Detailed studies of isolated walls of SchizophytZwn commune have

been made over the last few years by Wessels and his group (deVries

and Wessels 1972, 1973a, b; Wessels et aZ. 1972; Sietsma and Wessels

1977, 1979). A lytic enzyme preparation from Trichoderma viride grown

on isolated SchizophyZlun walls was found to be active against the known

substrates chitin and R- and S-glucan, thus giving a clue to the identity

of the wall components (deVries and Wessels 1972, 1973a, b). The most

external portion of the wall in S. commune is a water soluble layer of

mucilage. The basic structure of this component is a B1,3 glucan back-

bone with single glucose units linked through the sixth carbon atom of

every third glucose residue (Wessels et al. 1972; Sietsma and Wessels

1977). An alkali soluble al,3 linked chain, the S-glucan, lies adjacent

to the mucilage and next to this is the alkali insoluble R-glucan which

is: similar structurally to the mucilage except that it is more highly

branched (Sietsma and Wessels 1977). The R-glucan is closely associated

with the chitinous portion, identified by x-ray diffraction studies, of

the wall. A tentative model of the R-glucan complex was postulated in

which covalent linkages were suggested between the chitin and the

R-glucan portions (Sietsma and Wessels 1979). Exo- B1,3-glucanase

hydrolysis of the R-glucan followed by chitinase treatment yielded a

compound containing N-acetylglucosamine, glucose, lysine, citrulline,










glutamate, and glucosamine. The model drawn from these data describes

this portion of the wall as consisting of a linear chitinous chain

which is linked to an R-glucan oligomer by a bridge containing lysine,

citrulline, glutamic acid, glucose, and N-acetylglucosamine. In

summary, it was found that hyphal wall fragments consisted of 67.7%

glucose, 3.4% mannose, 0.2% xylose, 12.5% N-acetylglucosamine, 6.4%

amino acids, and 3.0% lipid. The mannose and xylose monomers are

associated with the S-glucan component.

Chemical analyses of the walls of Tremella mesenterica indicated

the presence of xylose, mannose, rhamnose, and fucose in addition to

glucose (Cameron and Taylor 1976). The amino acid content of these

walls was studied and, as mentioned previously, hydroxyproline was

found (Cameron and Taylor 1976). Polystictus and Ustilago walls have

been analyzed and the above listed monosaccharides were found; galactose

was also found in Ustilago walls (Crook and Johnston 1962).

Deuteromycete Walls

These are the imperfect fungi and some are known only by their

asexual states (Alexopoulos 1952). Enzyme degradation studies of

Aspergillus oryzae and Fusariuw solani indicated the presence of chitin

and 81,3 glucans. Wall degradation did not occur unless both enzymes

were present or unless there was a glucanase pretreatment, leading to the

speculation that the wall consists of a chitin-containing core masked by

the glucan (Skujins et al. 1965). Chemical analyses of the carbohydrate

content of isolated walls from AspergilZus sp. and A. niger showed that

50 to 60% of the wall is carbohydrate of which 4.3% is mannose, 5 to 14%

is galactose, and the remainder is glucose (Ruiz-Herrera 1967; Cole et aZ.









1979). The amount of chitin in both organisms is 15%. Studies of

several species of PeniciZlliu, F. oxysporum, and Botrytis cinerea

revealed the same proportion and types of monosaccharides as found in

Aspergillus, although mannose was not universally found (Crook and

Johnston 1962; Pengra et al. 1969).

Total protein measurements revealed between 7 and 8% of the dry

weight of the wall. Amino acid analyses were performed on both

Aspergillus organisms and the usual spectrum was found (Crook and

Johnston 1962; Ruiz-Herrera 1967; Cole et al. 1979). Hydroxyproline was

not found. The lipid content of readily extractable and bound lipids

was assayed for two Aspergillus species. Extractable lipids were found

to be present as 7.3% of the wall while bound lipids varied between 7

and 12% depending on the study (Ruiz-Herrera 1967; Cole et al. 1979).

Because some of the lipid could only be extracted after acid treatment

of the walls, some of the lipoidal material present in the wall is

probably completed with structural polysaccharides and/or proteins

(Ruiz-Herrera 1967). Ash was not found in the A. niger wall (Cole et

aZ. 1979), but was found in that of AspergiZlus sp. (4%) (Ruiz-Herrera

1967). Phosphorus content in both types of walls was found to be very

low (0.1%) (Ruiz-Herrera 1967; Cole et al. 1979).

Ascomycete Walls

An analysis of the neutral sugars isolated from Chaetomium globosum

and Neurospora sitophila indicated high glucose and low mannose and

galactose amounts. Glucosamine was found in the walls of both organisms,

and galactosamine was found in Neurospora (Crook and Johnston 1962). The

presence of chitin was established by the usual means. Treatment of the










wall with exo- and endo-81,3-glucanases indicated the presence of a

B1,3 glucan with some 81,6 linked glucose residues. From this datum it

was proposed that the wall consists of layers of 81,3 glucans overlying

a chitinous core (Potgieter and Alexander 1965). It was also noted in

this study that, although there was noticeable wall thinning after

exhaustive enzyme treatment, the characteristic hyphal morphology remained

unaltered (Potgieter and Alexander 1965).

More complete studies were performed on Neurospora crassa walls

where analyses of wild type and single gene morphological mutants

("colonial") walls were performed. Neurospora crassa walls were separated

into four fractions based on their solubilities in a variety of solvents

(Mahadevan and Tatum 1965). Changes in fraction I, basically consisting

of glucose, galactosamine, and glucuronic acids, were felt to be the

predominant factor in influencing colonial morphology (Mahadevan and

Tatum 1965). Higher amounts of uronic acids in wild type walls suggested

that these compounds have a role in regulating linear hyphal growth,

possibly due to the increased water content accompanying these compounds

which may increase wall plasticity (Cardemil and Pincheira 1979). An

increase in mannose and galactose in the mutants suggested that colonial

morphology may result from higher levels of branching mannan component

allowing increased bonding and therefore more rigidity (Cardemil and

Pincheira 1979).

Five peptide fractions extracted from N. crassa walls with weak

alkali indicated the presence of all normally occurring amino acids

(Wrathall and Tatum 1973). Quantitative differences were found, but

there were similarities in the ratio of acidic to basic components and

in the proportions of hydrophilic residues. 0-glycosyl-serine linkages










were discovered which indicated that this component was part of a glyco-

protein which did not appear to be covalently linked to any other major

wall constituent. It was felt that this was evidence for a separate

glycoprotein reticulum as a wall component, thus supporting the earlier

work of Hunsley and Burnett (1970).

The yeast cell wall has been studied in great detail and the follow-

ing will only briefly touch on the subject. Three fractions of the yeast

wall were obtained by extractions with anhydrous ethylenediamine (Korn

and Northcote 1960). Fraction A, soluble in water and ethylenediamine,

contained the total spectrum of amino acids plus mannose and glucosamine

(36% of the total amino sugar found). This fraction was felt to represent

a mannan-protein complex with the amino sugar serving as a link between

the polysaccharide and protein components (Korn and Northcote 1960).

Fraction B, insoluble in water but soluble in ethylenediamine, was

similar to A, except that glucose was present. Fraction C, insoluble

in both solvents, contained 58% of the glucosamine in addition to chitin.

Subsequent work has confirmed and extended these data. Both the glucan

and mannan components have been characterized more completely. The

major portion of yeast glucan is a S1,3 linked polymer with some i1,6

linkages and the minor portion is mainly 1,6 linked with a few 81,3

linked chains which may occur as interchain or interresidue linkers

(Manners et al. 1973a, b). It was thought that these glucans provide a

structural function with the B1,3 component forming an inner fibrillar

layer (Cabib 1975). Yeast mannan is a polymer of one protein and two

carbohydrate moieties, and may have both immunological and structural

functions (Cabib 1975).










Two models have been proposed for the yeast cell wall. In one

model (Lampen 1968) the wall is made up of phosphomannans which are

located in the outer layer of the wall. Wall-bound enzymes exist in this

portion of the wall and release of these enzymes or cleavage of this

fraction occurs by the action of an enzyme, the PR-factor, a "mannosidase."

A smaller mannan is linked to the phosphomannan complex and also to

glucan fibrils located in the inner portion of the wall. Protein

molecules bound together by disulphide bridges make up part of the glucan

portion of the wall. Observations by Kidby and Davies (1970) of enzyme

release by sonication or thiol treatment in addition to previous studies

by Bacon et al. (1965) led to a slight alteration of this model. In the

altered model enzymes are inserted between the outer and middle layers

and are held by non-chemical means. The structural integrity of the

external wall layer is maintained by disulphide bridges. In this model

the middle layer is a mannan-glucan associated with disulphide-linked

proteins which are bound to a glucan chain which lies just outside the

plasma membrane.

Phycomycete Walls

Initial wall analysis of Allomyces macrogynus (a uniflagellate

Phycomycete) (Aronson and Machlis 1959) indicated the presence of chitin,

glucan, ash, and protein, the latter depending, however, on the method of

wall purification. Chemically cleaned walls contain 68% chitin, 8% glucan,

and 10% ash, while walls cleaned with buffer and water contain 58%

chitin, 16% glucan, 8% ash, and 10% protein. It is obvious that the

two methods of cleaning resulted in modifications of the wall constituents.

Amino acid analysis of a polypeptide fraction revealed a wide range of









these compounds (Youatt 1977). al,4 and cl,6 linkages were found in the

hyphal walls and $1,3 in the walls of discharge plugs (Youatt 1977).

Rhiziomyces sp., another uniflagellate form, has both cellulose and

chitin in its walls as determined by x-ray diffraction studies (Fuller

and Barshad 1960).

Zygomycete walls

Zygorhynchus vuilleminii walls contain galactose, mannose, fucose

(the most abundant monosaccharide found), and glucosamine plus the usual

assortment of amino acids (Crook and Johnston 1962). Two acidic poly-

saccharides, mucoran and mucoric acid, were isolated and analyzed from

Mucor rouxii (Bartnicki-Garcia and Reyes 1968). Mucoran is made up of

2 fucose:3 mannose:5 glucuronic acid and mucoric acid is a homopolymer

of glucuronic acid. It was felt that these components made up a single

heteropolymer (Bartnicki-Garcia and Reyes 1968). Later studies of the

walls of M. mucedo revealed a glycuronan made up of 5 fucose:l mannose:

1 galactose:6 glucuronic acid non-covalently bound to glucosamine polymers

(Datemaet aZ. 1977a). The homopolymeric glucuronic acid part of the

isolated glycuronan is thought to be associated with the glucosamine

polymers (Datema et al. 1977a). Mucor walls also contain weakly

acetylated chitin, chitin, and chitosan (Bartnicki-Garcia and Nickerson

1962; Bartnicki-Garcia 1968; Datemaet al. 1977b). On a percentage w/w

basis the composition of the hyphal wall of M. mucedo is 7% neutral

sugar, 12% uronic acid, 16% phosphate, 32% hexosamine, 13% protein, 10%

amino acids, and 13% ash (Datema et aZ. 1977a, b).

Oomycete walls (Leptomitales)

Analyses of buffer-water washed sonicated walls of Sapromyces

elongatus indicated a typical Oomycete wall, containing 91% glucan,









4% protein, and 0.1% ash with glucose as the only monosaccharide (Pao and

Aronson 1970). 81,3, 81,4, and 81,6 linkages were found. Weakly

crystalline cellulose I was present but chitin was not detected (Pao and

Aronson 1970). The walls contained the full complement of amino acids

with aspartic acid, glutamic acid, serine, and threonine the most abundant

(46% of the wall protein). Hydroxyproline is 2.5% of the total amino

acid content. No lipids were found. ApodachZya sp. and A. brachynema

walls differ from those of Sapromyces in having glucosamine (Sietsma

et al. 1969; Lin et al. 1976). X-ray diffraction studies and stains

for chitin indicated the presence of both weakly crystalline cellulose I

and chitin in these walls (Lin and Aronson 1970). The walls of A.

brachynema contain phospholipids, fatty acids, and triglycerides (Sietsma

et al. 1969). Linkage studies of A. brachynema revealed that 4% of the

dry weight of the wall is soluble in Schweitzer's reagent and consists

solely of 81,4 linkages, and 52% was found to be a branched 81,3 and

81,6 linked glucan, and 32% a linear 81,3 linked glucan (Sietsma et al.

1968). Apodachyla sp. walls contain 67% total glucose, 18% chitin, 9%

cellulose, 6.4% protein, 1.5% acid-soluble hexosamine, and 3.1% alkali-

soluble hexosamine (Lin and Aronson 1970; Lin et al. 1976). Analysis

of the hyphal wall chemistry of Leptomitus lacteus indicated similarity

to the walls of Sapromyces and ApodachZya, especially in regard to the

linkage pattern (Aronson and Lin 1978).

Oomycete walls(Peronosporales)

Wall chemistry analyses of a number of species of Phytophthora

indicated the presence of 90% glucan, 4% protein (10% reported in one

species), 2% lipid, 0.4% phosphorus plus small amounts of mannose,

glucosamine, arabinose, xylose, galactose, rhamnose, ribose, and









galactosamine (Bartnicki-Garcia 1966; Bartnicki-Garcia and Lippman 1967;

Novaes-Ledieu et al. 1967; Tokunaga and Bartnicki-Garcia 1971). Weakly

crystalline cellulose I makes up about 25% of the wall (Novaes-Ledieu

et al. 1967). About 5% of the total amino acid content is hydroxyproline

(Bartnicki-Garcia 1966). Walls of various Pythium species have also been

characterized and similar compositions have been reported, although the

total glucan and cellulose is lower (82% and 20% respectively) and the

lipid is higher (8%) (Cooper and Aronson 1967; Novaes-Ledieu et at. 1967;

Sietsma et at. 1969). All the common amino acids including hydroxyproline

have been reported (Novaes-Ledieu et al. 1967). Low levels of chitin have

also been found (Dietrich 1973).

The same type of linkage pattern was found in Phytophthora and

Pythium walls as was described for the Leptomitales (Bartnicki-Garcia

1966; Bartnicki-Garcia and Lippman 1966, 1967; Aronson et al. 1967;

Cooper and Aronson 1967; Novaes-Ledieu et al. 1967; Eveleigh et al. 1968;

Novaes-Ledieu and Jimenez-Martinez 1969; Sietsma et at. 1969, 1975;

Zevenhuisen and Bartnicki-Garcia 1969; Tokunaga and Bartnicki-Garcia

1971; Yamada and Miyazaki 1976). The basic pattern which emerged from

a number of studies is that of a highly branched glucan of B1,3 and

81,6 linkages covering and firmly bound to a B1,4 linked linear glucan

(cellulose). There are varying opinions as to the degree of branching

and as to which linkage groups serve as main chains and which as branches

(Eveleigh et at. 1968; Novaes-Ledieu and Jim&nez-Martinez 1969; Sietsma

et al. 1969, 1975; Zevenhuisen and Bartnicki-Garcia 1969; Yamada and

Miyazaki 1976). Both components, the branched glucan and the cellulosic,

are reported to be slightly contaminated by linkages of the other (Novaes-

Ledieu and Jimenez-Martlnez 1969; Zevenhuisen and Bartnicki-Garcia 1969;









Sietsma et al. 1975). A 81,2 glucan was reported for the walls of one

species of Pythium (Mitchell and Sabar 1966) and an al,3 glucan was

reported for a species of Phytophthora (Miyazaki et al. 1974).

Oomycete walls (Saprolegniales)

The basic pattern which has been described for the Leptomitales and

the Peronosporales is also seen in the Saprolegniales, the principle

differences lying in the relative quantities of the various components.

The walls of four species of Saprolegnia have been analyzed. The

predominant monosaccharide of S. ferax is glucose while considerably

smaller amounts of glucosamine, mannose, rhamnose, and ribose have been

found (Crook and Johnston 1962; Parker et al. 1963; Novaes-Ledieu et at.

1967). Quantitative studies revealed 93 or 85% total carbohydrate, 3 or

1.1% protein, 1.7 or 2.7% hexosamines, and 1 or 5% lipids depending on

the study (Novaes-Ledieu et al. 1967; Sietsma et al. 1969). All the

studies indicated that cellulose is present in these walls; however, there

are vast quantitative differences ranging from 42% (Novaes-Ledieu et al.

1967) to 18% (Sietsma et al. 1969) to 15% (Parker et al. 1963). In one

study an attempt was made to estimate the proportion of linkages and it

was found that 18% are $1,4, 44% are branched 81,3 with 81,6 linkages,

and 20% are linear B1,3 (Sietsma et al. 1969). The usual amino acid

composition was found (Crook and Johnston 1962; Novaes-Ledieu et al.

1967). Other species of SaproZegnia, S. litoralis, S. monoica, and

S. diclina reveal essentially the same pattern, although uronic acids

were reported in S. litoralis (Parker et al. 1963) and S. diclina

(Cameron and Taylor 1976). Some quantitative differences were found in

S. diclina possibly reflecting differences in wall preparation. These









walls consist of 72.6% neutral sugars, 0.9% amino sugars, 3% uronic

acids, 8.5% protein, and 12% lipid (Cameron and Taylor 1976).

Wall chemistry of Achyla flageZZata, A. racemosa, A. ambisexuatis,

Brevilegnia unisperma var. delica, B. bispora, Dictyuchus sterilis and

Dictyuchus sp. is similar to SaproZegnia in all respects (Parker et at.

1963; Sietsma et al. 1969). The general linkage pattern common to all

these fungi was established for A. ambisexualis and D. steritis (Aronson

et al. 1967; Sietsma et al. 1969). An attempt to determine the proportion

of the linkages was made for D. steriZis and was found to be similar to

that of S. ferax (Sietsmaet al. 1969). Dietrich (1973), studying four

Oomycete genera, found hexosamine in all the walls (the three Achlya

species studied had the highest content: 2.4, 3.1, and 3.8%) and upon

treatment of these walls with snail gut enzyme, N-acetylglucosamine at

1 and 2% levels was obtained. These results led to the speculation of

the presence of a chitin/chitosan component in these heretofore considered

chitinless walls (Dietrich 1973). An indirect indication of the presence

of chitin in Achyla walls stems from the observation of Wang and LeJohn

(1974) of the absence of a UTP requirement for glutamic dehydrogenase;

UTP has been found to be necessary for activation of the enzyme in

organisms with chitinless walls. The walls of the marine fungus,

AtkinsieZZa dubia, have also been studied and in general these walls are

similar to those described above (Aronson et al. 1967; Aronson and

Fuller 1969). Notable differences are the protein content (13.7% in

these fungi) and a very high level of hydroxyproline (20.4% of the total

amino acid content and 2% of the dry weight of the wall)(Aronson and

Fuller 1969).










Fungal Walls, Physical Structure


The only polymers whose structures will be described here are those

which occur in Oomycete walls, i.e., those with 81,3; 01,4; and B1,6

linkages. Cellulose has been studied extensively. Glucose units in

cellulose are joined by 81,4 glycosidic bonds and it is because of this

type of linkage that the polymer can be described as a flat ribbon (Rees

1977; Preston 1979). The chain is stabilized by hydrogen bonds which

form between the third carbon of a glucose molecule and the ring oxygen

of the adjacent glucose unit (Preston 1979). Each ribbon-like chain has

numerous potentially hydrogen binding hydroxyl groups along each edge, so

when two chains come in contact many hydrogen bonds form creating a

stable structure (Preston 1979). In native cellulose,usually termed

cellulose I, it is thought that the chains lie parallel with each other

and parallel to the surface of the wall in staggered layers. In

regenerated cellulose, usually termed cellulose II, the chains are

antiparallel and lie in regularly stacked layers (Rees 1977; Preston

1979). The highly ordered arrangement of chains, known as a microfibril,

creates a crystalline structure amenable to x-ray diffraction and

polarizing light microscope studies. A microfibril is described as

containing a central crystalline core (5 to 7 nm wide in higher plants)

surrounded by a paracrystalline cortex. The cortex is made up of

molecular chains lying parallel to the microfibril length. The chains,

however, are not in a crystalline arrangement because of their mixed

cellulose and hemicellulose content (Preston 1974a). With the cortex

added to the core, the width of the microfibril is about 10 nm (Preston

1974a). It is felt that the chains in the cortex become increasingly









more hemicellulosic with increasing distance from the core

(Preston 1974a).

Polymeric glucan chains linked together by a1,3 bonds exist in a

hollow helical pattern (Rees 1977; Preston 1979). Three suggestions have

been made as to how the hollow area is filled: 1) by the formation of an

inclusion complex with appropriately sized molecules; 2) by the formation

of a double or triple helix with other 81,3 linked chains, and 3) by the

nesting of a number of 01,3 linked chains (Rees 1977). The structural

importance of these polymers is their ability to twist around each other

forming a network which is effective in entangling other polysaccharides

or in itself creating a strong, but flexible, assemblage of molecules

(Rees 1977; Preston 1979). The helical conformation will also exist, even

if the 01,3 bonds are interrupted by 81,4 bonds (Preston 1979).

Loosely jointed linkages and chains are formed when glucans are

held together by 01,6 bonds. There is a lot of freedom of rotation of

molecules involved in this type of bonding because of the separation of

the monomeric units by three bonds rather than two, placing the sugar

rings further apart (Rees 1977). Commonly individual glucans such as

these are not found in nature. Instead these glucans exist as branches

on other types of polysaccharides (Rees 1977). It is speculated that

the flexibility of these linkages may allow the molecules involved to

aid in various biological interactions such as the entry and exit of

enzymes (Rees 1977).



Fungal Walls, Morphology

There is general agreement that hyphal walls consist of an inner

microfibrillar core of randomly oriented chitinous or cellulosic fibrils










covered by an amorphous matrix of varying chemical constituency (Aronson

and Preston 1960). The wails of sporangial, spore, and the sexual

apparatus may differ either by having an outer microfibrillar component

or an outer melanin layer (Tokunaga and Bartnicki-Garcia 1971; Hegnauer

and Hohl 1978; Cole et al. 1979; Hawes 1979; Mendoza et al. 1979).

Information on the morphology of hyphal walls, treated chemically

and enzymatically, comes from ultrastructural studies of surface

replicas and thin sectioned material. The classic study of wall morphology

is that of Hunsley and Burnett (1970) who compared walls after sequential

enzyme treatments of three different fungi, each representing one of

the major taxa. Live hyphae were used for two reasons: 1) any artifacts

brought about by wall isolation were eliminated; and 2) confidence that

enzymatic digestion occurred from the outside in. Models for each of

the three major groups were then developed from these data.

Walls of Schizophyllum commune, the representative Basidiomycete,

have a four-layered structure which none of the three enzymes, laminarinase,

pronase, or chitinase, could hydrolyze. However, preliminary treatment

with KOH was effective in removing the outer protective (S-glucan) layer.

Subsequent treatment with laminarinase revealed a microfibrillar outline

which was clarified by the addition of pronase. Chitinase treatment

following that of laminarinase and pronase removed the microfibrillar

component. The model which was derived from this study depicts the wall

as having an outer S-glucan layer bounded internally by R-glucan. The

R-glucan is bordered on the inside by a thin, but discrete, proteinaceous

sheet which in turn is completed and intermixed with the chitinous micro-

fibrils (Hunsley and Burnett 1970. Van der Valk and Wessels (1977) using










isolated walls did a similar study and found that pronase had no effect

on the R-glucan-chitin portion of the wall. This led to the belief that

there is no protein layer and no protein-chitin complex. Carbon-

platinum replicas of the S-glucan revealed a surface composed of randomly

oriented parallel arrays of short rodlets and a filamentous surface of

the mucilage (Wessels et al. 1972).

Neurospora crassa is the representative Ascomycete which Hunsley

and Burnett (1970) studied. In thin section the wall appeared three-

layered. Laminarinase treatment removed the outer amorphous layer

revealing a coarsely stranded network, more clearly resolved by the

addition of pronase, filled with a matrix material. Chitinase in

conjunction with the other two enzymes resulted in dissolution of the

wall. Neither chitinase nor pronase added alone or in sequence had any

effect at all. The model derived from these data envisions the wall as

having an outer layer of 31,3, B1,6 glucan with an inner layer of protein

in which is embedded coarse strands of a glycoprotein (glucan-peptide-

galactosamine) reticulum. A discrete protein layer lies between the

reticulum and the chitinous microfibrils which are embedded in a protein

"matrix" and lie in the innermost part of the wall (Hunsley and Burnett

1970). A similar study by Mahadevan and Tatum (1967) indicated that the

wall consists of an outer coarse fibrillar layer (glucan-peptide-

galactosamine) and an inner layer of primarily 81,3 glucan with an

embedded core of fine chitin fibrils.

Phytophthora parasitica walls were studied in order to derive a

model for a Phycomycete wall. The two-layered wall has a finely granular

amorphous surface which is unaffected by treatment with cellulase or









pronase or both. Laminarinase treatment resulted in the exposure of

microfibrils whose outlines were more pronounced if pronase treatment

followed. Laminarinase and cellulase treatment resulted in almost total

digestion. The model of the wall designed from these studies describes

an outer amorphous layer of 81,3; 81,6 linked glucan and an inner layer

of cellulose embedded in protein. Similar layering has been seen in the

hyphal walls of Phytophthora palmivora and Pythium acanthicum (Tokunaga

and Bartnicki-Garcia 1971; Sietsma et al. 1975; Hegnauer and Hohl 1978).

Chemical removal of the outer amorphous layer of isolated walls of

Saprotegnia litoralis and Atkinsiella dubia and chemical and enzymatic

removal of this layer in Sapromyces elongatus revealed a distinctly

microfibrillar layer (Parker et al. 1963; Aronson and Fuller 1969;

Pao and Aronson 1970).

Isolated walls of Choanephora cucurbitarium, another Phycomycete,

were found to exist in two layers, an outer thick layer of randomly

oriented microfibrils made up of a mixture of chitosan, protein, and

lipids, and an inner thin layer of chitinous microfibrils oriented in a

parallel fashion (Letourneau et al. 1976). Microfibrillar orientation

of the hyphal walls of Linderina pennispora is longitudinal except in

the most interior portion of the wall where it is random (Young 1970).

Sporangiophore walls in this same organism are similar except for the

existence of spicules covering the outer surface of the wall (Young

1970).



Fungal Walls, Growth

Burnett (1968) presented a diagram of his views of apical and

subapical wall organization and how it is altered in response to growth.










The hyphal tip is thin-walled and non-extensible, but the area directly

behind the tip is thicker-walled and it is here that maximum intussusception

takes place. Distal to this zone lies a second thick-walled area known

as the region of maximum extensibility. In the most distal region

described, the wall reaches its maximum thickness and becomes rigid.

It has been suggested that the subapical wall in Phytophthora parasitica

has more protein in which the microfibrils are embedded and a greater

degree of microfibrillar aggregation than is found in the apical

(Hunsley and Burnett 1970), which may account for the increasing rigidity

of this part of the wall. In this most distally described area of the

wall the arrangement of the microfibrils is longitudinal as compared

to the transverse arrangement nearer the tip. Growth is explained by a

change in the balance between synthetic and lytic enzymes (Bartnicki-

Garcia 1973) which allows for turgor driven apical expansion (Thomas

1970; Bartnicki-Garcia and Lippman 1972).

Autoradiographic studies indicated that the sites of growth are

at the tip (Van der Valk and Wessels 1977), although some wall thicken-

ing and modification is seen subapically (Bartnicki-Garcia 1973). It

has been hypothesized that vesicles play a role in wall synthesis based

on the observation of their accumulation at growing tips (Heath et al.

1971; Van der Valk and Wessels 1976; Beakes and Gay 1978; Hawes 1979)

and at the sites of antheridial initials (Mullins and Ellis 1974). The

suggestion has been made that these vesicles carry wall degrading

enzymes (Mullins and Ellis 1974; Fevre 1977) and materials for plasmalemma

and wall synthesis (Bartnicki-Garcia 1973).














MATERIALS AND METHODS


Organism and Culturing Techniques


Strain E 87 male of Achlya ambisexualis Raper (Barksdale 1960)

obtained from Dr. J. T. Mullins was the organism used in this study.

Mycelia were grown on defined media (Mullins and Barksdale 1965; Kane

1971) on agar plates or in liquid culture on a reciprocating shaker

(100 rpm) at 250C. Two day old mycelium, grown on agar, was sporulated

in 0.5 mM CaC12 on a reciprocating shaker (100 rpm) for 20 hr at 250C

with one change of solution after the first 2 hr. An inoculum of 200 000

zoospores was added to 20 ml of defined liquid medium and grown for 48

hr. Mycelium was harvested by vacuum filtration and washed two times

with either 0.05 M potassium phosphate buffer pH 7.0 or 0.1 M tris-HCl

buffer pH 7.5 depending on the subsequent method of hyphal wall prepara-

tion. Harvested washed mycelium was quick frozen at -700C in a Revco

Ultra Low freezer.



Hyphal Wall Isolation and Purification


In all cases hyphal walls were isolated by grinding in a chilled

mortar and pestle 10 gm fresh weight frozen mycelial lots until a fine

powder was obtained. The entire procedure was performed at 0-4C. The

appropriate buffer (final amount 20 ml) was added and grinding was con-

tinued. The resultant "slush" was centrifuged (Sorval RC-2B Automatic

Refrigerated Centrifuge) at 1085 x g and the pellet was saved for

further purification.










Two methods of cleaning hyphal walls were followed. In one, the

walls were cleaned chemically by a modification of Tokunaga and Bartnicki-

Garcia (1971) and,in the other, they were cleaned by repeated washings

with buffer and water (Lin et al. 1976). In the first method the

pellet was washed with phosphate buffer and then sonicated in 10 ml

2% sodium lauryl sulfate for one minute at 15 watts (Heat Systems-

Ultrasonics Sonifier Cell Disruptor, Model W 185, fitted with a standard

microtip). After sonication the suspension was placed in a 90C water

bath for 30 min. It was then centrifuged and the pellet was treated with

60 ml of a 2 95% ethanol: 1 2 N KOH solution three times for 10 min

each in a boiling water bath. The resulting pellet was washed with

distilled water three or four times or until the washings showed a

neutral pH. Wall purity was determined by phase and electron microscopy

and the decreasing level of protein found in the washings. The second

method followed closely that described by Lin et al. (1976) with a 4 min

sonication at 30 watts. Cleaned walls were dried by lyophilization

(Virtis Research Equipment) and stored over desiccant until further use.

All subsequent analyses were begun with 100 mg samples of this material.

Total glucan was determined on each preparation of cleaned walls by

the anthrone method (Morris 1948; Dische 1962).



Chemical Fractionation of the Wall


The cleaned and freeze-dried walls were chemically fractionated by

three successive treatments. The first was acid (0.5 N HC1) (Aronson

et al. 1967; Sietsma et at. 1969) and the second was alkali (2 N KOH).

The third treatment used was either Schweitzer's reagent (personal










communication Dr. J. M. Aronson) or cadoxen (Jayme and Neuschaffer 1957;

Jayme and Lang 1963), both known cellulose solvents. The acid-soluble

fraction was obtained by five 30 min treatments at 700C of 100 mg wall

material in 50 ml 0.5 N HC1. The supernatants from each treatment were

pooled and brought to a final concentration of 85% ethanol and allowed to

stand overnight at 4C. The ethanol precipitated polysaccharide was

collected the next day by centrifugation, freeze-dried, and stored over

desiccant until further use. The pellet which remained from the acid

extraction was washed with distilled water until neutral, and then treated

with 2 N KOH in the same manner as the acid treatment. This became the

alkali-soluble fraction. The remaining pellet was dissolved in either

one of the two cellulose solvents, with cadoxen being favored because

of its colorless and odorless nature (Ladisch et aZ. 1978). The basic

procedure for cellulose dissolution was the same with both solvents and

both appeared equally effective. The pellet remaining from the acid-

alkali extractions was treated overnight under N2 with 40 ml of freshly

prepared reagent at room temperature with stirring. Two additional 2 hr

extractions were performed and finally the supernatants were pooled and

treated with glacial acetic acid until neutral. The solution was

centrifuged at 48 300 x g for 20 min in a Beckman J2-21 refrigerated

centrifuge. The pellet was washed once with 1 N acetic acid, twice with

distilled water, twice with 22% NH4OH for the Schweitzer's reagent or

30% ethylenediamine for the cadoxen reagent (the first time for 30

min), once with 1 N acetic acid, and finally with distilled water. The

pellet was freeze-dried and stored over desiccant for further study.

This is the Schweitzer's or cadoxen reagent-soluble fraction, and is

termed cellulose II (regenerated cellulose) ( Preston 1974a). The










pellet remaining was neutralized by washing with distilled water and

freeze-dried as above. This is the insoluble residuum.

Cellulose I or native cellulose and chitin were extracted from

frozen mycelia or isolated walls following the method of Aronson and Lin

(1978).



Chemical Analyses of Wall Constituents


Preparation of Material for Neutral Sugar Analyses

Enzyme hydrolysis

Lyophilized walls or their derived fractions were treated with

various enzymes and the products of hydrolysis were determined. The

enzymes used were laminarinase (E. C. 3.1.1.6, B1,3-glucanase ex mollusca,

B grade, CalBiochem), cellulase (E. C. 3.2.1.4, g1,4-glycanohydrolase

from Aspergillus niger, Type I, Sigma), chitinase (E. C. 3.2.1.14, chito-

dextrinase, poly(l,4-8-[2-acet-amido-2-deoxy]-D-glucoside) glycanohydrolase

from Streptomyces griseus, Sigma), protease (from S. griseus, Type VI,

Sigma), and lipase (448 from hog pancreas, Nut. Biochem. Co.). The

buffer used for cellulase and laminarinase was 0.05 M sodium citrate

pH 5.0; for chitinase 0.05 M phosphate pH 6.0; for protease 0.05 M HEPES

pH 7.6 or 0.05 M sodium phosphate-citrate pH 7.6; and for lipase 0.05 M

sodium phosphate-citrate pH 6.2 Reactions were allowed to run 24 hr unless

otherwise specified. The chitinase and lipase reactions were carried out

at 250C and the others at 370C. The concentration of enzyme was 500 pg/

ml and that of the substrate 2 mg/ml. Bacterial contamination was

prevented by the addition of 100 pg/ml streptomycin, 500 ig/ml merthiolate,

or when gas chromatographic analyses were to follow, a toluene layer










covering the reaction mixture. Tests for enzyme purity were performed

by reacting the enzyme in question with a known substrate. They were:

laminarin, from Laminaria digitata, A grade, anhydroglucose 94%,

CalBiochem; cellulose, carboxymethylcellulose or acid swollen Whatman

Ashless Powder for chromatography (Reese and Mandels 1963b; Green 1963);

chitin, purified powder from crab, Sigma; Tween 20; and bovine albumin

powder, Fr. V, 96-99%, Sigma. The conditions of enzyme hydrolysis are

compiled in Table 1. Two of the enzymes were found to be active against

more than one substrate. Laminarinase was found to be active against

both laminarin and cellulose, and cellulase was active against both

protein and cellulose (Whitaker 1970). Thus, it was necessary to purify

these two enzymes before use (Sietsma et aZ. 1968).

Assays for enzyme activity

The activity of laminarinase was measured by determining the increase

in total reducing sugar or in glucose over that of the enzyme or sub-

strate alone, using the anthrone method (Morris 1948; Dische 1962) or

the glucostat test (Worthington Biochemical Corp.). Cellulase activity

was determined viscometrically (Thomas and Mullins 1969). The production

of N-acetylglucosamine, as determined by the DMAB method (Reissig et al.

1955), was used to measure the chitinase activity. The activity of

protease was determined by the decrease in total protein, as measured

by the BioRad technique (BioRad Technical Bulletin #1051). The release

of fatty acids, as determined by a change in pH, was used to measure the

activity of lipase (Bier 1955).


1This substrate contained B1,3 linkages only. Another laminarin (source
unknown) was also used which was found to contain @1,6 linkages in
addition to the a1,3.









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Acid hydrolysis

Unfractionated walls and the wall fractions described previously

were hydrolyzed in sealed ampules for varying periods of time with 6 N

HC1 under N2 at room temperature. The resulting hydrolysates were then

analyzed for mono- and disaccharides. Before analysis the hydrolysates

were diluted with distilled water and either dried over NaOH pellets

in vacuo until they were neutral or were deionized by passage through

a Dowex carbonate column and then dried in vacuo. The sample size was

2 mg per 0.5 ml 6 N HC1. Unfractionated walls were treated for 48 hr,

the acid- and alkali-soluble fractions and the insoluble residuum for

4 hr, and the cellulose II fraction for 96 hr. The anthrone reagent

was used to determine total glucan in these samples.


Neutral Sugar Analyses

All neutral sugar analyses were performed on chemically cleaned

walls or the fractions prepared from these walls.

Acid and enzyme hydrolysates of unfractionated walls and their

fractions were analyzed by both paper and gas-liquid chromatography, the

latter by the formation of trimethylsilyl derivatives (Sweeley et at.

1963; Zanetta 1972). Single dimension descending paper chromatography

was done on Whatman #1 chromatography paper, 46 by 56 cm, in a solvent

saturated Chromatocab (Research Specialties Co.) at room temperature for

24 hr following known procedures (Kowkabany 1954; Block et al. 1958).

Material containing 100 pg or more carbohydrate was applied to the paper.

The solvent system used was 6 butanol: 4 pyridine: 3 water. Dried

chromatograms were developed in a 105C oven after having been sprayed










with aniline phthalate (Partridge 1944). R values were calculated from
g
the resulting spots and compared with known standards.

Derivatization of samples and standards for GLC was done in the

following manner. Solutions containing known amounts of carbohydrate were

first lyophilized and then volatilized by a 10 min treatment with 0.2 ml

dried pyridine, 0.1 ml hexamethyldisilazane and 0.1 ml trimethylchloro-

silane when only monosaccharides were present, or for 3 hr when disac-

charides were (Ishizuka et al. 1966; Bhatti et al. 1970). The

reactants were mixed on a vortex and after gentle warming (Yamakawa and Ueta

1964a, b-) the reactions were carried out at room temperature. At the

end of the reaction period, the mixture was dried with a stream of N2 in

a warm water bath, and the derivatives were extracted with 0.4 ml

methylene chloride (Mallinckrodt, nanograde). Sample size varied from

2 to 6 il depending on the amount of carbohydrate, and sample concentration

varied from 10 to 20 pg carbohydrate.

The gas chromatograph used was a Hewlett-Packard F & M 402 with

dual flame ionization detectors. The carrier gas was helium. The

columns were standard 1.8 m tubes with internal diameters of 3 mm. The

packing material was 3% (w/w) JXR on 100-120 mesh Gas Chrom Q. Two

different temperature programs were used. Monosaccharide separations

used a starting temperature of 170C for 5 min, followed by a rise of

2/min to 2100C where the temperature was held. Disaccharide separations

used a similar temperature regime, except that once 2100 was reached the

program was changed to a 100/min increase to 2400 where the temperature

was held. The internal standard which was incorporated at the time of

acid or enzyme hydrolysis was myo-inositol in the monosaccharide program









and sucrose in the disaccharide. Peak areas were determined by the use

of a K & E Compensating Polar Planimeter (620 005) and the relative

quantities of the disaccharide components found were calculated on the

basis of the internal standards (Davison and Young 1969; Clamp et al. 1971).

Linkage and branching analyses of the various polysaccharides

isolated from the wall were done by periodate oxidation. The following

procedure was modified from that of several previous ones (Dyer 1956;

Goldstein et aZ. 1965; Hay et al. 1965). A 36.7 mg sample was dissolved

in 25 ml of 0.04 M sodium metaperiodate (Sigma), which had been dissolved

in acidified water (pH 4.5), and placed in flasks which were covered

with black electric tape and aluminum foil. Aliquots were taken

immediately for TO and analyzed. The materials to be oxidized were

placed on a wrist-arm shaker at 40C and aliquots were removed for

analysis every 24 hr for a total period of 120 hr. Periodate ion

consumption was determined by UV absorption at 222.5 nm of 0.1 ml

samples after a 250-fold dilution. Formic acid liberation was determined

by titration with 0.01 N NaOH on 1 ml samples, to which 0.1 ml acid free

ethylene glycol and, after 10 min at room temperature, 0.5 ml 0.02%

methyl red had been added. The amount of base necessary for neutrali-

zation was then correlated with the amount of formic acid in the sample.

Appropriate controls were also analyzed.

Live hyphae with developing branches plus samples of isolated

cleaned walls and their fractions were observed under a polarizing

light microscope. The pattern of birefringence was noted.

Samples of both Schweitzer's and cadoxen reagent-soluble material

were subjected to x-ray diffraction analyses by Dr. J. M. Aronson of the










Department of Botany and Microbiology, Arizona State University, Tempe,

Arizona.


Amino Sugar Analyses


Solubility of the hexosamine component of the wall

The procedure used was that of Lin et al. (1976). The three major

fractions were first lyophilized and then hydrolyzed with 16 ml of 4 N

HC1 at 980C under N2 in sealed ampules. After a 16 hr reaction period,

the ampules were opened and the acid was removed by rotoevaporation.

The remaining contents were washed three times with distilled water

and finally dissolved in 5 ml 0.01 N HC1 for amino sugar analysis in

an automated Amino Acid Analyzer (Model JLC-6AH, Japan Electron Optics

Laboratory Co., Ltd., Tokyo, Japan).

Lugol's iodine detection of chitin

Lugol's iodine was prepared as a 1 iodine: 2 potassium iodide: 300

distilled water solution. The test material was placed in a depression

slide in a few drops of oxalate buffer ranging in pH from 1.6 to 4.0. A

few drops of the Lugol's iodine was added and the material was observed

under a light microscope to determine any color development (Prakasam and

Azariah 1975).


Uronic Acid Analysis

The procedure for isolating uronic acids follows that of Gancedo

et al. (1966). The presence and quantity of uronic acid was determined

by the carbazole test (Bitter and Muir 1962).










Protein and Amino Acid Analyses


Total protein was determined on wall samples which had been washed

in buffer and water. They were then homogenized with a glass tissue

grinder (Kontes Glass Co.) in 1 N NaOH and the resulting homogenate was

placed at 50C for 3 hr. Protein was determined by the BioRad method

(BioRad Technical Bulletin #1051 1977).

Amino acid profiles were determined on 200 and 20 mg samples of

chemically cleaned and of buffer-water washed walls, respectively, follow-

ing hydrolysis in 6 N HC1 at 105C. Similar profiles were also determined

on 20 mg samples of walls taken at various stages during chemical cleaning.

These stages were: (1) supernatant after initial pelleting subsequent

to grinding; (2) buffer washed once; (3) buffer washed once plus sonicated

and heated 30 min at 900C in 2% SLS; and (4) walls from (3) treated once

10 min in boiling water with ethanolic KOH. After treatment for 48 hr

the acid was removed by rotoevaporation as described previously and the

amino acids were analyzed by an automated Amino Acid Analyzer.

Lipid Analysis

The procedure of extracting readily extractable lipids was that of

Kanfer and Kennedy (1963). The dried extract was spotted on Silica Gel G

plates activated with iodine and detection was by double bond formation

with iodine (Whitehouse et al. 1958). The solvent used for ascending

chromatography was 65 chloroform: 25 methanol: 8 glacial acetic acid

(Ames 1968). The pattern of spots suggested phospholipid. A Fiske-

Subbarow solution (Bartlett 1959) plus 0.5 ml 10 N H2SO4 was sprayed

onto the dried plates to detect phosphorus.










Phosphorus Analysis

Total phosphorus was determined on wall samples which had been

combusted at 1600C in 10 N H2SO4 and H202 for 48 hr. Phosphorus was

measured by the Fiske-Subbarow method (Barlett 1959).



Hydrolysis of Buffer-Water Washed Walls
by A. ambisexualis Cellulase


A sample of the enzyme cellulase was extracted with acetone from

medium in which AchLya had grown on the enriched formula (Kane 1971) for

48 hr. Enzyme precipitation was achieved by adding 2 volumes of acetone

to the medium, followed by centrifugation at 12 100 x g for 20 min. The

pellet was then resuspended in distilled water at a ratio of 1 ml per gm

fresh weight of mycelium, and centrifuged at 18 800 x g for 15 min. The

supernatant was dialyzed for 24 hr against a 0.018 M citrate-NaOH buffer

pH 5.0 with 0.05% merthiolate to remove glucose present in the original

medium. Viscometric assay of this enzyme solution revealed an activity

of 5 units/ml (Thomas and Mullins 1969). Isolated buffer-water washed

walls were prepared as usual except that they were not lyophilized, and

a final washing with the above citrate-NaOH buffer was made. One ml of

the enzyme solution and 2.5 ml of the wall suspension were added to a

15 ml conical centrifuge tube and placed at 30C for 168 hr. Aliquots

of 0.2 ml were taken at 24 hr intervals and the total reducing sugar was

measured in the supernatant by the anthrone method (Morris 1948; Dische

1962).










Ultrastructural Studies


Surface structure of live hyphae, isolated walls, and wall fractions

was studied under a number of varying regimes of chemical and enzymatic

treatments.


Surface Structure of Chemically Treated Walls


Isolated walls were first treated with 0.5 N HC1 and placed in a

70C water bath for 30 min. This treatment was repeated five times.

Surface replicas were made of samples of the wall which remained. The

rest of the remaining wall was treated similarly but with 2 N KOH.

Samples of the wall left from this treatment were taken for surface

replication. The residual wall material was treated with cadoxen reagent

and surface replicas of the insoluble material were made. The cadoxen-

soluble material was treated with acid to regenerate cellulose II and

surface replicas were again made of this component.


Surface Structure of Wall Fractions


Surface replicas of each of the wall fractions described in the

section on Chemical Fractionation of the Wall were made.


Surface Structure of Chemically Treated Live Hyphae


Live hyphae were treated in the same manner as the isolated walls

and replicas were made of the wall surfaces of the hyphal samples after

each treatment. Replicas were not made, however, of material which was


solubilized in cadoxen.










Surface Structure of Enzymatically Treated Live Hyphae


Very small amounts of 48 hr old mycelium were placed in the wells

of a plastic Tissue Culture Cluster Chamber (Costar) and 0.2 ml of the

various enzyme solutions containing merthiolate were added. Sterile

cotton, soaked in sterile water, was placed in nearby wells to prevent

desiccation. The reaction mixtures were placed at 370C for 48 hr. At

the end of the incubation time hyphae were removed from the well, washed

with sterile water, and placed on freshly cleaved mica for drying and

eventual surface replication. Hyphae, which were to be treated with a

second enzyme, were washed and returned to the well and the second enzyme

was added. If a third enzyme was to be added, the same procedure was

repeated. Assay conditions were the same as those described in the

section on enzyme hydrolysis. The activities of the enzymes used were:

laminarinase (purified), 620 ig reducing sugar (as glucose) released

from 2 mg cell wall/ml enzyme; Aspergillus niger cellulase (purified),

12 units/ml; AchZya ambisexualis cellulase, 5 units/ml;

and protease, 500 Pg/ml. Controls for each sample contained boiled

enzyme.


Diameter of Microfibrils

The width of microfibrils was determined from negatives of carbon-

platinum surface replicas taken at 33 K and 50 K magnification under

varying preparative conditions. These conditions were treatment with

buffer (untreated), laminarinase, laminarinase-protease, and 0.5 N HC1

followed by 2 N KOH. The diameter of cellulose I microfibrils was also

determined.










Preparation of Replicas


Single-stage carbon-platinum replicas (Pease 1964; Bradley 1965)

were made of the wall surface by the following procedure. Samples of

the treated wall were air-dried on mica and were shadowed with platinum

at an angle of 450 in a Balzer's High Vacuum Coating Unit Micro-BA 3 or

a Balzer's BA 360 Freeze Etch Device. After shadowing, the specimens

were coated with carbon. Biological material and the mica were removed

from the replicas by floating on 40% chromic acid solution. The replicas

were washed twice with distilled water and allowed to sit overnight in

50% chlorox. The chlorox was washed off by two 15 min washes with

distilled water. The replicas were placed on 100 mesh copper formvar

coated grids and were examined by a Hitachi HU-11E or a Jeolco JEM-100

Cx electron microscope.














RESULTS


Criteria for Wall Purity


Observations of both chemically cleaned and buffer-water washed

walls with phase and electron microscopy revealed that they were

relatively free of cytoplasmic contaminants (Fig. 1 a and b). In

addition, there was a decrease in the protein content of the wall

washings during the successive stages in the chemical cleaning process

as shown in Fig. 2.

The two methods of wall cleaning gave quite different amounts of

dried cleaned walls per original gm fresh weight of mycelium. About

twice as much wall material was obtained after buffer-water washing as

after chemical cleaning, 6.82 mg and 3.20 mg dried walls/gm fresh weight

of mycelium. Total glucan content of both preparations, as measured by

the anthrone method, was quite similar:52.80 mg and 56.00 mg/100 mg dried

walls for buffer-water washed and chemically cleaned, respectively.



Chemical Fractionation of the Wall


Table 2 gives the results of the chemical fractionation of the

carbohydrate component of the wall. That portion of the wall soluble in

weak acid was 37.58%, while only 7.07% of the remaining wall was soluble

in alkali. Some 20.83% was soluble in cellulose solvents, leaving a

5.58% residuum. These four fractions account for 71.06% of the wall

andwere shown to consist of the following: (1) acid-soluble = 01,3


































Figure 1. Phase contrast photographs of cleaned walls.
(a) Chemical. X 800. (b) Buffer-water. X 800.








48



4-





0





2-














WASHES

1. Buffer suspension after grinding
2. Detergent treated, sonicated, and heated
3. 2 ethanol: 1 KOH
4. 2 ethanol: 1 KOH, 10 min boiling, 1X
5. 2 ethanol: 1 KOH, 10 min boiling, 2X
6. 2 ethanol: 1 KOH, 10 min boiling, 3X
7. Water, lX
8. Water, 2X
9. Water, 3X


Figure 2. Decrease in total proteina as a measure of wall purity.


determined by the Lowry method














Table 2. Carbohydrate fractions of A. ambisexualis wall.


Fraction


Acid-soluble


Alkali-soluble

Cellulose II

Insoluble residuum

Cellulose I

Chitin

Total glucanb


average of three determinations based
determined (as glucose) with anthrone
walls; calculated as anhydroglucose
buffer-water washed walls
chemically cleaned walls


on 100 mg samples
on unhydrolyzed


a
mg


37.58

7.07

20.83


5.58


19.06

0.63

52.8C,


56.0d









and 81,6 glucan; (2) alkali-soluble = 1,3; 81,4 and 81,6 glucan; (3)

cellulose II = 01,4 glucan; and (4) insoluble residuum = 81,3; 81,4

and 81,6 glucan.

The amount of cellulose I was 19.06% of the wall and this is 2%

less than the value for cellulose II. A very small portion of chitin was

found (0.63%) and some additional tests were made to support this

identification. A two week incubation of this component with chitinase

released about the same level of N-acetylglucosamine as did a known

chitin substrate. A 700 1g sample of wall material released 58 pg

of N-acetylglucosamine and a 2000 pg sample of crab chitin released

152 ug. A spot identified as N-acetylglucosamine was obtained with paper

chromatography from both enzyme hydrolysates (Table 3). Cytological

staining with Lugol's iodine compared favorably with known samples of

chitosan and deacetylated chitin.



Chemical Analyses of Wall Constituents


Neutral Sugars

The above fractions (Table 2) were then analyzed for their mono-

saccharide composition, types of glycosidic linkage, and pattern of

branching. The acid-soluble fraction was hydrolyzed and the products

separated by paper chromatography. They consisted of the monosaccharide

glucose and the disaccharides laminaribiose and gentiobiose (Table 4).

Positive identification of a probable trisaccharide spot was not possible

because of conflicting R values between laminaritriose and cellotriose.
g
The two disaccharides found indicated the presence of two different

linkages, 81,3 and 81,6, respectively. The alkali-soluble fraction


























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revealed glucose, laminaribiose, gentiobiose, and cellobiose. Both

the cellulose I and II fractions gave glucose and cellobiose with the

same analysis. The residuum, remaining after removal of the three

above fractions, produced small amounts of glucose, laminaribiose,

gentiobiose, and cellobiose upon hydrolysis. The unfractionated wall

produced all of the above sugars except laminaribiose, even after a

short (2 hr) hydrolysis period. Both the unfractionated wall and

cellulose II contained a compound which was unidentifiable by R value.
g
Before presenting the results of the enzyme hydrolysis studies,

some comments on the properties of the purified enzymes will be made.

The purified laminarinase (see Materials and Methods) when reacted with

a laminarin containing only 1,3 linkages, produced glucose, laminaribiose,

and laminaritriose. When, however, it was presented with a laminarin

containing mixed linkages of $1,3 and 01,6, gentiobiose appeared with

the above products. This enzyme preparation thus behaves like both an

exo-B1,3-glucanase and an endo-al,3-glucanase. The purified cellulase

preparation obtained from Aspergillus niger exhibited activity against

carboxymethylcellulose in the viscometric assay and is thus classified

as an endo- or random splitting enzyme (Reese and Mandels 1963a). When

this enzyme was reacted with wall fractions or the intact wall no

chromatographable compounds were formed in the reaction time used.

However, treatment of the wall or its fraction with unpurified enzyme

yielded glucose. The unpurified enzyme preparation also is able to

hydrolyze sucrose, as this disaccharide was never obtained in GLC

analysis, even though it was added as an internal standard at the time

of hydrolysis (Fig. 3e).






























GLC of the TMS derivatives of the monosaccharides released
by hydrolysis of the wall fractions or the total wall by
unpurified A. niger cellulase. (a) Acid-soluble. Glucose
is the only product. (b) Boiled enzyme control. Small
peaks of mannose and glucose are contaminants of the enzyme
and substrate preparations respectively. (c) Cellulose II.
Glucose is the only product. (d) Boiled enzyme control.
(e) Total wall. Glucose is the only sugar produced. The
fructose and some of the glucose result from the action of
8-glucosidase, present as a contaminant in the enzyme pre-
paration, on sucrose added as the internal disaccharide
standard. (f) Boiled enzyme control.


Figure 3.






































Gic


Glc


Mann


I 1 I I I I
0 5 10 15 20 25
TIME (min)


Mann


d.


Mann


ir I I I

f.







Mi

Mann Glc

) 5 10 15 20 25


TIME (min)










Table 3 gives the results of enzyme hydrolysis of the various

wall fractions. Neither lipase nor protease showed activity against

any of the wall fractions. Purfied laminarinase hydrolyzed the acid-

soluble portion of the wall releasing glucose, laminaribiose, and

gentiobiose. Glucose was released from all the fractions, except the

chitinous one. Laminaribiose was found in the unfractionated wall after

treatment with laminarinase. This disaccharide was also found in

laminarinase hydrolysates of the alkali-soluble fraction and the

insoluble residuum. Based on the R value, cellobiose was also found
g
with laminarinase treatment of this residuum.

In a number of cases an unidentified spot appeared with a variable

R value. It was always higher than laminaribiose but lower than
g
glucose. The R value of this spot was a little higher than that found
g
in some of the acid hydrolysates. All the control enzyme and substrate

solutions were chromatographed, and no spots were found. From the GLC

studies, a compound which cochromatographed with mannose, was found

associated with both laminarinase and cellulase; however,the R value
g
of authentic mannose is higher than glucose for this solvent system.

The data obtained from the GLC studies is similar to that found

with paper chromatography. Acid hydrolysates (Fig. 4a-c) of the acid-

soluble and cellulose II fractions plus the unfractionated wall yielded

a mono- and disaccharide pattern similar to that found in the paper

chromatographs. Some differences, however, were found. The hydrolysate

of cellulose II consisted of not only glucose and cellobiose, but also

small amounts of laminaribiose and gentiobiose (Fig. 4b). The gentio-

biose component of this fraction was small, but it was difficult




























GLC of the TMS derivatives of the mono- and disaccharides
released by acid hydrolysis of wall fractions and total
wall. (a) Acid-soluble. Products are glucose, laminaribiose
and gentiobiose. (b) Cellulose II. Products are glucose,
cellobiose, laminaribiose, and gentiobiose. (c) Total
wall. Products are the same as in (b).


Figure 4.














GIc l n
r\^ Ml
GIC ff 1


Suc

c Lam Gen


b. Mi Suc


Gic
GIc
Glc


Lam/Cel n


C.



Suc

GIc Mi

Lom L Gen
Celam
I I i I __ I I I I V
0 5 10 15 20 25 35 40 45 50 55
TIME (min)










to determine the amount of the laminaribiose portion because there was

an overlap between the two laminaribiose peaks and the second of the

cellobiose. Acid treatment of the unfractionated wall revealed a

pattern similar to that of the acid-soluble fraction, except for the

presence of a small cellobiose peak (Fig. 4c). Although laminaribiose

was not found in the paper chromatographic analysis of this hydrolysate,

it was found in the GLC analysis.

Laminarinase treatment of fractionated and unfractionated walls

is presented in Fig. 5a-f. Products from the enzymatic hydrolysis of

the total wall and the acid-soluble fraction were glucose, laminaribiose,

and gentiobiose (Fig. 5a and e). No products were obtained from

laminarinase hydrolysis of cellulose II. Control samples containing

boiled enzyme were also analyzed, and the results indicated that this

treatment produced inactivation (Fig. 5b, d and f). The only substrate

which had any residual sugar was the acid-soluble fraction which gave

a very small glucose peak upon analysis. Treatment of the various

fractions with cellulase gave results which were similar to the paper

chromatographic studies (Fig. 3a-f). Glucose was the primary product

found in these hydrolyses, and for this reason only the monosaccharide

portions of these chromatographs are included.

Both laminarinase and cellulase were reacted with the unfractionated

wall and only glucose was obtained (Fig. 6a and b). For some reason the

mannose component was absent.

Since glucose was the only monosaccharide found, this datum was not

quantified. The disaccharide components were quantitated based on their

peak areas and that of a known standard (sucrose). The molar relative

response factors for each of the components found were not calculated


































GLC of the TMS derivatives of the mono- and disaccharides
released by hydrolysis of the wall fractions or the total
wall by laminarinase. (a) Acid-soluble. Products are
glucose, laminaribiose, and gentiobiose. (b) Boiled enzyme
control. Mannose is a contaminant of the enzyme preparation.
(c) Cellulose II. (d) Boiled enzyme control. (e) Total
wall. Products are glucose, laminaribiose, and gentiobiose.
(f) Boiled enzyme control.


Figure 5.








Mann





Glc Mi
Gic Suc
Lam Gen


b. Mann Mi








Gic


C.
Mann



Mi
A Suc
fl Sue


35 40 45 50 55
(min)


0 5 10 15 20 25
TIME








































Figure 5. Continued.





63



*d Mann

Mi

Suc







e.Mi Suc







Gic
Gic




Mann
Mi Suc











0 5 10 15 20 25 35 40 45 50 55
TIM E (min)















a.


Gic


GIc


0 5 10 15 20 25

TIME (min)


Figure 6.


Mi


I I


) 5 10 15 20 25

TIME (min)


GLC of the TMS derivatives of the monosaccharides
released by hydrolysis of the total wall with
laminarinase and unpurified A. niger cellulase.
(a) Total wall. (b) Boiled enzyme control.









because of variabilities in the system observed from day to day. It

was felt that greater accuracy was obtained by comparing the areas of

the peaks in question to known standards run simultaneously. The ratio

of laminaribiose to gentiobiose in the acid-soluble fraction following

acid hydrolysis was 1:0.72 and following enzyme hydrolysis was 1:0.57.

In the unfractionated wall acid hydrolysis gave 1:1.02 and enzyme

hydrolysis 1:0.9.

Periodate oxidation studies were done in order to gain some knowledge

of the linkage and branching patterns in the various wall fractions. In

addition to the studies of the wall fractions, three standard poly-

saccharides of known linkage patterns were also analyzed. These were

cellulose powder (Whatman Ashless Powder, Chromatographic Grade) and

the two different laminarins described previously. Table 5 gives the

values of periodate consumption and format liberation for the various

wall fractions, and Table 6 for the polysaccharide standards.

Observations of unfractionated walls, wall fractions, and live

hyphae under polarizing light revealed strong birefringence in the

cellulose II fraction but none in the acid-soluble. Both live hyphae

and isolated walls showed birefringence.

The results of x-ray diffraction analysis of cellulose II isolated

by dissolution with Schweitzer's reagent or with cadoxen are presented

in Table 7 and Fig. 7a and b. The lattice spacings of both preparations

were the same as those found for the avicel cellulose II standard.

Amino Sugars

Because of the relatively large amount of glucosamine found in

the samples analyzed for amino acids, a more detailed study of this














Table 5. Periodate consumption and format liberation
of A. ambisexualis wall fractions.



Fraction Periodatea,b Formateab


Acid-soluble 0.917 0.427

Alkali-soluble 0.609 0.091

Cellulose II 0.329 0.031

Insoluble residuum 0.610c 0.031c

Total Wall 0.963 0.213

Cellulose I 0.366 0.061



amoles per mole glucose
b
values after 96 hr of treatment
values after 48 hr of treatment


























Table 6. Periodate consumption and format liberation of
known polysaccharides.



Polysaccharide Periodate'b Formateab


Whatman cellulose powder 0.370 0.031

Laminarin (l1,3 linked) 0.159 0.152

Laminarin (l1,6; 81,3
linked) 0.329 0.152


amoles per mole glucose
values after 96 hr of treatment

























Table 7. X-ray diffraction analysis of Schweitzer's
and cadoxen reagent-soluble fractions of
A. ambisexualis wall.


Samples Lattice spacings in A


Avicel standard 7.37 4.46 4.08

Schweitzer's 7.37 4.46 4.08

Cadoxen 7.37 4.46 4.08







































Figure 7. X-ray diffraction patterns of cellulose II isolated
from A. ambisexualis walls. (a) Cellulose dissolved
with Schweitzer's reagent. (b) Cellulose dissolved
with cadoxen.


~








70
















,,


.







r




















































-9 -











S.
'-










monosaccharide was done. The solubility characteristics of the glucosa-

mine component of the wall were studied and the results are given in

Table 8. A large portion, 98.5%, of the glucosamine was insoluble in

both dilute acid and base. The preliminary characterization of a small

chitinous component isolated from the wall has already been described.


Uronic Acids


Preliminary studies of the uronic acid content of the wall gave 0.03

mg/100 mg dried wall prepared by buffer-water washing. No attempt was

made to identify which uronic acids were present.


Protein and Amino Acids

Total protein of the untreated wall was 6 mg/100 mg dried wall

after preparation by buffer-water washing. If, however, these walls

were washed with 1 N NaOH and placed in a 500C water bath for 3 hr, the

total protein value increased to 10 mg/100 mg wall. Total protein was

also measured in the various wall fractions. The acid-soluble fraction

was the only one which showed the presence of measurable protein, 1.5 mg/

100 mg dried wall. Traces of protein were found in the cellulose II

fraction and in the insoluble residuum. Much of the protein was

probably destroyed or washed away during the fractionation processes.

Amino acid analyses performed on both types of wall preparations

showed distinct differences (Table 9). The chemically cleaned walls

contained very low levels of amino acids and in some cases certain

expected ones were missing, even when as much as a 200 mg wall sample

was used. Walls which were buffer-water washed contained the whole

spectrum of amino acids, including hydroxyproline (3%)(Table 9). A



















Table 8. Analysis of solubility of glucosamine from
unfractionated walls of A. ambisexualis.



Treatment mg Glucosamine


1 N NH4OH, 250C 0.039

1 N acetic acid, 980C 0.000

Insoluble 2.587


Total glucosamine 2.626


amg/100 mg dried wall, buffer-water washed










Table 9. Amino acid profile of the total wall of A.
ambisexuaZis after chemical or buffer-water
cleaning.


Amino Acid or
Amino Sugara Chemical Buffer-water

Glucosamine (average) 0.513 1.224
Lysine 0.009 0.298
Histidine 0.016 0.085
NH3 0.629 0.205
Arginine 0.015 0.207
Hydroxyproline --- 0.103
Aspartate 0.009 0.187
Threonine 0.002 0.308
Serine 0.002 0.222
Glutamate 0.007 0.419
Proline T 0.162
Glycine 0.007 0.166
Alanine 0.058 0.226
Cysteine --- 0.043
Valine 0.006 0.206
Methionine 0.004 0.045
Isoleucine 0.004 0.172
Leucine 0.009 0.296
Tryosine --- 0.104
Phenylalanine 0.008 0.168

Total amino acid 0.156 3.417



mg/100 mg dried wall
b200 mg sample used for hydrolysis
c20 mg sample used for hydrolysis










comparison of the total amounts of amino acids/100 mg dried wall revealed

that the buffer-water washed walls contained nearly 22 times more amino

acids than the chemically cleaned walls. The level of total protein

in the buffer-water washed walls was much higher than the total amino

acid content (10 mg and 3.42 mg respectively).

Examination of Table 10 and Fig. 8 indicates that the amino acid

profile of the wall changed drastically during the chemical cleaning

process. Detergent treated walls which were washed once with ethanolic:

KOH followed by a 10 min incubation in a boiling water bath had a greatly

reduced amino acid content. In a 20 mg sample only measurable amounts

of lysine and methionine were present, and only trace amounts of

aspartate, threonine, serine, glutamate, glycine, alanine, isoleucine,

and leucine. Even detergent treated walls, which theoretically contained

all covalently bound amino acids but not others, had a generally low

level. These walls were also missing hydroxyproline, an amino acid

generally found associated with cellulosic walls. The buffer washed

walls (one washing) did not appear clean and most likely contained a lot

of contaminating membrane proteins. The level of hydroxyproline was

very low in these walls. Observations of Fig. 8 indicate that the

profile in the buffer-water washed walls (washed until clean micro-

scopically) in general follows that of the single buffer washing. The

amino acid content of the supernatant after the initial pelleting

represented only those amino acids which were soluble, as this fraction

was not treated with any solubilizing agent. Significantly, hydroxy-

proline was missing from this sample. Strangely, histidine was also

missing, and proline and cyteine were present in only trace amounts.

In addition, glucosamine was not found.



















0 I I I I I I I
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Lipid

Only trace amounts of extractable lipids were found by the method

used. The pattern of spots seen on thin layer chromatography plates

suggested mainly phospholipids, and this was confirmed by a colormetric

spray test specific for these compounds.


Phosphorus


The level of phosphorus per 100 mg dried wall was 0.15 mg.


Total Wall Composition


The composition of the total wall is given in Table 11. When all

the components were added the total represented 86.44% of the wall. The

remaining 13.56% represented unidentified constituents or experimental

losses.



Hydrolysis of Buffer-Water Washed Walls by
A. ambisexualis Cellulase


The results of the hydrolysis of isolated walls by A. ambisexualis

cellulase can be seen in Figs. 9 and 10. In the first 24 hr of hydrolysis,

there was a significant increase in total reducing sugars in the treated

wall samples as compared with the controls. This increase continued for

another 72 to 96 hr, but at a reduced rate, and ceased between 96 and

120 hr.


Ultrastructural Studies


Surface Structure of Chemically Treated Walls

The replica of an untreated wall reveals a smooth surface (Fig.

lla). Walls treated with 0.5 N HC1 appear somewhat less smooth than the




















Table 11. Chemical constituents of the buffer-water
washed walls of A. ambisexualis.


Constituent % Dry Weight


1. Glucana 52.80

2. Glucanb 50.23

3. Cellulose II 20.83

4. Alkali-soluble hexosamine 0.04

5. Insoluble hexosamine 2.59

6. Protein 10.00

7. Total amino acids 3.42

8. Uronic acids 0.03

9. Phosphorus 0.15

10. Readily extractable lipids T


86.44%c



determined (as glucose) with anthrone on unhydrolyzed
walls; calculated as anhydroglucose
b
sum of acid- and alkali-soluble portions and the
insoluble residuum of the wall
sum of 1, 3, 4, 5, 6, 8, and 9

































>1



0
C;





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Figure 11.


Surface replicas of isolated walls treated chemically.
(a) Untreated. The surface is relatively smooth with a
few microfibrils. X26 000. (b) 0.5 N HC1 treatment.
Microfibrils are indistinct in both the control (inset)
and the treated. X 30 000. (c) Treatment with 0.5 N
HC1 followed by 2 N KOH. Microfibrils, not seen in the
control (inset), are evident after treatment. X 30 000.
(d) Sequential treatment of 0.5 N HC1, 2 N KOH, and
cadoxen. Control material (inset) appears intact and
smooth, while in the treated only scattered pieces of
amorphous material remain. X 27 000 (control), x 30 000
(treated). (e) Cadoxen-soluble wall material reconstituted.
No recognizable microfibrillar pattern is seen. x 30 000.












untreated, but no microfibrillar structure is revealed (Fig. lib). Walls

treated with 0.5 N HCI followed by 2 N KOH show a pattern of microfibrils

(Fig. llc). Sequential treatment of walls with acid, alkali, and cadoxen

results in a general disintegration with only pieces of amorphous material

remaining (Fig. lld). When the cadoxen-soluble material was reconstituted

as cellulose II, a definite linear pattern resulted (Fig. lle). This

pattern does not resemble the microfibrillar one seen after acid and

alkali treatment.


Surface Structure of Wall Fractions

A surface replica of the acid-soluble fraction has an amorphous

appearance (Fig. 12a), while that of the alkali-soluble fraction appears

weakly microfibrillar Fig. 12b). The surface of the cellulose II

fraction does not appear microfibrillar in Fig. 12c, but does in

Fig. 12d. Cellulose I has a microfibrillar pattern similar to that

seen after acid and alkali treatment (Fig. 12e). The insoluble residuum

reveals faint microfibrils (Fig. 12f). The chitinous portion of the

wall is microfibrillar, and it appears that the individual microfibrils

are bound together in bundles (Fig. 12g). These bundles are arranged

longitudinally, and there are a few microfibrillar groups which seem

to run perpendicularly to the longitudinal ones.


Surface Structure of Chemically Treated Live Hyphae

The smooth surface of an untreated hyphae is seen in Fig. 13a.

Mild acid (0.5 N HC1) treatment suggests an underlying pattern (Fig. 13b).

Treatment with 0.5 N HC1 followed by 2 N KOH produces a dramatic change

with microfibrils becoming very evident (Fig. 13c). The surfaces of
































Figure 12.


Surface replicas of wall fractions. (a) Acid-soluble
fraction. The surface is amorphous. X 30 000. (b)
Alkali-soluble fraction. Some microfibrils are visible.
X 30 000. (c) Cellulose II. No microfibrillar pattern
is evident. X 32 000. (d) Cellulose II. Microfibrils
are seen in apparent aggregations. X 32 000. (e)
Cellulose I. Microfibrils are evident. X 32 000 (f)
Insoluble residuum. A faint microfibrillar pattern is
seen. X 32 000. (g) Chitin. Distinct arrangements
of microfibrils are seen. X 32 000.






















12a


I Y'.


_A ..































Figure 13.


Surface replicas of live hyphae after chemical treatment.
(a) Untreated. The surface is smooth. X 27 000. (b)
0.5 N HC1 treatment. The surfaces of both the control
(inset) and the treated samples appear amorphous. Faint
microfibrils are seen in both samples. X 32 000. (c)
Treatment with 0.5 N HC1 followed by 2 N KOH. The control
(inset) surface is amorphous, while that of the sample
shows distinct microfibrils. X 30 000. (d) Sequential
treatment of 0.5 N HC1, 2 N KOH, and cadoxen. The surface
of the control (inset) is smooth, while that of the
sample is striated with a suggestion of fibrillar material.
X 30 000.




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