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ENVIRONMENTAL FACTORS AFFECTlNG
PREDATOR-PREY RELATIONSHIPS AMONG YEASTS
Ankica Pupovac-Velikonja
Department of Plant Sciences
Submitted in partial fulfilment of the requirements for the degree of
Master of Science
Faculty of Graduate Studies The University of Western Ontario
London, Ontario November 1998
O Ankica Pupovac-Velikonja 1999
National library 1+1 of,, Bibliothèque nationale du Canada
Acquisitions and Acquisitions et Bibliograp hic Services services bibliographiques
395 Wellington Street 395, me Welhgton OttawaON K1AON4 OtGawaON K1AON4 canada canada
The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Libmy of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/fïlm, de
reproduction sur papier ou sur format électronique.
The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor subsbntial extracts from it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation.
ABSTRACT
A novel type of contact necrotrophic relationship among yeasts, characterized
as yeast predation, has been studied. Predacious yeasts, the teleornorphs of which
were recently classified into a single genus (Saccharomycopsis), and their potentiai
prey from several yeast genera were grown separately and in CO-culture on vanous
media. Haustorium formation and cell penetration were obseived using Iight and
electron microscopy. Known predacious yeasts are sulfur auxotrophs. Therefore,
their predation dynamics was studied on synthetic media in which the content and
type of organosulfur source were varied. Contrary to what was expected, even very
low organosulfur concentrations (e.g., 2 1 pprn methionine) stimolated, rather than
inhibited predation with an eventual elimination of the prey. Endo-kglucanase, but
not a-mannanase or chitinase activity, increased during predation. Preliminary
experirnents with inorganic salts indicated that the presence of ammonium sulfate
inhibited predation and suppressed endo-blucanase activity.
Kevwords: A~thmascus, auxotrop h, beta-g lucanase, Candida, haustorium,
interfungal, methionine, organosulfur, necrotrophic, parasitic,
penetration, predacious yeast, predation, predator, predatory yeast,
prey, su lfu r, Saccharomycopsis, yeast.
iii
ACKNOWLEDGEMENTS
Herewith I express my most sincere gratitude to Professor Marc André
Lachance, my principal advisor. 1 consider myself honoured and very forninate for
having been chosen by him to work on a hitherto unexplored topic and ... well, boldly
to go where no one has gone before. It was a pleasure to work with him and under
his guidance (...well, most of the time), for he is a combination of a thoroughbred
scientist, an artist. a philosopher, a tnie gentleman, and a fnend - a rara avis,
indeed.
I am very grateful to rny other advisors, Professor Charles G. Trick and
Professor Priti Krishna. for their help, adviœ and encouragement. They indebted
me with generously offering their fime and showing interest in my work. They also
taught me that you c m cal1 your supervisor simply André (instead of sehr geehrter
Herr Professor Doktor Doktor honoris causa, as we fiorn Europe usually do).
My special thanks to Professor Jane Bowles for her guidance during our
yeast-sampling field trip in Southwestem Ontario; to Professor Carlos Augusto
Rosa, post-doctoral fellow from Bello Horizonte, for his optimism and the soond of
his enchanting Brazilian CDS in the lab; Mrs. Birgit Schlag-Edler Nienna, Austria)
for excellent electron microphotography; Mr. Tom Haffie and my fellow TAS for
making my teaching assistantship in PS 290 (Genetics) a tnily enjoyable
experience.
Also many thanks to the secretanal and technical staff (Donna Cheshuk,
Stefani Tichboume, Elizabeth Myscich) for their help and patience.
Finally, my love to Ogo and Joran. They know what for.
And to Nikka, who I brought into this world only two and a half days after
defending this thesis, 1 10 days ahead of tirne. She was patiently waiting for me to
finish and is now fighting her own silent battle to grow, breathe on her own and join
US.
TABLE OF CONTENTS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Certificate of Examination ii
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abstract and Keywords iii
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgernents iv
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table of contents v
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . List of TabIes viii
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . List of Figures ix
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . List of Plates xiv
. . . . . . . . . . . . . . . . . . . . . . . . . . Introduction the Scope of the Thesis 1
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fungi - A Unique Phylum 2
. . . . . . . . . . . . . . . . . . . . The Evidence of Fungi in the Fossil Record 4
. . . . . . . . . . . . . . Taxonomie and Phylogenetic Position of the Fungi 6
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fungal Cell Walls 9
. . . . . . . . . . . . . . . . . . . . . lnterfungal Relationships Mycoparasitism 15
. . . . . . . . . . . . . . . . . . . . Yeasts as Fungal Parasites and Predators 18
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Killer Yeast Phenornenon 19
YeastPredation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thesis Objectives 24
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and Methods 25
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microorganisms 25
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Culture Media 26
. . . . . . . . . . . Selection of Most Acüve Predator-Prey Combinations 27
Search for Media Components That Affect Predation . . . . . . . . . . . . 28
.............................. Light Microswpy of Predation 28
Light Microscopy of Post-Predation Viability ................... 29
Electron Microscopy of Interactions in Liquid Media ............. 29
Predation Monitoring on Selected Media (Colony Counting) . . . . . . 30 . . . . . . . . . . . . . . . . . . . . . . . . Effect of inorganic Salts on Predation 31
. . . . . . . . . . . . . . . . . . . . . . Predation-Associated Enzyme Activities 32
PrelirninaryTests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
EnzymeAssays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 . . . . . . . . . . Effect of Inorganic Ions on P(1+3)-Glucanase Activity 34
Thin-Layer Chromatography of Mono- and Oligosaccharides . . . . . 34
ExoglucanaseAssay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results and Discussion 36
. . . . . . . . . . . Selection of Most Active Predator-Prey Combinations 36
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Light Microscopy of Predation 39
. . . . . . . . . . . . . . . . . . . . . . Interactions on Solid Media . Predation 39
. . . . . . . . . . Interactions on Solid Media . Post-Predational Viability 42
Scanning Electron Microscopy of Interactions in Liquid Media . . . . . 45
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General Effect of Nutrients 53
. . . . . . . . . . . . . . . . . . . . . . . . . . Time Course (Continuous Growth) 53
. . . . . . . . . . . . . . . . . . Predaüon Dynamics (Discontinuous Growth) 67
. . . . . . . . . . . . Search for Media Components That Affect Predaüon 89
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Role of Organic SuMir 90
The Role of L-Methionine . Predation of Saccharomycopsis
. . . . . . . . . . . . . . . . . . . . . ja vanensis on Saccharomyces cerevisiae 92
The Role of L-Methionine . Predation of Saccharomycopsis
. . . javanensis and Candida strain 'W1 " on Metschnikowia hibisci 107
The Role of Other Organosulfur Sources . Predation of
.... Saccharomycopsis javanensis on Saccharomyces cerevisiae 124
Effect of Inorganic Salts on Predation of Saccharomycopsis
.................... ja vanensis on Saccharomyces cerevisiae 1 37
..................... Predation-Associated Enzyme Activities 138
....................................... Preliminary Tests 139
Total P(1-+3)-Glucanase and a-(l-+4>Mannanase Activities . . ; . 140
. . . . . . . . . Effect of Inorganic Ions on P(1-3)-Glucanase Acüvity 147
Hydrolytic Cleavage of P(1+3>Glucan to Glucose and
........................................ Oligoglucosides 148
Exo-Po-GlucanaseAssay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
........................................... Conclusions 158
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Literature 160
CumkulumVitae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
vii
LIST OF TABLES
1 2.1 Sugar Distribution in Fungal Cell Walls . . . . . . . . . . . . . . . . . . . . . . . 12
1.2.2 Polysaccharide Chernotypes of Fungal Cell Walls . . . . . . . . . . . . . . 13
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.3 Different PGlucans from Fungi 15
. 1 3.1 .1.1 Mycocinogenic Yeast Genera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
1 .3.1 .2.1 Yeast Teleomorphç with Demonstrated Predacious Behaviour . . . . 23
1.3.1.2.2 Yeast Anamorphs with Dernonstrated Predacious Behaviour . . . . . 24
Yeast Strains Studied As Potenüal Predators and Preys . . . . . . . . . 25
Basic Nutrient Media . composition and Properties . . . . . . . . . . . . . 26
Selective Media . Composition and Properties . . . . . . . . . . . . . . . . . 27
Potential Predator-Prey Combinations . . . . . . . . . . . . . . . . . . . . . . . 38
Effect of Various Complex lngredients of Nutrient Media on the
Predation of Saccharomycopsis javanensis Grown in Co-Culture
. . . . . . . . . . . . . . . . . . . . . . . . . . . . with Saccharomyces cerevisiae 89
Predator-Prey Pairs and LNB (Basal) Medium Supplements in
. . . . Experiments on the Role of Organic Sulfur in Yeast Predation 91
Effects of lnorganic Salts on Predation of Saccharomycopsis
javanensis on Saccharomyces cere visiae . . . . . . . . . . . . . . . . . . . . 1 37
viii
LIST OF FIGURES
Modern Classification of Organisms From the Former Phylum
Mycota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Polyphyly of the 'Traditional Fungi", Mostly According to SSU rDNA
Sequencing Data, and Their Position Among Other Taxa . . . . . . . . . 8
Types of Fungal Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 6
Mechanisrns of Fungal Combat . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Interface Types in Mycoparasitism . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Growth of Saccharomycopsisjavanensis and Saccharomyces
cerevisiae on GY Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 55
Growth of Saccharomycopsis javanensis and Saccharomyces
cerevisiae on GY Agar - Staüsticç . . . . . . . . . . . . . . . . . . . . . . . . 57
Growth of Saccharomycopsis javanensis and Saccharomyces
cerevisiae on Basal Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Growth of Saccharomycopsisjavanensis and Saccharomyces
cerevisiae on Basal Medium - Statistics . . . . . . . . . . . . . . . . . . . 61
Growth of Saccharomycopsis javanensis and Saccharomyces
cerevisiae on Rich Medium (YM Agar) . . . . . . . . . . . . . . . . . . . . . . . 63
Growth of Saccharomycopsisjavanensis and Saccharomyces
cerevisiae on Rich Medium (YM Agar) - Staüstics . . . . . . . . . . . 65 Predation Dynamics of Saccharomycopsis javanensis and
Saccharomyces cerevisiae on GY Agar with 0.1 gfL Yeast Extract . 69
Predation Dynamics of Saccharomycopsis javanensis and
Saccharomyces cerevisiae on GY Agar with 0.1 gl L Yeast Extract
.......................................... - Statistics 71
P redation Dynamics of Sacchammycopsis javanensis and
Saccharomyces cerevisiae on GY Agar with 1 glL Yeast Extract . . 73
Predation Dynamics of Saccharomycopsis javanensis and
Saccharomyces cerevisiae on GY Agar with 1 glL Yeast Extract
........................................... - Statistics 75
Predation Dynamics of Saccharomycopsis javanensis and
Saccharomyces cerevisiae on GY Agar with 10 glL Yeast Extract 77
Predation Dynamics of Saccharomycopsis javanensis and
Saccharomyces cerevisiae on GY Agar with 10 glL Yeast Extract
- Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Predation Dynamics of Saccharomycopsis javanensis and
Saccharomyces cerevisiae on Low Nitrogen Basal (LNB) Agar:
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . O. 1 glL (NH4),S04 8 1
Pred ation Dynamics of Saccharomycopsis javanensis and
Saccharomyces cerevisiae on Low N itrogen Basal (LN B) Agar:
. . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 gR (NH4),S04 - Statistics 83
Predatio n Dynamics of Saccharomycopsis javanensis and
. . . . . Saccharomyces cerevisiae on Basal Agar: 5 g1L (NH,),SO, 85
Predation Dynamics of Saccharomycopsisjavanensis and
Saccharomyces cerevisiae on Basal Agar: 5 glL (N H4),S04
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - Statistics 87
Influence of Organic Sulfùr on the Predation Dynarnics of
Saccharomycopsis ja vanensis and Saccharomyces cerevisiae on
. . . . . . . LNB Medium with Varying Concentrations of m me thionine 93
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1.1 - No Methionine (Control) 94
3.5.1 -1 -a Influence of Organic Sulfur on the Predaiion Dynamics of
to Saccharomycopsis javanensis and Saccharomyces cerevisiae on
3.5.1 -6-a LNB Medium with Varying Concentrations of L-Methionine
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - Statistics 100
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1.1 -a - No Methionine (Control) 101
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1 -2-a - 0.0001 g1L L-Methionine 102
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1.3.a - 0.001 glL m me thionine 103
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1.4.a - 0.1 g/L m me thionine 104
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1 -5-a - 1 g / l m me thionine 105
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1 -6-a - 10 glL L-Methionine 106
3.5.2.1 Influence-of Organic Sulf'ur on the Predation Dynamics of
to Saccharomycopsis javanensis and Metschnikowia hibisci on LN B
3.5.2.3 Medium with Varying Concentrations of L-Methionine . . . . . . . . . . 108
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2.1 - No Methionine (Control) 109
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2.2 - 0.001 g R L-Methionine 110
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2.3 - 10 glL m me thionine 111
3.5.2.1-a Influence of Organic Sulfur on the Predation Dynamics of
to Saccharomycopsis ja vanensis and Metschniko wia hibisci o n LN B
3.5.2.3-a Medium with Varying Concentrations of L-Methionine
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - Statistics 112
3.5.2.1 -a - No Methionine (Control) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 13 3.5.2.2-a - 0.001 glL L-Methionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
3.5.2.3-a - 10 glL m me thionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
3.5.2.4 Influence of Organic Sulfur on the Predation Dynamics of Candida
to strain 'WI " and Metschnikowia hibisci on LN B Medium with Varying
3.5.2.6 Concentrations of L-Methionine . . . . . . . . . . . . . . . . . . . - . . . . . . . 116
3.5.2.4 - No Methionine (Control) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 17
3.5.2.5 - 0.001 g1L L-Methionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
3.5.2.6 - 10 g/L L-Methionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 19
3.5.2.4-a Inff uence of Organic Sulfur on the Predation Dynamics of Candida
to strain '7N1 " and Metschnikowia hibisci on LNB Medium with Varying
3.5.2.6-a Concentrations of L-Methionine
- Statistics . . ........................................ 120
3.5.2.4-a - No Methionine (Control) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
3.5.2.5-a - 0.001 g/L m me thionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
3.5.2.6-a - 10 glL m me thionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
3.5.3.1 Role of Organic Sulfur in the Predation Dynamics of Saccharomycop-
sis javanensis and Saccharomyces cerevisiae on LN8 Agar with
1 glL DL-Methionine (Racemate) . . . . . . . . . . . . . . . . . . . . . . . - . . 125
3.5.3.1-a Role of Organic Suhr in the Predation Dynamics of Saccharomycop-
sisjavanensis and Saccharomyces cerevisiae on LNB Agar with
1 glL DL-Methionine (Racemate) - Statistics . . . . . . . . . . . . . . . 127
3.5.3.2 Role of Organic Sulfur in the Predation Dynamics of Saccharomycop-
sis javanensis and Saccharomyces cerevisiae on LN B Agar with
1 glL Sodium Thioglycollate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
xii
3.5.3.2-a Role of Organic Sulf'ur in the Predation Dynamics of Saccharomycop-
sis javanensis and Saccharomyces cerevisiae on LN B Agar with
1 g R Sodium Thioglycollate - Statistics ................... 131
3.5.3.3 Role of Organic Sulfur in the Predation Dynamics of Saccharomycop-
sis javanensis and Saccharomyces cerevisiae on LN B Agar with
1 g R L-Cysteine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
3.5.3.3-a Role of Organic SuIfUr in the Predation Dynamics of Saccharomycop-
sis javanensis and Saccharomyces cemvisiae on LN B Agar with
1 g/L L-Cysteine - Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
3.7.2.1 Beta-(1 -+J>Glucanase Assay 48 Hours After Co-Culture of Saccharo-
mycopsis javanensis and Saccharomyces cerevisiae on Methio n i ne-
. . . . . . . . . . . . . . . . . . . . . Supplemented Predation Medium (GY) 143
3.7.2.2 Beta-(1+3>Glucanase Assay 6 Days After Co-Culture of Saccharo-
mycopsis javanensis and Saccharomyces cerevisiae and in Pure
Culture of Saccharomycopsis javanensis on Methionine-
..................... Supplemented Predation Medium (GY) 145
3.7.3.1 Effect of lnorganic Salts on j glu cana se Activity from Co-Culture of
Saccharomycopsis javanensis with Saccharomyces cerevisiae on
~redatiofl Medium (GY) with and without Methionine
. . . . . . . . . . . . . . (Washed Cells from Co-Culture after 48 hours) 149
3.7.3.2 Effect of lnorganic Salts on P-Giucanase Activity from Co-Culture of
Saccharomycopsis ja vanensis with Saccharomyces cerevisiae on
Predation Medium (GY) with and without Methionine
. . . . . . . . . . . . . . . . (Supematant from Co-Culture alter 48 hours) 151
3.7.5.1 Exo-P~Glucanase Activity Assay in Pure Cultures and Co-Cultures
of Saccharomycopsis javanensis with Saccharomyces cerevisiae on
Predation Medium (GY)
. . . . . . . . . . . . . (Washed Cells and Supernatants affer 48 hours) 156
xiii
P redation of Saccharomycopsis fermentans on Saccharomyces
cerevisiae in Slide Culture on GY Agar . . . . . . . . . . . . . . . . . . . . . . 40
Post-Predational Recovery in Slide Culture of Saccharomycopsis
javanensis and Saccharomyces cerevisiae onYM agar . . . . . . . . . 43
Predation of Sacchamrnycopsis javanensis on Saccharomyces
cerevisiae in Liquid Predation Medium (GY): Scanning Electron
Microscopy of Predator-Prey Cell Contacts . . . . . . . . . . . . . . . . . . . 47
Details of Haustoria Formation and Penetration During Co-Culture of
Saccharomycopsis javanensis and Saccharomyces cerevisiae in
Liquid GY Medium (Scanning Electron Microscopy) . . . . . . . . . . . . 49
Predation of Saccharomycopsis javanensis on Schirosaccharomyces
pombe in Liquid Predation Medium (GY): Scanning Electron
. . . . . . . . . . . . . . . . . . . Microscopy of Predator-Prey Cell Contacts 51
Thin Layer Chromatogram of Laminarin Hydrolyzate Obtained with
Washed Cells Rom Co-Culture of Saccharomycopsis javanensis and
Saccharomyces cerevisiae on GY agar . . . . . . . . . . . . . . . . . . . . . 153
xiv
INTRODUCTION - THE SCOPE OF THE THESIS
The present thesis constitutes a continuation and an integral part of
studies of a new antagonistic interfungal phenomenon (LACHANCE and PANG, 1997)
exhibited by certain yeasts grown in co-culture with other yeasts. A relationship of
necrotrophic parasiüsrn, or predation. of those novel strains upon others, chosen as
hosts, became manifest under certain culturing conditions. This predatory
behaviour came as a surprise. as such a relationship, although known and well
documented for many other fungi, was hitherto unknown among yeast genera. In
fact, this phenomenon had been seen at least once in the past (KREGER-VAN RIJ and
VEENHUIS, 1973), but its true nature and significance remained unrecognized.
The faculty of being predacious appears to be associated with some other
characteristics, pertinent to the strains known and studied thus far:
al1 known predacious yeasts were collected from specific habitats, namely
wounded trees (Fagaceae) or biocoenoses of insects/flowers (Malvaceae); predation is not an obliaatory behaviour, sinœ the yeasts grow well when
cultured on suitable media in the absence of a host;
predation is manifested by the appearance of elonaated protuberances
(haustoria or penetration peas), with which the predator penetrates its host;
haustorial penetration is lethal to the host;
with only three anamorphic exceptions, the known strains are
ascos~oroaenic teleomomhs;
host s~ecificitv varies with each predacious species, and appears to be
confined to the fungi;
medium comoosition is of crucial importance, with certain standard nutrients
effectively inhibiting predaüon (e.g., yeast extract) and others having the
opposite effect (e-g.. trace amounts of L-methionine);
phvsical conditions of cultivation - temperature and agitation - have a
marked influence on the outcome and extent of predation;
metabolic deficiencies in the utilization of sulfate appear to be a characteris-
tic of al! recognized predacious strains;
phvlogenetic relatedness among the examined strains is high, allowing the
inclusion of al1 the teleomorphs into a single genus (KURTZMAN and R O B N ~ ,
1995);
The significance of the findings on predatory yeasts is twofold:
they open up a new cha~ter in the study of interfungal relations, with the
possibility of shedding new light on the ecology of yeast comrnunities;
they might eventually offer new insights and lead to new methods in the
biocontrol of plant pathoaens and the mycoflora involved in food s~oilaoe
(e.g., DEAK and BEUCHAT, 1996).
1 .l FUNGl - A UNIQUE PHYLUM
The systematic position of organisrns which customarily were called
"fungi" has been a fiuctuating one in the many past and modern attempts at
correctly positioning them in relation to other living creatures. This fluctuation is
refiected in historical attempts at their description, classification and the explanation
of their ongin (AINSWORTH, 1965).
Fungi have accornpanied humans from the very beginnings of individual and
collective consciousness. Most intuitively, one thinks of the edible or poisonous
fruiting bodies of macroscopic fungi. Some of those structures had different uses,
like the dried bracket fungi used as tinder, or the hallucinogen-containing fungi in
sharnanic rites. On the other hand, whether or not the organisms thernselves were
visible and perceived as such, the human population had benefitted or suffered from
thern. Examples of harm done by certain fungi are the destruction of crops, food
spoilage, diseases of plants, animals and man, rnycotoxicoses and deterioration of
many materials. Examples of benefits to man are the leavening of bread, the
production of various fermented and fungus-processed foods and alcoholic
fermentations. In spite of this large body of experience with fungi, the awareness
of the existence of those not visible to the unaided eye had to wait for the
microscope to be constnided by Antonie van Leeuwenhoek in the 17th century.
Traditionally, fungi were regarded and classified as plants rather than
animals. probably because mushrooms and toadstools were generally found in
associations with plants, did not run away and, as such, had been studied by
botanists. After the Gutenbergian revolution they were included in conternporane-
ous herbals and one such description, in "The Great Herbal" from 1526, portrays
them in Paracelsian and Aristotelian ternis, showhg a preoccupation with the
poisonous nature of some of them (in AINSWORTH, 1965):
Fungi ben musherons. They be colde and moyst in the thyrde degre and that is shewed by theyr vyolent moysture. There be two maners of them, one maner is deedly and sleeth hem that eateth of them and be called tode stooles, and the other dooth not.
Over the course of centuries fascination with fungi had been growing steadily,
inspiring the lives and works of outstanding nahiralists, who helped to promote the
study of fungi from the spheres of black rnagic and gastronomy to the rank of a
science. A systematic study of fungi started some two and a half centuries ago
with PietAntonio Micheli's [1679-1737 "Nova genera plantarum" (ALEXOPOULOS et
al., 1996). An introductory account of this development with further references is
given by AINSWORTH (1 965).
It is now estabfished that fungi are truly cosmopolitan organisms, a paradigm
of ubiquity in the biosphere. There is probably no ecological niche that could not be
colonized by. and no other organism that could not be host to a suitably equipped
fungus. In cornparison with other taxa (e.g., plants and animals), fungi were not
particularly innovative in the acquisition of new biochemical/physiological traits
(CARLILE, 7980). Moreover, their development often followed parallel, convergent
and retrograde evolutionary paths (SAVILE. 1968). Their recipe for success seems
to consist primarily in their formidable reproductive and survival capacities, their
propensity for associations with other organisms and the ability to adapt to highly
variable nutrient sources.
From an estirnated 1.5 million fungal species, only about 69,000 are
described (CS%), making the fun@ one of the least well known taxa of the living
world (HAWKSWORTH, 1997 ), comparable to the estimated known-to-unknown ratio
of 1 :30 for the postulateci 30 million arthropod species (ERWIN, 1982).
1.1.1 THE EVIOENCE OF FUNGI IN THE FOSStL RECORD
Fungi had developed presurnably in marine environments in the
Precambrian (>570 Ma b-p.), and rnany of them must have lived in close
associations with otherforms of life, particularly cyanobacteria and algae. A gradua!
colonization of aquatic habitats with decreasing salinity must have been a
prerequisite for terrestrialization (HALLBAUER and VAN WARMELO, 1 974; PIRONNSKI ,
1976). This transition is assumed to have taken place at the latest in the Silurian
(438-408 Ma b-p.) (SHERWOOD-PIKE and GRAY, 1985). within the protective interior
of early land colonizers from the plant kingdom (parasitic or symbiontic
associations). Some of these earliest known terrestrial filamentous fungal fossils
rnay already represent members of the Ascomycota.
In the course of evolution some fungi ihen developed strategies to wunter
desiccation, leave their hosts and start exploiting new ecoiogical niches, created by
the spreading of vegetaüon. Indeed, fossil evidence of what appeared to represent
vesicular-arbuscular mycorrhizal (VAM) fungi associated with Devonian (408-360
Ma b.p.) vascular plants would support argumentations that such fungus-plant
associations were the cause rather than a consequence of land colonization
(PIROZYNSKI and MALLOCH, 1975; PIROZYNSKI, 1976, 1981 ; MALLOCH et al., 1980;
LEWIS, 1987). However, later reevaluations of these fossils (e.g., PIROZYNSKI and
DALPC, 1989) make them more likely candidates of saprobic groups. Be that as it
may, fungi were early land colonizers and continued gradually to conquer the
diversifying ecological niches.
All fungi are heterotrophs, completely lacking photosynthetic pigments and
chloroplasts and feeding by way of uptake of dissolved nutrients. In this single
fundamental respect they differ from almost al1 plants. In fact, when considering the
Uiree phylogenetically most recent and most closely related kingdoms of the
taxonomie superkingdom Eukaryofae - the Plantae, Animalia and Fungi - the
crucial determinant for the assignrnent of organisms into one of these three crown
taxa is their mode of nutrition (MOORE. 1996):
Plan tae: autotrophic or closely related to autotrophs
Animalia: phagotrophic heterotrophs or closely related to phagotrophs
Fungk al1 lysotrophic heterotrophs
The ecological roles that these three kingdoms have played are those of
producers, consumers and degraders. Howewer, the thickness of coal deposits from
the Carboniferous (360-286 Ma b.p.), derived from the recalcitrant remains of
pteridophytes, bears witness to a world in which fungi could not yet fiourish
(CORNER, 1964; cited in MOORE, 1996). Only the advent of easily rotting flowering
plants enabled an explosion of the fungal biota, lasting up to the present time.
TAXONOMIC AND PHYLOGENETIC POStTfON OF THE FUNGl
Profound differences between fungi and other "tnie" plants have been
recognized early on and many mycologists in the past were unwrnfortable with fungi
being classifieci among plants- Their generaf lack of motility, absence of
photosynthesis. ingestion of dissolved nutrients, presence of ceIl walls and a
fonerly assumed (and later largely abandoned) descent from algae were sufficient
to regard them more closely related to plants than to animals (HICKMAN, 1965).
Consequently, fungi ended up in the taxonornic kingdom of plants at a time when
only two kingdoms were established. Atternpts to increase the number of these
largest taxa to three (HAECKEL, 1866) or even five (COPELAND, 1956), in order to
arrive at a natural, phylogenetic system of classification and to accomodate protists,
did not alter the taxonornic position of the fungi as a whole.
As late as three decades ago fungi still cunstituted the second phylum (or
division) - Mycota or Mycobionta - of the kingdorn Plantae. But a change was
imminent due to the following developments in biology (ALEXOPOULOS et al.. 1996):
realization that the hitherto employed kingdom classification is unnatural;
acknowledgment of fungal polyphyly;
acceptance of phylogenetic systematics;
application of methods molecular systematics;
discovery of new taxa;
increasing and re-evaluated fossil records.
Decisions about phylogenetic relatedness are complicated by the inherent
diffÏculty in differentiating between primitive and acquired characters. Nowadays,
fungi are categorized in a separate kingdom (WHITTAKER. 1969), not only because
of their differences from "true" plants (and their similariaes to animals), but also
because of their problematic and still incompletely resolved evolutionary origin.
Phylum Chytndiornycota Phylum Zygomycota Phyf um Ascomycota Ph y lum Basidiomycota
Phylum Oomycota P hy lurn Hyphohytnomycota Phylum Labynnthulomycota
Protists
Phylum Plasmodiophoromycota Ph y l urn Dictyosteliornycofa Phylum Acrasiomycota Phylum Myxomycota
Figure 1.1.2.1 Modem classification of organisms from the former phylum Mycota (according to HAWKSWORTH et al.. 1994).
The long overdue and finally formal dissociation of fungi from plants paved
the way for further separation of fungal taxa. The classical phylum of "fungi" is
definitely polyphyletic, which is most conclusively demonstrated by cornparison of
the small subunit ribosornal DNA (SSU rDNA) fragments (e-g., BRUNS et al., 1991 ;
1993). In order to account for this poiyphyly, HAWKSWORTH (1 991 ) proposed the
terni fungi (with lower case 9, to denote "organisms studied by mycologists". What
used to be the lowest subclass of the primitive, and abandoned taxon of
Phycomycetes - the Oomyceüdae, was promoted to the level of the newly
proposed kingdom Chrornista (FORSTER et al., 1990). This designation was not
8
accepted, however. and the previously proposed kingdom of Stramenopiia
(PATERSON, 1989) now includes oomycetes as well as other former fungi
(hyphochytrids and labyrinthulids) and some taxa traditionally belonging to algology.
The most recently accepted classification places "traditional fungi" into two separate
kingdoms and four protist phyla (Fig. 1 .1.2.1).
Plants
rl - Amoebofiagellates
"--- Euglenoids
Figure 1.1.2.2 Polyphyiy of the "traditional fungi" (boldface), mostly according to SSU rDNA sequencing data, and their position among other taxa (compiled frorn various authors by ALEXOPOULOS et al., 1996, q.v.).
9
The first two of the former sub-phyla of the Mycota, the cellular slime molds
(Acrasiomycota) and aie true slime molds (Myxomycota) have been split into four
phyla, but common ancestors are still elusive (PATTERSON and SOGIN,-1992).
Phylogenetic relationships of the Fungi with other organisms are shown in Figure
1 -1.2.2.
The taxonomy of the fungi is süll changing on al1 levels as new
molecular data become available. Therefore, it should not be assumed that the use
of the terni fungi in the continuation of this text automatically excludes those
organisms sensu HAWKSWORTH (1 991 ) which phylogenetically do not belong into the
kingdom Fungi, particularly since many quotations from literature pre-date or simply
disregard this still unresolved matter.
1.2 FUNGAL CELL WALLS
Any functional living ceIl is equipped with a cell membrane
(cytoplasmic membrane or plasmalemma) - a complex partition between self and
non-self, to separate and protect its metabolic machinery from instabilities and
hazards of the outside world and to exploit resources fully. lntracellular functions
and processes are compartmentalized by similar structures. Most cellular or
subcellular membranes are topologically stnictured, Ruid double layers of
amphiphiiic molecules, and structures enclosed within such membranes could be
compared to micelles of water-in-oil-in-water emulsions.
This combination of hydrophilicity on the intemal and extemal surfaces
with hydrophobicity in the interior of the membrane entails cnicially important
functionalities to the cell membrane. Some of these functionalities are:
maintenance of an aqueous cell interior in aqueous environments,
prevention of simple diffusion into aie ceIl of rnany low-molecular weight
compunds, including nutrients and potentially hazardous substances,
prevention of simple diffusion out of the cell of many products of cell
rnetabolism,
establishment of a physical barrier for macromolecules, including enzymes,
maintaining of appropriate concentrations and gradients of precursors,
products and electrolytes,
maintaining electric fields by ionic charge separation and thereby enabling
bioenergetic processes,
providing a matrix for the incorporation of membrane-bound and
transmembrane proteins, enzymes and enzyme complexes,
providing a matrix for structures of intercellular recognition and signal
transduction.
The most important functionality which the membrane cannot provide
is structural stability and mechanical resistance. These are the main properîies of
cell walls - physically n'gid outer envelopes separated from cell membranes by a
periplasmic space. Without such reinforcements. fungal protoplasts would lyse in
media of lower osmotic pressure. Other, l e s obvious funcüonalities associated with
cell walls may be defined as mu l t i f~n~ona l organelles of protection, shape, cell
interaction, signal reception, attachment and specialized enzymic activity (FLEET,
1991 ).
Not al1 cells are equipped with walls. Protobacteria are assumed to
have posessed cell walls as early as about 3.5 billion years ago (BARTNICKI-GARCIA,
1984), and al1 extant eubacteria and archaebacteria have them. Among the
eukaryotic taxa, cell walls are missing in animal and protist cells. With a
phagotrophic mode of nutrition characterizing the latter two taxa. cell walls would be
an impediment, whereas the photoautotrophism of plants and the lysotrophism of
fungi (both accompanied by the buildup of high osmotic gradients against the
exterior) mandate the presence of an external reinforcement (RUIZ-HERRERA, 1 992).
Prokaryoüc cell walls are very different from their eukaryotic analogues and their
further description lies beyond the scope and purpose of this chapter.
The main components of al1 fungal cell walls are polysaccharides
(typically -80%) and proteins (typically 3% to 20%). Polysaccharides may be linear
or branched, cross-linked or not via sugar moieties or other small molecules (e-g.
amino-acids). In addition to simple sugar monomers as building blocks (rnainly
glucose), sugar derivatives (e.g., amino-sugars and uronic acids), and substituent
groups (such as acetyl) may be present. Types and amounts of sugars found in cell
walls of individual taxa are given in Table 1.2.1.
f he principal component of plant ceIl walls is cellulose, Po-(1+4)-
glucan. Except for one known species from the phylum Chytndomycota (FULLER
and CLAY, 1992), cellulose is absent in Fungi, but is the main cell wall element in
Stramenopila (oomycetes and hyphochwds). Pectins, which are partially
rnethoxylated poly[l,4a-~-galacturonides] with varying degree of este rification, and
are present as a reinforcing cernent in the central larnellae between plant cells, have
not been found in fungi.
Fungal wall polysaccharides are a broad class of homo- and
heteropolymers, some of them in the fom of glycoproteins, with very different
funcüons, ranging from structural rigidity in al1 species, to virulence in pathogenic
fungi (e.g . . SAN-BLAS et ai., 1 977), to protedive capsule formation (e.g . , GQLUBEV,
1991).
BARTNICKI-GARCIA (1 968) identified eight chernotypes of fungal cell
walls according to their main polysaccharides. Only five of them characterize the
monophyletic fungal kingdom of today (Table 1.2.2).
-- 1 Chitosan-chitin 1 Zygomycota (except Tnchornycetes) - 1 Chytridiomycota, "Euascomycetes" (i .e., filamentous Ascomycetes). "Homobasidiomycetes" (i.e., Hymenomycetes and Gasteromyce ts) , Deuteromyce tes
Mannan-glucan
Mannan-chitin
Table 1.2.2 Polysaccharide chernotypes of fungal cell walls (after BARTNICKI- GARCIA, 1 968).
"Hemiascomycefes" (Le., Archiascomycetes and Saccharomycetales)
"Heterobasidiomyce fes" (i. e., Urediniomycetes, Usfilaginomycetes)
Polygaiactosamine- galactan
Fungal walls are ever-changing cornplex composites of different
materials, held together by non-covalent interactions and possibly covalent bonds
consisting of (a) polysaccharide fibrïls (chitin and glucans); and (b) a matn'x in which
Tkhomycetes
the fibrils are embedded, consisting of kand a-glucans, chitosan, polyuronides,
glycoproteins, Iipids, salts and pigments (RUIZ-HERRERA, 1992; p. 9).
One of the main constituents of fungal cell walls is chitin, poly[1,4-P-2-
acetamido-2deoxy-D-glucopyranoside], determined to contain about 2,000 sugar
monomers boa when obtained from crustaceans (MU~ARELLI, 1984) and when
enzymatically synthesized in vitro (CALVO-MENDES and RUIZ-HERRERA, 1 987). C hitin
is present in fungal cell walls in amounts of about 2% to 20% wall dry rnass. Low
values are generally found in yeasts, whereas the content in sorne fungi may be
considerably higher (-60%) (RUIZ-HERRERA, 1978). In the cell wall of S. cerevisiae
chitin accounts only for about 1.2% of the total dry mass (CABIB and BOWERS, 1971 ;
BERAN et al., 1972), but most of it is localized in bud scars (up to -70%; HOLAN et
aL, 1981).
Characteristic for the Zygomycetes is chitosan, poly[l,4-PZ-amino-2-
deoxy-o-g lucopyranoside], which is synüiesized by chitin deacetylation (DAVIS and
BARTNICKI-GARCIA, 1984) and does not appear to have any direct structural role.
Land plants lack chitin and chitosan completely, but chitin occurs in green algae,
diatoms chrysoflagellates, protozoans, coelenterates and nematodes, and iç the
major organic structural material in tf?e annelids, molluscs and arthropod
exoskeletons (RUIZ-HERRERA, 1992; p. 91).
In the context of this thesis the most important aspect is the presence
of homopolysaccharides that provide mechanical strength. These are characterized
predominantly by k(1-3) and P(1-+6) glycosidic Iinkages between the sugar
moieües. The natural conformation of a-glucans is an intramolecular spiral,
resulting in arnorphous and more readily soluble matrices (e.g., amylose in starch).
On the other hand, repeating P-glucosidic bonds irnpart to the poiymenc chains an
increased tendency to intermolecular hydrogen bonding , the consequence of which
is a higher crystallinity and a lower solubility of the polymer (e.g., cellulose). An
intertwined triple helical structure with six glucose subunits per tum has been
proposed for the unbranched P-1.3-glucan synthesized by recovering protoplasts
of Saccharomyces cerevisiae (KOPECKA and KREGER, 1986). A collection of
bonding types and properties of the main P-glucans is given in Table 1.2.3.
The differences in solubility between individual glucans shown in Table
3 may be due prirnarily to the size of the molecule. but also to the exclusion of water
from densely packed clusters of high crystallinity. In addition to that, evidence was
found of chitin-glucan covalent bonding. In the case of yeasts this was shown for
ceIl walls of Candida albicans (SURARIT, et al., 1988) and bud scars of S. cerevisiae
(MOL and WESSELS, 1987).
The most important type of branched glucans consists of chains of
el ,3 giucans wiUi significant proportÏons of pl ,ô-glycosidically linked side chains.
Among yeasts, the most thoroughly studied ones are those from S. cerevisiae.
Linkage type
p1,3 unbranched
Solubility
Alkali
pl ,3 with occasional pl ,6 branching of single glucose uni&
pl ,3 with significant Pl ,6 branching Alkali or 1 insolu bIe
Water
p1,3 m-1.4
&1,3 / ~ 1 , 4 / p-1,6
Occurrence
Synthesized by regenerating yeast proto plasts
Un known
Alkali
lntracellular mycolarni- narans, extracellular mucilage
-- -
Achlya , Anniilarfa - - - -
Most abundant celf watl glycan in yeasts
Fraction of yeast glucan
Table 1.2.3 Different Pglucans f'orn fungi (from RUIZ-HERRERA, 1992)
INTERFUNGAL RELATIONSHIPS - MYCOPARASITISM
Mycoparasitic fungal interactions (Figure 1 -3.1 ) are numerous and
many are well characterized. Both nectrotrophic and biotro phic hostlparasite
relationships between filamentous fungi exist (JEFÇRIES and YOUNG, 1994; pp. 46-
78). Combat strategies for the antagonizing fungi are outlined in Figure 1.3.2.
Contact necrotrophy can result from, or be induced by close proximity between
antagonistic mycelia with or without physical contact. Such interactions are also
known as hv~hal interference (IKEDIUGWU and WEBSTER, 1970). On the other hand,
destructive penetration of one mycelium by another is categorized as invasive
necrotrophy (LUMSDEN, 1992). The various kinds of antagonistic interactions at the
level of mycelial interfaces are represented in Figure 1.3.3. Common phenornena
in the destruction of the attacked rnycelium include marked alteration in the
16
pemeability of the plasmalemma. loss of turgor. granulation. vacuolation and
coagulation of the cytoplasm. In some of those interactions autolytic phenornena
in the host mycelium also appear to play a role. Interference can be rnediated by
hydrolases (e.g.. chitinases, ~1,3-glucanases, proteases) (RIDOUT et al., 1988). the
activity of which may increase in the presence of host cell wall, even in the forrn of
purified cell wall components (ELAD et al., 1 985). Low-molecular weig ht factors
which diffuse through cellulose membranes were also found to be responsible for
necrotrophic interactions (IKEDIUGWU and WEBSTER, 1970).
1 NEUTRALlSTlC 1 1 PRIMARY RESOURC 1 (COMM ENSALISTIC) 1 1 CAPTURE 1 - - - - -- -
Not detrimental to either, not Initial colonization of beneficial to both organisms. substrate by a fungus.
Beneficial to both I SECONDARY RESOURC CAPTURE
organisms. Cornpetifive replacement of one fungus by another within a partiwlar niche.
Figure 1.3.1 Types of fungal interaction (after COOKE and RAYNER. 1984).
1 YESYUISMS 1 o y e s o /.ROM/ OFCOMBAT Contact? a Total? MYCELiAi (ANTAGONIS INVOLVEMENT
(e.g., volatile and (e.g., parasitism and non-volatile antibiosis) hyphal interference)
Figure 1.3.2 Mechanisms of fungal combat (after RAYNER and WEBBER. 1984)
I Yes I
Speciatized interfaces
BIOTROPHS 810TROPHS
Figure 1.3.3 Interface types in mycoparasiüsm (from JEFFRIES and YOUNG, 1994)
YWSTS AS FUNGAL PARASITES AND PREDATORS
Yeasts do not occurr at random in the biosphere but form
communities of species (LACHANCE and STARMER, 1998). LACHANCE (1 990) defines
the yeast habitat as "a place or collection of places that share sufficient similarities
to result in their tendency to harbor similar yeast wmmunities". There are four
major terrestrial yeast habitat types - soil. plants, animals. and the atrnosphere (Do
CARMO-SOUSA, 1969). Soif, along with aquatic habitats, is problematic to a degree,
in that it is sometimes difficult to detemine whether a particular yeast is an
autochthonous or an allochthonous organism for a particular habitat. Yeast
communities and their natural habitats are cuvered in detail by PHAFF and STARMER
(1 987).
Yeasts are non-rnotile, stnctly chernoorganotrophic microfungi
(WALKER, 1998). They posess relatively modest physiological faculties (LACHANCE,
1990) and are consequently saprotrophic or, in a minonty of cases, parasitic on
animais and man. Members of both of these groups benefit from the ready
availability of nutrients in their respective habitats. Yeasts are not usually among the
eariy colonizers of more recalcitrant substrata.
Antagonistic interactions among yeasts have received much less
attention than those among filamentous fungi and lower fungal genera. It may be
argued that the opposite would be ramer surprishg because of their predominantly
saprotrophic way of life. One may expect cornpetition for nutrients to take place
arnong yeasts, as it normally happens when mixed microbial populations share
common resources. Other than that, only two tnily antagoniçtic phenornena among
yeasts are known as of now - the killer phenomenon and yeast predation.
THE KJLLER YEAST PHENOMENON
Certain yeast species produce extracellular, diffusible proteins
or glycoproteins (killer toxins) which are lethal to other (sensitive) yeast strains, but
to which the producers thernselves are immune. The phenornenon was first
observed with Saccharomyces cerevisiae strains 35 years ago (MAKOWER and
BEVAN, 1963), and was extensively reviewed since (e.g., YOUNG, 1987; WICKNER,
1992,1996). Similar toxins were later found to be produced by other fungi, namely
Ustilago (KOLTIN, 1988) and Polysphondylium, an acrasid slime mold (MIZUTANI et
al., 1990). Therefore, the name mycocins (coined after the bacteriocins) has been
proposed as preferable to the hitherto used term yeast killer toxins (GOLUBEV, 1998).
Mycocin production (killer activity) may be assayed (YOUNG, 1987)
by inoculating the suspected killer strain onto a n'ch nutrient agar containing
suspended cells (usually =4 x 10' ml'') of a mycocin-sensitive strain. The agar is
supplemented with methyiene blue (30 mg/L). Since the toxins are active in a
relatively narrow range of pH values (3 s pH s 6), the media are customarily
bufiered with sodium citrate (4.3 s pH s 4.7). Killer activity becornes manifest after
an incubation for 2-3 days, usually at 1 8-20°C (because of toxin thermolability). A
growth inhibition zone around the inoculurn is surrounded by a halo of blue-stained
dead cells of the sensitive strain. This zone of dead cells is indicative of a mycocin-
producing strain.
The discovery of S. cerevisiae rnycocins has ultimately led to an
intensive search for this trait in sorne existing yeast collections. The most
comprehensive of these surveys was the one in the National Collection of Yeast
Cultures (Colney Lane, Norwich, UK), done by PHILLISKIRK and YOUNG (1 975).
They found killer strains with a frequency of 6% (59 out of 964 strains from 28
genera) among commercial strains, and 31 % (27/86) among standard genetic
strains of the same collection. The study also revealed mat the killer trait was not
a peculiarity of Saccharomyces. Strains from the genera Candida, Debaryomyces,
Kluyveromyces, Pichia, Tonrlopsis (Candida), and, es pecially , Hansenula were
20
found to be kiiler toxin producers. The search for yeasts in natural habitats (STUMM
et al., 1977) gave an incidence of 17% (2611 57). Today, there are about 80 known
mycocinogenic species from about 20 di#ferent genera (Go~uew, 1998), as outlined
in Table 1.3.1.1 -1.
Yeast mycocins are protein or glycoprotein dimers or tnmers, usually
with a relative molecular weight of ICI-20 kDa. However, glycoprotein rnycocins
from Pichia and Khyveromyces are one order of magnitude larger (2 100 kDa).
Genes encoding Saccharomyces mycocins are based on Iinear
&RNA plasmids (WICKN ER. 1 996). Mycocins from KIuyveromyces laciis (STARK et
al., 1990) and Pichia acaciae (MCCRACKEN et al., 1994) are encoded on linear DNA
plasmids. A number of other killer yeasts inherits the mycocin genes
chromosomally, and for others still, the genetics of inheritance is unknown
(references in GOLUBEV, 1998, and WALKER, 1998).
Genus Genus Genus
Candida Hanseniaspora Rhodotorula
Saccharomyces
Kluyveromyces Cysto filobasidium
Trichosporon Debaryomyces
Table 1 .LI .1.1 Mycocinogenic yeast genera (after GOLUBEV. 1998)
All mycocins are divided into 12 classes (killer phenotypes, K,-KI,
and ha), according to cross-reactivity between species (YOUNG, 1 987) of which the
toxÏns Ki-K, and K, are secreted by Saccharomyces strains. One of thern (K,)
requires two viruses. MI and LA. The MI virus encodes a preprotoxh which is
hydrolyzed into the mycocin itself, an ap dimer (PALFREE and Busseut 197b), and
into a resistance factor, a giycosylated y-peptide (ZHU et al. 1993). The L-A virus
carries the gene for the capsid protein of both viruses (WICKNER, 1996).
The mode of action is diMerent for the individual mycocins. The best
studied system is the one of K, toxin (SKIPPER and BUSSEY, 1977; DE LA PENA et al.,
1981). About 1 o4 KI molecules are needed to kill one sensitive cell and the activity
reaches its maximum after 2 3 hours of exposure (SKIPPER and BUSSEY, 1977). It
starts with the attachment of the Psubunits to the j3-(1+6)-glucan chains in the cell
wall of the susceptible species. After mat, a-subunits are inserted through the cell
membrane. thereby forming channels which upset tJie transport of H' and K+ ions,
amino acids and ATP (MARTINAC et al., 1990).
No known mycocins are active against prokaryotes, plants or
anirnals. However, it has been shown that some mycocin-producing yeasts inhibit
the growth of a number of wood-rotang and phytopathogenic fungi (WALKER et al.,
1995).
YEAST PREDATION
As early as 1973 morphological feahires of anastornotic
interacüons between the filamentous yeast Saccharomycopsis (Althroascus,
Endomycopsis) javanensis and several other yeast genera had been studied
(KREGER-VAN RIJ and VEENHUIS, 1973). Electron micrographs revealed the
formation of denticles on hyphae and single cells of A. javanensis. These, in tum,
grew out into stalks which penetrated cell walls of other susceptible yeasts. It was
unclear, however, whether the process of penetration was parasitic in nature, or
whether it occurred only on dead cells.
Studies conducteci in this department (LACHANCE and PANG, 1997)
suggest that yeast penetration is, in fact, predation. The phenornenon appears to
be rare. Thus far, only eleven species have been shown to behave as predators
under certain conditions. Eight of them are teleomorphs (Table 1.3.1.2.1). The
remaining three are anamorphs belonging to the genus Candida (Table 1.3.1 -2.2).
The teleomorphs in Table 1.3.1.2.1 are phylogenetically related
(KURRMAN and R O B N E T 1995) and are currently classified as members of the
same genus Saccharomycopsis (KURTZMAN and FELL, 1998).
Interestingly, al1 these species share an unusual requirement for
organic sulfur. From an evolutionary aspect, a metabolic deficiency of SO,-uptake,
which per se would be quite a disadvantage in cornpetition with prototrophic
populations, could have becorne a seledive benefit to such auxotrophs in
environments where high levels of toxic sulphate analogues are encountered:
selenate(V1) (Seo?) and chrornate(V1) (~102') ions are both transported across the
cell membrane by Iow-specif~city S0:--permeases (BRETON and SURDIN-KERJAN,
1 977).
According to MHANCE and PANG (1997), the following general
prerequisites have to be fulfilled for predation to occur: (1 ) a nutrient medium poor
in organic sulfur, (2) growth on a solid support or, at least, in a stagnant and
convection-free layer of liquid, and (3) presence of a suitable organism as prey.
Yeast predation involves fomation of protuberances (haustoria) on
the cell surface of the predator grown in rnixed culture with its prey, subsequent
penetraüon of the tips of the haustoria into vegetative prey cells and/or spores. The
overall outcorne of this antagonistic relationship is a disniption of the cytoplasmic
integrity of the prey cells and, in sorne cases and under certain conditions. a
decimated prey population.
Predacious yeasts are not obligate parasites, as they grow
saprophytïcally on common media. In fact, n'ch, cornplex media or some simple
media with added methionine may suppress predation (LACHANCE and PANG. 1 997).
Moreover. it appears that the induction of hailstorium formation and the penetration
23
of prey cells is a function of organosulfur availability in the environment Because
predation-eliciting signais yield a positive response only when predator and prey
cells are in close proximity, sulfur-containing metabolites may act as chemotactk
signals for the induction of haustoflurn formation. The fact, however, that organisms
with radically different cell envelopes, e.g., the yeast-like alga Protofheca zopfii
(CONTE and PORE, 1973) failed to elicit a predatory response could mean that a
specific ledn-mediated dl-cel l recognition between predator and prey is needed.
Systematic name in use at time when predacious behaviour was demonstrated
Arfhroascus javanensis (Klocker) von Aix (1 972)
New classification as Saccharomycopsis (KURTZMANN and SMITH, 1998)
S. javanensis (Klocker) Kurtzmann 8 Robnett (1 995)
Arthmascus fermentans C.-F. Lee. F.- L. Lee, Hsu & Phaff (1994)
S. fermentans (C.-F. Lee, F A . Lee, Hsu & Phaff) Uurtanann & Robnett (1 995)
Afihroascus schoenii (Nadson 8 Krasil'nikov) Bab'eva, Vustin, Naumov & Vinovarova (1 985)
S. schoenii (Nadson & Krasil'nikov) Kurtzmann 8 Robnett (1 995)
Botryoascus synnaedendrus (D. B. Scott 8 van der Walt) von AIX (1972)
Saccharomycopsis crataegensis Kurtzmann 8 Wickerham (1 973)
S. synnaedendra D.B. Scott & van der Walt (van der Walt and D.B. Scott 1971)
--
Guilliermondella selenospora Nadson 8 Krasil'nikov (1 928)
1 UNCHANGED
- - -- -- -
S. synnaedendra (Nadson & Krasil'nikov) Kurtzmann & Robnett (1 995)
Sa ccharomycopsis fib uligera (Lindner) Klocker (1 924)
Table 1.3.1.2.1 Yeast teleomorphs with demonstrated predacious behaviour
Saccharomycopsis malanga (Dwidjoseputro) Kurhrnan, Vesonder & Srniley (1974)
UNCHANGED
1 Swcies name 1 1 Candida sp. [UWO-PSI 91-124.1 1 1 Candida sp. [UWO-PSI 91-127.1 1
Candida sp. [UWO-PSI 95-697.4 (Strain 'Wl")
Table 1.3.1.2.2 Yeast anamorphs with demonstrated predacious behaviour
1.4 THESIS OBJECTIVES
Based on what was said in the previous Section 1.3.1 -2, the following
study objectives were defined pnor to the beginning of experirnental work:
- To continue the study of phenomena associated with yeast predation by
selecting potential predator-prey pairs upon rnicroscopical and electron
rnicrosco pical examination of cultures.
To fmd growth conditions (environmental factors) underwhich penetration
does not take place, in order to help explain the reason for yeast
predation.
To investigate the production of hydrolases and the hydrolysis of prey cell
wall components associated with penetraoon.
MATERIALS AND METHODS
MICROORGANISMS
All microorganisms used in this study are preserved under liquid nitrogen
in the culture collection of the Department of Plant Sciences, University of Western
Ontario (UWO[PS]) and with only one exception (the alga Prototheca zopfi!' all of
them were yeasts or yeast-like fungi. They have been selected on the basis of their
predacious behaviour or their potential to serve as prey. The origin of isolates in
both groups is summarized in Table 2.1 .l.
1 Saccharomycopsis javanensis 1 82-52 ( Oak flux 1
1 PREDATOR
Saccharomycopsis ja vanensis
1 Saccharomycopsis synnaedendra 1 96-1 2.1 1 Oak frass 1 1 Saccharomycopsis selenospora 1 8 1 -1 08 1 Oak flux 1
1 UWO[PS] No.
92-247.1
1 ~accharomycopsi& fermentans 1 CBS 7830' 1 Orcahrd soi1 1
1 ORlGlN
Drosophila sp.
Candida strain "Wq" I 95-697.4
1 Aureobasidium pullolans 1 95879.1 1 Hibiscus fly 1
Hibiscus beetle
Rhodotorula M u t a
Schizosaccharomyces pombe
1 Prototheca zopW 1 95-917.2 1Opuntiarot 1
Table 2.1 -1 Yeast strains studied as potential predators and preys (* Centraalbureau voor Schimmelcultures, Julianlaan 67, 2628 BC Delft, The Nemerlands )
1 PREY 1
-
95-922.4
94-208.2
Saccharomyces cerevisiae 96.6
Me fschniko wia hibisci 1 95-747.4
Hibiscus fiy
Fermentation
Fermeniing birch sap
Hibiscus fl ower
Nutrient media were used for recovery of cultures after liquid hi, storage
(YM agar), maintenance of cultures in the course of study (GY agar and YE-fortifieci
GY agar), colony counts (selective media) and individual experiments (special
media designed for predation assessment).
The composition and properties of basic media (solidified form) are given
in Table 2.2.1. Selecüve media for wIony counts were used to detenine selectively
the cell numben of predators and preys. The composition and properties of the
latter are given in Table 2.2.2.
Composition Description
YM + 1 ppm
YM + Cyc cycloheximide
- -- - -
YM + 5 ppm YM +
cetyltrimethylammonium CTAB
bromide (CTAB)
Selective for predacious yeasts.
lnhibits growth of prey yeasts used in
this study.
Selective for Metschnikowia hibisci.
CTAB inhibits growth of predator.
Table 2.2.2 Selective media - composition and properties
2.3 SELECTION OF MOST ACTIVE PREDATOR-PREY COMBINATIONS
Nearly equal amounts of actively growing potenüal predator and prey
cultures were picked from maintenance medium (GY agar slants), combined on GY
agar plates (predation medium), and incubated at 25'C. After 24 hours, sarnples
of the mixed cultures were transfered to thin agar slabs (1.8% w/v) on microscope
slides, covered with coverslips and examined for haustorium formation and cell
penetration under phase contrast (oil immersion, magnification 1000x) with a Leitz
Orholux microscope.
W hen Schizosaccharomyces pombe was studied as the potential prey of
Saccharomycopsis javanensis. the predator was inoculated first onto GY agar,
incubated for 24 hours at 25°C. and then mixed with S. pombe and incubated for
another 24 hours before rnicroscopy.
2.4 SEARCH FOR MEDIA COMPONENTS THAT AFFECT PREDATION
To a 20-gR aqueous solution of yeast extract (Difcu) an equal volume of
97% (vEv) ethanol or acetone was added slowly and under agitation. The resulting
mixtures separated into a solid phase (precipitate) and a clear aqueous solvent
phase (supernatant). The supematants were carefully decanted and evaporated in
a water bath. These evaporates as well as the collected precipitates were
subsequently dried ovemight in a vacuum desiccator. Each of the four fractions
thus obtained was redissolved in the initial volume of water. These solutions were
supplemented with glucose (1 0 g/L) and agar (1 5 g/L), and after sterilization plates
were poured.
Another series of plates was prepared, containing the usual predation
medium (GY, see Table 2.2.1), suppiemented with one other complex nutrient
Chosen were malt extract, peptone and tryptone (20 glL each), and a vitamin
mixture (140 mgR) (al1 from Difco).
Predation on these media was assayed as describeci in Section 2.3.
2.5 LlGHT MICROSCOPY OF PREDATION
A thin slab of GY agar was placed on a sterile microscope slide,
inoculated with a mixture of Saccharomycopsis fennentans and Saccharomyces
29
cerevisiae, covered with a sten'le coverslip, and left at the microscope stage for up
to 3 days. The mixed culture was periodically inspected and photographed.
2 -6 LIGHT MICROSCOPY OF POST-PREDATION VlABILlTY
Microscopy was performed after predator and prey had grown together
for a period of tirne on predation agar (GY). Their subsequent recovery on rich
medium (YM) was studied microscopically as described in Section 2.5.
Approximately equal amounts of Saccharomycopsis javanensis and
Saccharomyces cerevisiae were transferred from GY agar maintenance slants.
mixed together on GY agar plates (predation medium) and incubated for ca. 15
hours at 25°C. The mixed culture thus obtained was suspended in sterile H,O,
transferred onto a thin slab of YM agar, placed on a sterile microscope slide. and
covered. Selected regions in the field of view were photographed ai diHerent
intervals to document changes (Plate 3.2.2.1-A).
2.7 ELECTRON MICROSCOPY OF INTERACTIONS IN LIQUID MEDIA
The following four predator-prey pairs were cultured in still and shaken
liquid predation medium (GY) and studied by scanning electron microscopy:
Saccharomycopsis javanensis - Saccharomyces cerevisiae
Saccharomycopsis javanensis + Schizosaccharomyces pombe
Saccharomycopsis synnaedendrus -r Metschnikowia hibisci
Candida strain "W 1 " -, Metschnikowia hibisci
Pure cultures were taken from maintenance slants and suspended in
small amounts of sterile water. The suspensions were vortexed at medium speed.
Predator suspensions (20-pL aliquots) were wmbined with prey suspensions (200-
pL aliquots) in 25 mL of liquid predation medium in 250-mL Erlenmeyer flasks.
Incubation was camed out at room temperature for 3 or more days.
Scanning electron microscopy was perforrned as follows:
Ceils Born liquid predation medium (204- aliquots) were placed on
Nuclepore membranes (0.45 Pm pore sire) resting on agar plates. The membranes
were floated onto 2.5% (wlv) glutaraldehyde in 0.1 M sodium cacodylate buffer.
ARer fixation for 15 min or more, the membranes were rinsed in cacodylate buffer,
dehydrated for 15 min in 1,Z-dimethoxypropane (propylene glycol dimethyl ether)
acidified with dilute HCI and critical-point dried. Sarnples were gold-coated by
plasma sputtering (5 min), and observed in a Hitachi F4500 Field Emission
Scanning Electron Microscope. Images were recorded electronically (Plates 3.3.1,
3.3.2 and 3.3.3).
2.8 PREDATION MONITORING ON SELECTED MEDIA
(COLONY COUNTING)
Cultures were grown on the surface of a piece of dialysis membrane
resting on agar medium. The membrane barrier serves as a nument-permeable
support for the growing cultures and allows the entire biomass to be removed from
the culture medium. Cell loss is thereby minimized and reliable colony counts can
be obtained.
Separate suspensions of predator and prey cells were prepared from pure
cultures grown on maintenance agar slants and vortexing in sterÎle &O. Celi
densities of these original suspensions were made to be roughly equal, and aliquots
of suspensions were mixed at a 1/10 predator-prey ratio.
Controls were prepared by diluting separate predator and prey suspen-
sions to the same respective concentrations as in the mixture.
Mixed suspensions and controls thus prepared were applied as 20-pL
aliquots into the centre of pieces of dialysis membrane (3 cm x 3 cm) lain flat on the
surface of a selected agar medium in a PetrÎ dish. Multiple (up to six) membrane
cultures of each predatodprey combination and control were initially prepared and
incubated at 25°C.
At selected intervals individual plates were withdrawn from the incubator
for counting. This consisted of lifong the membrane from the underlying agar,
submerging it into 3 mL of sterile surfactant-supplemented water (Tweeng 80. ca.
10 mgfL) and vortexing at medium speed. Without removing the membrane, a serial
dilution of the resulting suspension was made by transfemng 200 PL of the undiluted
suspension into 3 m l of surfactant-supplemented sterile H,O and repeating this
same dilution pattern 4 to 5 times as required.
Suitable dilutions were plated for colony counts. Twenty-microlitre
aliquots of each dilution were applied, in triplicate, ont0 the surface of YM agar with
or without selective agents (see below) and the liquid was alfowed to dry. A total of
12 spots per plate were thus obtained. The plates were incubated for 24 hours and
the small colonies were counted under a dissection microscope (1 0 x magnification).
Medium composition and incubation temperatures differed depending on
the yeast counted (Table 2.2.2). For Saccharomyces cerevisiae the plates (YM
agar) were incubated at 37°C. At this temperature S. javanensis did not grow.
EFFECT OF INORGANIC SALTS ON PREDATION
Predation of Saccharomycopsis javanensis on Sacharomyces cerevisiae
was studied on predation medium containing selected inorganic salts andlor L-
methionine. Predation on these media was assayed as described in Section 2.3.
A series of plates was prepared with predation medium (GY, Table 2.2.1).
supplemented with one of the following:
10 g/L ammonium sulfate, (NH&SO,
10 g R potassium sulfate, K2S04
10 g/L potassium nitrate, KNO,
10 gR ammonium sulfate, (NH,),SO, + 10 g R m me thionine
10 g/L bmethionine
no additive (control)
2.1 O PREDATION-ASSOCIATED ENZYME ACTIVITIES
2.10.1 PRELIMINARYTESTS
Preliminary tests were carrieci out to assess the presence or absence of
P-glucanase and chitinase adivities in predator-prey pairs of Saccharomycopsis
javanensis with Saccharomyces cerevisiae and Candida strain 'W1" with
Metschnikowia hibisci grown separately and together on predation agar (GY) and
on LNB agat with added DL-methionine (1 glL).
Plates with dialysis membranes were inoculated with suspensions of pure
cultures and their mixtures (cf. Section 2.8). Incubation was at 25°C. Cells were
harvested after 24 and 48 hours by washing the membranes in 0.5 mL of 50-mM
succinate buffer, pH = 5.2.
To determine extracellular enzyme activities. the ceIl suspensions were
immediately centrifuged (8 x 1 O3 min-') and the dl-free supematank were carefully
pipetted off and mixed with equal volumes of the required substrate solutions
(laminarin or chitin).
The reaction mixtures were incubated at (34 I 1)"C. Five-pL samples
were taken every W hour and applied directiy to the surface of filter paper (Whatman
No. 1) and air dried. The presence of reducing sugars was detected with the
alkaline silver nitrate reaction as described by LACHANCE and PHAFF (1975) (see
reagents in Section 2.9.3).
2.1 0.2 ENZYME ASSAYS
Cultures of Saccharomycopsis javanensis and Sacchammyces cerevisiae
grown separately or together were assayed for hydrolysis of laminarin and yeast
rnannan. Both activiües were assayed spectrophotometrically by measuring the
accumulation of reducing sugars using the Nelson-Somogyi colour reaction as
described by SPIRO (1 966).
The culture medium for this experiment was GY agar supplemented with
5 g/L me thionine (to stimulate predation). Multiple plates wSth dialysis membranes
were inoculated with suspensions of pure cultures or their mixture (cf. Section 2.8).
Incubation was at 25°C. Cells were harvested after 48 hours and 6 days by washing
the membranes in 1 .O ml of 50-mM succinate buffer, pH = 5.2.
To detemine extracellular enzyme activities, the resulting cell
suspensions were immediately œntrifuged (8 x lo3 min-') and the cell-free
supernatants were carefully pipetted off and mixed with equal volumes of the
required substrate solution.
For the assay of dl-bound acüvities, centrifugeci cell pellets were washed
3 times by resuspending thern with vortexing in fresh succinate buffer and
centrifuging. The washed cells were resuspended in 2.0 mL of the respective
su bstrate solution.
The reaction mixtures were incubated at (34 I 1 )OC (Figures 3.7.2.1,
3.7.2.2, 3.7.3.1, and 3.7.3.2). Samples were taken periodically. Those which
contained cells were œntrifuged (8 x IO3 min") and the clear supernatants were
pipetted off for further use.
The reaction in these sarnples was stopped by adding equal volumes of
the "modified copper reagenf' (SOMOGYI, 1952; as quoted in SPIRO, 1966) and
stored in a refrigerator until the end of sample taking, at which time the rest of the
procedure was applied.
Calibration graphs were prepared with varying concentrations of glucose
and mannose in 5&mM succinate buffer, pH = 5.2.
2.10.3 EFFECT OF INORGANIC IONS ON P(1+3>GLUCANASE ACTlVlN
The media described in Section 2.9 were used to grow CO-cultures of
Saccharomycopsis ja vanensis and Saccharomyces cerevisiae on dial ysis
membranes, as detailed in Sections 2.8 and 2.10.2, 2. The enzyme assays were
then camed out after 48 hours as outlined in Section 2.1 0.2, 3 sqq.
2.10.4 THIN-LAYER CHROMATOGRAPHY
OF MONO- AND OLIGOSACCHARIDES
The production dynamics of glucose and oligoglucosides from laminarin
by yeast 1 , b ~ ~ - g l u w n a s e s was monitored using thin layer chromatography.
Predator and prey were cultured and the reaction mixtures with laminarin
as substrate were prepared and incubated as outfined in Section 2.9.1. The only
difference was that the reacüons were stopped by appling samples ont0 the surface
of TLC-plates.
Prior to the enzyme assay tests were conducted to detemiine the best
chrornatographic conditions (stationary phase and solvent system, spot application
volume, single vs. multiple elution). Prefabricated Kieselgel60 TLC-plates, 20 cm
x 20 cm, 0.2 mm layer thickness (E. Merck, Darmstadt, Germany) were chosen.
Pre-drying of plates did not result in any improvement Best separations were
achieved with the temary eluent consisting of 3 parts n-butanoi + 1 part glacial
acetic acid + 1 part water (STAHL, 1969). Double development in a single direction
gave the most satisfactory results.
Individual sarnples were applied in 5-pL aliquots approx. 3 cm from the
lower plate edge, spaced 1.5 cm from each other (12 spots per plate). During
application the spots were dned with a hair drier. The duration of a single
developrnent in the above solvent system, up to about 1 cm from the upper plate
edge, was 5% houn. Pnor to the second development the plates were dried
ovemight in a vacuum oven (approx. 50°C).
The separated spots were visualized by spraying with Reagent 1 [0.5 mL
saturated aqueous Ag NO, in 100 mL acetone, solubilized by the dropwise addition
of water]. After drying, the plates were sprayed with Reagent 11 [2.3 mL 55% (wlw)
NaOH in 100 rnL 95% ethanol].
The staining procedure was repeated until the spots were dark. Spot
visibility was enhanced by heating the plates in an oven for 1 - 2 minutes at 120°C
after the final spraying.
2.1 0.5 EXOGLUCANASE ASSAY
Saccharomycopsis javanensis and Saccharomyces cere visiae were
grown separately and in co-culture on dialysis membranes resting on GY agar (cf.
Section 2.8). Cells were haniested and washed after 48 hours of incubation (cf.
2.1 0.2). The resulting centrifuged cells and supematants were assayed for exo-P
glucanase acüvity with a solution of pnitrophenyl-PD-glucoside (1 O glL). These
assays were carn'ed out in the same manner as those described in Section 2.1 0.2,
3 et sqq. The reacüon was stopped after appropriate time intervals with a solution
of sodium carbonate (40 glL Na,CO,), using 2 mL of this solution for 0.1 mL of
recation mixture. Thereafter the absorances of these solutions were measured in
a 1-cm spectrophotometer cell at A = 450 nm.
RESULTS AND DISCUSSION
SELECTION OF MOST ACTIVE PREDATOR-PREY COMBINATIONS
In order to find suitable combinations of cultures to study yeast predation,
the following criteria were applied:
presence of haustoriurn-mediated predation;
susceptibility of host to its predator;
predator and prey rnorphologically distinguishable from each other;
unicellular growth of both organisms on solid substrats (important for
counting ).
Five potentially predacious yeasts were tested against six prospective
prey species (Table 2.1.1). The prey included the hemiascomycetous yeast S.
cerevisiae, the holoascornycetous yeast M. hibisci, the heterobasidiomycetous yeast
Rh. minuta, the archiascomyœtous fission yeast Sz. pombe, the ascomycetous
yeast-like mould A. pullulans, and the achlorophyllous alga P. zopfii.
Tested were 23 predator-prey cornbinations. Observations from these
experiments are surnmarized in Table 3.1.1. Predation was most active with
Saccharomycopsis javanensis UWO[PS] 92-247.1 preying u pon S. cerevisiae.
Consequently, in the majority of later experiments this particular predator-prey
combination was used. An almost complete prey elimination on GY agar was
achieved after 24 houn. Morphological features characteristic for predators
(haustorkm formation, prey cell penetration) were clearly manifest, as well as the
progressively deteriorating status of the prey (details in Sections 3.2.1 and 3.2.2).
Similar results were obtained with Saccharomycopsisjavanensis UWO[PS] 82-52.
The othertwo tested rnembers of the genus Saccharomycopsis gave less
satisfacto ry results. W hen S. selenospora - fo merl y GuiMermondelia selenospora
Nadson et Krassilnikov- is grown in pure culture on GY agar, numerous thin lateral
branches can be seen along its hyphae, many of them bearing asci in the form of
ellipsoidal and ovoid structures. This corresponds closely to the description in the
systematic discussion of the species (KREGER-VAN RIJ, 1984). It was also reported
that S. selenospora produces lateral denticles which. when grown out to stalks, are
capable of penetrating its own mycelium and dead cells (KREGER-VAN RIJ and
VEENHUIS, 1973; KREGER-VAN RIJ, 1984). The filamentous growth of this organism
would preclude accurate counting of cell numbers.
S. synnaedendra - fonerly Bot~oascus synnaedendm (Scott et van
der Walt) von Arx - is known to produce a branched, septate mycelium with
sphencal or oval blastospores which conjugate with hyphal cells to produce
sphencal aval or elongated asci (KREGER-VAN RIJ, 1984). Morphologically similar
features were seen on GY agar in pure culture as well as in CO-culture with potential
prey and precluded a clear conclusion with regard to predation.
It should be noted that the species description of S. javanensis- formerly
Arthroascus javanensis von Am - also mentions the faculty of its myceliurn to
produce short lateral branches (denticles) which grow out to stalks and may then
penetrate dead cells (KREGER-VAN RIJ, 1984; KREGER-VAN RIJ and VEENHUIS. 1973).
However, with this species it is easy to distinguish between actual prey penetration
and other phenomena, as predation is perforrned by single cells that arise either
from budding or by hyphal fragmentation. In the conditions used in this study, true
hyphal growth was not oserved.
The alga P. zopfii, unrelated to the fungi (Huss and SOGIN, 1990) and with
a radically different cell envelope composition, did not induœ predatory behaviour.
Moreover, al1 tested predators were apparently outcompeted by the alga on GY
agar. This was particularly conspicuous with S. javanensis. growing poorly in the
f o m of sparse, very thin cells among the much more abundant P. zopfii.
Fission yeasts, including Schizosacchamyces pombe, differ from other
yeasts in many ways (SIPICZKI. 1995). Despite earlier daims that S. pombe was not
susceptible to penetration (KREGER-VAN RIJ and VEENHUIS, 1973; UCHANCE and
38
PANG, 7 997). active predation after 24 hrs was observed when S. pombe was added
to one-day old cultures of S. javanensis.
h
Saccharomycopsis ja vanensis
UWO[PS] 92-247.1
Saccharomycopsis javanensis
UWO[PS] 82-52
1 Candida strain 'W1"
LEGEND:
a predation very pronounced (n.t.) not tested a predation clearly visible (7) predation uncertain
predation modest O no predation observed
+ prey overgrew its predator
Table 3.1.1 Potential predator-prey combinations.
3.2 LIGHT MICROSCOPY OF PREDATION
3.2.2 INTERACTIONS ON SOLlD MEDIA - PREDATION
Predation was studied by light rnicroscopy and microphotography of
predator-prey interactions between Saccharomycopsis fermentans and
Saccharomyces cerevisiae on predation agar (GY). Observations in slide culture
require that both predator and prey be capable of growth under reduced oxygen
tension. Therefore, S. fementans, a facultative anaerobe (Iike S. cerevisiae), was
used as predator in this experiment.
Predation of S. fermentans on S. cerevisiae growing on GY agar was
easily observed afier 72 hours. Haustona penetrating prey cells were seen at sites
of close contact (Plate 3.2.1.118. arrows). Abundant growth of S. cerevisiae was
seen only in regions away from predator mycelium or cells. Rernnants of markedly
altered prey cells could be seen trapped among the cells of S. fementans (Plate
3.2.1.118, bottom).
Plate 3.2.1 .i Predation of Saccharomycopsis fermentans on
Saccharomyces cerevisiae in slide culture on GY agar.
Phase contrast. Bar length = 5 prn
(A) t = O h
Prey cells (Sc) with adhering predator (S9. (6) t = 72 h
Growing rnycelium of S. fementans (S9 penetrates its
prey (arrows);
Unaffected region (U).
42
3.2.2 INTERACTIONS ON SOLID MEDIA - POST-PREDATIONALVIABILITY
Plate 3.2.2.1 shows a mixture of Saccharornycopsis javanensis and
Saccharomyces cerevisiae after transfer from conditions that favor predation (GY
agar) to a slide culture of YM agar, to assess the viability of penetrated cells. In A,
unpenetrated cells of S. cerevisiae developed nomally and after 20 hours overgrew
the predator. In B. a cluster of penetrated cells rernained inactive for 20 hours at
which point S. javanensis had multiplieci considerably. However, it is not clear
whether every S. cerevkiae cell in the cluster had been actually penetrated.
Without further expen'ments it is not possible to conclude with certainty whether
arrested growth of S. cerevisiae is caused by penetration, contact, or sirnply close
proximity to S. javanensis.
Plate 3.2.2.1 Post-Predational recovery in slide culture of Saccharomycopsis
ja vanensis and Saccharomyces cerevisiae on Y M agar.
Phase contrast. Bar length = 5 pm
(A) Control region (predorninantly prey cells, Sc).
Photographed at t = O h (A-1); t = 4 h (A-2); t = 20 h (A-3).
(BI Region of intense predation (growing predator cells, Sj).
Photographed at t = O h ( -1) ; t = 4 h (B-2); t = 20 h (5-3).
3.3 SCANNING ELECTRON MICROSCOPY
OF INTERACTIONS IN LIQUID MEDIA
With a suitable predator-prey combination, predatory behaviour is best
observed on solid substrats (GY agar). The occurrence of predation in liquid GY
medium is a much more uncommon phenornenon and a considerably longer time
is needed for it to take place. When the medium was agitated, no predation
whatsoever was observed. However, the best electron micrographs of predation
were obtained from samples of still Iiquid cultures and examples of these are shown
in this section.
The following four pairs were tested:
Saccharomycopsis javanensis 4 Saccharomyces cerevisiae
Saccharornycopsis javanensis -+ Schizosaccharomyces pombe
Saccharornycopsis synnaedendra -, Metschnikowia hibisci
Candida strain " W 1 " -+ Mefschniko wia hibisci
Predation was observed after 3 to 15 days, sornetimes longer. In still
cultures of S. ja vanensis with S. cerevisiae, active predation was seen after 1 5 days
(Plates 3.3.1 and 3.3.2). It appears that the contact between predator and prey
occurs exclusively via the penetration pegs (Plate 3.3.1). Areas of S. javanensis
without denticles do not appear to adhere to S. cerevisiae cells. Large clumps were
formed in the liquid medium. The latter might be the result of iectin-rnediated
agglutination (BARAK et al., 1 985; BARAK and CHET, I W O ) . The penetrated cells
ulümately collapse (Plate 3.3.2JB. arrows).
Intense predation of S. javanensis on Sz. pombe came somewhat as a
surprise, because introductory work on yeast predation did not reveal Sz. pombe as
susceptible to penetration (KREGER-VAN RIJ and VEENHUIS, 1973; LACHANCE and
FANG, 1997). The predator-prey cell contact appears much more intimate here
(Plate 3.3.3). The slender predator cells may adhere completely to their prey (Plate
3.3.3, upper right and lower left). Dense predator ceil aggregations with fdl
envelopment of the prey cell may be seen (Plate 3.3.3, lower right), reminiscent of
the entrapment of nematodes by fungal parasites. Patches of a rnucous deposit on
the surface of attacked prey cells are commun (Plate 3.3.3, lower left). The origin
and nature of these deposits are unclear.
The remaining two predator-prey pairs exhibited very lime predation in
liquid medium. This may be due to the fact that these predators are generally less
effective (Section 3.1 and Table 3.1 -1). No predation was detected in shake-flask
cultures of any of the four pain tested.
Plate 3.3.1 Predation of Saccharomycopsis javanensis on
Saccharomyces cerevisiae in Iiquid predation medium (GY):
scanning electron microscopy of predator-prey cell contacts.
Bar length = 2 Pm
Plate 3.3.2 Details of haustonum formation and penetration during CO-culture
of Saccharomycopsis javanensis and Saccharomyces cerevisiae
in liquid GY medium (scanning electron microscopy).
Bar length = 5 prn
(A) Hausto ria (a rrows)
(B) Collapsed prey cells (arrows)
Plate 3.3.3 Predation of Saccharomycopsis ja vanensis on
Schizosaccharomyces pombe in Iiquid predation medium (GY):
scanning electron microscopy of predator-prey ceIl contacts.
Bar length = 5 pm
3.4 GENERAL EFFECT OF NUTRIENTS
3 -4-1 TlME COURSE (CONTINUOUS GROWTH)
In this phase of the study experiments were done with Saccharomycopsis
javanensis and Saccharomyces cerevisiae in order to:
compare predator-prey interactions on three very different media;
find growth curves (log N vs. time) for pure and mixed cultures;
determine the interrelatedness of growth and predation;
establish a general correlation between nutnents and predation.
Experiments were carried out on nutrient-restricted medium (GY agar;
Figure 3.4.1.1 ), organosulfur-free medium (Basal agar; Figure 3.4.1.2), and
cornplete medium (YM agar; Figure 3.4.1.3).
According to the data in Figure 3.4.1.1, active predation occurred on GY
medium. When grown in pure culture, the cell yield was somewhat higher in the
predator, but in CO-culture predator cell numbers exceeded those of the prey by
almost three orden of magnitude (Figure 3.4.1.1 ).
On organosulfur-free basal medium (Figure 3.4.1 -2) S. javanensis clearly
benefited from the presence of S. cerevisiae. On the other hand, the reduction in
prey yield was much less dramaüc than on GY.
The remarkably sirnilar predator-to-prey cell density ratios of the mixed
and separated cultures on YM agar (rich medium) are shown on Figure 3.4.1 -3. In
both cases there was a 40- to 50-fold increase in ratio, due probably to a faster
growth rate of S. javanensis and possibly also to the larger cell volume of S.
cere visiae.
Maximum growth in al1 media was reached after approx. 48 hours. The
serni-logarithmic plot of the predator-to-prey ceIl ratio on GY agar (Figure 3.4.1 .l)
reveals that it is the period of exponential growth of both cultures in which predation
is most prominent
The decline of S. javanensis on medium devoid of organic sulfur was
expected, as this yeast requires sulfur in the organic fom. In the presence of prey,
the predator-prey ratio was not comparable to that observed on GY. The reason
may be that predatory activity depends on the release of organic sulfur into the
medium by growing prey cels. Whereas YM agar grown cells showed no sign of
predatory behaviour in the microscope, both GY and basal medium grown predators
formed abondant infection pegs.
Figure 3.4.1 -1 G rowth of Saccharomycopsis javanensis and Saccharomyces
cerevisiae on GY agar.
LEGEND:
Upper graph:
Lower graph:
Logarithm of wlony-foning units,
log N vs. time
Predator, grown separately
Predator, grown together with prey
Prey, grown separately
Prey, grown together with predator
Logarithm of predator-prey cell ratio,
log (NDmaw I N,,,) vs. time
Predator and prey grown separately
Predator and prey grown together
3 Time ? days 5
Figure 3.4.1 .l a Growth of Saccharomycopsis javanensis and Saccharomyces
cerevisiae on GY agar.
~ t a t i s k .
Logarithrn of colony-foming units, log N vs. time.
Error bars represent 99% confidence intewals (i2.576 O).
Each data point is a mean of 3 separate platings.
Numerical values in graphs are coefficients of variation.
Upper left graph: predator, grown separately
Upper right graph: predator, grown together wioi prey
Lower left graph: prey, grown separately
Lower right graph: prey, grown together with predator
Figure 3.4.1.2 G rowth of Sacchammycopsisjavanensis and Saccharomyces
cerevr'siae on basal medium.
LEGEND:
Upper Graph: Logarithm of colony-fomiing units,
log N vs. time
-0- Predator, grown separately
-@- Predatoi, grown together with prey ---O--- Prey, grown separately
---*--- Prey, grown together with predator
Lower Graph: Logarithm of predator-prey cell ratio,
log (Np-, I Np,,) vs. tirne
Predator and prey grown separately
Predator and prey grown together
Figure 3.4.1.2a G rowth of Saccharomycopsisjavanensis and Saccharomyces
cerevisiae on basal medium.
Statisücs.
Logarithm of colony-foming units, log N vs. time.
Error bars represent 99% confidence intervals (iî.576 O).
Each data point is a mean of 3 separate platings.
Numerical values in graphs are coefficients of variation.
Upper left graph: predator, grown separately
Upper right graph: predator, grown together with prey
Lower leff graph: prey, grown separately
Lower nght graph: prey, grown together with predator
Figure 3.4.1.3 Growth of Saccharornycopsis javanensiç and Saccharomyces
cerevisiae on rich medium (YM agar).
LEGEND:
Upper Graph:
Lower Graph:
Logarithm of colony-forming units.
log N vs. time
Predator, grown separately
Predator, grown together with prey
Prey, grown separately
Prey, grown together with predator
Logarithm of predator-prey cell ratio,
log (Np,,/ IVpmy) vs. time
Predator and prey grown separately
Predator and prey grown together
Figure 3.4.1.34 Growth of Saccharomycopsis javanensis and Saccharomyces
cerevisiae on n'ch medium (YM agar).
Statistics.
Logarithm of colony-foming units, log N vs. time.
Error bars represent 99% confidence intervals (I2.576 o).
Each data point is a mean of 3 separate platings.
Numerical values in graphs are coefficients of variation.
Upper left graph: predator, grown separately
Upper right graph: predator, grown together with prey
Lower left graph: prey, grown separately
Lower nght graph: prey, grown together with predator
3.4.2 PREDATION DYNAMICS (DISCONTINUOUS GROWTH)
Experiments in this phase were designed to clarify the predatoiy response
of Saccharomycopsis javanensis p reying u pon Saccharomyces cerevisiae in the
presence or absence of yeast extract as a predation suppressor.
Because predation appears to occur during active cell growth, and in
order to exclude effects of nutrient limitation and aging. transfers to fresh media had
to be done between the late exponential and the early stationary growth phase.
Young mycelia are more susceptible to invasive necrotrophy (LAING and DEACON,
1990) and the same may be true for yeast cultures. If predation occuw in nature,
then it must be associated with movement of yeasts to fresh substrates. Indeed,
yeasts associated with plants are consumed by insects, and periodically are
transferred to new environments (PHAFF and STARMER, 1987).
Five media were used:
GY agar with 0.1 glL yeast extract
GY agar with 1 glL yeast extract
GY agar with 10 glL yeast extract
Basal agar with 5 g/L (NH,),SO,
LNB agar with 0.1 g R (NH,),SO,
The first three media were designed to verify the suppressive effect of
yeast extract on yeast predation. Here, the amount of yeast extract was increased
1 O and 100 times.
The other two media did not contain any yeast extract at all. The nitrogen
source, ammonium sulfate, was added at two widely different concentrations to see
whether nitrogen (or sulfate) may play a regulatory role with respect to predation.
On GY agar with 0.1 g/L YE predation was so intense that after the third
day (one day after the first transfer) the predator-to-prey cell density ratios changed
alrnost 10' times, and no prey could be found in subsequent transfers (Figure
3.4.2.1). In repeated experiments the prey was eliminated af€er the first or second
transfer, depending on the initial population density (not shown).
With lglL YE, predation was still obvious and subsequent transfers
resulted in a steady decline in prey nurnbers (Figure 3.4.2.2). Predator-to-prey cell
ratio changed about Io4 times after 8 days (end of third transfer).
With 10 glL YE, predation had hardly any detectable effect on the
predator-to-prey cell ratio over an 84ay period. The ratios in mixed cells and cells
grown separately were essentially the same (Figure 3.4.2.3).
In thetwo organosulfur-free media (basal and LNB) the predator benefited
greatly and to an alrnost equal extent from the presence of prey. This can be judged
by the oscillating but elevated (one to two orders of magnitude) predator-to-prey cell
ratios (Figures 3.4.2.4 and 3.4.2.5). In controls (separate growth) the predator-to-
prey ceil ratio declined until the predator eventually disappeared. This decline was
more rapid at the higher ammonium sulfate concentration.
Figure 3.4.2.1 Pred ation dynamics of Saccharomycopsis javanensis
and Saccharomyces cerevisiae on GY agar with
0.1 gl L yeast extract.
The cultures were transferred at 2day intervals.
LEGEND:
Upper Graph: Logarithm of coiony-foming units,
log N vs. tirne
-0- Predator, grown separately
-a- Predator, grown together with prey --..O--- Prey, grown separately
---a--- Prey, grown together with predator
Lower Graph: Logarithm of predator-prey cell ratio,
log Np$ vs. tirne
Predator and prey grown separately
Predator and prey grown together
4 6
Time 1 days
Figure 3.4.2.1 a Predation dynamics of Saccharomycopsis javanensis
and Saccharomyces cerevisiae on GY agar with
0.1 gl L yeast extract.
Statistics.
Logarithm of colony-forming units, log N vs. üme.
Error bars represent 99% confidence intervals (kZ.576 O).
Each data point is a mean of 3 separate platings.
Numerical values in graphs are coefficients of variation.
Upper left graph: predator, grown separately
Upper right graph: predator, grown together with prey
Lower left graph: prey, grown separately
Lower right graph: prey, grown together with predator
Time /days T h e / days
Figure 3.4.2.2 Predation d ynamics of Saccharomycopsis javanensis
and Saccharomyces cerevisiae on GY agar wiih
1 g R yeast extract
The cultures were transferred at 2day intervals.
LEGEND:
Upper Graph: Logarithm of colony-forming uni&,
log N vs. time
-0- Predator, grown separately
-@- Predator, grown together with prey ---O--- Prey, grown separately
---a--- Prey, grown together with predator
Lower Graph: Log arithrn of predator-prey ceil ratio,
log (Alpdaw / N,,) vs. time
Predator and prey grown separately
Predator and prey grown together
4 6
Time / days
Figure 3.4.2.24 Predation dynamics of Saccharornycopsisjavanensis
and Saccharomyces cerevisiae on GY agar with
1 g/L yeast extract Statistics.
Logarithrn of wlony-forming units. log N vs. üme.
Error bars represent 99% confidence intervals (I2.576 a).
Each data point is a mean of 3 separate platings.
Numerical values in graphs are coefficients of variation.
Upper left graph: predator, grown separately
Upper right graph: predator, grown together with prey
Lower left graph: prey, grown separately
Lower right graph: prey, grown together with predator
Time f days
Figure 3.4.2.3 Predation dynamics of Saccharomycopsis javanensis
and Saccharomyces cerevisiae on GY agar with
1 0 gIL yeast extract.
The cultures were transfemd at 2-day intervals.
LEGEND:
Upper Graph:
Lower Graph:
Logarithm of colony-forming units,
log N vs. time
Predator, grown separately
Predator, grown together with prey
Prey, grown separately
Prey, grown together with predator
Logarithrn of predator-prey cell ratio,
log I N,,) vs. time
Predator and prey grown separately
Predator and prey grown together
4 6
Time / days
Figure 3.4.2.3-a Predation dynamics of Saccharomycopsis javanensis
and Saccharomyces cerevisiae on GY agar with
1 O g/L yeast extract
Statistics.
Logarithm of colony-forming units, log N vs. tirne.
Error bars represent 99% confidence intervals (12.576 a).
Each data point is a mean of 3 separate platings.
Numerical values in graphs are coefficients of variation.
Upper left graph: predator, grown separately
Upper right graph: predator, grown together with prey
Lower left graph: prey, grown separately
Lower right graph: prey, grown together with predator
Tirne / days Time ldays
Figure 3.4.2.4 Predation dynarnics of Saccharomycopsis javanensis and
Saccharomyces cerevisiae on low nitrogen basal (LNB) agar:
0.1 gIL (NH&S04.
The cultures were transferred at 2day intervals.
LEGEND:
Upper Graph: Logarithrn of colony-forrning units,
log N vs. tirne
A- Predator, grown separately
-0- Predator, grown together with prey - - - O--- Prey, grown separately
- - - a--- Prey, grown together with predator
Lower Graph: Logarithm of predator-prey cell ratio,
log (N'Ma, 1 Npy) vs. time
Predator and prey grown separately
Predator and prey grown together
4 6
Time I days
Figure 3.4.2.4-a Predation dynamics of Saccharomycopsis javanensis and
Saccharomyces cerevisiae on low nitrogen basal (LNB) agar:
0.1 g/L (N H&SO,.
Statistics.
Logarithrn of colony-forming units. log N vs. time.
Error bars represent 99% confidence intervals (I2.576 O).
Each data point is a mean of 3 separate platings.
Numerical values in graphs are coefficients of variation.
U pper left gra p h: predator, grown separately
Upper right graph: predator, grown together with prey
Lower left graph: prey, grown separately
Lower right graph: prey, grown together with predator
Time I days
Figure 3.4.2.5 Predation dynamics of Saccharomycopsis ja vanensis and
Saccharomyces cerevisiae on basal agar:
5 g/L (NH&SO,.
The cultures were transferred at 2-day intervals.
LEGEND:
Upper Graph:
Lower Graph:
Logarithm of colony-forming units,
log N vs. üme
Predator, grown separately
Predator, grown together with prey
Prey. grown separately
Prey, grown together with predator
Logarithm of predator-prey cell ratio,
log (AlpMaw / Aimy) vs. time
Predator and prey grown separately
Predator and prey grown together
4 6
Time / days
Figure 3.4.2.54 Predation dynamics of Saccharomycopsis ja vanensis and
Saccharomyces cerevisiae on low nitrogen basal (LN B) agar:
5 g/L (NHJ2SO4.
Statistics.
Logarithm of colony-forming units, log N vs. time.
Error bars represent 99% confidence intervals (i2.576 a).
Each data point is a mean of 3 separate platings.
Numerical values in graphs are coefficients of variation.
Upper left graph: predator, grown separately
Upper right graph: predator, grown together with prey
Lower left graph: prey, grown separately
Lower right graph: prey, grown together with predator
Time I days Time idays
3.4.3 SEARCH FOR MEDIA COMPONENTS THAT AFFECT PREDATION
As was shown in Sections 3.4.1 and 3.4.2, rich media (YM agar) and
higher concencentrations of yeast extract in otherwise poor media tend to inhibit
predation. Therefore, an atternpt was made to find the sources of assumed
inhibiting factors in complex nutient ingredients. To thateffect, media with only one
such complex ingredient were prepared (cf. Section 2.4).
Also, a simple fracüonation of yeast extract solutions with ethanol and
acetone was performed. as outlined in Section 2.4, and the fractions were tested for
their respective predation-inhibiting potential.
The obtained results (Table 3.4.3.1) are a semiquantitative assessrnent
of rnicroscopic observations of Saccharomycopsis javanensis grown in co-culture
with Saccharomyces cerevisiae.
#
Cornplex ingredient
Acetone preci pitate
Haustorium formation
- -
-
-
- - - - - - - -
Evaporated aqueous
acetone
Ethanol precipitate
Evaporated aqueous ethanol
Peptone
Tryptone
1 Vitamins I +++ I +++ 1
Penetration
- -
-
-
1 Malt extract
Table 3.4.3.1 Effect of various complex ingredients of nutrient media on the predation of Saccharomycopsis javanensis g rown in CO-culture wi th Saccharomyces cerevisiae.
The intensity of haustona formation and penetration was evaluated on a subjective scale from negative (-) to very high (+++).
+ 1 - - +
+++
- -
+++ I
3.5 THE ROLE OF ORGANIC SULFUR
The role of organic sulfur in yeast predation was studied. based on the
fact that al1 known predacious yeasts are organosulfur auxotrophs (LACHANCE and
PANG. 1997). The rationale was that a sufficient concentration of L-methionine
added to an organosulfur-free medium would cover the needs of the auxotroph and
that the predatory response to the preçence of a susceptible prey organism would
not be elicited. The following predator-prey combinations were chosen:
Saccharornycopsis javanensis -r Saccharomyces cerevisiae
Saccharomycopsis javanensis + Metschnikowia hibisci
Candida strain "Wl" 4 Metschnikowia hibisci
The apparent importance of organic sulfur in the phenornenon of yeast
predation and an attempt to demonstrate a causative link between predacious
behaviour and sulfur auxotrophy prompted for an expansion of the model system.
Therefore, the second and third pair consist of a relatively large predator with a
small-celled prey and a system with both small-celled organisms, respectively.
In addition to L-methionine, some other organosulfur compounds were
tested with S. javanensis preying on S. cerevisiae. These included L-cysteine. the
racemic DL-methionine (in which only one half of the mixture is biochemically active)
and sodium thioglycollate (or rnercaptoacetate, HSGH,-COO-Na'), the anion of
which is easily taken up by yeastç. The predator-prey pairs and their respective
media are summarized in Table 3.5.1. Selective counts of Metschnikowia hibisci
were obtained by plating on YM agar + CTAB (see Table 2.2.2).
fl Cr)
V>: C')
3.5-1 THE ROLE OF L-METHIONINE - PREDATION OF
Saccharornycopsis javanensis ON Saccharomyces cerevisiae
The following expenments were based on the hypothesis that the
predatory behaviour had developed as a means to overcome an obligate
requirement for organic sulfur, and a preliminary observation that methionine
inhibited predation in Candida sp. W1. The specific objective was to determine the
lowest amount of methionine suficient to prevent predation. Concentrations of L-
methionine in the LNB medium were varied in a broad range of five orders of
magnitude (plus methionine-free LW) . The findings as a whole were rather
unexpected.
On methionine-free LNB agar the predator was gradually lost in
successive transfers (Figure 3.5.1.1). In the CO-culture the predator grew in an
oscillatory pattern of coexistence with its prey.
Only the lowest tested methionine concentration (0.0001 g/L, Le., c l PM)
promoted coexistence rather than antagonism in CO-culture (Figure 3.5.1.2). A
slightly oscillatory pattern was still observed. Contrary to what occurred in
methionine-free medium, the growth of the predator was stable even in absence of
prey. This suggests that even traces of bmethionine (=0.1 ppm) are suffident to
sustain the predator's viability, but insufficient for significant predation to take place.
When the concentration of kmethionine equalled or exceeded 0.001 glL.
(1 ppm) the prey was no longer countable 24 hrs after the first transfer (three days
after start) (Figures 3.5.1 -3 to 3.5.1 -6). Even at a concentration four orders of
magnitude higher, methionine had no inhibitory effed
Contrary to the expected outcorne, m me thionine stimulated rather than
inhibited the predatory response. Therefore, one should conclude that sulfur
auxotrophy and predation are coincidental.
Figures 3.5.1 -1 Influence of organic sulfur on the predation dynamics of
to 3.5.1.6 Saccharomycopsis javanensis and Saccharomyces cemvisiae
on LN6 medium with varying concentrations of m me thionine.
The cultures were transferred at 2-day intervals.
LEGEND:
Upper Graph: Logarithm of colony-foming units,
log N vs. time
-0- Predator, grown separately
-a- Predator, grown together with prey
---O--- Prey, grown separately
---a--- Prey, grown together with predator
Lower Graph: Logarithrn of predator-prey cell ratio,
log (Npdaw I Npm,,) vs. time
-V- Predator and prey grown separately
-V- Predator and prey grown together
Figure 3.5.1.1
Figure 3.5.1 -2
Figure 3.5.1.3
Figure 3.5.1 -4
Figure 3.5.l -5
Figure 3.5.1.6
No methionine (Control).
0.0001 glL L-methionine.
0.001 g/L L-methionine.
0.1 g/L L-methionine.
1 glL me thionine.
10 g/L me thionine.
4 6
Time 1 days
4 6
Time 1 days
4 6
Time I days
2
Time l days
Time I days
Time l days
Figures 3.5.1.l-a influence of organic sulfur on the predation dynarnicç of
to 3.5.1.6-a Saccharomycopsis javanensis and Saccharomyces cerevisiae
on LNB medium with varying concentrations of me thionine.
Statistics.
Logarithm of colony-forming units, log N vs. time.
Error bars represent 99% confidence intervals (I2.576 O).
Each data point is a mean of 3 separate platings.
Numerical values in graphs are coefficients of variati on.
U pper lefl graph: predator, grown separately
Upper right graph: predator, grown together with prey
Lower left graph: prey. grown separately
Lower right graph: prey, grown together with predator
Figure 3.5.1 .+a No methionine (Control).
Figure 3.5.1 - 2 9 0.0001 g R L-methionine.
Figure 3.5.1 - 3 4 0.001 glL L-methionine.
Figure 3.5.1 - 4 4 0.1 glL me thionine.
Figure 3.5.1 .5-a 1 glL m me thionine.
Figure 3.5.1 .6a 10 g/L m me thionine.
Time l days
64 O Q, p g 2 - z q x m 9;-3-:g
!. 5 % g o a o o o 8 6
O 2 4 6 8 1 0
7ïme ldays
O 2 4 6 8 1 0
Tfme 1 days
Time I days
. . - -. . -- -
I I I I i
O 1 2 3 4
Time l days
O 1 2 3 4
3 9 O 1 2 3 4
Time idays
O 1 2 3 4 9 3 - l
I I I I . .-- - - - . - - . -
. . ... . . . . ... . .- . . .- - . . . . - . .. .-. . - ...F::: - .--... -.*- ....... . . . - ....
. .- --.. -- . - r.: - - 2 - 1
- .. : . -y . . . . . . .
-. 1
2- O 1 2 3 4
Time /days
O 1 2 3 4 9 1 . 1 - I . - I 1 I
Time I days
1 I 1
1 2 3
Time ldays T h e I days
3-5.2 THE ROLE OF L-METHIONINE - PREDATION OF
Saccharomycopsis javanensis AND Candida strain 'W1"
O N Metschnikowia hibisci
Only two extrerne concentrations were tested: 0.001 glL (1 ppm) and
1 O glL (1 %).
The main feature of these two systems was a lower susceptibility of the
prey to both predators. Unlike in the system described in Section 3.5.1 above,
Metschniko wia hibisci suwived and grew in association with both predators even
after three consecutive transfen on both concentration extremes (Figures 3.5.2.2,
3.5.2.3, 3.5.2.5 and 3.5.2.6). The characteristic oscillatory growai pattern in CO-
culture on methionine-free LNB medium was seen with both pairs (Figures 3.5.2.1
and 3.5.2.4). although it was more pronounced with Candida strain 'W1" as the
predator (Figure 3.5.2.4).
Saccharomycopsis javanensis gradually disappeared in pure culture on
organosulfur-free LN6 agar but survived well when grown with the prey (Figure
3.5.2.1). On the other hand, Candida str. 'W1" had only a growth reduction on the
same medium, eventually going into oscillations (Figure 3.5.2.4). This leads to the
conclusion that the latter strain is not a true suifur auxotroph.
Figures 3.5.2.1 Influence of organic sulfbr on the predation dynamics of
to 3.5.2.3 Saccharomycopsis javanensis and Metschnikowia hibisci on
LNB medium with varying concentrations of L-methionine.
The cultures were transferred at 24ay intervals.
LEGEND:
Upper Graph:
Lower Graph:
Logarithm of colony-forming units,
log N vs. time
Predator, grown separately
Predator, grown together with prey
Prey, grown separately
Prey, grown together with predator
Logarithm of predator-prey cell ratio,
log (Al,,,, I Al,,,,) vs. time
Predator and prey grown separately
Predator and prey grown together
Figure 3.5.2.1 No methionine (control).
Figure 3.5.2.2 0.001 g/L m me thionine.
Figure 3.5.2.3 10 g/L m me thionine.
4 6
Time I days
4 6
Time 1 days
4 6
Time / days
Figures 3.5.2.14 Influence of organic sulfur on the predation dynarnics of
ta 3.5.2.34 Saccharomycopsis javanensis and Metschnikowia hibisci on
LNB medium with varying concentrations of L-methionine.
Statistics.
Logarithm of colony-forming units, log N vs. time.
Error bars represent 99% confidence intervals (I2.576 a).
Each data point is a mean of 3 separate platings.
Numerical values in graphs are coefficients of variation.
U pper left graph: predator, grown separately
Upper right graph: predator, grown together with prey
Lower left graph: prey, grown separately
Lower right graph: prey, grown together with predator
Figure 3.5.2.1-a No methionine (control).
Figure 3.5.2.2-a 0.00 1 gR L-methionine.
Figure 3.5.2.3-a 10 g R m me thionine.
Time /days Tirne l days
O 2 4 6 8
lime I days
O 2 4 6 8
Time /( lays Time l days
Figure 3.5.2.4 influence of organic sulfur on the predation dynarnics of
to 3.5.2.6 Candida strain 'W1 " and Metschniko wia hibisci
on LNB medium with varying concentrations of ~~rnethionine.
The cultures were transferred at 2-day intervals.
LEGEND:
Upper Graph:
Lower Graph:
Logarithm of colony-forming units,
log N vs. time
Predator, grown separately
Predator, grown together with prey
Prey, grown separately
Prey, grown together with predator
Logarithm of predator-prey cell ratio,
log (Alpdam l NPmJ vs. time
Predator and prey grown separately
Predator and prey grown together
Figure 3.5.2.4 No methionine (control).
Figure 3.5.2.5 0.001 glL m me thionine.
Figure 3.5.2.6 10 glL L-methionine.
4 6
Time / days
4 6
Time l days
4 6
Time I days
Figure 3.59.4-a Influence of organic sulfur on the predation dynamics of
to 3.5.2.64 Candida strain 'W1" and Metschniko wia hibisci
on LN6 medium with varying concentrations of me thionine.
Statistics.
Logarithm of colony-forming units, log N vs. time.
Error bars represent 99% confidence intervals (î2.576 0).
Each data point is a mean of 3 separate platings.
Numerical values in graphs are coefficients of variation.
Upper left graph: predator, grown separately
Upper right graph: predator, grown together with prey
Lower left graph: prey, grown separately
Lower rïght graph: prey, grcwn together with predator
Figure 3.5.2.4-a No methionine (control).
Figure 3. J.2.S-a 0.001 g/L L-methionine.
Figure 3.5.2.6-a 10 g/L L-methionine.
Time I days
- -
4 I 1 I l 1 ' I I I
O 2 4 6 8
Time idays
O 2 4 6 8
0 m.. a..- h-- Q.- O- ICZ- Q) hl
ts- m- nt- m - - m w
8 f Z g s . - y g
Time l days
Time l days Time fdays
3.5.3 THE ROLE OF OTHER ORGANOSULFUR SOURCES - PREDATION OF Saccharomycopsis javanensis ON
Saccharomyces cere visiae
The effect of added DL-methionine in LNB medium (Figure 3.5.3.1) was
not difTerent from that of pure L-methionine (Figures 3.5.1 -3 to 3.5.1.6). With
sodium thioglycollate (Figure 3.5.3.2) the prey survived only one day longer in co-
culture (48 houn after the first transfer). When L-cysteine was added as the sole
organosulfur source, the predator cell density only slowly exceeded that of the prey
by a factor of about 102 after the second transfer (Figure 3.5.3.3).
It is interesting to note that the steadily decreasing predator-to-prey cell
ratio in the controls with ~~cysteine revealed the incapacity of S. javanensis to use
mis amino acid as an external source of sulfur (Figure 3.5.3.3) under the conditions
used here. The latter may be due to the low solubility of this amino acid in LNB
medium, but it is more probable that it was oxidized to L,L-cystine, its dirner, and
could no longer be taken up by the yeast.
Figure 3.5.3.1 Role of organic sulfur in the predation dynamics of
Saccharornycopsisjavanensis and Saccharomyces cerevisiae
on LNB agar with 1 glL o~~methionine (racemate).
LEGEND:
Upper Graph:
Lower Graph:
Logarithm of colony-foming units,
log N vs. time
Predator, grown separately
Predator, grown together with prey
Prey, grown separately
Prey, grown together with predator
Logarithm of predator-prey cell ratio,
log (Ai,,, I N,,) vs. time
Predator and prey grown separately
Predator and prey grown together
3 4
T h e 1 days
Figure 3.5.3.1-a Rote of organic sulfur in the predation dynamics of
Saccharomycopsis javanensis and Saccharomyces cerevisiae
on LNB agar with 1 gfL DL-methionine (racemate).
Statistics.
Logarithm of colony-forming units, log N vs. tirne.
Error bars represent 99% confidence intervals (e.576 a).
Each data point is a mean of 3 separate platings.
Numerical values in graphs are coefficients of variation.
Upper left graph: predator, grown separately
Upper right graph: predator, grown together with prey
Lower left graph: prey, grown separately
Lower right graph: prey, grown together with predator
Time / days Time Idays
Figure 3.5.3.2 Role of organic sulfur in the predation dynamics of
Saccharomycopsisjavanensis and Saccharomyces cerevisiae
on LNB agar with 1 g/L sodium thioglycoltate.
LEGEND:
Upper Graph:
Lower Graph:
Logarithm of colony-foming units,
log N vs. time
Predator, grown separately
Predator, grown together with prey
Prey, grown separately
Prey, grown together with predator
Predator and prey grown separately
Predator and prey grown together
2 3 4
Time I days
Figure 3.5.3.2a Role of organic sulfur in the predation dynamics of
Saccharomycopsk javanensis and Saccharomyces cerevisiae
on LN8 agar with 1 g/L sodium thioglycollate.
Statistics.
Logarïthm of colony-foning units, log N vs. time.
Error bars represent 99% confidence intervals (I2.576 a).
Each data point is a rnean of 3 separate platings.
Numencal values in graphs are coefficients of variation.
Upper left graph: predator, grown separately
Upper right graph: predator, grown together with prey
Lower left graph: prey, grown separately
Lower right graph: prey, grown together with predator
Time I days Time Idays
3 4
Time l days
Figure 3.5.3.3- Role of organic sulfur in the predation dynamics of
Saccharomycopsis javanensis and Saccharomyces cerevisiae
on LNB agar with 1 glL ~ q s t e i n e .
Statistics.
Logarithm of colony-foming units, log N vs. time.
Error bars represent 99% confidence intervals (f2.576 O).
Each data point is a mean of 3 separate platings.
Numerical values in graphs are coefficients of variation.
Upper left graph: predator. grown separately
Upper right graph: predator, grown together with prey
Lower left graph: prey. grown separately
Lower right graph: prey. grown together with predator
Time I days
..-. .. : . : :::l:.. 6 : - . - ..:-f-- - - . 2- - . . ... - - .... - ... . ... . . . ................. . . - -.
...+.. A- k
Time ldays
3.6 EFFECT OF INORGANIC SALTS ON PREDATION OF
Saccharomycopsis javanensis ON Saccharomyces cerevisiae
As will be explained in Section 3.7, the influence of common inorganic ions
(K+, NH,', NO, SOC) on predation was studied (Section 3.7.3). This was inspired
by a study on the influence of inorganics on the production of amylases by
Endomyces (Hnrro~i and IIDA. 1964; see below). The results appear in Table 3.6.1
below, complernented with data on P-glucosidase activities on the same culturing
media (cf. Section 3.7.3 below).
Media 1 Haustoriurn 1 Penetration 1 PGlucanase formation activitv
GY + methionine +++ +++ -+ GY + (NH4),S04 + methionine 1 - 1 - 1 -
GY + KNO,
Table 3.6.1 Effects of inorganic salts on predation of Saccharomycopsis javanen-
sis on Saccharomyces cerevisiae.
The intensity of haustorium formation and penetration was evaluated on a subjective
scale from negative (-) to veiy high (+++).
Beta-glucanase activity assessrnent is an interpretation of results in Fig . 3.7.3.1 .
3.7 PREDATION-ASSOCIATED ENZYME ACTlVlTlES
Host cell wall penetration by parasitic fungi is normally accompanied by
some elevated enzymic activity on the part of the parasite (RIDOUT et al., 1988). The
integrity of the host's cell wall has to be weakened or disrupted before penetration
structures (e.g., haustoria) can enter the cytoplasm or periplasmic space. It was
therefore reasonable to expect elevated levels of hydrolases in predator-prey
interactions where penetration plays a role, particularly an increase in
polysaccharide hydrolases (glucanases, mannanases, chitinases).
As was rnentioned in Chapter 1, manifestations of interfungal antagonism
rnay require direct contact between mycelia or they rnay be mediatecl at a distance
via difisible agents (toxins, enzymes). In some cases both strategies play a role
(JEFFRIES and YOUNG, 1994; p. 53).
After a preliminary screening for enzymic activities capable of hydrolyzing
ceil wall components (Section 3.7.1), more in-depth experiments were devised
(Section 3.7.2) so as to give an appraisal of the individual contributions of both
extracellular and cell-wall bound predator hydrolases in enzyme-assisted cell wall
penetration. Therefore, samples taken from CO-cultures of predator and prey grown
on solid media were brought into suspension, and the resulüng suspensions were
immediately separated into a biomass portion and a ceIl-free supernatant. Control
samples of pure predator cultures were treated correspondingly.
In addition to such experiments on predation media (GY) with or without
added ~œrnethionine as predation activator, the influence of common inorganic ions
(K', NH,', NO;, S0:') on predation was also studied (Section 3.7.3). The latter was
motivated by an early study on the production of amylolytic enzymes by a strain of
the former mycelial yeast genus Endomyces (HATTORI and IIDA, 1964). Amylase
formation by washed cell suspensions of Endomyces IF0 01 11 was found to be
efficient with most inorganic potassium salts except with K2S04 and KNO,. The
latter two suppressed this enzyme activity. Since some species formerly assigned
to the genus Endomyces - e.g., E. fibuliger, E. javanensis - are predacious and
now belong to the genus Saccharomycopsis (KURTZMANN and R o e ~ m , 19951, it
was inferred that predator-prey interactÎons and the production of hydrolases of the
three main groups of yeast cell-wall polysaccharides - &lucanases. a-
mannanases and chitinases - also might be affecteci by inorganic ions.
Finally, laminarin hydrolysates obtained by the action of P(1-3)-
g lucanase(s) tom predator-prey cultures were chrornatog rap hically separated into
individual sugars (glucose and ~1igoglucosides). This was done in an attempt to
estimate the contribution of endoglucanases vs. exoglucanases in the disruption of
prey cell walls.
3.7-1 PRELIMINARY TESTS
Preliminary tests were conducted with the predator-prey pain:
Candidastrain"Wi"-+Metschnikowiahibisciand
Saccharomycopsis javanensis -, Saccharomyces cerevisiae.
Both pairs did not reveal any chitinase activity. Consequently, no further
chitinase assay was wrried out. Beta-(143)-glucanase was found in cultures of
both pairs, as evidenced by the coloured spot reactions obtained with culture
samples (Section 2.8.1 ).
However, in the case of Candida strain 'W1 " -+ Metschnikowia hibisci,
this enzymic activity was expressed in both pure cultures (predator and prey) as well
as in their mixture. This pair was therefore rejected from further studies.
With S. javanensis -r S. cerevisiae P(l-3)-glucanase activity was
regularly and clearly seen only in coculture. Occasionally it appeared also in older
cultures of the predator grown alone. Pure cultures of S. cerevjsjae showed no P glucanase activity whatsoever. Based upon the results of these tests, and taking
into consideration the fact that the main ceIl wall constituents of S. cerevisiae are
P(1-.3)-glucan and a-(1 +4)-mannan, only the latter pair was accepted for further
investigation.
3.7.2 TOTAL P(1-3>GLUCANASE AND a-(1 -4)MANNANASE ACTlVlTlES
These experiments were an attempt to assess freed (dissolved) and œll-
wall bound extracellular g lycan hydrolases individually du ring predation of S.
javanensis on S. cerevisiae. Curves shown in Figures 3.7.2.1 and 3.7.2.2 represent
the concentration of reducing sugar moieties liberated from the polysaccharide
substrate as a function of reaction time. In reality, the concentration of the
accumulated reducing sugars is a cornplex funcüon of many variables.
After 48 hours of growth on methionine-sup plemented predation agar
(GY), ~(l-+3)-glucanase activity could be measured only in predator-prey CO-
cultures, both as cell-wall bound activity and in cell-fiee supernatants (Figure
3.7.2.1). The rate of reducing sugar accumulation was exponential rather than
linear. It is significant to observe the absence of any measurable activity in two-day
old predator cultures (controls).
After six days of cultivation the situation changed (Figure 3.7.2.2).
Beta-(1+3)-glucanase activity emerged now in both washed cells and cell-free
supernatant of pure predator cultures. However, in both cases the rate of laminarin
hydrolysis was lower than in the corresponding samples from mixed cultures.
No mannanase activities were found, either in pure cultures or in mixtures.
The following explanations of the above findings are offered. Beta-(l+3)-
glucanase observed in No-day old mixed cultures (but not in pure predator cultures)
is an inducible acüvity produced in response to the presence of prey. Its
appearance in pure predator cultures after a longer incubation period (6 days) may
substantiate the fact thaï Saccharomycopsis javanensis penetrates its own
myceliurn (KREGER-VAN RIJ and VEENHUIS, 1973), probably in the stationary phase
or in conditions of prey depletion.
The simultaneous occurrence of predator-cell associated and freely
soluble P(1-+3)-glucanase is by no means a proof of the existence of two different
activities. The enzyme (or, possibly, several isozymes) may be essentially
extracellular but catalytically active pn'or to its complete detachment from the cell
wall. It cauld also be predominantly locaiized on the surface of cells or haustoria,
its presence in the liquid phase being possibly an artefact due to sample
manipulation or part of normal turnover. Under the conditions used here, S.
javanensis forms asci that deliquesce due to the action of glucanases and other
hydrolases. This could also cause the release of enzymes.
Laminann degradation curves obtained after 6 days with washed cells of
predator alone and predator-prey mixtures have a sigmoidal shape and differ greatly
from the exponential shape of the con-esponding supernatant fractions (Figure
3.7.2.2). it is possible that this sigmoidal shape, a characteristic of allosteric
enzymes, is lost in cell-free samples due to an alteration of the native conformation.
In other words, the binding sites for allosteric effectors rnay become ineffective once
the enzyme molecule is detached from the cell envelope and brought into solution.
It is important to note that the predator cell densities affer 6 days of
cultivation were about five times higher than in the culture suspensions obtained
after 2 days. It is reasonable to assume that the living cells themselves consume
some of the low-molecular weight sugars initially produced, which can not be the
case in cell-free supernatants.
The exponentiai appearance of most sugar accumulation curves reveals
another important fact. Polysaccharide chains may be broken down by two diffefent
classes of enzymes. Exoglucanases cleave off terminal sugar molecules. If the
reaction rab of such an enzyme does not change with time, there would be a linear
increase in reducing sugar concentration, as only one enzyme molecule can act
upon any polysaccharide chain regardless of the chain length. In other words.
reducing sugar accumulation is a linear function of time. On the other hand,
endoglucanases hydrolyse randomly along the polysaccharide chain. A disruption
of the prey cell wall must predorninantly rely upon the action of endogiucanases.
since there are few, if any, free chah ends. If an endoglucanase is to act alone on
a polysaccharide chah in solution, the accumulation of reducing sugar ends would
also be linear in the event of simple enzyme kinetics. Only when the chain is
eventually split into fragments too small to allow formation of an enzyme-substrate
cornplex, one would see a gradua1 decrease in sugar accumulation rate. The
opposite, raising dope of the obtained curves in Figure 3.7.2.2 - if not caused by
an allosteric enhancement of enzyme activity - indicates the simultaneous action
of bath endo- and exoglucanases. The acüon of the former creates an ever
increasing number of sites for the latter, and an exponential curve profile would
result.
Figure 3.7.2.1 Beta-(l-i3)-glucanase assay 48 hours after CO-culture of
Saccharomycopsisja vanensis and Saccharomyces cerevisiae
on methionine-supplemented predation medium (GY).
The concentration of reducing sugars liberated frorn laminan'n as substrate, in
millimoles per litre, was measured spectrophotometrically at A = 520 nrn against
glucose as standard (SPIRO, 1966).
Legend: O - Washed, centrifugeci cells --- O - - - Cell-free supernatant
60 90 120
Time / min
Figure 3.7.2.2 Beta-(l+3)-glucanase assay 6 days after CO-culture of
Saccharomycopsis javanensis and Saccharomyces cerevisiae
and in pure culture of S. javanensis on methionine-supple-
mented predation medium (GY).
The concentration of reducing sugars iiberated from laminah as substrate, in
millimoles pet litre, was measured spectrophotometncally at A = 520 nm against
glucose as standard (SPIRO, 1966).
Legend : O - Co-culture: washed, centrifuged cels --- 0 --- Coculture: cell-free supernatant
-V- Predator alone: washed, centrifugeci cels ---v--- Predator alone: dl-free supernatant
3.7.3 EFFECT OF INORGANIC IONS ON P(1+3}-GLUCANASE ACTlVlTY
This series of assays, carried out after 48 hours of growth, started with
tests on ordinary predation agar (GY), containing traces of endogenous methionine,
and on L-methionine supplemented GY, followed by expenments with added salts.
Here, too, no P(1+3)-glucanase activities were seen with pure predator cultures.
Assay results obtained with washed cells are represented in Figure 3.7.3.1 , those
with celf free supernatants in Figure 3.7.3.2. Two features are striking: different
curve profiles (sugar accumulation curves were not exponential) and a total absence
of glucanase activities in celbfree supematants with al1 of the inorganic salts tested.
The rather linear appearance of curves in Figure 3.7.3.1 could indicate that only one
of the two classes of glucanases was now active.
Potassium salt additions (74 mM K#04 and 99 mM KNO,) had an almost
identical effect: a slight reduction in laminafin degradation rate with respect to
cultures grown on simple GY agar and the same, almost linear (very slightly
sigrnoidal) degradation curves. indicative of only one class of glucanases.
Obviously, NH,+ plays an important role here, narnely that it fulfills the
predator's requirement for nitrogen. The inability of Saccharomycopsis javanensis
to assimilate nitrate could explain the dramaüc difference between the curves
obtained with KNO, and (NH4),S0,, respectively.
The absence of ~(1+3)-glucanase activity in cell-free supernatants when
any of the salts was present during cultivation is probably due to electrostatic
phenornena. Cells cultured in an electrolyte-rich medium with high ionic strength
can have a different overall charge on the cell envelope. This charge may, in turn,
be responsible for an anchoring of nascent enzyme molecules, due to electrostatic
interactions (ionion, ion-dipole) between the charged andlor polar groups of the
enzyme and the ionic double layer around the ceIl.
It is also possible that these salts affected ascus maturation and therefore
the reiease of enzymes into the medium.
3.7.4 HYDROLYTIC CLEAVAGE OF P(1+3>GLUCAN
TO GLUCOSE AND OLlGOGLUCOSlDES
Thin layer chromatography of laminarin hydrolyzates produced by washed
c d ls fro rn mixed cultures of Saccharomycopsis ja vanensis and Sacharomyces
cerevisiae on simple GY agar revealed a simultaneous appearance of glucose and
oligoglucosides in the reaction mixture. Oligomers with up to about eight glucose
subunits could be effectively separated (Figure 3.7.4.1 ). The detedon of 2 and 3
unit oligomers in early samples in addition to glucose points to the presence of
endo-P(1-3)-glucanase activities in conditions of predation. The presence of
glucose indicates exo-&g lucanase activity.
Figure 3.7.3.1 Effect of inorganic salts on Pglucanase acüvity
fro m co-cu ltu re of Saccharomycopsis ja vanensis wi th
Saccharomyces cerevisiae on predation medium (GY)
with and without methionine
(washed cells from co-culture after 48 hours).
The concentration of reducing sugars iiberated from laminarin as substrate. in
rnillimoles per litre, was measured spectrophotometrically at A = 520 nrn against
gfucose as standard (SPIRO, 1966).
Legend: 4- GY agar (no added methionine)
M - GY agar + 10 glL me thionine
-AS-- GY agar + 10 g/L (NH4)2S0,
-AS+M- GY agar + 10 g/L (NH4),S04 + 10 g/L r-methionine
P S - - GY agar + 10 glL K,SO,
P N - GY agar + 10 glL KNO,
Figure 3.7.3.2 Effect of inorganic salts on pglucanase activity
from co-culture of Saccharomycopsis javanensis with
Saccharomyces cerevisiae on predation med iurn (GY)
with and without methionine
(supernatant from CO-culture after 48 houn).
The concentration of reducing sugars liberated frorn laminarin as substrate, in
millimoles per litre, was measured spectrophotometrically at A = 520 nm against
glucose as standard (SPIRO, 1966).
Legend: -O- GY agar (no added methionine)
M - GY agar + 10 gfL kmethionine
Plate 3.7.4.1 Thin layer chromatogram of lamina rin hydrolyzate obtained with
washed cells from CO-culture of Sacchammycopsis javanensis
and ~acchammyces cerevisiae on GY agar.
DP = degree of polymerization.
Stationary phase:
Eluent:
Development:
Visualization:
Kieselgel 60 plates, 20 cm x 20 cm, layer thickness 0.2 mm,
not pre-dried
3 vol. pts. n-butanol + 1 vol. pt. acetic acid + 1 vol. pt. water
Double, one-dimensional elution, 5% hours each, in closed,
paper-lined chamber at RT; drying at 50°C overnight between
etutions
Repeated spraying with alkaline silver nitrate reagent (Section
2.8.3); 1-2 minutes at 120°C after final spray
3.7.5 BO-PD-GLUCANASE ASSAY
This experiment involved cultures of Saccharomycopsis javanensis and
Saccharomyces cerevisiae. It was aimed at assessing aie wntri bution of exo-P
glucanase actMty in the overall laminarin hydrolysis measured in previous
experiments (Sections 3.7.2-3.7.4). Here, laminarin as the enzyme substrate was
replaced by the chromogenic pnitrop henyl-po-glucoside, which is hyd rol yzed to
glucose and the yellow pnitrophenol only by ~o-glucosidases and exo-PD-
glucanases.
Measurements were carrieci out with both the washed cells and the first
washings (supernatants) of harvested cells.
The resultç are represented in Figure 3.7.5.1. It follows that activities are
found in pure cultures of both predator and prey, as well as in their mixtures.
The main finding was that there was no measurable ~lucanase/glucosi-
dase activity in supernatants of either of the pure cultures. Their respective washed -
cell fractions were, however, able to utilize the substrate via one or more cell-bound
activities. When predator and prey were grown in mixed culture and harvested in
an identical manner, ~glucanaselgluwsidase acüvity was readily measurable in the
supernatant. It may be hypothesized that in co-culture one or more oher P-glucana-
se/glucosidase activities are induced.
Figure 3.7.5.1 Exo-PD-glucanase activity assay in pure cultures and co-cultures
of Saccharornycopsis ja vanensis with Saccharomyces cerevisiae
on predation medium (GY)
(washed cells and supematants after 48 hours).
The activities were assayed spectrophotornetrkally at A = 450 nm by measuring the
accumulation of pnitrophenol liberated from gnitrophenyl-P~-glucoside.
Legend: 5- Cell fraction, CO-culture
-0- CelI fraction, prey culture
-A- Cell fracion, predator culture
-- - O --- Supernatant, CO-culture
0.3 .
0.2 .
O.? .
0.0 -
CONCLUSIONS
As has been ernphasised in the introductory sections, predacious
yeasts are not obligatory predators. Rather, this type of behaviour is elicited as a
response to certain conditions in the predators' environment. Experiments in this
thesis were therefore designed to elucidate at least some environmental parameters
conducive to the predatory response.
Based on the s u l ~ r auxotrophy of al1 known predacious yeasts, it was
hypothesized that predation was a means to acquire organic sulfur and that an
addition of suitable organosulfur wmpounds into the growth media would eliminate
the need for predation. Various metabolizable organosulfur (OS) compounds were
added to the LN8 nutrient medium in order to establish the threskold at which
predation would be stopped (Section 3.5). In most of the tests L-methionine was
used and the media always contained some inorganic sulfur (1s) in the fom of
ammonium sulfate. Quite wntrary to the expected outcorne, the following was
found:
No added OS + low IS 10.1 gIL INH4)7so~l. - Predator alone was not able to grow. - Predator and prey coexisted in CO-culture without predation.
Minimal OS 10.1 ma/L methioninel + low IS 10.1 g/L (NH4)2&J.
- Predator alone grew normally.
- Predator and prey coexisted in co-culture without predation.
m h e r OS II mq1L to 10 q/L methioninel + low IS 0.1 qlL INH4),SOAl. - Predator eliminated prey in CO culture within 3 to 4 days.
It can be concluded that an addition of organic sulfur stimulates rather
than inhibits predatory behaviour of methionine-auxotrophic yeasts. Methionine at
concentrations above 1 mglL is even necessary for predation to take place.
Predation is thus obviously not a strategy used by methionine auxutrophs to satisfy
their need for organic suIfW.
Later expenments with inorganic salis (Section 3.6) showed that
ammonium sulfate played an important role in suppressing predation. High
concentrations (10 g R ) in predation medium (GY), with or without added
methionine, entirely inhibited haustorium formation, prey cell penetration and
Pglucanase activity (Table 3.6.1 ). Initially, it was assumed that high concentrations
of the sulfate ion were responsible for the observed suppression of predation. But
since K2S04 (10 glL) did not have the same effect as (NH,),SO,, it appears that the
ammonium ion is strongly correlated to the regulation of predatory behaviour.
Further experiments in that direction are necessary.
Other desirable research should be directed at studying in more detail the
enzymatic mechanism of prey ceIl wall penetration, as it became apparent that
elevated levels of Pglucanase are associated with this phenornenon.
The observed differences in the extent of predation achieved with various
predator-prey pairs indicates that some fom of recognition. possibly at the level of
cell envelope, is involved. Therefore, the search for a lectin-type recognition
between predator and prey might yield interesting resulk
As far as the potenüal application of predacious yeasts is concerned.
studies with yeasts pathogenic to man and animais should be conducted.
It would also be of interest to CO-culture predacious yeasts with yeasts
commonly associated with food spoilage. These expenments should be conducted
in media prepared from or simulating actual food.
Interactions of predacious yeasts with other fungi have not been studied.
It would be very interesting to commence such studies with filamentous fungi
responsible for food spoilage and crop deterioration.
Hopefully, progress in the understanding of the mechanism of predation
will prove useful in its enhancement and commercial exploitation.
160
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