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TTTn SENSITIVITY OF TO KILLER TOXINS: \ryITH FOCUS ON THE KILLER YEAST MEMBRANIFACIENS

by

NICHOL¡.S ANNNNW Y¡.P

Thesis submitted for the degree of

DOCTOR OF PHILOSOPHY

July 2000

The Department of Plant Science Faculty of Agricultural and Natural Resource Sciences TTTN UNIVERSITY OF ANNT,NTON Ansrn¡,cr

The yeast killer phenotype is defined by a yeast's ability to secrete a toxin that is lethal to other yeast strains, but to which they are themselves immune. An investigation was undertaken to identify a yeast with broad spectrum killer activity towards indigenous non- Saccharomyces yeasts of the wine ferment. The growth of these indigenous yeasts during wine fermentation may result in inappropriate sensory properties to the wine.

The sensitivity of tester strains characteristic of the wine ferment microflora to 14 killer yeasts were assayed at pH 4.5, revealing a total of I47 killer-sensitive reactions. At a pH comparable to a wine ferment (pH 3.5), only 287o of these 147 h'tller-sensitive reactions were observed. Intraspecific differences in killer susceptibility were identified for strains of a number of yeast species, whilst intraspecific differences in killer activity were identified for strains of Pichia anomala, Kluyveromyces lactis (two strains) and Pichia membranifaciens.

To gain further insight into the killer phenotype of Pichiamembraniføciens, the killer activity of ten Pichia membranifaciens strains was assayed towards 15 tester strains. Intron primer PCR confirmed the ten Pichia membranifaciens strains to be related, but different to, the type strain of Pichiamembranifaciens.Based on their killer activity each Pichia membranifaciens strain was allocated one of four possible killer types.

The killer phenotype of the two strains of the Class C killer type, Pichin membranifaciens CBS 638 and the type strain CBS 107, was found to be encoded by nuclear genes. In contrast, the killer strains of the Class B and D killer types harboured an extrachromosomal element of the same molecular weight. For Pichin membraniþci¿r¿s CBS 7374 of Class D this extrachromosomal element (pPM01) was determined to dsRNA in nature, however, its not known whether pPM01 is associated with the killer phenotype.

Of the ten Pichia membranifaciens strains investigated, strain CFS 7374 displayed the broadest killing range. The Pichiamembranifaciens CBS7374ktller toxin was found to be a heat liable protein with an acidic pI. Using a purification protocol developed in this study, a protein of 20.5 kDa was identified as a candidate for the Pichin membraniþciens CBS 7374 killer toxin

Investigating the sensitivity of tester strains to killer yeasts further revealed a petite of Saccharomyces cerettisiae AWRI 1360 (p+ks), strain AWRI 1361 (p-kn), to be resistant to ten killer yeasts to which the parent was sensitive. This included resistance to the killer yeasts K2, Kluyveromyces lactis var. lactis and Williopsis saturnus var. mrakii, where the primary and mode of action differs for each killer ll protein. This is the first known report of a mutant displaying resistance to more than one killer type. Characterisation of this petite revealed that its resistance to these killer toxins is attributed to a partially dominant, nuclear mutation. This mutation was found to be independent of oxidative-phosphorylation and yet, conferred resistance only in the presence of non-functional mitochondria. This study also revealed that for some strains, petites of sensitive parents showed a reduction in sensitivity to killer yeasts, and that this reduction in sensitivity was independent of oxidative-phosphorylation. lll

DECLARATION

This work contains no material which has been accepted for the award for any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where reference has been made in the text.

If accepted for the award of Docûor of Philosophy, this thesis will be available for loan and for photocopying.

Nicholas Andrew Yap

11 December 2000 lv

ACKNOWLEDGMENTS

I would like to give my sincere thanks to my supervisors Professor Peter Langridge, Dr Paul Henschke and Dr Miguel de Barros Lopes for their suppoft, advice and critical thought. In particular, I would like to pay special thanks to Dr de Barros Lopes for his extra ordinary time and effort in assisting me in my studies.

I would like to acknowledge the Cooperative Research Centre for Viticulture for financing my postgraduate scholarship. It has been a privilege to be involved with the Research Centre, and its associated links with the Australian wine and grape growing industry.

I would like to give my sincere thanks to the all the staff and students of The Australian Wine Research Institute for their kind support and friendships. In particular, I would like to pay tribute to Mr Holger Gockowiak for his continued assistance during my studies. I would like to say thankyou also to the staff and students of the Peter Langridge laboratory of the Department of Plant Science.

I would like to acknowledge Dr Elizabeth Vy'aters of The Australian Wine Research Institute, and Mr Jella Lahnstein of the Nucleic Acid and Protein Chemistry Unit of the University of Adelaide, for their kind assistance and technical advice on the subject of protein purification.

I am grateful for my family for their support during my university studies.

Finally,I would like to say thankyou to Mr Sam Verco, Ms Toni Paterson, Mr Nick Pannell, Ms Helen-Lucy Moss, Dr Roger'Woods, Ms Julie Ucinek, Mr Todd Ballinger, Ms Naomi Lindsay and Ms Ann Keeler for their warmth, laughter, kindness and humour. Thankyou. V

PUBLICATIONS

Part of the work described in this thesis of Nicholas Andrew Yap has been published:

Yap, N. 4., de Balros Lopes, M., Langridge, P., and Henschke, P. A. (2000). The incidence of killer activity of non-Søcchnromyces yeasts towards indigenous yeast species of grape must: potential application in wine fermentation. fournal of Applied Mcrobiology 89, 381-389. TABLE OF CONTENTS

Abstract I Declaration üi Acknowledgments 1V

Publications...... v

CHAPTER 1 INrnonucTIoN AND PROJECT ATUS 1

CHAPTER 2 LrrBn¡.tuRE REvrEw.... 3 2.I INTRODUCTION. 3 2.2 YSaSTS AND FUNGI WITH DSRNA ENCODED KILLER TOXINS 3 2.2.I The killer yeast Saccharomyces cerevisiae ...... 3 2.2.1J The L-A and M dsRNAs...... 3 2.2.I.2 The killer toxins of Saccharomyces cerevisiae ...... 4 2.2.I.3 The self-immunity factor of the Kl killer toxin ...... 5 2.2.2 The killer Ustilago maydis .6 2.2.3 The killer yeasts Zygosaccharomyces bailü and Hanseniaspora uvarLtm .6 2.3 LINEAR DNA ENCODED KILLER YEASTS 7 2.3.1 The killer yeast Kluyveromyces lactis 7 2.3.IJ The linear DNA of Kluyveromyces lactis ...7 2.3.1.2 The killer toxin of Kluyveromyces lactis 8 2.3.2 The killer yeast Pichia acaciae.. 9 2.4 Ku,TBn YEASTS WITH NUCLEAR ENCODED TOXINS .9 2.4.I The killer yeastWilliopsis saturnus HM-1 .9 2.4.2 The killer yeast Pichiafarinosa 10 2.5 THE ECOLOGY OF KILLER YEASTS. 10 2.6 CHENRCTSRISATION OF MUTANTS RESISTANT TO KILLER TOXINS...... 11 2.6.I Resistant mutants with wall defects t2 2.6.2 Resistant mutants and functional mitochondria .-....I2 2.6.3 Cell walls defects of petite mutants...... 13 2.6.4 Mitochondrial driven 13 2.7 APPLICATIONS FOR KILLER YEASTS...... t4 2.8 I¡IoICBNOUS YEASTS OF TI{E GRAPE MUST FERMENT...... 16 2.9 TTm TNU FORMING YEAST PTCUTI, MEMBRANIFACIENS .,,..I7 2.IO KuBR STRAINS OF PICHIA MEMBRANIFACIENS IN FOODS. .,,,..L7 2,II THE IDENTIFICATION OF SPOILAGE YEASTS 18

CHAPTER 3 TTTn TNCTDENCE OF KILLER ACTIVITY OF NON-S¿CCHAROMYCES YEASTS TOWARDS INDIGENOUS YEÄST AND BACTERIA SPECIES OF THE GRAPE MUST 19 3.T INTRODUCTION.... 19 3.2 EXPERIMENTAL. 19 3.2.1 Yeast and bacterial strains T9

3.2.2 Agar killer assay for testing yeast strains...... 20 3.2.3 Agar killer assay for testing bacteria strains. 20 3.3 RESULTS ..... 2L

3.3.1 Killer yeast activity. . . . . 2L 3.3.2 Yeast strain susceptibility..... 22 3.3.3 Intraspecific differences in killer activity .22 3.3.4 Intraspecific differences in killer susceptibility .... .23

3.3.5 Reduction of killer yeast activity in an acidic environment ' ...... 24 3.3.6 Assaying bacteria susceptibility to killer yeasts .24 3.4 DISCUSSION .25

CHAPTER 4 MOI,BCUI,AR TYPING OF PICNII, MEMBRANIFACIENS STRAINS ÄND THEIR KILLER ACTIVITIES...... 31 4.I INTRODUCTION 3t 4.2 E)GERINßNTAL. 3l 4.2.I Yeast strains and media 31

4.2.2 Total nucleic acid preparations...... 32 4.2.3 Mini-preparations of nucleic acids. .32 4.2.4 Nucleic acids gel electrophoresis .. .33 4.2.5 Treatment of total nucleic acids with DNase I or

Ribonuclease A...... 33 4.2.6 Cycloheximide treatment of P ichia membranifaciens CBS 7374 .33 4.2.7 Ultra-violet light treatment of Pichia membraniþciens CBS 7374 .34 4.2.8 PCR-intron fingerprint technique... .34 4.3 Rnsul,rs. .34 4.3.I Differentiating strains of the Pichia membranifaciens species. . . .34 4.3.2 Putial characterisation of killer activities. .35 4.3.3 Detection of extrachromosomal elements in Pichia membranifaciens .36 4.3.4 Characterisation of the extrachromosomal element of Pichia

membranifaciens CBS 7374. . .36 4.3.5 Curing Pichia membraniþciens CBS 7374 of pPM01. .37 4.4 DrscussroN...... 37

CHAPTER 5 CTTNN¡,CTERISATION OF Tl¿r. PICHIA MEMBRANIFACIENS CBS 7374 KILLER TOXIN .43 5.1 INTRODUCTION. .43 5.2 EXPERIMENTAL...... 43 5.2.L Yeast strains and media .43 5.2.2 The agar plate well diffusion killer assay. .43 5.2.3 Killer activity from different media. .44 5.2.4 Optimum pH for killer toxin production and activity .44 5.2.5 Assaying killer activity at different stages of cell growth...... 44 5.2.6 Pronase E digestion...... 45 5.2.7 Temperature sensitivity.... .45 5.2.8 Optimal storage conditions 45 5.2.9 HPLC reverse phase chromatography .. .45 5.2.10 Ultrafiltration of the toxin supernatant. .46 5.2.L1 HPLC anion exchange chromatography .46 5.2.12 SDS polyacrylamide gel electrophoresis .47 5.3 RESULTS .48 5.3.1 Optimising toxin production in liquid media culture of Pichia membraniþci¿r¿s CBS 7374 ...... 48 5.3.2 Determining the optimum pH .49 5.3.3 Effect of stage of cell growth on killer activity .49 5.3.4 Protease digestion and temperature sensitivity .49 5.3.5 Optimal storage conditions .50 5.3.6 IIPLC reverse phase chromatography. .50 5.3.7 HPLC anion exchange chromatography. .51 5.3.8 SDS-PAGE of the active fractions from AEC .52 5.4 DISCUSSION ... .52

CHAPTER 6 TTTE TTT.T.ER YEAST SENSITIVITY OF PETITE MUTÄNTS 57 6.L INTRODUCTION. 57 6.2 E)GERIMENTAL. 57 6.2.I Yeast strains and media. 57 6.2.2 Inducing petites via treatment with ethidium bromide. 57 6.2.3 Selection of spontaneous petites. 58 6.2.4 Killer assays 58 6.2.5 Forming diploids .58 6.2.6 Concentration of the Pichis membranifaciens CBS 737 4 þ,tller toxin supernatant. .58 6.2.7 mtDNA isolation and restriction analysis...... 59 6.3 RESULTS. .59 6.3.1 Saccharomyces cerevisrø¿ AWRI 136I, derived from Sacchnromyces cerevisla¿ AWRI 1360, harbours deletions in its mitochondrial genome .59 6.3.2 The petite Saccharomyces cerevislø¿ AWRI 1361 displays killer toxin resistance .. 60 6.3.3 Reduction in yeast killer toxin sensitivity displayed by petite

Saccharomyces cerevisiae strains . . . . .61 6.3.4 The killer resistant phenotype of Saccharomyces cerevisiae A\ryRI 1361 is partially attributable to a nuclear mutation. 62 6.3.5 Estimating the level of resistance of Saccharomyces cerevisiae AWRI 1361.. 63 6.4 DISCUSSION..... 63

CHAPTER 7 GpNBn¡,r. DISCUSSION AND CONCLUSIONS.... 70

BTSLTOCnAPHY .74 Chapter L

INTRODUCTION AND PROJECT AIMS

The yeast killer phenotype is defined by a yeast's ability to secrete a toxin that inhibits growth of other yeast strains, but to which they are themselves immune. Although this phenotype was first observed for Saccharomyces cerevisiae @evan and Makower, 1963), it is now known to be associated with a wide rarìge of yeast species and genera (Young and Yagiu, 1978; Magliani,1997). Killer yeasts can be grouped conveniently into three classes, based on the type of genetic determinant encoding their killer toxin. The first class is composed of those toxins encoded by double stranded (ds) RNA , and includes the well documented Saccharomyces cerevisiae Kl killer yeast. The second class, such as Kluyveromyces lactis, have their killer toxins encoded by linear DNA plasmids. The third class includes those killer yeasts having toxins encoded by nuclear genes, such as the HM-l killer toxin of Williopsis saturnus var. saturnus.

To date, all known killer toxins have been found to be proteinaceous in nature. With a few exceptions, most killer toxins characterised have a low pI, and display maximum killer activity in an acidic environment. Killer toxins target a primary receptor in the yeast cell wall, and it appears that the type of receptor differs for each toxin. Furthermore, the killer toxins from different species, and even the toxins from strains of the same species, differ significantly in structure and function. The characterisation of mutants resistant to killer toxins has provided insights into various aspects cell biology. In particular, due to the specific nature of killer toxins with various components of the yeast cell wall, characterisation of resistant mutants has revealed insights into cell wall biosynthesis and structure.

The observation that killer toxins induce the death of sensitive yeast cells, has led to the exploitation of the yeast killer phenotype for various clinical and industrial applications. Such applications include the use of antiidiotypic antibodies of the Pichia ønmnla killer toxin to induce cell death of fungal parasites responsible for human infections (Seguy et al., 1996); and the of Ustilago maydis killer toxin from transformed crop plants for protection against fungal infections (Kinal et a1.,1995). The killer toxin of Saccharomyces cerevisiae has been employed also, for the biocontrol of unwanted indigenous Saccharomyces cerevisiqe strains present in the wine ferment (Boone et aI., 1990a; Michalcakovâ' et al., 1994). 2

Previous to this study, the identification of a yeast killer toxin with a broad spectrum of killer activity towards a variety of indigenous non-Søcchnromyces cerevisiae strains of the wine ferment, has not been documented. Unconffolled growth of these non-Saccharomyces cerevisiae yeasts during fermentation can produce spoiled wine with off flavours and aromas (Sponholz, 1993). At present, the conventional method for controlling the growth of such yeasts is by the addition of the antimicrobial compound sulphur dioxide (SOÐ. With the aim of replacing or reducing the use of SO2, this study first surveyed known killer yeasts for antagonistic activity towards a broad range of non-Søccharomyces cerevisiae yeasts typically associated with the wine ferment. The results of this initial investigation have been reported by Yap et al. (200O).

This survey also revealed intraspecific differences in the killer activity of the yeast Pichiú membranifaciens. This species is commonly associated with the spoilage of various foodstuffs and beverages, such as cheese, beer and wine. To date, however, there is no quick and reliable method for the identif,rcation of this spoilage yeast in an industrial context. Consequently, the second aim of this study was to evaluate a molecular typing technique for the identification of Pichin membranifaciens isolates of the wine ferment, and for strain differentiation.

To gain further information on the little known Pichia membranifuciens killer phenotype, the intraspecific differences in killer activity between the strains was further investigated. PiÊhin membranifaciens CBS 7374 was identified as displaying the strongest killer activity towards a variety of yeast species. As this killer activity had not previously been characterised, a further aim of this study was to develop a method for the purification of the killer toxin.

During the characterisation of the Pichin membranifaciens CBS 7374 ktller phenotype, a Saccharomyces cerevisiae mutant resistant to the Pichin membranifaci¿r¿s CBS 7374 þ'tller toxin was identified. Characterisation of this mutant revealed that it was resistant to a number of killer yeast species. This is the first report of a yeast mutant resistant to a number of structurally distinct killer toxins with different mechanisms of action. Characterisation of the mutant demonstrated that the alteration was in a nuclear gene, but also depended on the yeast lacking functional mitochondria. 3

Chapter 2

LITERATURE REVIEW

2.1 lNrnooucrroN

Since the first report by Bevan and Makower (1963), killer activity has been described for over 100 species of yeast. Being so widely distributed in the fungi, killer yeasts have been isolated from a variety of natural habitats, including decaying plant and fruit material, soil and water , and insects such as Drosophila (Stumm et al., 1977; Morais et aI., 1995; Vadkertiova and Slavikova, 1995). Killer yeasts have also been isolated from fermented foods such as miso, soy sauce and salted vegetables, and from commercial fermentations of beer, wine and sake (Taylor and Kirsop,1979; Shimizu, 1993). Killer activity has also been detected in a number of potentially pathogenic strains (Kandel and Stern, L979; Kandel, 1988).

Killer yeasts can be classed into three categories based on the genetic determinants encoding the killer activity. These determinants may be chromosomal, or extrachromosomal elements in the form of linear DNA plasmids, or double stranded (ds) RNA encapsidated in viruslike particles. Each killer toxin described at the molecular level has been found to have its own novel system for toxicity. Primary interaction with the sensitive cell, uptake, and the subsequent mechanisms of inducing cell death differ from toxin to toxin (Maglian et al., 1997). Greater understanding of the action of killer toxins not only provides information on protein processing and secretion, viral propagation, and the biosynthesis of the yeast cell wall, but it also results in the exploitation of killer systems for practical applications.

2.2 YP¡,STS AND FUNGI WITH DSRNA ENCODED KILLER TOXINS

2.2.1 The killer yeast Saccharomyces cerevísíae

2.2.1J The L-A and M dsRNAs

Being the first yeast to be identified with killer activity, Saccharomyces cerevisiae has become the most investigated of all reported killer yeasts (Woods and Bevan, 1968; Wickner, 1996). There are five killer types in Saccharomyces cerevisiae. Three derive their toxins from dsRNAs and are referred to as K1, K2 and K28. Two killer types, called KHR (Goto et a1.,1990) and KHS (Goto et aI., 1991), derive their toxins from chromosomal 4 genes. Kl, K2 and K28 each have two dsRNAs, L-4, with M1, lll2 or M28, respectively. These dsRNA replicate stably in the cells, neither lysing them or antagonising their growth, implying the cell and the live in a synergistic state (Wickner, 1996) .

The L-A virus (4.6 kb) is responsible for maintaining the stable replication of the M virus, which encodes the toxins responsible for killer activity. Its replication is conservative (parental strands stay together), asynchronous [the plus (+) and minus (-) strands are made at different points of the replication cyclel and intraviral [both the (+) and (-) strands are synthesised within the virus-like particlesl. The (+) strand encodes two proteins: the major coat protein, called gag, and the gag-pol fusion protein. The gag-pol fusion protein is thought to bind the (+) strand during viral packag\ng. PoI is only expressed as a gag-pol fusion protein, formed by a -1 ribosomal frameshift event in the region overlapping the gag andpol ORFs. This event occurs at a frequency of about 27o dunng translation of the LA (+) strand transcript. Nine chromosomal genes have been identified which are responsible for maintaining the -1 frameshift. Mutations in these mof (maintenance of frameshift) genes results in the reduction or absence of the M virus [for review see Wickner (1996)]. There have been extensive studies into the frameshifting event of the L-A virus, motivated in part by the importance of this process in the propagation of HIV and other retroviruses @inman and Wickner,1994) .

Tlhe mof genes are not the only chromosomal genes required to maintain killer activity in Saccharomyces cerevisiae.There are a number of chromosomal genes involved in regulating viral propagation (MAK genes) and the translation of viral mRNA (SKI genes). The MAK (maintenance of killer) genes are essential for the propagation of the Ml and M2 dsRNA viruses. MAK3, MAKL9 and PETL8 have also been found to be essential for LA propagation. Mutants defective in MAK genes showed decreased levels of free 605 ribosomal subunits critical for Ml propagation, and to a more modest extent, LA propagation (Wickner, 1993).

The SKI(super killer) genes negatively control the copy number of L-A and M dsRNAs. Six chromosomal genes, SKI2, SKI3, SKI4, SKI6, SKIT and SK18 have been identified. It appears the SKI2, SKI3, and SK/8 gene products are part of a cellular system which specifically blocks the translation of viral mRNA by recognising the absence of a 5' cap and/or a 3' poly(A) tail (Wickner, 1993). All the SKI genes identified are not essential for cell growth. However, ski mutants with an M dsRNA are either sick or lethal (Schmitt and Tipper, 1992). Mutations in many of the MAK genes are suppressed by sÉi mutations (Toh-

e and Wickner, 1980) .

2.2.L.2 The kiJler toxins of. SaccharoÍnyces cerevisiae 5

The Sacchnromyces cerevisiae killer types KI, K2 and K28, were first classified into three groups based on differences in their killing profiles, and the lack of immunity towards each other (Rogers and Bevan, 1978; Young and Yagiu, 1978). It was not until the killer toxins corresponding to each group were cloned and sequenced that researchers realised the difference in the killing patterns was due to each group encoding a novel toxin. Although Kl, K2 and K28 vary in structure and function, their respective toxin precursors undergo similar processing events (Bussey, 1991; Dignard et al., I99I; Schmitt and Tipper 1995). Processing of the Saccharomyces cerevisine toxin precursor starts with it entering the , where the signal peptide is removed, and the remaining precursor N- glycosylated. The final processing event before secretion is the proteolytic cleavage of the precursor by the endoprotease Kex2 and the carboxypeptidase Kexl (Bussey, 1991).

The Kl toxin, the most studied of three toxins, is composed of two distinct disulphide- bonded subunits, termed a (9.5 kDa) and 0 (9 kDa) (Table 2.1). The cr subunit is multifunctional, being involved in binding to the cell wall receptor, forming a toxin channel, and self-immunity. In contrast, the B subunit has only one function, which is the binding of the toxin to the primary receptor (Bussey, l99I;Zhu andBussey, 1991). Once bound to the primary receptor, p-1,6-glucan, the Kl toxin targets the plasma membrane (Schmitt and Compain, 1995), where it induces aberrant activity of the TOK1 potassium selective channel. This results in the TOK1 channel failing to regulate potassium homeostasis and subsequent cell osmolysis occuring (Ahmed et a1.,1999).

The overall structure of the K2 toxin precursor resembles that of the Kl precursor but shares little sequence similarity @ignard et aI., 1991). The K2 toxin is a heterodimer (28 kDa) consisting of the two subunits, cr lI72 amino acids (aa)l and p (140 aa), and appears to induce cell death via osmolysis (Table 2.1) (Meskauskas and Citavicius, 1992; INf.agliani et aL,1997). Like the Kl and K2 killer toxins, the K28 toxin is a heterodimer, composed of an cr, (11 kDa) and a P (10.5 kDa) subunit (Table 2.1) (Pfeiffer and Radler, 1982; Schmitt and Tipper, 1995). However, in contrast to the primary receptor of the Kl toxin, the primary receptor for the K28 toxin is the cell wall mannoprotein (Schmitt and Radler, 1987). Furthermore, the K2 toxin appears to induce cell death, not via osmolysis, but via inhibiting DNA synthesis (Schmitt et a1.,1989), and arresting the cell cycle in S phase (Schmitt et al., tee6).

2.2.1.3 The self-immunity factor of the K1 killer toxin

Insertion mutations of the Kl toxin ORF revealed that the domain required for immunity includes the sequence encoding two hydrophobic regions of the ø subunit, but extends into the sequence encoding for the N-terminal part of the y subunit (Boone et al., 1986). Although not well understood, immunity is thought to be conferred to the killer cell by a Table 2.1

Mode of action Yeast dsRNA Toxin structure Size (kDa) Primary receptor p-1,6-o-glucan Disurption of the ce s c erev isiae L-A (4.6 kb) a disulphide-bonded 19.0 Saccharonry channel and K1 heterodimer composed of a (9 TOK1 Ml (1.8 kb) subsequent osmolYsis kDa) and F (9.5 kDa) p-1,6-o-glucan Osmolysis Saccharonryces cerevisiae L-A (4.6 kb) an 0p heterodimer glYcoProtein 2t.5 K2 M2 (1.5 kb)

Inhibition of DNA Saccharomyces cerevßlae L-A (4.6 kb) a disulfide-bonded heterodimer 2r.5 c-1,3-Mannose composed of synthesis and cell K28 , M28 (1.8 kb) cycle arrest at S phase cr (10.5 kDa) and Ê (11lcDa) Osmolysis Ustílago mnydisKPl N'{L{MZ (1.4 kb) heterodimer composed of 19.0 u, (I2kDa) and Ê (13 kDa) Osmolysis Ustilago maydisKP4 M2 (0.e8 kb) single polypetide 11.1 Osmolysis Ustilago maydisYtP6 M2 (1.2 kb) heterodimer composed of 17.7 cr (8.6liDa) and I (9.1 kDa)

Zy g o s ac charomy c e s b ailií L (4.s kb) M (1.8 kb) p-1,6-glucan Hanseniaspora uvarurn L (4.s kb) M (1/1.4 kb)

Table 2.1: Killer yeasts and fungi with dsRNA virus encoded killer toxins 6 component of the toxin precursor interacting with the toxin's primary receptor during secretion. This may lead to either toxin loss by shunting off the precursor toxin-receptor complex to a vacuole, or by masking the receptor by the tight binding of the precursor toxin (Boone et a1.,1986; Sturley et a1.,1986).

2.2.2 The killer fungus Ustilago maydís

There are three closely related killer types of Ustilago maydis, KPl, KP4 and KP6 (Table 2.I). Like those of Sacchnromyces cerevisiae, these killer types were based originally on their killer sensitivity/resistance towards each other (Puhalla, 1968). Based on their molecular weights, the dsRNAs encoding these killer toxins can be grouped into three size classes: H (heavy), M (medium), and L (light). The H dsRNA of KPl, KP4 and KP6 encode for structural and non-structural proteins essential for viral maintenance, such as the capsid polypeptide, viral replicase and transcriptase, whereas the M dsRNA encodes the killer activity (Koltin, 1988; Shelbovrr' et a1.,1988). The genes encoding the KPl, KP4 and KP6 toxins have been cloned and sequenced. However, researchers have yet to elucidate how the toxins induce cell death.

The KP6 toxin precursor is encoded by a single ORF, which when processed by Kex2Jike and other protease-processing events, gives a heterodimer comprising of an cr (8.6 kDa) and a 0 (9.1 kDa) subunit (Table 2.1). Both subunits are essential for toxicity, and are both able to interact with the cell wall independently as monomers (Peery et aI. 1987; Tao et al. 1990; Ginzberg and Koltin , 1994). Unlike the Kl killer system of Saccharomyces cerevisiae, the immunity factor of KP6 is encoded by a recessive allele of a nuclear gene @inkler et al., 1992). The KPl toxin is also encoded by a single ORF. The KPl toxin precursor is processed to give separate subunits, of which only the p peptide can cause cell toxicity (Park et al.I996b). The KP4 toxin is encoded by a single ORF. However, this toxin differs from KPl and KP6 in that it is composed of a single polypeptide only (Park et al., 1994; I(tnal et a1.,1995).

2.2.3 The killer yeasts Zygosaccharomyces baílíí and, Hanseniaspora avarum

Like the Saccharomyces cerevisiae and Utilago maydis killer systems, the killer systems of Zygosaccharomyces bailü andHanseniaspore uvarum are directed by encapsidated dsRNAs (Table 2.1). The Zygosaccharomyces bail.iiY,tller yeast harbours three dsRNAs, L (4 kb), Z (2.9 kb) and M (1.8 kb), and the Hanseníaspora uvarum killer yeast two, L (4.5 kb) and M (l or 1.4 kb) (Schmitt and Neuhausen, 1994). For both the Zygosacchnromyces bailü and Hanseniaspora uvarum killer yeasts the M dsRNA encodes the killer toxin and the L dsRNA l the maintenance of M. The M dsRNA virus of the Hanseniaspora uvarum killer can vary in size, being either I kb or 1.4 kb (Zorg et a1.,1988). This type of variation is seen also in the Saccharomyces cerevisiae ktller system, where the size of the M dsRNA differs for each killer type. Whether the difference in size of the M dsRNA virus determines two different killer types for Hanseniasporauvarum has yet to be determined. Very little is known of the biochemistry or the action of the Hanseniaspora uvarum and the Zygosaccharomyces bailü toxins, although, it appears that B-1,6-glycosidic linkages are important as a receptor for the Hanseniaspora uvarum toxin (Radler et a1.,1990).

It is interesting to note that Sacchnromyces cerevßiae has been successfully transfected with the encapsidated L and M dsRNAs of Zygosaccharomyces bailü. The resultant transformant gave a two fold increase in killer activity as compared to the Zygosaccharomyces bailü wild type killer. Furthermore, it was found that the M dsRNA of Zygosaccharomyces bailü was stably maintained by the L-A dsRNA of Saccharomyces cerevisiae. This last result suggests that the killer systems of Saccharomyces cerevisiae andZygosaccharomyces bailü may sharc a common ancestor (Schmitt and Neuhausen, 1994).

2,3 LTNpln DNA PLASMID ENCODED KILLER YEASTS

2.3.I The killer yeast Kluyveromyces lactís

2.3.1.1 The linear DNA plasmids of Kluyverornyces lactís

A notable characteristic of the killer system of Kluyveromyces lactis is the presence of two linear DNA plasmids. This discovery not only led to research into the killer system of Kluyveromyces lactis, but generated considerable interest into the structure of linear DNA plasmids. The two linear DNA plasmids of Kluyveromyces lactis, pGKLI and pGKL2, aÍe located in multiple copies in the cell's cytoplasm. Both plasmids carry terminal inverted repeats (TIRs), where initiation of plasmid replication takes place (Tommasino, l99l; Schaffrath and Meacock, 1995).

The smaller plasmid of Kluyveromyces lactis, pGKLI (8.9 kb), contains four ORFs encoding the heterotrimeric killer toxin subunits (pGKL1-ORF2 and 4) and the immunity determinant (pGKL1-ORF3) (Stark et aI., 1990). The function of pGKLI-ORFI is unknown, being dispensable for killer plasmid replication and maintenance (Schaffrath et al., L992). The larger plasmid, pGKI-2 (13.5 kb), provides essential functions for the gene expression and maintenance of both plasmids. pGKL2 contains ten ORFs. However, specific functions can only be attributed to some ORFs. These include pGKL2-ORF2, which 8 encodes the plasmid specific DNA polymerase, and pGKL2-ORF6, which encodes an RNA polymerase specific for translation of the plasmid transcripts (Schaffrath et a1.,1995).

All 14 genes encoded by the linear DNA plasmids are under the control of a unique gene expression system. Upstream of each gene are conserved cis-acting sequences (UCSs), which allow expression in the cytoplasm only. Nuclear genes under the conffol of UCS promoter sequences fail to be expressed. This specificity for cytoplasmic expression has be exploited, by expressing nuclear genes in the cytoplasm of both Kluyveromyces lactis and Saccharomyces cerevisiae (Kamper et al., l99I1' Scründer and Meinhardt 1995; Schickel ¿r al.,1996).

2.3.I.2 The killer toxin of Kluyverornyces lactís

The Kluyveromyces lactisktller toxin is a]arge, heterotrimeric glycoprotein (156.5 kDa), which has been found to act on a variety of yeast genera. The toxin is comprised of three subunits, u (99 kDa), 0 (30 kDa)and y (27 .5 kÐa), where cr and p are encoded by pGKLI- ORF2, and y is encoded by pGKLI-ORF4 (Table 2.2). It appears the oP precursor is processed by a Ktuyveromyces lactis I(FXI endopeptidase, to give separate c and p subunits (Stark and Boyd, 1986; Stark ¿r aI.,l99}).The Kluyveromyces lactis KEXL gene shows low but significant homology to the KEX2 gene of Saccharomyces cerevisiae (Wesolowski-Louvel et a1.,1983). It is unclear how the subunits interact to form the toxin, but it appears they are disulphide bonded in an cr1p1y1 form (Stark and Boyd, 1986; Stark et a1.,1990).

Much interest has focused on the y subunit because of its ability to cause Gl arrest in sensitive Saccharomyces cerevisiae cells. Secretion of the 28 kDa y subunit from the host cell, and access inside the sensitive cell, is dependent upon the interaction with the other subunits (Stark et a1.,1990; Butler, et al. I99Ia: Butler et al. l99Ic; Takita and Castilho- Valavicius, 1993). Intracellular expression of pGKLI-ORF4, under the control of a GALT promoter in Saccharomyces cerevisiae, resulted in Gl arrest. This cell cycle arrest does not cause cell death, and is fully reversible when expression is stopped. For cells in Gl arrest to be killed, the cr subunit of the Kluyveromyces lactis toxin must also be present. The molecular mechanisms controlling this Gl arrest and eventual killing have yet to be elucidated (Tokunaga et a1.,1989; Butler et a1.,1991c).

The immunity factor of Kluyveromyces lactis is encoded by pGKLI-ORF3. The biochemical basis of immunity is unknown, with the predicted ORF3 product showing no significant similarity to any known protein sequence. It appears to be a cytoplasmic protein since it has neither an amino-terminal hydrophobic signal peptide, nor any other strongly hydrophobic regions (Stark et a1.,1990). Table2.2

Yeast linear DNA Toxin structure Size (kDa) Primary receptor Mode of action Kluyveromyves lactis pGKLI (8.4 kb) heterotrimeric glycoprotein 156.5 Chitin Cell cycle arrest in the pGKL2 (13.4 kb) composed of subunits a (99 kDa), Gl phase and chitinase ß (:O lrOa) andY Q7.5þ,Ða) acti Pichia acaciae pPacl-l (13.6 kb) heterotrimeric glycoprotein -190 Chitin Blocks completion of pPac l-2 (6.8 kb) composed of subunits ø (110 the cell cycle

kDa), ß (39 kDa) and Y (38 kDa)

Table 2.2:y KllLer yeasts with linear DNA encoded killer toxins.

Table 2.3

Yeast Toxin Structure Size ftDa) Primary Receptor Mode of Action Williopsis saturnus var. mrakii HM-l monomer (10.7 kDa) r0.7 p-1,6-glucan Inhibition of 1,3-

saturnus var. saturnzs HYI monomer (10 kDa) 10.0 Pichiafarinosa heterodimer protein composed of t4.2 subunits a(6.9 kDa) and B (7.9 kDa) cerevisiaeKlß. monomer 1 8 cerevisiaeKÍlS monomer 75

Table 2.3: Killer yeasts with chromosomally encoded killer toxins. 9

The linear DNA plasmids of Kluyveromyces lactis have been transferred to Saccharomyces cerevisiae by protoplast fusion, where they were stably maintained, and conferred killer activity and immunity (Gunge and Sakaguchi, 1981; Gunge et al., 1982). The killer activity of Kluyveromyces lactis was successfully transferred to Kluyveromlces marxianus, by mating the two closely related yeasts. The resultant transformant was shown to produce approximately 17 times the amount of killer toxin produced by Kluyveromyces lactis. Killer activity was also successfully transferredto kefyr by protoplast fusion, resulting in a six-fold increase in production of the killer toxin. The killer activity of the Cqndidn kely, transformant declined from generation to generation, however, because the plasmids were not stably maintained (Sugisaki et a1.,1985).

2.3.2 The killer yeast Píchia. acacíøe

The killer systems of Pichia acacine and Kluyveromyces lactis share significant similarities, the most obvious being the two linear DNA plasmids. Like that of Kluyveromyces lactis, the smaller plasmid of Pichin acaciae, P-PacI-2 (6.8 kb), encodes the killer and immunity phenotype Qable 2.2). The larger plasmid, P-Pacl-L (13.6 kb), is responsible for replication of P-PacI-2.The Píchia acaciae toxin (-190 kDa) is composed of three subunits, with molecular weights of approximately 110, 39 and 38 kDa, and exhibits chitinase activity (Worsham and Bolen, 1990). A 1473 bp ORF was cloned from pPacl-2, which was found to share regions of homology with the N-terminal region of the Kluyveromyces ladis a subunit. Furthermore, a UCS-like promoter sequence was also identified upstream of the pPacL-21473bp ORF. However, it is not known whether the polypeptide encoded by this ORF is involved in toxicity (Bolen et al., 1994). The majority of Saccharomyces cerevisiae strains found resistant to the Kluyveromyces lactis toxin were sensitive to the Pichia acaciae toxin. This would suggest that even though the two toxins share similar characteristics, they differ in their mechanisms for inducing toxicity (McCracken et aI.,1994).

2.4 KTIINN YEÄSTS WITH NUCLEAR ENCODED TOXINS

The killer toxins of some yeast species, such as Williopsis saturnus, Pichia farinosa, and as mentioned above, the Saccharomyces cerevisiae killer types KHR and KHS, are encoded by nuclear genes (Table 2.3). However, this is not to say that killer yeasts whose toxins are encoded chromosomally, do not contain extrachromosomal elements. An example is the halophilic yeast Debaryomyces hansenü. This salt-tolerant yeast encodes its toxin chromosomally, yet it harbours three cryptic linear DNA plasmids (Gunge et a1.,1993).

2.4.1 The killer yeast WíIlíopsís saturnus HM'1 l0

The HM-1 (HMK) toxin of Williopsis saturnus var. mrakii strain IFO 0895 is synthesised as a large precursor (135 aa), with a N-terminal propeptide of 37 aa (fable 2.3). Processing of the precursor gives a mature monomeric toxin of 88 aa, with an estimated size of 10.7 kDa (Yamamoto et aL,1986b; Kimura et aI., 1993). In contrast to other well characterised yeast killer toxins, the HM-l toxin has a basic isolelectric point (pI 9.1), and displays a high level of thermostability attributed to its many disulphide bonds (Yamamoto et al., 1986b). By employing electron microscopy it was found that the HM-l toxin develops pores on the growing point of the bud, giving rise to an out-flux of cytosolic materials and subsequent cell death (Komiyama et a1.,1996). The osmolysis of sensitive cells by the HM-l toxin can be extenuated, when assayed in hypotonic conditions (Komiyama et al., 1996). The primary receptor for the HM-l toxin has yet to be clearly identified, although, it appears the toxin can bind to the nascent p-glucan chains formed at the budding cell sites (Takasuka et al., L996; Kimura et a1.,1997).

Southern analysis suggests the gene sequences encoding killer toxins from different strains of Williopsis saturnus var. mrakü show strong homology to the HM-l toxin sequence, with western analysis suggesting these toxins are structurally similar (Kimura et al., 1995). Kimura et aI. (L993) also report that the HYI (HSK) toxin of Williopsis saturnus var. saturnus is a single polypeptide (87 aa), whose encoding sequence shows 82Vo homology to the HM-l toxin sequence.

2.4.2 The killer yeast Píchía farinosa

Like the HM-l toxin of Williopsís saturnus, the killer toxin of the halotolerant Pichia farinosa is nuclear encoded (Table 2.3). Interestingly, this particular toxin exhibits maximum activity in the presence of 2 M NaCl (Suzuki and Nikkuni, 1989), and subsequently, was named the salt-mediated killer toxin (SMKT). The SMKT toxin is translated as a 222 amino acid precursor, which when processed, gives a mature heterodimer toxin consisting of an cr

(6.3 aa) and a P (7.8 aa) subunit (Suzuki and Nikkuni, 1994; Suzuki, 1999). Its not known how the SMKT toxin induces cell death, however, the topology of this toxin is identical to the KP4 toxin of Ustilago maydis (Gt et a1.,1995).

2,5 THE ECOLOGY OF KILLER YEASTS

The widespread occurrence of the killer phenotype in yeasts suggests that it is a mechanism for interference competition, whereby toxin production prevents a competitor from gaining access to resources (Stumm et al., 1977). Because yeasts are saprophytic and colonise decaying substrates during the initial phase of necrosis, selection for mechanisms 11 that confer an advantage during the early stages of population growth would be expected (Starmer et a1.,1987). The toxins produced by killer yeasts appear to be suited to this role. They are optimally produced during exponential growth when resources are abundant, and in general, have a low pH activity profile (Magliani et al., 1997). This is precisely the natural condition of most yeast substrates during microbial colonisation, especially decaying fruit (Starmer et a1.,1987; Morais et al. 1995; Abranches et al.,2000).

A study into yeast succession in fallen affßpa fruit revealed a great diversity in yeast species in the early phase of the fruit's necrosis (l-2 days), attributed to the intense visiting by Drosophila and other insects. However, during the intermediate stage of fruit deterioration(4-6 days) yeast diversity had decreased whilst the incidence of killer yeasts increased. V/ithin six to ten days of necrosis yeast diversity had again increased, as did the pH of the rotting fruit from pH 4.0 to pH 6.0. This increase in pH was found to inactivate the toxins of the killer yeasts found prevalent during the intermediate stage. These results suggest the decrease in yeast diversity is due to the exclusion of some species by killer toxin-producing yeasts (Morais et a1.,1995).

Ecological studies of yeast communities in the decaying fruit of cacti revealed that killer- sensitive interactions are minimal within or between fruits of the same locality, and that most killer-sensitive interactions occr¡r between yeasts from different localities (Starmer er a1.,1987). Likewise, killer strains isolated from lake water or sediment samples displayed a broader killer range against strains isolated from different lakes or in a different time (Vadkertiova and Slavikova, 1995). This result suggests a level of adaptation has occurred in response to local selective pressures for killer resistance (Starmer et al., 1987; Morais e/ a1.,1995; Vadkertiova and Slavikova, 1995).

The limited killing range of yeasts within a single location may also be attributed to selective pressures for the yeast's distribution vectors, such as for the Drosophila fruit fly (Starmer et a1.,1987; Morais et al.,1995; Vadkertiova and Slavikova, 1995; Abranches e/ al., 2000). Mixed yeast cultures provide a greater nutritional and fitness benefit to Drosophila, as opposed to a single culture. Consequently, a killer yeast which tends to eliminate all other yeast species in that habitat, will reduce the fitness of the Drosophila. This in tum reduces the probability of the killer yeast being transferred from one habitat to another. For this reason, the yeast communities of rotting fruit and other natural habitats investigated were not composed of a single 'super-killer' strain displaying a broad killing range.

2.6 CTT¡,n¡CTERISATION OF MUTANTS RESISTANT TO KILLER TOXINS T2 2.6.1 Resistant mutants with cell wall defects

The characterisation of mutants resistant to killer toxins has revealed information on various aspect of cell biology, with much focus on the synthesis of the yeast cell wall. This is consistent with the observation that the primary receptors for all killer toxins characterised to date are located within the cell wall.

The yeast cell wall is composed mostly of mannan and glucan polysaccharides (80-90%), protein, and some chitin (I-27o). The cell wall mannan is covalently attached to proteins, forming mannoproteins. These mannoproteins provide structural integrity for the cell wall, or act as enzymes in various extra-cellular activities. Some mannoproteins are carried on the cell wall surface and are involved in cell aggregation during cell mating and flocculation. The cell wall glucans are polymers of glucose, containing either P-1,3- or p-1,6- linkages. Crosslinking with chitin, these glucans provide the structural framework for the cell wall (for review see Cid et a1.,1995).

The KRE (Killer REsistance) genes characterised from mutants resistant to the Saccharomyces cerevisiaeKl toxin have revealed insights into the p-1,6-glucan biosynthetic pathway. This includes KRE6, which encodes a putative type II membrane protein (Roemer and Bussey, 1991), and KREI, whose gene product extends linear p-1,6-glucan chains within the cell wall (Boone et a1.,1990b). Other KRE gene products characterised include Kre9p, an O-glycosylated secretory protein (Brown et al., 1993), and Kre2p, a mannosyltransferase responsible for adding the third mannose residue on O-linked mannose carbohydrate chains (Hausler et aI., L992). Ceh4lp protein, whose gene deletion confers a sensitive cell Kl resistance, was found to be an integral membrane N-glycoprotein involved in an early step of p-1,6-glucan assembly (Jiang et al., L996).

Mutants resistant to the HM-l toxin of Wittíopsis saturnus, have resulted in the isolation of genes involved in the synthesis of B-l-3-glucan (Hong et al., L994; Yabe et al., L996; Kimura et aI.,1997). Also, mutants hypersensitive to Calcofluor White have been found to be resistant to either the HM-l toxin (Ram et al., 1995), or the Kl killer toxin (Ram et al., L994), representing either p-l,3-glucan or p-1,6-glucan defects, respectively. Mutants resistant to the Kluyveromyces lnctis toxin in the extracellular environment, were found defective in the synthesis of chitin (Kawamoto et al., 1992; Takita and Castilho-Valavicius, 1e93).

2.6.2 Resistant mutants and functional mitochondria

Although the majority of mutants found to be resistant to killer toxins harboured cell wall defects, some studies have reported the resistance of sensitive cells lacking functional 13 mitochondria. Both White et al. (1989) and Butler et al. (I991b) found a percentage of spontaneously arising mutants resistant to the Kluyveromyces lactis toxin were petiæ in nature, that is, the mutants lacked functional mitochondria.

Petite (p-) yeast cells are characterised by large deletions in their mtDNA. In some instances petite mutants may be devoid of all mtDNA (po). Although nearly all of the proteins located in the mitochondria are encoded by the nuclear genome, 13 polypeptide-components of the multisubunit erzyme complexes of the electron-transport chain present in the inner mitochondrial membrane are encoded by the mitochondrial genome. As a consequence, petite cells have an inactive electron transport chain, and thus, cannot carry out oxidative phosphorylation. Subsequently, these p-cells rely exclusively on glycolysis for their energy requirements (Grivell, 1995).

The petite state is stably inherited by progeny cells during vegetative growth, without reversion to wildtype (wt). The cross of two haploids, a p- mutant with a wt, gives a diploid heteroplasmic for two types of mitochondria; p- and wt. However, subsequent divisions of this diploid during vegetative growth sees the exclusion of one type of mitochondria, resulting in the diploid being homoplasmic for either p- or wt mitochondria only. Referred to as vegetative segregation, this transition from a heteroplasmic to a homoplasmic state, usually occurs within four generations of zygote formation (Gingold, 1988; Piskur, 1994).

2.6.3 Cell walls defects of petite mutants

Although not clearly understood, it appears the mitochondria is associated with cell wall synthesis. This is exemplihed with the observation that flocculation, which is the aggregation of cells into flocs, is reduced for petite mutants of some highly flocculent strains (Iung et al., 1999). Flocculation involves a number of cell surface components, and is thought to occur via a lectinlike interaction, between a cell wall sugar-binding protein and cell surface mannan (Bony et al., 1997). These findings relate to those of Evans et aI. (1980), which showed that the ability of cells to agglutinate via treatment with plant lectin concanvalin A is reduced for petites. It is thought the mitochondria plays a direct role in cell wall synthesis, and that defects in its respiratory chain leads to changes in the cell wall structure. Alternatively, the mitochondrial genome may influence the expression of nuclear genes encoding the biosynthesis of the cell wall (Evans et aI., 1980; Wilhe and Evans, I982;Iung et al., 1999).

2.6.4 Mitochondrial driven apoptosis

Recent research has revealed that the mitochondria plays an active role in mammalian apoptosis, an intrinsic suicide program characterised by membrane blebbing, cell shrinkage, T4 chromatin condensation, and nuclear and cellular fragmentation (for review see Green and Reed, 1993). Mammalian apoptosis is initiated by the action of pro-apototic proteins, such as Bax and Bak, which when expressed, trigger the release of cytochrome c from mitochondria. Cytochrome c is a nucleo-encoded protein, which resides between the outer and inner membranes of the mitochondria (Reed, 1997). The release of cytochrome c from the mitochondria is mediated through a polyprotein channel called the permeability transition (PT) pore (Narita et al., 1998; Juergensmeier et al., 1998), and occurs independently of respiration @skes et al., 1998; Matsuyama et al., 1998). Upon its release, cytochrome c binds to the caspase-activating protein Apaf-l. This association results in the activation of caspases, a family of proteases whose sequential activation and cleavage of key target proteins, dismantles the cell, and culminates in apoptosis (Adams and Cory, 1998).

The pro-apoptotic proteins, such as Bak and Bax, have structural similarities with the channel-forming toxins of bacteria, such as the ditheria toxin and colicins, and can form ion channels when added to synthetic membranes (Green and Reed, 1998). These findings have raised the possibility that the apototic family of proteins and the bacterial colicins have a coÍtmon ancestral origin, or represent an example of convergent evolution. This is particularly evident when one considers that mitochondria are believed to have descended from intracellular bacteria that developed a symbiotic relation with eukaryotic cells. Thus, the pore-forming colicins may have provided the framework for the subsequent emergence of the apoptotic family of proteins, ffiffiy of which are localised to mitochondrial membranes and which (like the colicins), are inextricably involved in the regulation of cell death (Schendel et aL,1997; Green andReed 1998).

The expression of pro-apototic genes such as Bax induces cell death in Saccharomyces cerevisiae in a similar manner to that of mammalian cells (Juergensmeier et al., 1997; Ligr et a1.,1998). Like the mammalian studies, Bax-induced cell death in Saccharomyces cerevísiae is independent of respiration (Matsuyama et a1.,1998), and is rescued with the coexpression of anti-apoptotic genes such as BcI-2 and Bcl-xL (Tao et al., 1997; Minn ¿r al., 1999). However, unlike mammalian systems, the apoptotic Bax protein does not require a pre- existing mitochondrial channel such as a PT pore, but instead, creates de novo channels in the yeast's outer mitochondrial membrane (Priault et al., 1999). Furthermore, the Bax- induced cell death in yeasts occurs in the absence of caspases, suggesting that this form of cell death predates this family of proteolytic enzymes (Adams and Cory, 1998).

2.7 AppIIC¿,TIONS FOR KILLER YEASTS

As work into killer yeasts continued, researchers began to realise the potential killer systems could have in various facets of medical and scientific research. One exploitation has been the 15 use of the killer system to secrete foreign proteins. The leader sequences of both the Sacch.aromyces cerevisiae and Kluyveromyces lactis killer toxins have been utilised for directing the secreting expressed polypeptides in Saccharomyces cerevisine. The þ'tller system of Saccharomyces cerevisiae has also been adapted for use as a cloning vector, utilising killer activity as the dominant selectable marker @aldari et al., 1987; Cartwright er aI., L992).

Another application of killer yeasts is in identifying unknown yeast strains based on their resistance/sensitivity to killer yeasts. By assuming a killer yeast will kill one strain but not another, yeast taxonomists can obtain a unique resistance/sensitivity profile for each strain of interest (Polonelli et al., 1983; Vaughan-Martiri et al., 1996). This method of strain identification has been applied for the differentiation of species of a single genus (Golubev and Boekhout, 1995; Provost et a1.,1995).

Clinical applications include the use of antiidiotypic antibodies of a killer toxin directed towards fungal parasites responsible for human infections. The killer yeast Pichi"ø arcmsla displays killer activity towards Pneumocystis carinü, a eukaryotic parasite which causes pneumonia in immunocompromised patients. Antiidiotypic antibodies of the Pichia arcmal.a killer toxin have been raised in a hope they can be used for the treatment and prophylaxis of

Pneumocystis carinii infections (Cailliez et a1.,1994; Seguy et a1.,1996) .

The KP4 toxin has been expressed successfully in tobacco plants under the control of the cauliflower mosiac virus 35S promoter. The concentration of the secreted toxin found on the leaves of transformants was sufficient to inhibit the growth of sensitive strains of Ustilago maydis. This illustrates the possibility of the biocontrol of Ustilaginales infections seen in many economically important crop plants, such as maize, wheat, oats, and barley (Park et al.,l996a). The fact that tobacco plants can express active KP4 toxin from a full length ORF suggests that the toxin precursor produced in tobacco cells is processed by mechanisms analogous to those in Ustilago maydis. (Park et al., I996a).

Lowes et al. (2000) investigated the potential of protecting maize silage and yoghurt from yeast spoilage by exploiting the anitmicrobiol properties of the HM-l toxin of Williopsis saturnus var. mrakii. The HM-l gene sequence was fused to the glucoamylase protein gene for the successful expression in the fungus Apergillus niger. This heterologous HM- l/glucoamylase toxin displayed similar physiological properties to the authentic HM-l toxin, and reacted also with HM-l-specific antibodies. The application of the partially purified heterologours toxin fromA. niger ferments, to mature marzr- silage prior to exposure to air, delayed the onset of aerobic spoilage. In yoghurt, introduced spoilage yeasts were eliminated by the addition of the heterologous toxin with no resurgence of resistant yeasts (Lowes et a1.,2000). L6

Biocontrol research has also focused on the possibility of employing a killer toxin to control the growth of spoilage yeasts found contaminating industrial ferments. A number of studies have looked at the potential biocontrol of yeast growth in breweries, and more recently, soy sauce making (Taylor and Kirsop 1979; Young 1981; Gunge et al., 1993). Other studies have investigated the possibility of employing the Sacchnromyces cerevisiae killer system for the control of indigenous Saccharomyces cerevisiae strains in wine ferments (Seki et aI., 1985; Boone et aI., l99}a; Petering et al., I99l; Sulo ¿f al., 1992; Michalcákovâ et al., L994). To date, however, there have been no reports of engineering a wine yeast to express a killer toxin which targets a broad range of yeast species indigenous to the wine must. By engineering such a wine yeast, the use of antimicrobial chemicals in wine making could be reduced.

2.8 INOTCNNOUS YEASTS OF THE GRAPE MUST FERMENT

Indigenous yeasts refers to those species of yeasts that originate from the grape berry or the winery equipment. The predominant species of the grape berry is Kloeckera apiculata, and its perfect form Hanseniaspora uvarum. Smaller yeast populations of the grape berry include Metchnikowia pulcherrima, Cand,i.dn stellata, species of Cryptococcus and Kluyveromyces, the film forming species Pichia and Hansenula, and pigmented species such as Rhodotorula and Sporobolomyces. Saccharomyces cerevisiae and to a much lesser extent, species of the film forming yeasts Pichia, Candida and Hansenula, make up the winery micro flora (Martini and Martini, 1990). Indigenous yeast species have been of much interest to winemakers for many years. However, this interest declined since the introduction of inoculating the grape juice with a single yeast strain, commonly referred to as the starter culture.

The advantage of inoculated ferments over the uaditional spontaneous ferments is the reduced risk of spoilage from indigenous yeasts due to the starter culture dominating the ferment. Furthermore, with the addition of the antimicrobial agent sulphur dioxide (SOZ) to the grape must, winemakers in general believe that indigenous yeasts play little or no role in the sensory properties of the final product. However, the assumption that the starter culture, plus the addition of an appropriate concentration of SO2, suppresses the growth of indigenous yeasts may not be strictly correct. Studies undertaken by Heard and Fleet (1985, 1986 and 1937) report that strains of Kloecl

2.9 Tnn nrr,vr FoRMING YEAsr Píchia membranifacíens

As mentioned above, the film forming Pichia membranifaciens is indigenous to the grape berry and winery equipment. The growth of this yeast is most prominent on the surface of wines found exposed to air during the maturation period, resulting in an unattractive yeast film present on the wine's surface. Strains of this yeast have, however, exhibited significant growth during the early stages of fermentation. The spoiled wine can show off odours, such as acetic acid, ethyl acetate and iso-amylalcohol (Rankine, 1966; Thornton, L99I; Sponholz, ree3).

Pichia membranifaciens has also been found to be responsible for the spoilage of various foodstuffs such as soft and brick cheeses (V/estall and Filtenborg, 1998; Valdesstauber ¿r al., 1997), bread doughs (Alrneida and Pais, 1996), and processed olives (Kotzekidou, 1997). Whilst in beer, the growth of Pichia membranifaciens can result in turbid beverage and having a sauerkraut off aroma (Thomas, 1993).

2.IO KTT.Inn STRAINS OF PICHIA MEMBRANIFACIENS IN FOODS

The characterisation of the yeast flora of 33 home-made corn and rye bread doughs, collected from locations in the north of Portugal, yielded 73 isolates belonging to eight different species (Alrneida and Pais, 1996). Of these 73 isolates twelve were strains of Pichia membranifaciens. When screend for killer activity against 7 tester strains, six of the twelve Pichia membranifaciens strains displayed killer activity. The role these Pichia membranifacienskíller strains may play in growth interefernce \ryas not investigated.

A similar study in the characterisation of the yeast flora of olive brines revealed 25 strains, six of which were Pichia membranifaciens (Marquina et al., 1992). Five of the six strains displayed killer activity when assayed against 15 tester strains. The concentration of

sodium chloride can effect the production of killer toxin by yeasts isolated from fermented foods, consequently, toxin production for two of the fwe Pichia membranifaciens kjller strains was further investigated (Suzuki et a1.,1989; Llorente et al., 1997). However, the toxicities of these strains did not vary significantly with an increase in concenration of

sodium chloride used in their cultivation media. 18

2,II TTTn TnnNTIFIcATION OF SPOILAGE YEASTS

The prominence of Pichin membranifaciens and other spoilage yeast species found contaminating various processed foodstuffs and beverages has borne the need for identifying suspect yeast isolates in an industrial context. At present, the conventional method for yeast identification, is based on a range of morphological and biochemical characteristics. The phenotypic characters required for yeast identification, however, can be absent or inherently variable, thus requiring a large number of time-consuming and labour intensive tests for accurate identification.

As a consequence, a number of molecular methods have been developed for the more rapid means of yeast classification and identification. These include DNA-DNA reassociation, characterisation of the ubiquinone type present on the respiratory chain, molar percentage of guanine and cytosine present on whole genomic DNA, the restiction analysis of different DNA regions, isoenzyme electrophoresis, electrophoretic karyotyping, the random amplification of DNA fragments by PCR, and DNA sequencing (for reviews see Kurztman and Fell, 1998 and Valente et a1.,1999).

Some molecular methods have been developed focussing on the identification of the spoilage yeast Pichia membranifaciens. Pearson et al. (1995) have developed a PCR fingerprint technique which uses retrotransposon long terminal repeat elements for the primer sequences. Although this method could successfully differentiate strains of Pichin membranifaciens, it failed to identify isolates at the species level. In contrast, the typing method developed by Kosse et aI. (1997), which uses 18S rRNA-targeted oligonucleotide species-specific probes, could identify isolates as belonging to the Pichia membranifaciens species, but not differentiate individual strains. Noronha-da-Costa et al. (1996) have, however, developed a typing method based on a isolates long-chain fatty acid composition, which is both strain and species specific. This method has been employed to successfully trace the source of a Pichia membranifaciens contarnination in a commercial winery (Malfeitoferrerira et al., 1997). 19

Chapter 3

TTTB INCIDENCE OF KILLER ACTIVITY OF NON.SACCHAROMYCES YEASTS TOWARDS INDIGENOUS YEAST AND BACTERIA SPECIES OF THE GRAPE MUST

3.1 INTRODUCTION

The growth of indigenous yeasts during wine fermentation can be highly undesirable, as they may introduce inappropriate sensory properties to the wine (Sponholz, L993). For this reason, the wine fermentation is induced with a highly active Saccharomyces cerevisiae stafter culture in the presence of sulphur dioxide (SOz). The addition of SO2 to the grape juice or must prior to yeast inoculation suppresses indigenous yeast growth, thereby facilitating the growth and dominance of the starter culture. An alærnative method for preventing unwanted yeast growth is to employ a starter culture which produces a killer protein active against various indigenous yeast species. The use of a killer toxin could reduce or replace the use of anti-microbial chemicals, such as SO2, in winemaking.

The aim of this investigation was to identify a killer yeast which displayed broad killer activity to species of yeasts commonly associated with the grape juice and must fermentation. Yeasts diverse in killer activities were selected from the published literature, and a range of species known to be associated with the grape juice and must were selected as the test strains. The poûential for killer yeasts to antagonise the growth of undesirable bacterial strains in wine making was also investigated. To identify a yeast with broad killer activity in a winemaking environment, yeast killer activity was assayed at pH comparable to a wine ferment.

3.2 EXPERIMENTAL 3.2.1 Yeast and bacterial strains

The killer and sensitive yeast strains used in the work described in this chapter are listed in Table 3.1 and 3.2, respectively. Strains were sourced from the following culture collections: the Centraalbureau voor Schimmelcultures (CBS), Delft, The Netherlands; the National Collection of Yeast Cultures (NCYC), Norwich, England; the American Type Culture Collection (ATCC), Manassas, VA; The Australian Wine Research Institute (AWRI), Adelaide, Australia. Saccharomyces cerevisiøe AWRI838 is an isolate from the commercial wine strain ECl118 (Lallemand fty. Ltd, France). Yeast strains were subcultured in YEFD fl%o (wtv) yeast extract,2To (w/v) peptone, 27o (w/v) glucosel at 25"C for 24-48 h before Table 3.1

Killer Yeast Original Substrate Origin basis of killer toxin

C andida glabrata NCYC 3 88 * nuclear D eb aromy c e s v anrij iac CB S 4072 slime flux of oak tree USSR Kluyveromyces lactis var.Iactis CBS 2359 crcamery USA linearDNA Kluyveromyces lactís var. drosophilarum CBS 2896 slime flux of oak tree USSR nuclear Kuyveromyces marxianzs NCYC 587 souring figs Pichin anomnla CBS 1982* Pichia anamaLa NCYC 434 PichiabisporacBs 1890 tunnel of bark beetle Ausfria dsRNA Pichia subpelliculosa NCYC 16 Pichin membranifac¿¿ns CBS 107 nuclea¡

Pichia memb ranifaciens CBS 7 37 3 draught beer

P ichia memb rønifaciens CBS 7 37 4* draught beer Williopsis saturnus var. mrakü CBS 107* soil Papua New Guinea nuclear Zy gosaccharoftiyces bisporus AWRI 784 wine ferment Australia

Table 3.1 : Killer yea,sts assayed for their ability to kill, or inhibit the growth of, species of yeasts representative of the wine fementation. * Yeast was assayed for killer activity towards bacterial strains found problematic to winemaking. Table 3.2

Sensitive Strain Original Substrate Origin Candidnkrusei A\ryRI863 wine ferment Austalia Candidakrusei AWRI873 wine ferment Australia

Candida søk¿ AWRI751 wine ferment Austalia

Candida søke CBS 159 x sake-moto Japan Candida stellata CBS 157 wine grapes Germany

Cand.ida stellata CBS 2649 x grape juice France, Medoc

D ekkera bnnellensis CBS 7 2 lambic beer Belgium, Brussel

D ekkera bruxellensis CBS 73 graPe must France Dekkera bruxellensis CBS 74 lambic beer Belgium, Brussel Dekkera bnnellensis CBS 75 lambic beer Belgium, Brussel Dekkera brwellensís CBS 49 14 tea-beer Mozarrbique

H ans eni asp o ra uv arutn CB S 3 14 muscatel grape USSR, Crimea

H ans eniasp o ra uv arurn AWRI 865 wine ferment Austalia Hanseniaspora uvarurn AWRI 866 wine ferment Austalia

Kuyv eromyce s thermotolerans CBS 2803 gfaw Italy

Kluyv eromyce s thermotolerans CBS 6340 plum conserve USSR

Metschnilcowia pulcherrima CB S 5 833 Concord grapes USA, California

Pichia mexícana CBS 58 15 grape must Italy

S ac clnromy c e s c e rev isine AWRI 7 29 grape juice Austalia Saccharornyces cerevisiae ATCC 46273 * sake - wine yeast hybrid Japan

Saccharomyces cerevisiae CBS 1395 Saccharomyces cerevisiae CBS 1907

Sacchnromyces cerevisr¿¿ AWRI 838 commercial wine yeast France Saccharomycodes ludwigií CBS 821 Torulaspora delbrueckü CBS 817

Torulaspora delbrueckü CBS 1 146

Table 3.2: Tester yeast strains assayed for their killer resistance/sensitivity. 20 being transferred to YEPD-agar IYEPD, 2Vo (wlv) agar] slopes, which were incubated at 25oC for 48 h, before storage at4"C until required.

The bacterial strains tested for sensitivity to the kil\er yeasts are listed in Table 3.3. The bacterial strains were sourced from the bacteria culture collection of The Australian Wine Research Institute (AWRI), Adelaide, Australia. Bacterial strains were subcultured in liquid

MRS medium 14.6%o (v/v) MRS (Amyl Media), supplemented with 207o (vlv) preservative free apple juice,O.O27o (w/v) magnesium sulphate, O.0O57o (w/v) maganese sulphate, O.LVo Tween 801 at 20oC for 2-3 days before being transferred to MRS-agar IMRS medium,ZVo (w/v) agarl slopes, which were incubated at 20oC for 2-3 days, before storage at 4oC until required.

3.2.2 Agar killer assay for testing yeast strains

The killer assay employed in this study is essentially that of Stumm et al. (1977). YEPD-agar containing 0.003Vo (w/v) metþlene blue was buffered to pH 4.5 or pH 3.5 with 0.1 M citrate-phosphate buffer. Strains tested for sensitivity to killer yeasts were subcultured in liquid YEPD to exponential growth phase, before being diluted in YEPD, and spread onto the surface of the assay medium at a concentration of 10s cells/plate. Disposable agar plates of 50 mm in diameter were employed for the assay. Killer yeasts were then streaked onto the surface of the plates, which were then incubated at25"C for 48-96 h.

Killer activity was scored positive when the killer strain was sulrounded by a region of bluish stained cells, or by a clear zone of growth inhibition bounded by stained cells. For determining the strength of activity, the radius of the killer halo was measured in mm, from the edge of the streaked killer yeast, to the point where the zone of growth inhibition ceased. Killer activity was expressed in terms of minimal activity (appearance of a dark blue halo), weak (up to 1.5 mm), medium (1.5 - 2.5 mm) or strong killer activity (> 2.5 mm).

3.2.3 Agar killer assay for testing bacteria strains

YEPD/\4RS-agar medium Í2Vo (wlv) YEPD, 4.6Vo (vlv) MRS broth (Amyl Me.dia Pty. Ltd.), 20Vo (vtv) preservativefren apple juice,0.02Vo (w/v) magnesium sulphate, O.OO57o (w/v) maganese sulphate, O.I7o Tween 801 containing O.ÙO3Vo (w/v) methylene blue was buffered to pH 4.5 or pH 3.5 with 0.1 M citrate-phosphate buffer. MRS broth, when added

to the media at the recoÍtmended concentration 14.67o (w/v)1, gives I7o (w/v) bacæriological peptone, 0.87o (wlv) beef extract, 2Vo yeast extract, 27o (wlv) di-potassium hydrogen orthophosphate and 2Vo (wlv) tri-ammonium citrate. Bacterial strains tested for their sensitivity were subcultured in liquid MRS medium to exponential growth phase before being spread onto the assay medium. Killer yeasts were spread onto the plates, which were Table 3.3

Tester strain

Lac t ob ac illus b r ev i s Ll1 aI

I-actob acillus c ellobío sus LI6a Inct ob ac illus hil g ardi I-5i

I-ac t ob ac illus pl ant arwn Ll L a

I¿uc ono st o c amelilío sum Lactic acidbacteria Leuconostoc cremoris

(Gram +ve) Leu c o n o s t o c fall a"r 40 18

I-euc ono sto c me s ent e roide s l*I d Oenococcus oeni Snb Oenococcus oenil-úz Pediococcus cerevßiae Ia

P e dio c o c cus c erevi sine Ib Pediococcus 6b

Acetic acid bacteria Ac et ob act e r p ast e uri anus Ab 1 -ve

Table 3.3: The bacteria strains assayed for sensitivity to killer yeasts. 2t then incubatedat25"C for 48-96 h. Killer activity was scored positive when the killer strain was sutrounded by a region of bluish stained cells, or by a clear zone of growth inhibition bounded by stained cells.

3.3 RESULTS

3.3.1 Killer yeast activity

To identify a killer toxin with potential use for winemaking, 14 killer strains representing ûen species of yeast were assayed for their ability to antagonise the growth of yeast species characteristic of the wine ferment microflora. These killer yeasts, which were mostly selected on the basis of their known killing activity (Young and Yagiu 1978), originate from a variety of environments (Iable 3.1). A total of 26 yeasts representing twelve species, typically associated with wine ferments, were tested for their sensitivity to the 14 killer yeasts. Half of the tester strains had originally been isolated from wine grapes or wine ferments. The other 13 strains were isolated from sake and beer ferments, or are of unknown origin Clable 3.2). Initially, the assay of killer activity was undertaken with the solid medium buffered at pH 4.5, a pH value which is near optimal for killer activity for the majority of killer yeast species being studied (Young and Yagiu, 1978).

Using this assay, 147 killer-sensitive interactions involving the 14 killer yeasts and the 26 tester strains were identified (Iable 3.4). An example of this killer yeast-sensitive yeast interaction is shown in Figure 3.1. Of the 14 killer yeasts, Píchia anomala NCYC 434 displayed the broadest killing range, killing 2I of the 26 strains tested (Iable 3.5). A broad killing range was also exhibited by Williopsis sa.furnus var. mraki, CBS 1707 and Kluyveromyces Inctis var. drosophilarum CBS 2896 which killed 19 and 18 of the 26 strains, respectively. All 14 killer yeasts displayed activity to at least one of the 26 strains assayed, Pichiabisporus CBS 1890 being the most selective in only hlling Saccharomyces cerevisiae ATCC 46273.

To identify a killer yeast with broad killer activity in a winemaking environment, the solid agar assays were undertaken at pH 3.5. Compared with the assay made at pH 4.5, the killing ability of strains was markedly reduced, with only 41 of the 147 killer-sensitive reactions being observed (Iable 3.4). This decrease in killer-sensitive interactions was due to a reduction in the killing range for all 14 killer yeasts. Pichia atnmaln NCYC 434, which displayed the broadest killing range at pH 4.5 by killing 21 yeasts, only killed nine of these yeasts at pH 3.5 (Table 3.5). The killing activity of Kluyveromyces lactis var. drosophilarumCBS 2896, the third most effective killer at pH 4.5, was even more affected by the pH change, with only five of the 18 sensitive tester strains remaining susceptible to Table 3.4

2 d I ! o Eo .B ,s 59 È =E 3 È È =E q'Í E È+ !¡ 9'; .!^ € 5È !ç Lts I Es :\F 'ìh Ès ots È -ô È6 Êo ÊF ÊF È" o SF 7å 7ã ù È3 Èd Èd å¡ Iø IØ Iø ¡ !ø ¡\t Ê8 9 lø sø ltø È8 sã Es õ3 Èla Êa0 tcQ €È És e o .6 Èo ñ< ü z QO Vo vo vz a:O \z {o {o À:o {o { z pH 4.5 pH35 o/t 3.5 3.5 3.5 4.5 3.5 4.s 3.5 4.5 3.5 4.5 35 4.s 3.5 4.5 3.5 4.5 3.5 T '/e T Tester 4.5 3.5 4.5 3.5 4.s 3.5 4.s 3.5 4.5 4.5 4.5 + -4 29 ) 8ó3 ++ ++ + ++ 214 I 7 krusei ê¡WRJ 873 + Candida 121 I 7 CandifusaÍ¿ AWNI751 +- +- + + + + + 321 0 0 Cotdtfu saL¿ CBS 159 214 0 0 Caùitu *ellata CBS 157 +- +- + 429 I 1 Candida stellata CBS 2649 + +- ++ 1 + + ++ +- 429 l4 Del:k¿ra brutellezsis CBS 72 + 00 0 0 Ðel*zra bruxclle¡sis CBS 73 + ++ ++ ++ 750 5 36 Dehtcra bruxclleruis CBS 74 ; ;; ;; + ++ ++ ++ E57 4 29 De|era brwllez¡i¡ CBS 75 + + ; ++ + ++ ++ 429 3 2l Del:bra btucllezsis CBS 4914 ++ + + +- 857 0 0 wan¡n CBS 3 14 + + +- Hanseniospora :'_ ++ 321 I 7 Haweniaspora waruz AWRI 865 + + t: 17 0 0 Haucniospora wan¡¡ø AWRI 866 +- ,| + + + +- ++ 750 l4 Klulneromyce s t he rmotolerans CBS 2803 +- ++ + +- +- 750 1 7 thermotolerans CBS 6340 + +- ++ ., Kl4ncromyces 14 + + ++ 429 Met sclnikowia pulc henitru CBS 5 833 + t: +- ++ +- ++ ; ++ 53ó 4 29 PichianeJ,icoføCBS 5815 + +- + t57 0 0 cerevisiae AINRI' 729 ; + +- +- +- Saccluronryces + 79 I 7 + + + ;. ++ 11 Saccharonycu ceratisiae ATCC 46273 + + +- +- +- 1 + +- + ++ 643 t4 fucclaronyces cerevisiae CBS 1395 + + +- + + +- + ++ + ll 79 I 7 cerevisiae CBS 1907 + + +- +- +- Soccluronyces +- 536 I 7 Sacclummyces c¿revi¡i¿¿ AWRI 838 + + +- ++ + + +- + ++ t2 8ó I 7 fuccharomycodes ludwigii CBS E2l + + + +- ;: +- + ++ + ++ : 964 u l4 Tortlupora delbruechii CBS 817 + + +- +- +- + + ++ 964 4 29 delbrueckii CBS 1146 + + + +- ++ +- ++ 9 ) 0 t2 I t2 {. 13 4:_ 19 t4 4 0 t47 40 4t 11 20 6 I 14 1 l8 5 8 0 t4 I 21 l0 4 46 l9 50 15 73 54 15 0 40 #& ll Kllled 80 23 4 54 4 69 l9 3l 0 54 4 80 3s 40 t2 0 46

Table 3.4: The resistant/sensitive interactions between 26 tester strains and 14 killer yeasts; *: sensitive; -: resistant.

Percentage Killed (%o): the percentage of tester strains showing sensitivity to the killer yeasts' Figure 3.1

killer yeast P. membranifaciens CBS 7 37 4

non-killer yeast S. cerevisiae AWRI729

seeded lawn D. bruxellensis CBS 74

Figure 3.1: The agar plate killer diffusion assay. The killer yeast Pichia membranifaci¿ns CBS 7374 and the non-killer yeast Saccharomyces cerevisiae AWRI 729 (control) were innoculated (streaked) onto the YEPD-agar medium which had been seeded with Dekkera bruxellensls CBS 74. Killer activity is identified by a zone of growth inhibition fringed with a dark blue halo of dead cells. Table 3.5

pH 3.5 pH 4.5

Killer yeast No. of Vo No. of 7o sensitive sensitive strains strains

W. saturnus var. mrakii CBS 1707 I4 54 19 73

P. anomnla NCYC 434 9 35 2T 80

K.lactis var. drosophilarum CBS 2896 5 l9 18 69

P. membranifaciens CBS 7374 5 L9 T2 46

P. subpelliculosa NCYC 16 4 15 13 50

K.lactis var.Iactis CBS 2359 1 4 t4 54

P. anomala CBS 1982 1 4 t4 54

P. membranifaciens CBS 7373 1 4 T2 46

D. varijiae CBS 4072 1 4 6 23

K. marxian¡¿s NCYC 587 0 0 8 31

Z. bisporus A}VRI784 0 0 4 15

P. mernbranifaciens CBS 107 0 0 3 L2

C. glabrataNcYc 388 0 0 2 8

P. bispora CBS 1980 0 0 1 4

Total 4T 11 r47 40

Table 3.5: The effect of pH on the pattern of sensitivity/resistance to killer yeasts as assayed on buffered solid medium.

\ 22 the killer activity. The effect of pH on the killer activity of Willíopsis saÍurnrus var. mrakü CBS 1707 was least affected, with 14 of the original 19 tester strains remaining sensitive, making this the only yeast to kill more than 5OVo of the strains tested at the lower pH. Five of the 14 killer yeasts, Cattdída glabrafa NCYC 388, IQuyveromyces marxianus NCYC 587, Pichin bispora CBS 1890, Píchin membranifac¡¿zs CBS 107 and Zygosaccharomyces bísporus AWRI 784 failed to show killer activity at pH 3.5.

3.3.2 Yeast strain susceptibility

Of the 26 strains assayed at pH 4.5, Saccharomycodes ludwigii CBS 821 displayed sensitivity to the greatest number of killer yeasts, twelve of the 14 strains tested (Iable 3.6). Two strains of Saccharomyces cerevisiae, ATCC 46273 and CBS L9O7, were also highly susceptible, showing sensitivity to eleven of the 14 killer yeasts. Only one strain, Deld<¿ra bruxellensis CBS 73, was found to be resistant to all 14 killer yeasts. The effect of pH on the strains assessed for killer sensitivity varied greatly (Table 3.6). The th¡ee strains that were susceptible to the highest number of killer yeasts at pH 4.5, Saccharomyces cerevßiae strains ATCC 46273 and CBS 1907 and Saccharomycodes ludwigü CBS 82I, were resistant to all but one killer yeast at pH 3.5, Williopsis saturnus var. mrakü CBS 1707. The effect of pH on killer sensitivity was less apparent in other strains. The type strain of Dekkerabrw,cellensis, CBS 74, displayed sensitivity to seven and five killer yeasts at pH 4.5 and pH 3.5 respectively. At pH 3.5, Pichia mexicanø CBS 5815 remained sensitive to four of the five killer yeasts found to be sensitive at pH 4.5.

At pH 4.5, the number of killer-sensitive interactions involving the 13 testers strains whose origin were from the grape or wine ferment only, was 52 in total, whereas, the number of killer-sensitive interactions involving the remaining 13 tester strains totalled 95. This two fold difference in the number of killer-sensitive interactions involving tester strains which originated from grape or wine ferments, and the tester strains that did not, was observed also for pH 3.5. Four of the five strains of yeast that were found to be resistant to all fourteen killer yeasts at pH 3.5, Candidn stellata CBS 157, Deklcera bruxellensis CBS 73, Hanseniaspora uvarurz AWRI 866 and Saccharomyces cerevisiae AWRI 729, were originally isolated from wine grapes or wine ferments.

3.3.3 Intraspecific differences in killer activity

Intraspecific variation in killing range of strains of a species was investigated with two killer strains of Píchia atnmala and Kluyveromyces lactis, and three killer strains of Pichia membranifaciens by assaying for their killing activity towards the 26 tester strains. These yeasts and strains were choosen for furtherinvestigation based on the known killer activity. Table 3.6

Killer yeast sensitivity Tester strain pH 3.5 pH 4.5 D. bruxellerzsis CBS 74 5 7 T. delbrueckiiCBS LI46 4 9 D. bruxellensis CBS 75 4 8 P. tnexiccut¿ CBS 5815 4 5 D. bruxellerzsis CBS 4914 Ja 4 T. delbruecktt CBS 817 2 9 K. tlrcrnrctol.erans CBS 2803 2 7 S. cerevisia¿ CBS 1395 2 6 C. kruseiAWRI 863 2 4 D. bruxellensis CBS 72 2 4 M. pulchenimaCBS 5833 2 4

S.Iudwigii CBS 821 1 12

S. cerevisiae ATCC 46273 1 11

S. cerevisia¿ CBS 1907 1 11

K. thermotolerans CBS 6340 1 7

S. cerettisict¿ AWRI 838 1 5 C. stellata CBS 2649 I 4

C. sake AWRI 751 1 3

H. L;urunt AWRI 865 1 J

C. krusei AWRI 873 1 2 H. t.ntúrturt CBS 314 0 8 S. cerettisia¿ AWRI729 0 8 C. sake CBS 159 0 3 C. stellcttct CBS 157 0 2 H. uvaruutx AWRI 866 0 I D. bn.txelle¡zsls CBS 73 0 0

Total 4I 1 41

Table 3.6: Sensitivity of tester strains at pH 3.5 and pH 4.5. Tester strains originally isolated from wine grapes or ferments are highlighted in red. 23

The killing range of Pichiamembranifaci¿ns strains CBS 7373 and CBS 7374 was the same at pH 4.5, killing the same twelve yeasts (fable 3.7). The third Pichin membranifaciens sffain, CBS 107, had a more limited killing range however, displaying activity against three tester strains only. These three sensitive strains, Deld

As for the intraspecific variation in the killing range of Pichia membranífaciens strains, the killing range of Kluyveromyces Lactis var. lactis CBS 2359 was a subset of that for Kluyveromyces hcrts var. drosophilarum CBS 2896, both at pH 3.5 and pH 4.5 (Iable 3.8). Although the different killer patterns were compatible with the two different varieties, to date there exists no genetic defînition for separating the species Kluyveromyces lactis into two varieties, with the division based solely on the yeast's ability to assimilate lactose (Kurtzman and Fell, 1998).

The two Pichin unmala strains differed in their killing range in that each strain displayed activity towards yeasts the other strain did not (Iable 3.9). This example of intraspecific variation in killer activity differs to that of Kluyveromyces lactß and Pichiø membranifaciens,whereby the killing range of one strain was a subset of the other. At pH 4.5 Pichia anomala CBS 1982 displayed killer activity towards two strains that Pichia atwmalaNcYc 434 dtd not, Candida krusei AWRI 873 and Metschnikowia pulcherrimt CBS 5833. Whereas Pichia anomaln NCYC 434 could kill several strains Pichin arcmaln CBS 1982 did not, such as Candida kruseí AWRI 863, Candi¿n søk¿ AWRI 75I, Candida stellata CBS 2649, Hanseniaspora uvarum AWRI 866, and several strains of Deld

3.3.4 Intraspecifïc differences in killer susceptibility

Of the tester strains studied, it is apparent that strains of the same species have different patterns of sensitivities to the 14 killer yeasts. The two strains of Issatcltenkia orientalis, Candida sake, Cøndida stellata, and Kuyveromyces thermotolerans, and the three strains of Hanseniaspora uvarum all displayed a different resistance/sensitivity pattern (table 3.4). Infaspecific differences in killer susceptibility were further investigated by determining the susceptibility of several strains of Del

Killer yeasts P. membranifaciens P. m¿mbranifaciens P. mcmbranifaciens Tester strains CBS 107 CBS 7373 CBS 7374 pH 3.5 pH 4.5 pH 3.5 pH 4.5 pH 3.5 pH 4.5 D. bnnellensrs CBS 74 + + + + + D. bruxellensrs CBS 75 + + + D. bruxellensis CBS 4914 + + + H. uvarum CBS 314 + + K. thermotolerans CBS 2803 + + K. thermotolerans CBS 6340 + + S. cerevisia¿ ATCC 46273 + + S. cerevisia¿ CBS 1395 + + S. cerevisia¿ CBS 1907 + + + S.ludwigii CBS 821 + + + T. delbrueckit CBS 817 + + + T. delbruecktt CBS 1146 + + +

Total Killed 0 3 1 T2 5 T2 Percentage Killed (7o) 0 25 8 100 42 100

Table 3.7: A comparison of the killing patterns of the three strains of Pichia membranifaciens. 24 flavour and volatile phenols (Sponholz 1993), these five yeasts were chosen because their identification and genetic similarity has been confirmed using different techniques @oekhout et aI. 1994; Yamada et al. 1994). Each strain of Dekl

3.3.5 Reduction of killer yeast activity in an acidic environment

The level of sensitivity of the four tester strains Delclczra bruxellensis CBS 74, Pichin mexicana CBS 5815, Saccharomyces cerevisiae CBS 1395 and Torulaspora delbrueckü CBS 817 were assayed to the two killer yeasts Pichie anamala NCYC 434 and Pichia subpelliculo,sa NCYC 16 using the agar plate killer yeast assay buffered at pH 4.5 and pH 3.5 (Table 3.10). These yeasts were chosen randomly for further investigation.

All four tester strains displayed the same medium level of sensitivity to Pichin arcmala NCYC 434 (table 3.10). At pH 3.5, however, the sensitivity of these four þster strains varied. At this lower pH both Saccharomyces cerevisiae CBS 1395 and Torul'aspora detbrueckä CBS 817 were resistant to Pichia anomaIaNCYC 434, whereas Pichia mexicana CBS 5815 displayed a weak level of sensitivity. Del

This variation in the reduction in killer yeast sensitivity due to a change in the pH level was not confined to the one killer yeast (Iable 3.10). Both Pichia mexicana CBS 5815 and Saccharomyces cerevisia¿ CBS 1395 displayed a strong level of sensitivity to Píchia subpelliculos¿ NCYC 16 at pH 4.5. At pH 3.5, however, the sensitivity level of Pichia mexicanacBs 5815 was reduced to + (up to 1.5 mm), whereas Saccharomyces cerevisiac CBS 1395 was found to be resistant. Torulaspora delbrueckü CBS 817 also showed resistance to Pichia subpelliculo,sa NCYC 16 at pH 3.5, when it had previously shown greater sensitivity (++, 1.5 - 2.5 mm) at pH 4.5. Like its sensitivity to Pichin arcmala NCYC 434, Dekkera bntxellensis CBS 74 displayed no change in ie level of sensitivity to Pichia subpelliculosø NCYC 16 when assayed at a lower pH.

3.3.6 Assaying bacteria susceptibility to killer yeasts Table 3.8

Killer yeast

K. lactis var.lactis K. lactis var. drosophilarum Yeast tester strain CBS 2359 CBS 2896 pH 3.5 pH 4.5 pH 3.5 pH 4.5

C. krusei AWRI863 + + + + C. sake A}VRI751 + +

C. stellata CBS 157 + + C. stellsta CBS 2649 + + + D. bruxeller¿sis CBS 75 + + H. uvarum CBS 314 + H. uvarum AWRI865 + + K. thermotolerans CBS 2803 + + K. thermotolerans CBS 6340 + M. pulcherrimaCBS 5833 J- +

S. cerevisiae AWRI729 + +

S. cerevisiae ATCC 46273 + +

S. cerevisia¿ CBS 1395 + +

S. cerevisiø¿ CBS 1907 + +

S. cerevisiaø AWRI838 + + S. Iudwigü CBS 821 + + T. delbruecftit CBS 817 + + T. delbruecfrti CBS 1146 + + +

Total killed 1 I4 5 18 Percentage killed (7o) 6 78 28 100

Table 3.8: Kluyveromyces lactis var. drosophílarum CBS 2896 displayed a greater killer range at both pH 3.5 and pH 4.5 as compared to Kluyveromyces lactis var. lactis CBS 2359. Table 3.9

Killer yeasts Yeast tester strain P. anomala CBS 1982 P. anomalaNcYc 434 pH 3.5 pH 4.5 pH 3.5 pH 4.5 C. krusei AWRI863 + C. krusei AWRI873 + C. sake A\ryRI751 + + C. sake CBS 159 + + C. stellataCBS2649 + D. bruxellensis CBS 72 + + + D. bruxellensis CBS 74 + + D. brwrellerzsis CBS 75 + + D. bruxeller¿sis CBS 4914 + + H. uvarum CBS 314 + + H. uvarum AWRI866 + K. thermotolerans CBS 2803 + + + K. thermotolerans CBS 6340 + + + M. pulcherrimaCBS 5833 + P. mexican¿ CBS 5815 + + +

S. cerevisia¿ AWRI729 + +

S. cerevisia¿ ATCC 46213 + +

S. cerevisia¿ CBS 1395 +

S. cerevisia¿ CBS 1907 + +

S. cerevísia¿ ECl118 + +

S.Iudwigü CBS 821 + + T. delbruecfrit CBS 817 + + T. delbrueckü CBS 1146 + +

Total Killed 1 1 4 9 2I

Table 3.9: Killing pattems of the two Pichia arnmala strains. Yeast sensitivity to Pichin atnmala NCYC 434 or Pichin anomnla CBS 1982 only, for either pH 3.5 or pH 4.5, are highlighted in different colours. Table 3.10

Killer yeast

Tester strain P. anomalaNcYc 434 P. subpelliculosa NCYC 16

pH 4.5 pH 3.5 pH 4.5 pH 3.5

D. bruxeller¿sis CBS 74 ++ ++ + +

P. mexicanø CBS 5815 ++ + +++ +

S. cerevisia¿ CBS 1395 ++ +++

T delbruecktt CBS 817 ++ ++

Table 3.10: The level of sensitivity of Dekl

The killer activity was scaled according to the radius of the killer halo (mm) surrounding the tester strarn: - : no visible sign of killer activity + : weak killer activity (up to 1.5 mm) ++ : medium killer activity (1.5 - 2.5 mm) +++: strong killer activity (more than 2.5 mm) 25

To determine whether killer yeasts could be inhibit the growth of bacteria strains commonly found in wine fermentation, a solid agar bacteria-killer yeast assay was developed. Developing the assay involved devising a medium that would sustain the growth of both the killer yeasts and bacterial strains. This investigation included bacterial strains of Oenococcus, Lactobacillus, Pedicoccus, Leuconostoc and Acetobacter (lable 3.3), and the four killer yeasts Williopsis saturnus var. mraki, CBS I7O7, Pichia atwmala CBS 1982, Delclæra v anrij iae CB S 4072 and P ichia membranifaciens CBS 7 37 4.

Initially, a medium comprising of the known growth requirements for both the bacterial strains and killer yeasts was trialed. This medium, referred to as Medium One (Figure 3.2), was found to sustain the growth of all 14 strains of bacteria and the four killer yeasts. At pH 3.5, however, Medium A inhibited the growth of three of the four killer yeasts: Williopsis saturnus var. mrakü, Pichin anomnla andDebaryomyces vanrijiø. By the process of omitting various medium components (see Medium Two and Three, Figure 3.2),it was found that the sodium acetate ÍO.SVo (w/v)l present in the MRS supplement (Amyl Media Pty. Ltd.), a supplement required for growth of the bacterial strains under test, was the cause of growth inhibition.

The MRS supplement, when added to the medium at the recoÍtmended dosage, gives 0.02Vo (w/v) magnesium sulphate, 0.005Vo (w/v) manganese sulphate, O.l%o (vlv) sorbiton mono- oleate complex and0.57o (w/v) sodium acetate. Eliminating the MRS supplement from the medium, while adding trace amounts of magnesium sulphate, manganese sulphate and Tween 80 (Medium Four), allowed the growth of both bacteria and the killer yeasts (Figure 3.2). This same medium supported Pichiamembranifaci¿ns CBS 7374ktller activity towards the sensitive Deklcera brwellensi.s CBS 74 at comparative level as the yeast medium employed in3.2.2. The use of Medium Four was preferred over that of Medium Two for the benefit of bacærial growth. Employing Medium Four for the bacteria-killer yeast sheak assay, it was found that at both pH levels, the four killer yeasts failed to display activity towards any of the fourteen bacterial strains.

3.4 DISCUSSION

Ecological studies on the role of killer sensitive interactions indicate that sensitive interactions occur frequently among yeasts isolated from different localities and habitats (Maule and Thomas 1973; Imamura et at. 1974; Stumm et al. 1977; Starmer et al. 1987; Motus et al. 1995; Vadkertiova and Slavikova 1995). With the aim of identifying a yeast with broad killer activity towards yeast strains found indigenous to the wine ferment, the activity of nine killer yeasts was investigated. These yeasts were originally isolated from habitats other than wine grapes orferments (Iable 3.1). Another six killer yeasts were also employed in this study, Figure 3.2

Medium One Medium Two Medium Three Medium Four

Yl\d Y\4 Y]VI Yl\d Íz so (wtv)f Íz qo (wtv)f lzEo (wtv)l lz so (wtv)) MRS MRS MRS MRS f+.e uo çvtv¡l l+.a vo lvtv¡l Í+.0 co (vtv)l Í+.e so (vtv)l Apple juice Apple juice Apple juice Apple juice lzo co (vtv)f lzo co (vtv)) lzo qo (vtv)f Ízo co (vtv)l MRS supplement Methylene bluex Sodium acetate Magnesium sulphater l+.+ so (vtv)f lo.s co (wtv)l Methylene blue* Yeast grolvth Magnesium sulphatet Manganese sulphate$ Gowth inhibition Manganese sulphate$ Tween 801

Tween 801 Methylene blue*

Methylene blue* Yeast grorvth Gowth inhibition

Figure 3.1: Identification of 0.5 Vo (wlv) Sodium Acetate as the component of the MRS supplement which inhibited the growth of Williopsis saturnus var. mrakü, Pichia anomnla CBS 1982, Pichia membranifaciens CBS 7374 and Debaromyces vanriiiae CBS 4072. on media buffered at pH 3.5. * Methylene blue was added to a final concentration of 0.003 Vo (wlv) t Magnesium sulphate was added to a final concentration of 0.02 Vo (wlv) $ Manganese sulphate was added to a final concentration of 0.005 7o (wlv) $[Tween 80 was added to a fînal concenffation of 0.1 Vo (vlv) 26 five of which originated from unknown sources, and the sixth, a killer yeast sourced from a wine ferment.

Previous studies on the bio-control of indigenous yeasts, have focussed solely on Saccharomyces cerevisiae, which is only one of the many yeasts species responsible for wine spoilage (Boone et a1.,1990a; Martini and Martini, 1990; Seki ef al., 1992; Sulo et aI., 1992; Mchalcakova et al., 1994). To further extend these studies, six Saccharomyces cerevisine and 2L non-Saccharomyces cerevisìae yeast strains representative of the indigenous yeast wine ferment micro-flora (Table 3.2), were assayed for their sensitivity to the 14 killer yeasts (Table 3.4).

The dependence of killer activity on pH was first observed by Woods and Bevan (1968). Since then the optimum pH for killer activity has been defined for a number of killer toxins, with the majority of them having an optimum pH between 4.2 and 4.7 (Rogers and Bevan 1978; Hens cltke, 1979; Pfeiffer and Radler 1984; Magliani et al., 1997). On plates buffered topH 4.5, atotalof L47 of apossible364killer-sensitiveinteractionswereobservedwith the yeasts studied (table 3.4). Pichia atnmala NCYC 434 (ongin unknown) displayed the greatest killing range (table 3.5), closely followed by Williopsis saturnus var. mrakü CBS 1707 (soil) and Kluyveromyces l.actis var. drosophilarum CBS 2896 (slime flux). The killer yeast known to be indigenous to the wine ferment, Zygosaccharomyces bisporus AWRI 784, showed the least activity, killing only one of the 26 yeast strains tested. Candida glabrataNcYc 388, noted for its limited activity and for its broad sensitivity to killer toxins (Young and Yagiu L9/8;Starmer et al. 1987;Bonilla-Salinas et al. 1995; Morais et al. 1995; Abranches et at. L997). displayed killer activity to two of the 26 strains.

Adjusting the pH of the solid agar diffusion assay to pH 3.5 in this study resulted in a72%o decline in total killer-sensitive interactions. Furthermore, in no c¿rse was there a killer- sensitive interaction at pH 3.5 that did not occur at pH 4.5. Of the three yeasts showing a broad killing range at pH 4.5,Wiltiopsis saturnus var. mraktt CBS 1707 displayed the most resilience to the pH change, remaining active to more than 50Vo of the 26 strains tested. The Williopsis saturnus var. mrakü CBS 1707 killer toxin shows strong homology to the Witliopsis satumus var. mrakü IFO 0895 toxin (tIM-l) (Kimura et al. 1995). The HM-l toxin exhibits exceptional temperature and pH stability, attributed to the many disulfide bonds present in the cysteine rich protein (Yamamoto et aI.1986b; Yamamoto et al.1988).

At pH 4.5 Saccharomycod.es lud.wigü CBS 821 was found to be the most susceptible yeast' showing sensitivity to twelve of the 14 killer yeasts. The change in pH, however, reduced this susceptibility to just one. This reduction in susceptibility is evident for all tester strains, although the sensitivity of strains Deldæra brwcellensis CBS 74 and Pichia mexicana CBS 5815 appeared less affected by pH. The simplest explanation for the reduction in sensitivity 27

is that the activity of the killer toxin is reduced in the acid media. However, the reduction in sensitivity due to the change in the pH was found to differ for each tester strain (Table 3.10). This is best illustrated with the observation that the four tester strains Delclæra bnnellensis CBS 74, Pichia mexicanacBs 5815, Sacchnromyces cerevisi¿¿ CBS 1395 and Torulnsporø delbrueckü CBS 817 displayed the same medium level of sensitivity to Pichin arcmala NCYC 434 at pH 4.5. At pH 3.5 the level of sensitivity of Pichia mexícata CBS 5815 to Pichia arcmaln NCYC 434 was reduced to a weak level, both Saccharomyces cerevßiac CBS 1395 andTorulaspora delbrueckit CBS 817 displayed resistance, and yet the level of sensitivity of Del

Of the 15 tester strains that displayed susceptibility to one or less of the killer yeasts at pH 3.5, ten originated from wine grape or ferments thereof (table 3.6). These results suggest that strains from different localities sharing the same habitat, such as wine ferments, can in general be more resilient to killer activity, as compared to strains of the same species originating from different environments (Yap et a1.,2000). This is further illustrated with the observation thatWilliopsis saturnus var. mrak t CBS 1707 displayed killer activity to 3OVo of the tester strains originating from wine ferments and grape berries, and activity to7l%o of the tester strains originating from other sources (Table 3.4). A more comprehensive investigation, involving a greater sample size of strains sourced from wine ferments and other known environments, would need to be undertaken to confirm this observation.

Strain differences in susceptibility to killer toxins have been established for a number of natural yeast communities. The pleiotropic nature of killer susceptibility is not unexpected if we consider an environment where killer yeasts are present, giving rise to strong selective pressure for resistant strains (Starmer et al. 1987;Vadkertiova and Slavikova 1995). Walker (1995) has proposed that resistance may be conferred by acquiring the ability to secrcte exffacellular protease, which could degrade proteinaceous killer toxins. Of the 26 tester strains employed in this study, only Sacchnromyces cerevisiae CBS 1907 has previously been shown to have extracellular protease activity (Rosi, 1937). However, of the five Saccharomyces cerevísiae tester strains investigated, Saccharomyces cerevisiae CBS 1907 displayed sensitivity to the most number of killer yeasts (Table 3.4).

In systems that have been studied genetically, it is apparent that mutations in one of a number of genes can confer resistance towards a killer yeast - many of these affecting the cell wall toxin receptors. It is likely that mutations in these different genes, or even different mutations in a single gene, will give rise to the observed variation in toxin susceptibility (Hutchins and 28

Bussey 1983; Finkler et aI. L992; Takita and Castilho-Valavicius 1993; Butler et aI. 1994; Hong et at. 1994). Apan from acquiring resistance to killer, the consequence of the modification to the yeast's ability to compete in it's environment remains to be determined. Acquiring resistance may impose negatively on'the yeast's fitness, analogous to the reduction in fitness seen in bacteria when acquiring bacteriocin, antibiotic or phage resistance (Riley 1998).

Yeast taxonomists have previously exploited this phenomenon of strain differential sensitivity for the typing of pathogenic yeast strains of , Candida glabraÍa, Candidú parapsilosis, Can¿inn pseu.dotropicalis, Can¿ida tropícalis and Cryptococcus neoþrmans @olonelli et aI. 1983, Morace et aL L984). Vaughan-Martini et al (1996) employed the use of 24 killer yeasts, belonging to 13 species of six genera, to differentiate eleven wine yeast strains of Saccharomyces cerevisiae. In this study we \ryere able to successfully differentiate strains of Deld

Intraspecific differences in killer performance, although well documented, remain poorly understood (Young and Yagiu 1978; Polonelli et aI. 1983; Morace et al. 1984;Walker et al. 1995; Vaughan-Martini et at. 1996). The two Pichia atnmaln killer strains CBS 1982 and NCYC 434 exhibited very different killing abilities (Table 3.9), suggesting that the toxins of each strain are biochemically distinct. This would be similar to that observed in Saccharomyces cerevisiae where four killer types have been identified, each expressing a biochemically distinct toxin. These four killer toxins, which are either chromosomally encoded or encoded by cytoplasmic double-stranded RNA viruslike particles, bear no sequence similarity to each other and kill cells by different mechanisms. For example, the K1 toxin forms membrane pores resulting in the loss of ions and cellular metabolites (Martinac ef at. 1990) and the K28 toxin blocks DNA synthesis (Schmitt et aI. 1989).

Strain variation in killing range was also observed with the Kluyveromyces lactis and Pichia membranifaciens yeasts. For these two species, unlike the Pichia arcmala yeasts, the tester strains susceptible to the less potent killer yeast are a subset of the sensitive strains killed by the more effective yeast. The different killing ranges of these yeasts may be attributable to the strains expressing biochemically distinct toxins. For Kluyveromyces lactis this appears likely, since the genetic basis of the killer system of Kluyveromyces lactß vat. lactß CBS 2359 is encoded by linear DNA plasmids, whereas the Kluyveromyces Incrts var. drosophilarum CBS 2896 killer system appears to be nuclear encoded (Gunge and 29

Sakaguchi 1981; Gunge et al. L98l;Fukuhara 1995). The finding could also be explained if a yeast expressed more than one toxin, although the expression of multiple toxins has not been observed in species where the killer phenotype has been well studied. Alternatively, the strains analysed might share the same toxin, with the distinct killer pattern being atftibutable to different levels of expression or secretion. Finally, modification of the toxin protein itself, for example by differential , may affect its specificity.

The killing range of Pichin membranifaci¿¿s CBS 7373 and CB,S 7374 are the same at pH 4.5, and only at pH 3.5, was the killing range of Pichia membranifaciens CBS 7373 found to be a subset of Pichia membranifaciens CBS 7374.In similar experiments Polonelli et al. (1983) increased their resolution of strain differentiation, by employing variations in ttre media and temperature requirements of their killer assay. Divergence of the gene sequences encoding the CBS 7373 and.CBS1374 toxins could generate differences in their stability under varying growth conditions. As a consequence a change in the environments pH would be reflected by differences in the killing range of the strains. The intraspecific differences in killer activity of strains of Pichia membranifaciens are further investigated in Chapter 4.

The possibility of killer yeasts antagonising the growth of bacterial strains found indigenous to the wine ferment was investigated. Included in this panel of bacterial strains were strains of the Gram positive species of Pediococcus, I-actobacillus and Oenocoocus and a Gram negative species Acetobacter pasteurianus.Líke the indigenous yeasts of the wine ferment, these bacterial strains can contribute to wine spoilage when grown in sufficient numbers (Iable 3.3). Common winemaking taints and faults associated with lactic acid bacteria include acidification during alcoholic fermentation, ropiness and both mannitol and mousy taint. Acetic acid bacteria are primarily responsible for vinegary taint (Sponholz, 1992; Bartowsky and Henschke, 1995).

The aforementioned bacterial strains of Table 3.3 were assayed against the four killer yeasts, employing the agar bacteria-killer yeast assay. Initially, a constituent of the MRS supplement required for growth of the bacterial strains, was found inhibitory to the four killer yeasts. The offending constituent, was omitted from the medium while still maintaining trace elements of magnesium sulphate, manganese sulphate and Tween 80 @gure 3.2). All four killer yeasts failed to display killer activity to any of the 14 Gram positive bacterial strains, or the Gram-negative Acetobacter pasteurianus AbL, at either pH 3.5 or 4.5.

To generate a wine yeast with effective killer capacity it may be necessary to express more than one killer toxin. This investigation revealed that the combination of the killer yeasts Pichin arcmaln NCYC 434 and Williopsis saturnus var. mrakü CBS 1707 would see a combined killing range of over gOVo at pH 4.5. However, due to the ineffectiveness of the killer yeasts at a reduced pH, this combined killing range would be reduced to less than 6OVo 30 at pH 3.5. Other factors of the wine ferment, such as high titatable acidity, proûeolytic activity, and binding to grape derived phenolic compounds may further reduce killer activity. Tlne Williopsis saÍurnus var. mrakü toxins FIY1 and HM-l have been noted for their exceptional temperature and pH stability, features which make them attractive for use as new food preservatives (Yamamoto et aI. I986b; Kimura et al. L993; Komiyama et al. 1995; Lowes et al., 2000). With the increased understanding of the molecular interactions that maintain protein structure at low pH (Mortensen and Breddam, L994), it may be possible to engineer killer proteins which maintain activity at wine pH. Combining these strategies may provide a yeast with a broad killer range at an acidic pH. 31

Chapter 4

MOLECULAR TYPING OT PICHIA MEMBRANIFACIENS STRAINS AND THEIR KILLER ACTIVITIES

4.L INTRODUCTION

To date there exists a wealth of information on the killer phenotype for a number of yeast species, however, there is limited information available regarding the killer phenotype of Pichiamembranifaciens. A frlm-forming yeast known for spoiling various food stuffs and beverages, the growth of Píchia membranifaciens in wine can result in various off-odours, such as acetyaldehyde andethyl acetate (Rankine, 1966 Thornton, I99I; Sponholz, 1993). This chapter aims to further investigate the strain characteristics of Pichiamembranifaciens.

The first aspect of this study was to investigate the diveristy of ten strains of Pichia membranifaciens, using an intron-PCR fingerprint technique which has previously been shown to provide a differentiation of strains of other yeast genera (de Barros I-opes et aI. 1993). The application of this molecular typing method, for the identifîcation of strains of Pichia membranifaciens inthe food and beverage industries, is further discussed.

The second aspect of this study was to investigate the killer activities of the ten Pichia membranífaciens sftains, since the genetic basis of this character has not previously been characterised. This investigation revealed that the ten strains could be divided into four sepa.rate killer classes, based on their individual killing patterns. Likewise, previous studies have revealed that strains of the killer yeast Saccharomyces cerevisiae couldbe classified into different classes, based on their killing activity and immunity towards each other (Rogers and Bevan, 1978; Young and Yagiu, 1978). Each killer class of Saccharomyces cerevisiae were found to express a unique toxin, which is encoded by dsRNA viruslike particles (Magliani, IggT).In contrast, this study revealed that the killer phenotype of one Píßhin membranifacíer¿s killer class is nuclear encoded, whilst strains of the other Pichia membranifacienskiller classes were found to harbour extrachromosomal elements.

4.2 EXPERIMENTAL

4.2.I Yeast strains and media

The Pichiq membranifaciens andtester strains employed in this study are listed in Tables 4.1 and 4.2, respectively. Strains were sourced from the following culture collections: the Table 4.1

P. mernbranífacíens Original Country of Origin Substrate strain

AWRI443 unknown unknown

AWRI T5O unknown unknown

A\ryRr 752* wine Barossa Valley, Australia

ATVRI753* wine Barossa Valley, Australia

AWRI745* wrne B arossa Valley, Australia

AWRI732 wlne Berri, Australia

CBS 1O7T unknown unknown

CBS 638 wlne Germany

CBS 7373 draught beer unknown

CBS 7374 druaght beer unknown

Table 4.1: The Pichiamembraniþcines strains employed in this investigation. * denotes strains isolated from the same wine. t Strain CBS 107 is the type strain of Pichia membranifaciens. 32

Centraalbureau voor Schimmelcultures (CBS), Delft, The Netherlands, and The Australian Wine Research Insti¡¡te (AWRI), Adelaide, Australia.

Yeast strains were subcultured in YEPD tl% (w/v)Veast extract,2To (w/v) peptone,2Vo (w/v) glucose] at 25oC for 2448 h before being transferred to YEPD-agar [YEPD,27o (wlv) agarl slopes at 25oC for 48 h, then stored at 4oC until required. For YEPD-agar containing cycloheximide, the appropriate dilutions of 0.17o (w/v) cycloheximide (made up in 95Vo ethanol and stored in the dark) were added to cooling YEPD-agar.

4.2.2 Total nucleic acid preparations

Total nucleic acid extracts to be used in enzyme digests is essentially that of Gunge et al. (1994). An exponentially growing yeast culture was streaked onto YEPD-agar, and incubated at25oCuntil cell growth became visible. Approximately 0.1 g of yeast cells were washed in 400 pl of protoplast buffer (0.8 M KCl, 10 mM EDTA, 0.1 M KHzPO+, 0.1 M Na2HPO4, buffered to pH 7.0 using sodium hydroxide), and then resuspended in 400 pl of protoplast buffer. Cell were converted to protoplasts with the addition of 25 pl of a zymolase solution lIVo (wlv) zymolase (Sigma),50 mM sodium phosphate buffer pH 7.5, 5O7o (vlv) glyceroll and O.57o (v/v) p-mercaptoethanol (Sigma), and incubated at RT for 30 minutes. Protoplasts were washed and resuspended in 400 pl of protoplast buffer, and then lysed by (Sigma), incubating at the addition of 40 ¡t"l of LOVo SDS and 2 ¡t"l of 2Vo (wlv) proteinase K 37oC for 30 minutes. This incubation was followed directly by a second incubation at 65oC for 15 minutes. The cellular debris were pelleted by centrifugation for 2O minutes at 13,000 rpm. The Supernatant was retained, and an equal amount of phenol:chloroform:isoamylalcohol (25:24:I) was added, mixed by inversion, and centrifuged for 3 minutes at 13,000 rpm. The top layer was removed to a fresh tube, where the nucleic acids were precipitated by the addition of a one in ten volume of 3 M sodium acetate and two volumes of 70Vo (v/v) ethanol, incubating at -2OoC for 60 minutes. The nucleic acids were pelleted by centrifugation at 10,000 rpm for 10 minutes at4oC, desiccated using a speedi-vac, and resuspended in 100 pl of TE buffer (10 mM Tris-HCl pH 8.0, 1 mMEDTA).

4.2.3 Mini-preparations of nucleic acids

Mini-preparations of nucleic acids (Cong et aI., L994) from Pichin membranifaci¿ns CBS 7374 wereundertaken, for the quick identification of pPM01 post curing treatments. A L-2 mm sized colony was suspended in 10 pl of a zymolase solutiot l47o (w/v) zymolase (Sigma), 50 mM Tris-HCl pH 8.0, 5 mM EDTA), and incubated for 60 minutes at 30oC. One microlihe of SVo (wtv) SDS and 3 ¡rl of 27o (wtv) proteinase K (Sigma) were then Table 4.2

Tester Strain Source (Strain)

D. bruxellensis CBS 74 CBS

D. bruxellensis CBS 75 CBS

D. bruxellensis CBS 4914 CBS

S. cerevisiae CBS 1907 CBS

S. cerevisia¿ AWRI 1360 I. Dawes* (Y3)

S. cerevisia¿ AWRI 1361 I. Dawes (Y3P)

S. cerevisia¿ AWRI 1363 I (rvv¿gz)

S. cerevisia¿ AWRI 1365 R. Davis$ (YP52)

S. cerevisiae AWRI 1367 (638)

S. cerevisiae L5da

S. cerevisia¿ 15d q

S. cerevisiae Jl6D

S. cerevisiaeYMl2l

S. cerevisiae Y357

S'codes ludwigii CBS 821 CBS

Table 4.2: The tester strains employed in this study. * Dr Ian Dawes, The University of New South Wales, Australia. t Dr Miguel de Barros Lopes, The Australian Wine Research Institute, Australia. $ Dr R Davis, Stanford University, USA. CBS - Centraalbureau voor Schimmelcultures (CBS), Delft, The Netherlands, Australia. 33 added prior to a 60 minutes incubation at 65oC. The cellular debris were centrifuged for 3 minutes at 13,000 rpm, and 15 pl of the supernatant removed for gel electrophoresis.

4.2.4 Nucleic acids gel electrophoresis

Nucleic acids were resolved by submerged, horizontal gel electrophoresis. One part of DNA gel-loading buffer l0.25%o (w/v) bromophenol blue,4O7o (w/v) sucrosel was added to five parts of the nucleic acid sample prior to loading. Samples were electrophoresed in a O.8Vo (w/v) agarose gel @harmacia Agarose NA) [27o (w/v) agarose for PCR products] immersed in TAE running buffer (40 mM Tris-HCl, 20 mM sodium acetate, 2 mM EDTA, buffered to pH 7.8 using glacial acetic acid), for approximately 60 minutes at 65 volts. After electrophoresis, gels were stained with ethidium bromide for 15 minutes (0.5 pglml), and visualised using a UV light box (254 nm). Gels were photographed with a Gel Cam Documentation System (Sony), and the photographs \ryere scanned to produce computer images (Hewlett-Packard Scan Jet 1ICXIT), using the Adobe Photoshop software program (Adobe Systems).

4.2.5 Treatment of total nucleic acids with DNase 1 or Ribonuclease A

DNase digests of total nucleic acids were performed in 20 ¡rl, with 5 pl of total nucleic acid extracts from Pichia membranifaci¿¿s CBS 7374 and O.2 ttg of DNase 1 (Promega), and incubated at37oC for 60 minutes. Ribonuclease (RNase) digests of total nucleic acids were performed in 20 pl, with 5 pl of total nucleic acid extracts from Pichia membranifaciens CBS 7374 and 0.4 pg of RNase A (Boehringer Mannheim), and incubated at 37oC for 60 minutes. Both types of digests were resolved by gel electrophoresis.

To determine whether the RNA extrachromosomal element of Pichía membraniþci¿zs CBS 7374 was single-stranded (ss), or double-stranded (ds), total nucleic acid extracts were treated with RNase A in varying salt concentrations. A 5 pl sample of total nucleic acid extracts ftom Pichia membranífaci¿ns CBS 7374 were incubated at 37oC for 60 minutes, with 5 ng of RNase A, in the presence of 0.2 M, 0.4 M or 0.6 M NaCl. Treated nucleic acid extracts were then resolved by gel electrophoresis.

4.2.6 Cycloheximide treatment of Pichíø membranifaciens CB,S 7374

The cycloheximide teaftnent employed in this study is essentially that of Fink and Styles (1972). An exponentially growing culture of Pichia membranifaci¿ns CBS 7374 was diluted to 1x107 cells/ml, then serially diluted onto either YEPD-agar plates containing cycloheximide (Sigma), at concentrations of 0.5 mg/I, I mdl and 5 mg/I, or without cycloheximide (control). Plates were incubated at 25oC for five days. A total of 96 colonies 34 arising from the treatment of 1 mg/l of cycloheximide, were randomly selected from plates, and assayed for pPM01, using the method outlined in Section 4.2.3.

4.2.7 Ultra-violet light treatment of Píchia 4lnembrønífacíens CBS 7374

The ultra-violet (uV) light treatment employed in this study is essentially that of Worsham ¿r ø1. (1983). An exponentially growing culture of Pichia membranifac¡ens CBS 7374 was diluted to 5x107 cellVml using an isotonic saline solution lO.85Vo (ilv) NaCll, and irradiated for period of 1 minute, 10 minutes, 20 minutes, or not at all (control), using a Gelman UV germicidal lamp (254 nm). Exposure to the UV light was improved by magnetic stirring of the yeasts as a thin liquid, in a sterile 100 mm petri dish. Treated cultures were serially diluted, before being plated onto YEPD-agar for viable counts. A total of I2O colonies arising from the 20 minute treatrnent were randomly selected from plates, and assayed for pPM01, using the two method outlined in Section 4.2.3.

4.2.8 PCR-intron fÏngerprint technique

The PCR-intron fingerprint technique employed in this study is essentially that of de Barros Lopes et al. (1996). For each Pichin membranifaciens strun of interest, DNA was prepared using a fræzplboil method. Liquid cultures were streaked onto YEPD-agar plates for single colonies, incubated at 25oC for 3 days, and then stored overnight at 4oC. A single yeast colony was then resuspended in 100 pl of sterile water, frozen in liquid nitrogen, and then boiled for 10 minutes.

PCRs were performed in 50 pl with 50 pmol of the intron primer 5'-

CTGGCTTGGTGTATGT-3', 2 ¡t"I of ttre DNA template, 32 ¡t"M of each dNTP, 2.5 mM MgCl, and 0.2 lJ of Taq polymerase (Advanced Biotechnology). The reactions were run for 33 cycles: denaturation was at 94oC for one minute, annealing at 45oC for 2 minutes, and extension at74oC for 1.5 minutes. An initial 3 minute denaturation at 94oC and a final 5 minuteextension at7$oC were used. Products of each amplification reaction were resolved as outlined in Section 4.2.4.

4.3 RESULTS

4.3.1 Differentiating strains of the Píchia metnbranífaciens species

Using the intron PCR method of de Barros Lopes et al. (1996), the amplification fingerprints were obtained for ten Pichiamembranifac¡¿ns strains sourced from wine and beer ferments, or of unknown origin (Table 4.1). When compared to the amplified fingerprint of the type 35 strain, Pichia membranifaciens CBS 107, the fingerprints of each Pichia membranifaciens strain was found to be unique, whilst still sharing a number of common amplified fragments (Figure 4.1).

The polymorphisms separating these fingerprints from one another were most evident when comparing Pichia membranifacl¿ns AWRI 732 and AWRI 745, with the other eight strains (Figure 4.1). The fingerprints of Pichia membranifacier¡s AWRI 732 and AWRI 745 could be differentiated from one another by a doublet at approximately 450 bp (lane 9). The amplified fingerprints of the eight remairung Pichia membranifací¿¡¿s strains, were relatively similar, but could be differentiated from each other by the presence or absence of one or more amplified fragments.

4.3.2 Partial characterisation of killer activities

The ten Pichia membranifaci¿ns strains of Table 4.1 were assayed for their killer activity towards one another, and for their killer activity towards 15 non-Pichia membrattifaciens tester strains (fable 4.2). Nl ten Pichia membranifaciens struns failed to display activity towards each other, and four of the ten strains, Pichia membranifaclens AWRI 750, AWRI 752, AWRI 753 and AWRI 443, fuled to display killer activity to any of ttre 15 tester strains. These four strains were, subsequently, grouped into Class A (Table 4.3).

Pichín membranifaci¿ns AWRI 732 andAWRI 745 both displayed weak activity (+) towards Dekkerabruxellensis CBS 74 and Sacclnromyces cerevisiae J16D, and consequently, these two Pichia membranifaciens strains were grouped into Class B. The type strain Pichia membranifaciens CBS 107, and Pichiamembranifaciens CBS 638, both displayed a broader killing range as compared to the two strains of Class B, displaying activity towards twelve of the 15 tester strains. Both these Pichin membranifaciens klller strains displayed the same level of killer activity to the same twelve tester strains, and therefore, were grouped together into Class C.

Of the ten strains assayed, Pichiamembranifacl¿ns CBS 7373 and CB,S 7374, displayed the broadest killing range, showing activity to all but one of the 15 tester strains. Pißhin membranifaci¿ns CBS 7374,however, displayed a gteater level of killer activity to eight of the 14 sensitive tester strains as compared to Pichia membranifacielrs CBS 7373. As a consequence, Pichia membranifaci¿r¿s CBS 7373 and CBS 7374 were subdivided into Classes D1 and D2, respectively.

Sacclnromyces cerevisr.d¿ AWRI 136L, the only tester strain found resistant to Pichia mcmbranifacr¿ns CBS 7373 and CBS 7374, is a petite of the wildtype (wt) respiratory competent Saccharomyces cerevßíø¿ AWRI 1360. This petite strain also displayed resistance Figure 4.L

bp Ml234r567 8910 bp 2r49

830

1090

489

33r

Figure 4.1: Intron primer PCR amplification fingerprints of strains of Pichia membranifaciens. Lane 1: CBS7374 Lane 6: AWRI752 Lane2: CBS 7373 Lane7: AWRI750 Lane 3: CBS 638 Lane 8: A}VRI745

Lane 4: CBS 107T (gpe strain) Lane 9: AWRI732 Lane 5: AWRI753 Lane 10: AWRI443 M: Double digest of pTZl8u (United States Biochemical) with the restriction enzymes DraI and RsaI.

The black brackets on the right hand side of the gel indicate the amplified fragments common for Pichia membranifaciens. The black affow at 450 bp highlights the doublet in Lane 9 which differentiates Pichiamembranifaci¿ns AWRI 745 andAWRI732, whilst the red ¿uïo\rys indicate the five distinct polymorphisms between Pichia membranifocl¿ns AWRI 745 andAlVRI 732has with the other eight strains. P. membtanifaciens strains Tester strains Class A Class B Class C Class Dl Class D2 AÌVRI75O A\ryRI752 A\ryRI753 AWRI443 AWRI732 AWRI745 CBS 107 CBS 638 cBS 7373 CBS 7374 D. bruxellens,r CBS 74 + + ++ ++ +++ +++ D. bruxellensis CBS 75 + ++ D. bruxellens¿s CBS 4914 + + S. cerevisiae CBS 1907 + + + + S. cerevisiae I5da + + + + S. cerevisiae 15da + + + + S. cerevisiae AWRI 1360 ++ ++ + + +++ S. cerevisiae AWRI 1361 S. cerevisine AWRI 1363 + + + ++ S. cerevisiae AIWRI 1367 + + + ++ S. cerevisiae JI6D + + + + + ++ S. cerevisiae YM121 + + + ++ S. cerevisiaeY35T + + + ++ S. cerevisine AWRI 1365 + + + ++ S'codes cBS 821 + + + + P. membranifac¿¿ns AWRI 750

P. membranifaciens AWNI 7 52 P. membranifaciens AWRI 753 P. membranifacien s AWRI 443 P. membranifaciens AWRI 7 32 P. membranifaciens AWRI 7 45 P. membranifuciens CBS 107 P. membranifaciens CBS 638 P. membranifaciens CBS 7 37 3 P, cBs7374

Table 4.3: The killer activity of strains of P. membranifacines.. The killer activity was scaled based on the radius of the killer halo (mm): ' : no visible sign of killer activity; + : weak killer activity (0.05 - 1.5 mm); ++ : medium killer activity (1.5 - 2.5 mm); #+: strong killer activity (2.5+ mm). 36 to both Pichin membranifaci¿ns CBS IO7 and CBS 638 (Class C), while the wt Sacchnromyces cerevísløe AWRI 1360 was sensitive.

4.3.3 Detection of extrachromosomal elements in Pichia membranífaciens

To further investigate the genetic basis of the Pichia membranifacr¿r¡s killer phenotype, the total nucleic acids of each of the ten Pichiø membranifaciens struns making up the four different killer types, were resolved using gel electrophoresis. Nucleic acid preparations of known killer strains harbouring extrachromosomal elements were also prepared. These were Kluyveromyces lactis var.Iactis CBS 2359 and the K2 killer type Saccharomyces cerevisiae CBS 6505, whose killer phenotypes are encoded by dsRNA and linear DNA plasmids, respectively.

I-ane 1 of Figure 4.2 shows the two dsRNA plasmids of Saccharomyces cerevisiae CBS 6505 of sizes of 4.6 kb (L-H) and 1.5 kb (K2). Separation of the nucleic acid preparations of Kluyveromyces lactß var. Iactís CBS 2359 Q-ane 2) also revealed its two linear DNA plasmids of size 13.4 kb (pGKL2) and 8.8 kb (pGKLl). Lane 3 revealed that Pichin membranífaciens CBS 7374 harbours an extrachromosomal element of 4.6 kb, whilst extrachromosomal elements of similar size were found also for Píchin mcmbranifaciens strains AIVRI 732 Q-ane 7), A\ryRI 745 Q-ane 9) and CBS 7373 (Lane 11). The Class C Pichin membranifaciens strains CBS 107 (Lane 6) and CBS 638 (I-ane 10) lacked this extrachromosomal element, as did the non-killer Pichia membranifoci¿r¿s strains of Class A (Lanes 2,4,7 and 8, Figure 4.3).

4.3.4 Characterisation of the extrachromosomal element of Pichia membrøníføcíens CBS 7374.

To determine whether the extrachromosomal element of Pichia membranifaciens CBS 7374 is DNA or RNA, nucleic acid preparations of this strain \ryere separated by gel electrophoresis following treatrnent with either RNase A or DNase 1. As expected, the DNase tneatrnent degraded the high molecular weight genomic DNA. However, the 4.6 kb extrachromosomal element remained undigested (Figure 4.4, part A). In contrast, fteatrnent with RNaseA at a concentration of 1 pglml, completely digested the extrachromosomal element, while leaving the genomic DNA intact.

To identify whether the RNA extrachromosomal element is double or single stranded, nucleicacidpreparationsof PiclríamembranifaciensCBsT3T4were digested with RNase A in higher concentrations of NaCl. Double stranded RNA is resistant to RNase degradation under high-salt conditions (Radler et al., 1993; Schmitt and Neuhausen, 1994). The 4.6 kb RNA extrachromosomal element remained undegraded to 0.5 pglml of RNase A in the Figure 4.2

kb Mr234 567 891011M

23.0- ,,¿þ{l¡t. -FfÇ.! Fç*.'. ' 9.4 - 6.6- 4.4 -

2.3 2.O

Figarc 4.2: Identification of the presence of extrachromosomal elements in strains of Píchia membranifaciens, by seperation of total nucleic acids using gel electrophoresis. Lane 1: Saccharomyces cerevisiae CBS 6505

Lane 2: Kluyv eromy c e s lacti s v ar. lactisCB S 23 59 Lane 3: Pichiamembranifaciens CBS 7374 (Class Dl) Lane 4: Pichia membranifaciens CBS 7374 digested with 10 ¡r g/ml DNase I Lane 5: Pichia membranifaciens CBS 7374 digested with 20 ¡t glml RNase A Lane 6: Pichia membranifaciens CBS 107 (Class C)

Lane J : Pichia membranifaci¿ns AWRI 732 (Class B) Lane 8: Pichiamembranifaciens AWRI443 (Class A) Lane 9: Pichia membranifaci¿ns A'WR[ 745 (Class B) Lane 10: Pichia membranifaciens CBS 638 (Class C) Lane 11: PichiamembraniÍaciens CBS7373 (Class D2) M: Hindlllrestriction digest of IDNA Figure 4.3

kb M1234 567 8

23.0- 9.4 - 6.6- 4.4 -

2.3 2.0

Figure 4.3: The absence of extrachromsmal elements in Pichia membranifaciens

strains AWRI 750, A\ryRI 7 52 and AV/RI 753. Lane 1: Pichiamembranifaci¿ns CBS 7373 (Class D2) Lane2: Pichia membranifoci¿ns AWRI753 (Class A) Lane 3: Pichiamembranifaci¿ns AWRI745 (Class B) Lane 4: Pichia membranifaciens AWRI 750 (Class A) Lane 5: Pichiamembranifaciens CBS 638 (Class C) Lane 6: Pichiamembranifaci¿ns AWRI 732 (Class B) Lane 7: Pichia membranifaciens AWF.I 443 (Class A) Lane 8: Pichiamembranifaci¿ns AWRI752 (Class A) M: HindIII restriction digest of ÀDNA Figure 4.4

A. B. Mt 2 3 r 234

.-,*ïfr :'åit":,',81Y3"1,

Figure 4.4: Characterisation of the extrachromosomal element of Pichia

membranifac iens CBS 7 37 4.

A. Total nucleic acids of Pichia membranifaci¿ns CBS 7374: Lane 1: undigested Lane2:. digested with 10 t¡ g/ml DNase I Lane 3: digested with 1.0 tt glmIRNase A M: IfindIII restriction digest of ÀDNA

B. Total nucleic acids of Pichia membranaefacíens CBS 7374 digested with 0.5 p dml RNase A in the presence of: Lane 1: 0.8 M NaCl Lane 2;0.6 M NaCl Lane 3: 0.2 M NaCl Lane 4: no sodium chloride 37 presence O.2M,0.6 M and 0.8 M NaCl (Figure 4.4, Part B). This RNA extrachromosomal element, however, was partially degraded by the RNase A treaünent in the absence of salt, suggesting that it is double-stranded (ds) in nature (Lane 4). For the remainder of the investigation, this 4.6 kb dsRNA is referred to as pPM01.

4.3.5 Curing Píchía membranífaciens CBS 7374 of pPM01

To determine whether pPM01 is associated with the killer phenotype of Pichia membranifacl¿ns CBS 7374, an investigation was undertaken to cure this strain of its dsRNA plasmid. Liquid cultures of Pichia membranifaciens CBS 7374 were grown in the presence of cycloheximide for 3 days at25oC. rWhen grown in the presence of 0.5 mg/l and 1 mg/l cycloheximide, Pichia membranifacier¿s CBS 7374 exhtbited a survival rate of l.4%o andI.LTo respectively (Table 4.4).Treating the yeast with a cycloheximide concentration of 5 mdl was found to be lethal. Resolving mini-preparations of nucleic acids by gel electrophoresis, all 96 isolates arising from the culture grown in the presence 1 mg/l of cycloheximide, were found to still harbour pPM01.

In a second attempt to cure Pichiamembranifaci¿ns CBS 7374 of pPM01, a liquid culture of Pichia membranifacier?s CBS 7374 was exposed to IJV light for a period of 1, 10 and 2O minutes, resulting in a survival rate of 4I7o, I47o and2vo rcspectively (Iable 4.4). When assayed, all 120 isolates arising from the culture exposed to UV light for 20 minutes, wele found to still harbour pPM01.

4.4 DISCUSSION

Pichia membranifacienshas been documented as a source of spoilage for various food stuffs (Deak and Beuchat, 1993) and beverages, such as beer and wine. The growth of Pichia membranifacíens inbeer is known to give a sauerkraut taint (Thomas, 1993), whilst in wine, spoilage includes off odours such as acetyaldehyde, ethyl acetzte, and isoamylalcohol (Rankine, 1966;Thornton, I99I; Sponholz, 1993). The ten Pichinmembranifacl¿r¿s strains employed in this investigation, were sourced from beer and wine ferments, as well as unknown sources (Table 4.1).

An investigation was undertaken to determine whether these strains could be differentiated, using a quick and reliable PCR f,rngerprint technique previously shown useful for yea.st of other genera (de Balros Lopes, 1993). Presently, conventional methods of yeast are based on a range of morphological and biochemical characteristics. As a consequence, the accurate identification of some yeast strains may involve a large number of time consuming and labour intensive tests. V/ith the advent of molecular typing techniques, there Table 4.4

Treatment Viable cell Survival Cured number ml'r rate* isolatesf

control 2x 107

0.5 mg/ml 2.8 x 10s I.4Vo nla

Cycloheximide control 2xI01

1mg/ml 2.1x I}s L.IVo ot96

control 2xI01

5 mglrnl 0 0 nla

control 6.8 x 10ó

l min. 2.8 x 10ó 4l7o nla

UV control 3.2 x 106

10 min 4.4 x I}s l47o nla

control 6.6 x 10ó

20 min 1.4 x 10s 27o 01r20

Table 4.4: Liquid cultures of Pichia membranifaciens CBS 7374 were treated with either IJV or cycloheximide and the survival rates calculated. Isolates arising from treated cells were assayed for the presence of the dsRNA pPM01. * The viable number of treated cells divided by the viable number of untreated cells x 100.

T The number of isolates cured over the number of isolates assayed. rVa - cells from this treatment was not assayed for the presence of pPM01. 38 exists the opportunity to develop a more rapid and reliable technique, for the identification of spoilage yeasts.

The PCR fingerprint technique developed by de Ba¡ros Lopes et aI. (1996) was employed in this study. The primer sequences used in this PCR technique are complementary to the intron splice sites of yeasts. These intron splice site sequences are highly conserved because they arc essential for the removal of introns during mRNA synthesis. In contrast, the intron sequences themselves are highly variable. Consequently, amplification of these variable sequences provides an effective method for differentiating strains, and has been employed successfully to differentiate strains of Sacchnromyces cerevisiae, Dekleera bruxellensis, ToruIaspora delbrueckü, Issatchenkia orientalis, Hanseniaspora uvantm, Hanseniaspora guilliermondü and Metschnikowia pulchcnimn (de Barros Lopes et aI., 1996; de Barros Lopes et aI.,1998).

Using the same primer set which successfully differentiated strains of Saccharomyces cerevisiae (de Barros Lopes et al., 1996; de Barros Lopes et aI., 1998), the PCR-intron fingerprints of all ten Pichia membranifaciens strains, including the type strain CBS 107' were found to be unique (Figure 4.1). Interestingly, the amplified fingerprints of Pichia membranifaci¿ns AWRI 732 andAWRI 745 were more similar to one another, than with the other eight Pichiø membranifaci¿ns strains. This result suggests that these two strains are genetically more distant to the other Pichia membranifaci¿r¿s strains, including the type strain CBS 107.

Although each Pichia membranifaciens strain, including the type strain, displayed a unique fingerprint, all ten strains were found to have a number of amplified fragments in common with one another (Figure 4.1). This result suggests the amplification of these fragments is characteristic for strains of this species. Likewise, de Barros Lopes et al. (1996 and 1998) have shown the PcR-intron fingerprint technique to be specific for many species. Therefore, this study has shown that this technique can both, differentiate strains of Pichia membraniþciens, as well as identify strains as belonging to the Pichin mcmbranifaciens species. As a result, this method of strain identification could identify suspected strains of Pichía membranifaciens isolated from spoiled foodstuffs and beverages.

The second aspect of this study investigated the possibility of grouping ten strains of Pichia membranifaciens into different classes, based on their intraspecific differences in killer activity. Preliminary investigations into the killer phenotype of Saccharotnyces cerevisiac, revealed that strains of this species could be classified into three types, KI, K2 and K28, based on their killing activity and immunity towards each other (Rogers and Bevan, 1978; Young and Yagiu, 1978). Further studies revealed that the killer phenotype of all thrce killer types, were encoded by extrachromosomal dsRNA virus like particles. However, the size of 39 these dsRNA virus like particles differed for each killer type. Subsequent investigations at the molecular level revealed that each killer type expressed a unique toxin, accounting for the infaspecific differences in killer activity (Magliani, 1997).

To determine whether the strains of Table 4.1 could be grouped into classes, based on their intraspecific differences in killer activity, the Pichía membranifaciens strains were first assayed for their ability to inhibit the growth of one another. However, no killer activity was detected (Table 4.3). Subsequently, the ten Pichia membranifociens strains were assayed for theirkiller activity towards 15 non-Pichia membranifaciens tester strains (fable 4.2). TfueÊ, strains of Deldæra bruxellensis were included in the panel of tester strains, which had previously shown intraspecific variation in susceptibility to three Pichia membranifaciens killer strains (Chapter Three). Eleven strains of Saccharomyces cerevísiae were also incorporated into the panel, including the respiratory competent wt Saccharomyces cerevisiae AWRI 1360, and a petite of this strain, Saccharomyces cerevisiac AWRI 1361. Sacchnromycodcs ludwrgfi CBS 821 was also included since this strain displayed the broadest level of sensitivity of the 26 tester strains assayed previously (Chapter Three).

The intaspecif,rc differences in killing activity allowed the ten Pichia membranifacier¿s strains to be grouped into the four classes A, B, C and D (Iable 4.3). Class A was composed of four Pichin membranifac¡¿ns strains, which failed to display activity to any of the 15 æster strains. The observation that these four strains failed to display killer activity suggests they lack the killer phenotype. It may be the case, however, that these Pichia membranifaciens strains have a killer phenotype, but they lack the ability to kill the tester strains employed in this study, or their killer activity is inactivated at pH 4.5.

The three remaining killer classes of Pichin m.embranifaciens were composed of strains which differed in their killing range. However, the killing range of the two classes, B and C, were a subset of Class D. As discussed in Chapter 3 and Yap et al. (2OOO), this subset in killing may be attributable to strains of each class expressing the same killer toxin, but differing in their level of toxin expression or secretion. Alternatively, the differences in killing range of the three classes, is due to divergence of the gene sequences encoding the toxins of each class. Otherwise, each killer class may be expressing biochemically distinct toxins.

Interestingly, the killer strains of both Class C and D displayed activity to the respiratory competent wt Saccharomyces cerevisia¿ A\ryRI 1360, however, the petite Saccharomyces cerevisiae AWRI 1361, remainedresistant (Iable 4.3). Initially, it was thought that these strains may share a common mechanism of killing, which was perhaps linked to functional mitochondria in the sensitive strain. Further investigations revealed, however, that resistance of the petite Saccharomyces cerevisiae AWRI 1361 was not confined to killer strains of 40

Pichia membraniÍaciens, but displayed resistance also to a number of different killer yeasts species, whose mechanisms of killing appear to be unrelated. These results are discussed further in Chapter Six. r To determine whether the killer phenotype of the Pichin membranifaci¿ns strains is plasmid encoded, total nucleic acid preparations of the ten strains were separated by gel electrophoresis @igures 4.2 and4.3). The four non-killer strains of Class A were found not to harbour any type of extrachromosomal element (Figure 4.1). Likewise, the two strains of Class C lacked extrachromosomal elements, yet these strains displayed strong killer activity (Figure 4.2).Therefore, the killer phenotype of Pichia membranifaciens CBS 107 and CBS 638 is probably nuclear encoded. Other killer yeasts species whose toxins are nuclear encoded include W. saturnus, Pichin anomala, Pichin kluyveri, Pichin farinosa, and the KHR and KHS killer types of Saccharomyces cerevisiae (Magliani et a1.,1995).

In contrast to the strains of Class A and C, the two Pichia membranifacietd strains of Class D both harboured a single extrachromosomal element of approximately 4.6 kb in size. The two strains of Class B, whose killer range was much reduced as compared to the Class D strains, also harboured an extrachromosomal element of similar size. Further investigation revealed that the extrachromosomal element of Pichia membranifaci¿r¿s CBS 7374, referred to as pPM01, was dsRNA in nature (Figure 4.4,Part A and B).

To date there have been no reports of a yeast killer phenotype being attributed to a single dsRNA element. As mentioned in Chapter Two, the killer yeasts whose toxins are borne from a dsRNA element, also harbour at least a second dsRNA element which encodes for viral maintenance and/or toxin immunity. The killer types of Saccharomyces cerevisiae, KI, K2 and K28, each harbour two dsRNA virusJike particles (VLPs), with the M dsRNA encoding the toxin, and the LA dsRNA encoding for viral replication and encapsulation. Likewise, the three killer types of Ustilago maydis, KPl, KP4 and KP6, each harbour three size classes of dsRNA VLPs, with the medium (M) dsRNA class encoding the killer toxin, and the heavy (H) dsRNA class encoding the proteins essential for viral maintenance (Finkler et al., 1992;Park et al., L994;Park et aI., I996b). In a similar fashion, the killer yeast Zygosaccharomyces bailü harbours three dsRNA VLPs, L, Z and M, where the M dsRNA andZdsRNA encoding the toxin and the yeasts'immunity respectively (Schmitt and Neuhausen, lgg4). The Hanseniaspora uvantm M dsRNA encodes its killer toxin. However, it is not known whether the second dsRNA of Hanseni.aspora twarum, the L dsRNA, has an association with its killer phenotype (Zotg et a1.,1988).

To determine whether pPM01 is associated with the Pichia membranifaciens CBS 7374ktller phenotype, this study investigated the possibility of curing the yeast of the extrachromosomal element. Two methods which have previously been proven successful in 4T curing the extrachromosomal elements from a number of yeast species, were employed in this study. The first method employed the use of cycloheximide, which at a higher concentration, results in a greater percentage of cured isolates @ink and Styles, L972; Radler et a1.,1993). At a cycloheximide concentration of 1 mg/I, which resulted in a survival rate of l.l%o, all 96 isolates still harboured pPM01.

At this same concentration of cycloheximide (1 mg/ml), Zorg et ø/. (1988) found that all isolates arising from fteated cultures of Saccharomyces cerevisiae K28, were cured of their two dsRNA particles. Moreover, at a cycloheximide concentration of 0.1 mg/I, a minimum of 50Vo of the isolates arising from three treated strains of Saccharomyces cerevisiae K2, were cured. It would appear from this study, that pPM01 is essential for the viability of Pichia membranifaci¿ns CBS 7374. However, both Zygosacclnromyces bailü @adler at al., 1993) and Hanseniaspora uvarum (Zorg et al., 1983) were only successfully cured of their dsRNA particles at a cycloheximide concentration of 10 mg/I. Pichia membranifacíens CBS 7374 is comparatively less tolerant to cycloheximide than Zygosacchnromyces bailü and Hanseniaspora uvarum, showing no growth after the treatment of 5 mgll of cycloheximide.

If pPM01 is essential for Pichia membranifaciens CBS 7374, this would explain why 120 isolates arising from a culture treated with UV light for 20 minutes, which resulted in a 27o survival rate, still harboured the extrachromosomal element. Worsham and Bolen (1990) found that more than half the isolates arising from liquid cultures of Pichia acacia.e, which had been treated with UV light to give a 77o stxvival rate, were successfully cured. Likewise, 28 isolates from a UV-treated culture of Pichia inositovora. only three retained detectable plasmid DNA (Hayman and Bolen, 1991).

Different treatments other than those methods mentioned previously, have had some limited success in curing yeast species of their killer phenotype. Incubating cultures at elevated temperatures (37-40oC) has been proven successful in curing Saccharomyces cerevisiae strains of their Kl killer type, and some Saccharomyces cerevisi.ae strains of their K2 killer type, but failed to cure strains of Hanseniaspora uvarum, Zygosaccharomyces bailü and Saccharomyces cerevisiaeK2S (Zorget a1.,1988; Petering et aI.,I99l; Radler et al., 1993). Treating cultures with S-fluorouracil or acridine orange have proven successful also, in curing killer strains of Saccharomyces cerevisiac (Mitchell et al., 1973; Cansado et aI., 1939). It may be the case that one of these less successful methods may cure Pichin membranifaciens CBS 7374 of pPM01, if the extrachromosomal element is not essential for its survival.

From these results its difficult to discern whether the genetic basis of the Pichia membranifaci¿r¿s CBS 7374 ktller phenotype, is chromosomal, or associated with pPM01. Interestingly, an extrachromosomal element of the same size as pPM01, was found for 42 strains of the Class B killer type. These strains displayed a significantly reduced killing range, as compared to the killer strains of Class D, although, the killing range of Class B is a subset of Class D. As aforementioned, this subset in the killing range may be explained if Pichiamembranifacíens strains of different killer classes are expressing the same toxin, but differ in their level of toxin expression or secretion. If true this would be interesting, as it appears that the Class B strains are genetically more distant to the other Pichia membranifacr¿r¿s strains. Cloning the gene sequences encoding the Pichin membranifaciens CBS7374 toxin would provide a tool, for determining whether the same or different toxin, is being expressed by the strains of Class B, as well as the other Pichia membranifaciens strains investigated in this study. The toxin's gene sequence would also provide a means, for determining whether the toxin is encoded chromosomally, or by pPM01. 43

Chapter 5

CIr^I.ru.CTERISATION OF THE Pichiø membrønifaciens CBS 7374 KILLER TOXIN

5.1 INTRODUCTION

Evidence for inftaspecific differences in killer activity between strains of the Pichia membranifaciens yeast species was investigated in Chapter Four. This study revealed that strains of this spoilage yeast could be grouped into four distinct killer groups, based on the patterns of killer activity that these strains displayed towards various tester strains. To date, however,little is known of the killer toxin of Pichia membranifactens CBS 7374, the strain which displayed the widest killing range of the ten Pichia membranifaciens strains investigated. To gain an understanding of the biology of this novel toxin, conditions for its production in liquid culture was investigated, the toxin characterised and a protocol for its purification from a culture supernatant developed.

5.2 EXPERIMENTAL

5.2.L Yeast strains and media

Both the killer yeast Píchia membranifaciens CBS 7374, and the killer sensitive Deldcera bnnellensis CBS 74, were sourced from Centraalbureau voor Schimmelcultures (CBS), Delft, The Netherlands. Yeast strains were subcultured in YEPD lITo (wlv) yeast extract, 2Vo (wlv) peptone, 2Vo (wtv) glucosel at 25"C for 24-48 h, before being transferred to YEPD-agar IYEPD, 2Vo (wlv) agar] slopes, at 25oC for 48 h. Strains were stored on slopes at 4oC.

5.2.2 The agar plate well diffusion killer assay

The agar plate well diffusion killer assay employed in this study is a modification of that described by Radler et al. (199O). A liquid culture of the killer sensitive Delcl

5.2.3 Killer activity from different media

Media employed: 27o (wlv) YM medium[0.6Vo (w/v) malt extract, 0.6Vo (wlv) yeast extract, l7o (wlv) bacteriological peptone, 27o (w/v) dextrose; Amyl Medial; minimal media [10x (w/v) Bacto-Yeast nitrogen base without amino acids @ifco),27o (w/v) dextrosel; minimal media (N,ilyt) plus 0.67o (ilv) malt extract (Amyl Media); MM plus O.6Vo (wlv) yeast extact (Amyl Media); MM plus IVo (wlv) bacteriological peptone (Amyl Media); MM plus 27o (w/v) Bacto Casamino acids @ifco); and MM plus 2Vo (wlv) YM medium. All media types were buffered to pH 6.2 with a I M cifric-phosphate buffer, and then filter sterilised (0.22 MM).

The inoculant to be used for all media types, was Pichia membranifaciens CBS 7374 cultured in YEPD to an exponential phase, which was washed and resuspended in an isotonic saline solution. The sterile media were inoculated to give a total cell count of 1x106 cells/ml. Cultures of 20 ml in volume were undertaken in duplicate, agiøting at 100 opm at 24oC, in 50 ml cotton plugged, fermentation flasks. The supernatant for each culture was prepared at late exponential phase, by centrifugation at 5,000 rpm for 5 minutes at 4oC. Supernatants were assayed as outlined in Section 5.2.2

5.2.4 Optimum pH for kitler toxin production and activity

Pichiamembranifaciens CBS T3Tfwascultured in MM plus 2Vo casamino acids (MM+Cas) buffered to pH 4.0, pH 4.5, pH 5.0, pH 5.5, pH 6.0 or pH 6.5, using a 1 M citic- phosphate buffer. The procedure outlinedin5.2.3 was then followed, with the supernatants prepared when the growing cultures had reached 5x107 total cells/ml. Supernatants were then assayed as outlined in Section 5.2.2, except in this case, the YEPD-soft agar employed in the agar plate well diffusion killer assay was buffered to pH 3.0, pH 3.5, pH 4.0 or pH 4.5.

5.2.5 Assaying killer activity at different stages of cell growth

Cultures were undertaken in MM+Cas buffered to pH 6.0, following the method of 5.2.3, except in this instance, 100 ml volumes were employed, using cotton plugged half liû''e fermentation flasks. The procedure for sampling the culture supernatants for activity was essentially thatof Hodgson ¿t at.(1995). Every 12 hours for up to 60 hours, one ml of the 45 culture was removed from the ferment, and the supernatant prepared as outlined in 5.2.3. Killer activity was assayed as outlined in 5.2.2, with the YEPD-soft agar used in the agar plate well diffusion killer assay buffered to pH 4.0. This was the preferred pH level for the agar plate well diffusion killer assay when employed in the experimental procedures 5.2.6 through to 5.2.LI.

5.2.6 Pronase E digestion

A culture supernatant was obtained using the procedure outlined in 5.2.3, with the supernatants prepared when the total cell count had reached 1.5x108 cells/ml. The supernatant was filtered through a Millex-GV O.22 ¡tm ready to use filter, prior to being treated with either 0.5 mg/ml, I mg/ml or 2 mglml of Pronase E (Sigma), using the procedure recommended by the manufacturer. Activity of the treated supernatants were assayed using the agar plate well diffusion killer assay.

5.2.7 Temperature sensitivity

Culture supernatants obtained as outlined in 5.2.3 and 5.2.6, were incubated in duplicate 45 pl aliquots, at24oC,37oC or 65oC, using a PTC-100 PCR thermocycler (MJ Research, Mass., USA). At specific time intervals of 15 and 30 seconds, and at 1, 5, 10, 30, 60, 120 and 180 minutes, the activity of the treated samples was assayed using the agar plate well diffusion killer assay.

5.2.8 Optimal storage conditions

Supernatants from 100 ml cultures were prep¿ìred as outlinedin 5.2.6, and then divided into 10 rnl aliquots. The aliquots were stored in polystyrene or polyethylene tubes, or glass containers, for 48 hours at24oC. Activity of these supernatants were then assayed using the agar plate well diffusion killer assay.

For determining the optimal temperature for toxin storage, 10 ml aliquots of a supernatant were stored in glass containers at 24oC,4oC and -20oC. At time intervals of 4, 6, L2, 18, 34 and 61 days, the supernatant was assayed for activity using the agar plate well diffusion killer assay. A 10 ml aliquot was sampled only once, to avoid repeated cycles of freezing and thawing.

5.2.9 HPLC reverse phase chromatography

Culture supernatants obtained as outlined in 5.2.6, and the liquid medium MMrcas alone, were dilute d 40Vo, 50Vo, 60Vo and TOVo using HiPerSolv grade methanol (BDH Laboratories 46

Supplies). Culture suprnatants were also diluted 407o usingHiPerSolv grade ethanol (BDH Laboratories Supplies), and diluted O.O57o with HiPerSolv grade tifluroacetic acid (BDH Laboratories Supplies). The culture supernatants were diluted at the same concentrations using MM+Cas also. Diluted culture supernatants wexg incubated at room temperature for 60 minutes before being assayed for activity.

A 200 ¡.r,1 volume of a culture supernatant obtained using the method outlined in 5.2.6, was fractionated using a C-18 column (Protein and Peptide, 250 mm x 4.6 mm, Vydac, USA), on a 1090 IIEWLETT-PACKARD IIPLC system operated at 40oC. Before loading the sample, the C-18 column had been equilibrated with 0.057o TFA. For elution a gradient concentration of methanol of }to 50Eo (v/v) over a period of 18 minutes was used, with the elution rate set at one mVminute, before being returned to z.ero for 3 minutes. Prior to elution, 0.O5Vo TFA was passed through the column for two minutes (1 mUminute) to displace the void. Half ml fractions were collected in glass containers every 30 seconds, with the killer activity of each fraction assayed using the agar plate well diffusion killer assay.

5.2.10 Ultrafiltration of the toxin supernatant

Stock culture supernatant obtained as outlined in 5.2.6, \t/ere passed through Centricon fractionating columns (Amicon Corporation, MA, USA) with MW cut-offs of 10 and 50 kDa, using the manufacturer's recommended method. The subsequent retentates were diluted back to a stock concentration before they, and the fîltrates, were assayed for killer activity.

For concentrating killer toxin a culture supernatant of 900 ml was prepared by the method outlined 1n 5.2.6. This volume was reduced to 50 ml with a 200 ml capacity stirred cell, using an ultrafiltration membrane with an exclusion limit of 10 kDa (Sartorius, C-18), at 4oC, under a nitrogen pressure of 350 KPa. The 50 rnl concentrate was then reduced to 8 ml using a 50 ml capacity stirred cell, again using a 10 kDa cut-off ultrafiltration membrane (YM-10 membrane, Amicon Corporation, MA, USA), at 4oC under a niffogen pressure of 350 KPa. The concentrated toxin supernatant was then diluted 1 in 5 with 50 mM diethanolamine pH 8.8, prior to being fractionated by IIPLC anion exchange chromatography.

5.2.11 HPLC anion exchange chromatography

Culture supematants obtained as outlined in 5.2.6, and the liquid medium MMrcas alone, were diluted one in ten with sodium chloride to give final concentrations of 0.1 M, 0.3 M and 1 M. The culture supernatants were diluted to the same concentrations with MM+Cas also. Diluted culture supernatants were incubated at room temperature for 60 minutes before being assayed for activity. 47

A one ml volume of a 22.5 fold toxin supernatant concentrate (see 5.2.10) was fractionated using a MonoQ column (Pharmacia), on a 1090 IIEWLETT-PACKARD I{PLC system operated at24oC. Before loading the sample, the MonoQ column had been equilibrated with 50 mM diethanolamine pH 8.8. The elutant employed in the mobile phase was I M NaCl with 50 mM diethanolamine, both buffered to pH 8.8 with HCl. All solutions were degassed and filæred prior to use. For elution a concentration gradient of NaCl of 0 to 1 M over a period of 18 minutes was used, with the rate of elution set at one mVminute, before being returned to zero for 3 minutes. Prior to elution, 50 mM diethanolamine (pH 8.8) was passed through the column at arate of one mUminute for two minutes, to displace the void. Half rnl fractions were collected in glass containers every 30 seconds, with the activity of each fraction assayed using the agar plate well diffusion killer assay.

5.2.L2 SDS polyacrylamide gel electrophoresis

Discontinuous SDS polyacrylamide gel electrophoresis (SDS-PAGE), based on the method of Laemmli (1970), was performed at room temperature in a Bio Rad Mni-PROTEAN tr system. Separating gels were prepared by combining 2.25 ml of an acrylamide solution l30%o (wtv) acrylamide,0.87o (w/v) bisacrylamidel with 3.13 rnl 0.75 M Tris-HCl pH 8.8, 0.27o (wlv) SDS, 0.88 rnl H20, 8.75 pl Temed and 23.5 pl IÙVo (wlv) ammonium persulphate. The components were mixed, and then poured between two glass plates which were supported by the BioRad casting stand. The separating gel was overlayed with 1 ml H20, and left to polymerise for approximately 30 minutes.

The stacking gel was prepared by combining 0.25 rnl of an acrylamide solution (as above) with 1.25 ml 0.25 M Tris-HCl pH 6.8, O.2Vo (wlv) SDS, 0.98 ml H20 and 2.75 ¡t'l Temed. This solution was degassed for 10 minutes prior to polymerisation, by the addition of 11 pl l\Vo (wtv) ammonium persulphate. The stacking gel was poured on top of the separating gel with a comb set in place, and left to polymerise for at least 45 minutes. After polymerisation, the comb was removed, and the wells were washed at least twice to remove any unpolymerised acrylamide. Gels were covered with running buffer [0.1 M Tris-HCl, O.IVo (w/v) SDS , O.2 Mglycinel, and electrophoresed under constant voltage, stacking at 50 V and running at 100 V.

Prior to electrophoretic separation 300 pl of each fraction, as well as 300 pl of stock toxin supernatant, were concenffated up by ultrafiltration to 30 pl using a C-18 cartridge, centrifuged at 5000 rpm at 4oC. These 30 pl samples were further concentrated to 10 ul by evaporation using a speedi-vac. Prior to loading each 10 pl sample received a 1.5x loading buffer (950 pI0.5 M Tris-HCl pH 6.8, 200 ¡rl glycerol, 400 pl IOVo (wlv) SDS' 200 pl 2- ME, 100 ¡tlO.OSVo bromophenol blue). 48

After electrophoresis, the proteins were fixed and stained by incubating the gel in a fixative (methanol:acetic acid:water at a ratio of 4:1:5) containing O.IVo (wlv) Coomassie Brilliant Blue R-250 for 30 minutes, and then destained with either the fixative or water. The molecular weight of the protein band was determined from a calibration line obtained from a plot of the molecular weight (plotted on a loglg scale) against distance migrated from the interface of the stacking and separating gels. The molecular weight markers employed in this study were SDS-PAGE low range molecular weight standards (Bio-Rad Laboratories, CA, usA).

5.3 RESULTS

5.3.1 Optimising toxin production in liquid media culture of Píchia membranifacíens CBS 7374

An investigation was undertaken to identify a protein free culture medium, for the optimal production of the Pichia membranifacie¡¿s CBS 7374 L,tller toxin. Included in the seven different media evaluated, was the chemically complete YM medium. Also evaluated was minimal medium (MM), and MM supplemented with either O.67o (wlv) malt extract, O.67o (w/v) yeast extract, l%o (wlv) bacteriological peptone,2To (w/v) casamino acids or 27o (wlv) YM medium. Killer activity was assayed using the agar plate well diffusion killer assay buffered to pH 4.5, with the supematants of the liquid cultures being prepared at late exponential phase.

Supernatants from cells grown in the protein fiee MM, a media containing 0.O57o total nitrogen, displayed 3 killer units (kU) of activity (Figure 5.1). The addition of 0.6Vo (w/v) malt extract to the MM resulted in an increase in killer activity to 4 kU, with a corresponding increase in level of total nitrogen of 0.OIVo (Amyl Media). The addition of 0.67o (w/v) yeast extract to the MM increased the level of total nitrogen by 0.O77o (Amyl Media), with the supernatants from this protein rich medium displaying 5 kU of activity.

A further increase in killer activity to 8 kU, was observed when the MM was supplemented with l%o bacteriological peptone (Amyl Media), with this addition increasing the concentration of total nitrogen by 0.L7Vo @igure 5.1). The supplementation of MM with 2Vo casamino acids @ifco) increased the concentration of total niftogen four-fold, to a level of O.257o, with the supernatants of this medium displaying 8 kU of activity. Likewise, YlVl alone had a total nitrogen concentration of O.257o, yet supernatants prepared from this protein rich medium displayed 9 kU of activity. The supplementation of MM with 2Vo (w/v) YM increased the concenhation of total nitrogen to O.37o, and resulted in supernatants Table 5.1

Mean values

Time Total Killer (hrs) cells ml-t activity

0 8.6x104 1x106

T2 2.00x105 1.85x106

24 6.83x106 8.47xIO7 7

36 1.04x107 1.53x108 I

48 I.22xI07 1.54x108 2

60 1.15x107 1.53x108

Table 5.1: Killer activity of the Pichia membranifaciens CB,S 7374 toxin at different stages of cell growth.

Table 5.2

Temperature Time 24',C 37'C 65"C 100"c

15 sec I I )

30 sec 8 8 1 l min 8 I 5 min I 8 10 min I I 30 min 8 I thr 8 6 2hr I 4 3hr 8 2

Table 5.2: Killer activity of the Pichia membranifaciens CBS 7374 toxin supernatant incubated at different temperatures for different lengths of time. Figure 5.1

10 0.4

8 0.3 ct) èe H Ê = c) >r 6 èo I o o 0.2 tr d li E o 4 Fo g tr 0.1 2

0 0.0

,x s"

Figure 5.1: Killer activity of the Pichia membranifaciens CBS 7374 toxin supernatant, harvested from cells cultured in different medium with varying levels of total nitrogen. The killer toxin activity (clear bars) is expressed in arbituary units (kU), where 10 kU corresponds to the amount of killer toxin required to cause an inhibition zone (radius) of 10 mm. This scaling system was employed for the remainder of Chapter Five. The level of total nitrogen (hatched bars) is given as a percentage of the total medium. 49 displaying 9 kU. The protein free MM plus 2Vo casamino acids was the preferred medium for the remainder of the study.

5.3.2 Determining the optimum pH

To identify the optimal pH for the production of active killer toxin, both the protein free culture medium (MM+Cas), and the YEPD agar of the agar plate well diffusion killer assay, were buffered to different pH values using a cinic acid-phosphate buffer. Neither the killer yeast Pichia membranifaciens CBS 7374, nor the sensitive Del

5.3.3 Effect of stage of cell growth on killer activity

To identify the stage of cell growth at which killer activity was maximal, supernatants of growing cultures were assayed for activity at twelve hourly intervals (Iable 5.1 and Figure 5.3). At time zero, the liquid media were inoculated with 1x106 cellVml @igure 5.3). Killer activity was not detected, however, until 24 hours into fermentation, when the total cell count had reached 8.47x107 cellVml (Table 5.1). At this stage of cell growth, killer activity was 7 kU. This observation concurs with the result of Figure 5.2 (5.3.2), where a maximum activity of 7 kU was found for supernatants prepared from cultures grown to 5x107 total cells/ml.

The killer activity had increased to a maximum of 8 kU 36 hours into fermentation, with a total cell count reaching 1.5x108 cells/ml. As stationary phase continued, however, there was a rapid decline in killer activity. By 48 hours the killer activity of the supernatant had declined to 2 kU, with no detectable activity at 60 hours. For the remainder of the study, culture supernatants were prepared when the total cell count had reached 1.5x108 cells/ml.

5.3.4 Protease digestion and temperature sensitivity

To verify the proteinaceous nature of the toxin, a supernatant prepared from a liquid culture was feated with the protein degrading enzyme Pronase A. Concentrations of Pronase A of 0.5 mg/ml, 1 mg/ml and2 mg/Írl reduced the killer activity of the supernatant to 6 kU, 5 kU and 3 kU, respectively, after treatment for one hour at 30oC (Figure 5.4). The addition of 2 mg/ml of inactivated Pronase A had no effect on the activity of the supernatant. Figure 5.2

8

6 €U) pH level of the >r 4 well test assay €o (c pH 3.0 li I o S pH 3.5 g 2 I pH a.0 Ø pEa.5

0 4.0 4.5 5.0 5.5 6.0 6.5 pH level of the culture medium

Figure 5.2: Killer activity of the Pichia membranifaci¿ns CBS 7374 toxin supernatant, harvested from cells cultured in mm plus casamino acids buffered to different pH levels. Likwise, the solid agar well test killer assay was buffered to different pH levels. Peak activity was found when the culture medium was buffered to pH 6.0, and the solid agar well teast assay buffered to pH 4.0. Figure 5.3

9 10

8 8 çt) o # bo o ¿ viable cell growth 7 6 Þ * growth olr o total cell Ê crl (n ¡i 6 4 c) -4t- 0) killer activity O E

5 2

0 4 01224364860

Time (hours)

Figure 5.3: The killer activity of the Pichia tnembraniÍaciens toxin supernatant at different stages of cell growth. Total and viable cell growths, as well as killer activity, are the mean avaerages of two replica ferments (Table 5.3). .t) 8

= I 6

9 li (.) 4 g 2

."-:"f "s Concentration of Pronase E

Figure 5.4: Killer activity of Pichia membranifaciens CBS 7374 toxin supetnatant, which had been treated with increasing concentrations of the protein degrading enzyme Ponase E.

8 cA ) 6 I>'

o C€ 4 o¡i g 2

,rç'

ge

Figure 5.5: The killer activity of the Pichia membranifaciens CBS 7374 toxin supematant, after being stored in different types of containers, for 48 hours at 4oC. Starting activity refers to the toxin supernatant being assayed directly after being removed from the growing culture. 50

An investigation was undertaken to determine the effect that incubation temperature had on 'When the activity of the toxin containing supernatant. incubated at24oC, the supernatant had retained full maximum activity after three hours Clable 5.2). Incubation at 37oC for one hour, however, resulted in a reduction in killer activity to 6 kU, with a further reduction to 4 kU and 2kIJ, when incubated for two and three hours, respectively. When incubated at 65oC for 15 and 30 seconds, the supernatant displayed 2 kU and 1 kU of activity, respectively. No detectable killer activity was detected when incubated at this temperature for 60 seconds or longer.

5.3.5 Optimal storage conditions

To ensure that maximal killer activity was retained during the purification procedure, the stability of killer activity under different storage conditions was studies. Two aspects of storage were investigated, with first considering the importance of the storage container material. No change in killer activity occurred when the supernatant was stored in a glass container for 48 hours at 4oC (Figure 5.5). However, activity had dropped to 4 kU when stored in either polyethylene or polystyrene plastic. As a consequence, glass was the preferred material for storing the toxin. Moreover, because contact with plastic rapidly reduced killer activity, glass containers were used during the entire purification process, such as for the collection of fractions during the chromatography and the ultrafiltration steps.

The effect of temperature on killer activity during storage was the second aspect studied -20oC, @igure 5.6). No activity was lost when stored in a glass container, at either 4oC or for up to 12 days. For both these temperatures, however, the activity of supernatant was reduced to 7 kU after 18 days. At -z}oc,this reduction in activity continued with the loss of 2 kU and 3 kU of activity at 34 and 61 days of storage, respectively. In contrast, at 4oC the activityof the supematant remained constant until day 31, but by day 61 some activity had been lost. At 24oC the supernatant displayed a loss in activity Ñer 4 days, and no activity remained by day 12.

5.3.6 HPLC reverse phase chromatography

To evaluate the use of HPLC reverse phase chromatography (RPC) for the purification of the killer toxin, supernatant activity was first assayed in the presence of solvents used commonly in RPC (fable 5.3). Treatment with 407o (vlv) ethanol, a solvent used frequently in the mobile phase of RPC, reduced activity of the supernatant to 1 kU. Furthelrnore, 40Vo (vlv) ethanol alone (negative control), inhibited the growth of the sensitive lawn (Delclæra bnnellensis CBS 74) used in the agar plate well diffusion killer assay. Figure 5.6

10

8

cA )É 6 >l Storage temperature I (!C) 4 zzoc tr I c.) @ 4"c g -2ooc 2 I

0 04 612 18 34 6r Time (days)

Figure 5.6: The Pichia membranifaci¿z¿s CBS 7374 toxin supernatant was assayed at various intervals over a period of 61 days, whilst being stored at either 23oC,4oC or

minus 20oC. Table 5.3

Killer activity Chemical treatment Treated Positive Negative control control

ÙVo n.a. I n.a

4O7o 4 7

Methanol 507o 7

607o 6

707o 6

Ethanol 4O7o L 7 1

Trifluroacetic Acid 0.057o 6 I

Table 5.3: The killer activity of the Pichia membranifaciens CBS 7374 toxin supernatant assayed in the presence of different concentrations of methanol, 4OVo (vlv) ethanol, or with 0.057o (v/v) trifluroacetic acid. The positive controls refers to the toxin supernatant being diluted 0.057o,407o. 40Vo,6OVo and707o withMM+Cas, whilst the negative controls refers to MM+Cas being assayed in the presence of different concentrations of methanol, 4O7o (v/v) ethanol, or with 0.057o (v/v) trifluroacetic acid. n.a.: not applicable The effect of methanol, a second solvent used commonly as an elutant in stability of killer activity was determined by assay (Iable 5.3). In this instance, methanol reduced the activity from 8 kU to 4 kU, with a 5O7o (vlv) concentration reducing the activity to an undetectable level.In contrast to ethanol, a 4OVo concentration of methanol alone (negative control), had no effect on the growth of Dekl

Subsequently,20O ul of stock supernatant was fractionated on a C-18 column, buffered with 0,05Vo (v/v) TFA and eluting with a linear gradient of O-SOVo (v/v) methanol. All 46 fractions obtained from this RPC process, however, displayed no detectable killer activity

5.3.7 HPLC anion exchange chromatography

To determine the viabitity of HPLC anion exchange chromatography (AEC) for the purification of the toxin, activity of the supernatant was assayed in the presence of various salts and buffers used in the AEC's mobile phase. The elutant to be used in this instance, was sodium chloride, rising in concentration from zero to 1.0 M, with the AE column being buffered to pH 8.8 using 50 mM diethanolamine (as recommended by the columns' manufacturer, Pharmacia).

The killer activity of the supernatant was unaffected in the presence 0.1 M and 0.3 M NaCl, however, its activity had increased from 8 kU to 10 kU in the presence of 1.0 M NaCl (Table 5.4). At thís concentration, the sodium chloride alone, had no affect on the growth of Delcl

Prior to fractionation by AEC, the toxin supernatant was concentrated by ultrafiltration. To determine the appropriate molecular weight (lvfW) cut-off filter to be employed, a culture supematant was ultrahltered with a l0 and 50 kDa MW cut-off filter (Table 5.5). The filtraæ and the retentate of the supernatant fractionated with a 50 kDa MW cut-off filter, measured 4 kU and 5 kU of activity, respectively. Whilst only the retentate, and not the filtrate, of the supernatant fractionated with a 10 kDa cut-off filter, displayed activity. As a consequence, 10 kDa MW cut-off filters were employed in the concentration of the toxin supernatant.

Using a MonoQ column buffered with 50 mM diethanolamine pH 8.8, one ml of a22.5 fold supernatant concentrate was fractionated with a linear gradient of 0-1.0 M NaCl. A peak of activity was measured over fractions 2I-24 of the 46 fractions @igure 5.7), with the highest Table 5.4

Killer activity

Chemical treatment Treated Positive Negative control control

0.1M I 8 NaCl 0.3M I I 1M 10 I

Diethanolamine 50mM 8 8

Table 5.4: The killer activity of the Pichia membranifaciens CBS 7374 toxin supernatant assayed in the presence of 0.1 M, 0.3 M or 1 M NaCl, 50 mM diethanolamine and 20 mM L-Histidine.

Table 5.5

MW cut-off Killer activity

10 kDa Retentate I

Filtrate

50 kDa Retentate 5

Filtrate 4

Table 5.5: Killer activity of the Pichiamembranifaciens CBS 7374 toxtn supernatant which had been fractionated with Centricon fractionating columns (Amicon Corporation, MA, usA.). 500

400 a 11 ,- 1.0 oo c.ì 4 rõ € rd (€ 300 I tz o t- C) I (! U) lo.s à I l€i li o I ) L¡.t6) U) a t+i I 200 lo.o .ã I lc o ¡O I 5 l9 É ()¡i t¿ I lo.+ F \ I g IE 100 o LÉ I J to tiJ I LO.2 E I rE 1 lcl-o 0 rv 0 L6 L) min 0 5 15 20 Fractíon22 (10.5-11.0 min.)

Figure 5.7: The Pichia membranifaciens CBS 7374 toxin supernatant was fractionated by anion exchange chromatogrophy. The column was buffered at pH 9.0. As indicated by the atïow, peak activity of 11 killer units was dected for the 22ndfraction, taken at 10.5 - 11 minutes. The chromatograph of the concentration of eluted protein versues time, revealed a small peak at the 10.7 minute mark, which correlated with the eultion of peak activity. The estimated concentraion of NaCl at I0.7 minutes was 0.48 M. 52 level of activity of 11 kU detected for the 22nd fract¡on @gure 5.8). The estimaæd concentration of NaCl contained within the22ndfraction was 0.61 M.

A total of 26 of the 46 fractions eluted displayed actiyity, however, 19 of these 26 fractions displayed approximately I kU of activity. This included fractions 28 through to 40, where the estimated concentration of NaCl was 0.78-1.0 M. Fractions 41-43, obtained after the concentration of NaCl in the elutant was returned from 1.0 M to zeÍo, also displayed approximately I kU of activity.

5.3.8 SDS-PAGE of the active fractions from AEC

Fractions from the AEC which displayed strong killer activity, along with the fractions which displayed the same or less activity as the stock supernatant, were resolved by SDS- PAGE (Figure 5.9). When resolved the 22nd fraction, which had the highest activity of 11 kU, revealed a protein band of approximately 20.5 kDa (Lane 3). Likewise, the following fraction which displayed 10 kU of activity, Fraction 23 (Lane 4), also harboured this 20.5 kDa protein band, although with less intensity. The protein band was absent in the 21st fraction (Lane 2) andthe stock supernatant (Lane 1), both of which had displayed the same level of killer activity (8 kU). Likewise, this protein band could not be detected in fractions 24 and 25 (Lanes 5 and 6), both of which displayed less killer activity than the stock supernatant. Present in all lanes, including the lanes resolving the molecular weight markers (L,ane M) and the loading buffer alone (Lane 7), were some high molecular weight protein bands of 50-70 kDa.

5.4 DISCUSSION

In developing a protocol for the purification of the Pichia membranifaciens CBS 7374 ktller toxin, it was necessary to first confirm the toxin was excreted extracellulady. This was done by verifying that a cell free supernatant prepared from a liquid culture, harboured killer activity (data not shown). The agar plate well diffusion killer assay employed in this preliminary experiment, was developed to facilitate purification.

Sensitivity of the assay was optimised by several means. Del

Control Fractlon24 Fraction22

Figure 5.8: Using the sensitive well test killer assay, fractions obtained from the anion-exchange chromatography of the Pichia membranifaciens CBS 7374 toxin supernatant, were assayed for their killer activity. In this instance, fraction 24 displayed four killer units, whilst the 22nd fraction displayed eleven killer units. The control, being 1 M NaCl plus 50 mM diethanolamine, failed to display activity. Figure 5.9

kDa 1M234567 97.4- I 66.2- t 45- 3r- 2t.5- * + 14.4- ì

^l'.r ö. >_¡, þ- l-

Figure 5.9: Fractions from the AE chromatorgraphy of the Pichia membranifaciens toxin supernatant, which displayed killer activity, were resolved by SDS gel 'When elctrophoresis. resolved, the fraction with the highest level of killer activity, fraction 22 (lane 5), revealed a20.5 kDa band (arrow). This 20.5 kDa band was present also in the 23rd fraction, which dsiplayed the second highest level of killer activity.

Lane 1: Stock concentration of the toxin supernatant (8 kU) Lane2; Fraction 21 (8 kU) Lane 3: Fraction 22 (llkU) Lane 4'. Fraction 23 (10 kU) Lane 5: Fraction 24 (4kU) Lane 6: Fraction 25 (2kU) LaneT; Loading buffer only M: SDS-PAGE low range molecular weight standards (Bio-Rad Laboratories, cA, usA). 53

In the production and purification of the Wiltiopsis mrakü K-500 toxin, Hodgson et al. (1995) used a minimal medium supplemented with ammonium sulphate and magnesium sulphate. Although the production of the K-500 toxin was slightly reduced when compared to cultures grown in YEPD, these workers preferred the use of the supplemented minimal medium, because of the absence of extraneous proteins. McCracken et al. (1994) also used minimal medium supplemented with casamino acids, for the production and subsequent purification of the Pichia acaciae killer toxin.

The favoured medium used to culture Pichia membranifaci¿ns CBS 7374 for toxin production, was a minimal medium supplemented with 2Vo (w/v) casamino acids (MM+Cas). Although cultures grown in this medium displayed slightly less killer activity, as compared to those grown in YM medium (Figure 5.1), MM+Cas is free of contaminating protein. In contrast, 677o (wlv) of the yeast extract component of the YM medium is protein, some of which may have hindered in the purification of the toxin. The addition of 27o (w/v) casamino acids was required, because the supernatant from minimal medium alone, had low killer activity.

Although not directly assessed in this investigation, it appeared that the killer activity of Pichiamembranifaciens CBST3T4increased, when cultured in a medium with an increased concentration of total nitrogen (Figure 5.1). It is difficult to discern, however, the real effect the level of nitrogen had on killer activity, due to the complexities of the various media used in this investigation.

To further maximise killer activity, the culture medium was buffered at pH 6.0 (Figure 5.2). At this pH level, maximum activity was detected when the agar plate well diffusion killer assay was buffered to pH 4.0. This second result suggests the optimum pH for the activity of the Pichiamcmbranifací¿ns CBS 7374toxin, is close to pH 4.0. Most characterised yeast killer toxins display maximum activity at an acidic pH (Young and Yagiu, 1978; Henschke, I979;Pfieffer and Radler, 1984; Radler et a1.,1985; Suzuki and Kikkuni, 1989; Goto et aI., 1990; Hayman andBolen, I99l;Radler et a1.,1993). An exception to this is the killer toxin of Pichia acaciae, which displayed maximum killer activity towards Saccharomyces cerevisiae at pH 7.O-7.s.This same killer toxin, however, displayed maximum killer activity towards Debaromyces tamarü at pH 5.3-6.6. The optimum pH for killer activity, is therefore also dependent on the test strain (McCracken et a1.,I994).In this study, the killer activity of the Pichia membranifaci¿ns CBS 7374 supernatant to Delcl

The activity of the Pichia membranifaci¿r¡s CBS 7374 culloxe supernatant was found to rapidly increased during the exponential growth phase, prior to peaking at early stationary phase at 1.5x10S total cellVml (Figure 5.3). The rapid increase in killer activity during the 54 exponential phase suggests that its formation is growth associated, when protein synthesis is high.

After peaking at 8 kU at the end of the exponential phese, the killer activity of the supernatant rapidly declined to an undetectable level. Likewise, Sugisaki ef al. (1984) had found ttre activity of the Kluyveromyces lactis toxin supernatant peaked at late exponential phase, but within 10 hours of stationary phase, this activity had decreased three-fold. In contrast, the killer activity of the K-500 toxin of Williopsis mrakü (Hodgson et al., 1995), the IfYI toxin of Wiltiopsis saturnus (Ohta et al., 1984), and the Kl toxin of Saccharomyces cerevísiae (Palfree and Bussey,1979), remained high during stationary phase.

All yeast killer toxins characterised, hitherto, have been found to be proteinaceous in nature, with most being temperature sensitive (Young and Yagiu, 1978; Radler et al., 1985; Suzuki and Kikkuni, 1989; Goto et al., L990; Butler et al.,l99Ia; Hayman and Bolen, 1991; Radler et a1.,1993). To confirm the proteinaeous nature of the Pichio membranifaciens CBS 7374 toxin, the culture supematant was treated with the protein degrading eîzyme Pronase E (Figure 5.4). As the concentration of the added Pronase E was increased, the killer activity of the supernatant was reduced. Furthermore, the killer toxin was rapidly inactivated when incubated at 65oC (table 5.2). It is not known whether the decline in killer activþ of the culture supernatant, when incubated at 37oC for an hour or more, is due to the temperature denaturing the toxin, or due to the activity of indigenous extracellular proteases.

During the purification process, optimal storage conditions for maintaining toxin activity were identified. In some cases, plastic tubes (polyethylene more so than polystyrene), as opposed to glass containers, have been found more satisfactory in maintaining protein stability (Linn, 1990).In this instance, however, glass and not plastic, was the best type of material for storing the toxin @gure 5.5). To further ensure the stability of killer activity, all purification steps, and the subsequent storage of the killer protein, were undertaken at 4oC (Figure 5.6).

The first chromatography process trialed, in an attempt to purify the toxin was reverse phase chromatography. An advantage with this method, is the sharp peak relative to other chromatography methods. However, the organic solvents used to elute proteins from the column's stationary phase, can often result in the denaturing of the protein, as seen in this instance (Table 5.3). Some activity did remain, however, when the culture supernatant was treated with a methanol concenffation of 40Vo (vtv). Subsequently, an attempt was made to fractionate 200 pl of stock toxin supernatant, however, the RFC process proved to harsh for the killer protein, with no killer activity detected in the fractions. A second chromatography method was employed, that of ion exchange. 55

To date, 13 of the 14 yeast killer proteins that have been partially, or wholly purified, have isoelectric points of less than pH 6.5 (Ohta et aI., 1984; Radler et al., 1985; Suzuki and Kikkuni, 1989; Goto et al., 1990; Radler et al., 1993; Komiyama et al., 1995). Only the HMK killer toxin of Williopsis mraküvar. mrakü has a basic isoelecric point (Yamamoto er al.,l986b). Therefore, in an attempt to purify the Pichin membranifaciens CBS 7374 toxin, an anion exchange column buffered at pH 8.8 was employed.

In determining whether the elutant to be used in the AEC process, would have any negative effect on killer activity, the supernatant was assayed in the presence of increasing concentrations of sodium chloride (Table 5.5). The activity of the stock supernatant remained unchanged in the presence of 0.1 M and 0.3 M NaCl, but when assayed in the presence of 1.0 M NaCl, killer activity had increased. A similar observation was seen for the Williopsis mrakävar. mrakü HMK toxin, whose activity had increased as the assay medium become less isotonic. The HMK toxin crcates pores on the growing point of the bud of sensitive cells, resulting in its death via osmolysis. Subsequently, the increase in osmotic pressure increased cell sensitivity to the HMK toxin (Komiyama et aI., 1996). The Pichia membranifaci¿ns CBS 7374 ktller toxin may also induce cell death via osmolysis, by increasing the permeability of the sensitive cell's membrane.

Although the killer activity of the supernatant increased in the presence of a high salt concentration, the elutant and the column's equilibration buffer (50 MM diethanolamine) did not reduce the supernatant's activity. As a consequence, a MonoQ column equilibrated to pH 8.8, was employed to fractionate one ml of a22.5x supernatant concentrate, with peak killer activity being successfully eluted at the 22nd fraction (Figure 5.7), which corresponds to approximately 0.61 M NaCl. As seen in the chromatogram, a small shoulder was present at the point of peak killer activity. The estimated concentration of NaCl required to elute this peak activity (0.61 M) suggests the killer toxin has an acidic pI.

Although a sharp peak of activity was obtained over fractions 2I-24, killer activity was eluted over another 2I fractions (Figure 5.7). Elution over many fractions is not uncornmon when a linear gradient is used. Its likely, however, that the high salt concentration found in these fractions, is responsible for extenuating killer activity. There also appears to be some non-specific exchange of the killer toxin, as fractions 41-43, which were obtained afær ttre elutant's NaCl concentration was returned to zero, displayed killer activity (Figure 5.7).

When resolved by SDS-PAGE, a single protein band of 20.5 kDa was found in the 22nd fraction (Figure 5.19). A single protein band of this size, but in a reduced concentration, was found also for the 23rd fraction, which displayed slightly less killer activity. Couple these observations with the obsêrvation that the 20.5 kDa band was absent in the stock supernatant, and absent in the fractions displaying killer activity equivalent to, or less than, 56 the stock supernatant, suggests this protein is the Pichia membranifaclens CBS 7374 toxin. A small number of high MW protein bands were seen in all lanes, including the loading buffer alone (I-ane 7). This would indicate that the presence of some of these high lvlW protein bands is an artefact of the gel, attributable tci þpurities in the loading buffer.

To confirm the 20.5 kDa protein as the killer toxin, the protein band in question would need to be assayed for killer activity, using a method similar to that of McCracken et aI. (1994). This would first involve visualising the proûein by native gel electrophoresis. The lane from this electrophoretic gel would then be cut into thin strips, placed on a YEPD-agar plate that had been pre-seeded with a sensitive strain, and assayed for killer activity.

Visualising the toxin by native gel electrophoresis would also provide information on its structure. The SDS PAGE of the 2Zndfraction suggests the toxin is a monomeric protein of 20.5 kDa. Other killer yeast toxins monomeric in structure include the 20 kDa KHR killer toxin of S. cerevisiae (Goto et a1.,1990) and the 19 kDa toxin of Pichia Huyverí (Magliani er a1.,1997). Alternatively, the Pichiø membranifaci¿ns CBS 7374 ktller toxin may consist of more than one subunit of, or approximately, 2O.5 kDa in size. Using a single anion exchange chromatography process, Pfeiffer and Radler (1982) successfully purified the Sacchnromyces cerevisiae K28 killer toxin, with SDS-PAGE analysis suggesting the toxin was a monomeric protein of 11 kDa. However, native gel electrophoresis, and subsequent gene sequencing, revealed the K28 toxin to be a heterodimeric 20.5 kDa protein, consisting of two subunits of 11 kDa and 10.5 kDa (Schmitt and Tipper, 1995).

It is difficult to extrapolate information on the size of the killer toxin from the ultafiltration results (Table 5.5), as passage through a molecular weight cut-off filter is dependent on the protein's three-dimensional structure, as well as its size (Pohl, 1990). Size exclusion chromatography could be used to determine the toxin's molecular weight.

Previous studies have isolated and characterised mutants found resistant to purified yeast killer toxins (Kasahara et al., 1994; Suzuki and Shimma, L999). By employing the purification protocol outlined in this study, resistant mutants could be isolated against the Pichia membranifaci¿¿s CBS 7374 toxin Further characterisation of these mutants would help to reveal the toxin's biological mechanism for inducing cell death. To date, the majority of yeast killer toxins investigated, induce cell death by osmolysis (Magliani et a1.,1997), and indeed this may be the case for the Pichía membranifuci¿ns CBS 7374 toxin. In consequence, as a first step it may be instructive to assay the sensitivity of mutants found resistant to previously defined yeast killer toxins, in a hope that they may also be resistant to

the Pichia memb ranifacrens CBS 7 37 4 tonn. 57

Chapter 6

TTTn KILLER YEAST SENSITIVITY OF PETITE MUTANTS

6.I INTRODUCTION

A petite strain of Sacclnromyces cerevisiae AWRI 1360, strain AWRI 1361, displayed resistance to two Pichin membranifaciens killer strains to which the parental was sensitive (reported in Chapter Four). A percentage of spontaneously arising mutants resistant to the Kluveromyces lactisvar. lactis CBS 2359 toxin were reported to have a petite phenotype (White et a1.,1989; Butler et a1.,1991b). However, both White et aI. (L989) and Butler øf al. (l99Ib) concluded that the petite phenotype per se was not responsible for toxin resistance. In a separate study, Polonelli et al. (1992) reported the resistance of some sensitive strains to four different killer yeasts, including Kluyveromyces lactß var. lactß CBS 2359, when assayed in an anaerobic environment.

The aim of this chapter was to further investigate the killer resistance of Saccharomyces cerevisiae AWRI 1361, by assaying the sensitivity of this strain to killer yeasts whose toxins differ in their cell wall primary receptor and method of killing. The genetics of this killer resistance were also investigated, as was its association with non- functional mitochondria. To gain further insight into killer sensitivity and functional mitochondria, the sensitivity of a number of strains deficient for functional mitochondria was assayed to a variety of killer yeast.

6.2 EXPERIMENTAL

6.2.L Yeast strains and media

The sensitive and killer yeast strains used in this study are listed in Table 6.I and 6.2, respectively. Yeast strains were subcultured in YEFD ÍL%o (wlv) yeast extract,2Vo (w/v) peptone, 2Vo (wtv) glucosel at 25oC for 24-48 h before being transferred to YEPD-agar IYEPD, 2Vo (wlv) agar] slopes at 25oC for 48 h, then stored at 4oC until required. For the selection of growth of yeast strains auxotrophic for a particular growth requirement, drop- out media were employed as outlined by Adams et al. (1997).

6.2.2 Inducing petites via treatment with ethidium bromide Table 6.1

Strain Genotype Source (Strain)

ATWRI1360 MATap* adel his4leu2-2 ura3 L Dawes* (Y3) AWRI1361 MATap- adel his4leu2-2 ura3 I. Dawes (Y3P) AWRI1362 MATap adel his4leu2-2 ura3 This study

A\ryRr 1363 MATap'his3-Ll leu2 ura3-52 tpl H. Richardsont lIvY+ez¡ AWRI1364 MATu.p'his3-L,l leu2 ura3-52 tpl This study AWRI1365 MATap* Ieu2 ura3 lys2 trpl R. Davis$ (YP52) A\ryRI1366 MATap- leu2 ura3 lys2 tpl This study AWRr 1369 MATa MATU p* ade)/ADEI HIS3/ his3-Ll his4/HI34 1360 x 1363 Ieu2-2/leu2 ura3/ ura3-52 TRPI/ tpl AWRr 1370 MATa MATa pt adel/ADEI HIS3/ his3-L,I his4/HI54 1360 x 1364 leu2-2/Ieu2 ura3/ ura3-52 TRPI/ tpl AWRI1371 MATa MATa p' adel/ADEI HIS3/ his3-L1 his4/HI54 1361 x 1363 leu2-2/leu2 ura3/ ura3-52 TRPI/ tpl AWRI1372 MATa MATa p adel/ADEI HIS3/ his3-L,I his4/HI54 This study leu2-2/leu2 ura3/ ura3-52 TRPI/ tpl AWRI1373 MATa MATa p adel/ADEl HIS3/ his3-L,1 his4/HI54 This study leu2-2/leu2 ura3/ ura3-52 TRPI/ tpl AWRI1374 MATa MATa p adel/ADEl HIS3/ his3-L,I his4/HI54 136I x 1364 leu2-2/leu2 ura3/ urs3-52 TRPl/ tpl

AWRr 1375 MATap adel his4leu2-2 ura3 This study AWRI1376 MATap- adel his4leu2-2 ura3 This study AlvRI1377 MATap- adel his4leu2-2 ura3 This study

15d cr MATap* adel hisl leu2 ura3 trpl B. Futcherr

Table 6.1: List of sensitive strains used in this study. * Dr Ian Dawes, The University of New South Wales, Australia. t Dr Helena Richardson, The University of Adelaide, Australia. $ Dr R Davis, Stanford University, USA. t[Dr Bruce Futcher, Cold Springs Harbour Laboratories, USA. Table 6.2

Killer yeast Genetic basis of killer phenotype

Debaromyces vanrijiae NCYC 388 nuclear

Kluyveromyces lactis var. lactis CBS 2359 linearDNA

Kluyveromyces lactis var. drosophilarum CBS 2896 nuclear

Kluyveromyces marxiar¿øs NCYC 587

Pichiø anomnla CBS 1982

Pichia anomnla NCYC 434

Pichia subpelliculosø NCYC 16

P ichia memb ranifac iens CBS I07 nuclear

P ichia memb ranifacl¿ns CB S 63 8 nuclear

P ichia membranifaciens CBS 7 37 3

Pichia membranifaciens CBS 7374

Sacharomyces cerevisiø¿ CBS 6505 ds RNA

Williopsis saturnus var. mrakii CBS 1707 nuclear

Table 6.2: The killer yeasts employed in this study 58

The petite Sacchnromyces cerevisrd¿ AWRI 1361 (Collinson and Dawes, 1992), was sourced from Dr Ian Dawes of the University of New South Wales. The remaining petiæs employed in this study were constructed using the method of Crow et al. (1998). Approximatety 107 yeast cells were inoculated into 5vnl of SD medium lO.67Vo (w/v) yeast nitrogen base without amino acids, 27o (wlv) glucosel supplemented with 27o (wlv) Casamino Acids and 10 ttúml of ethidium bromide. This culture was then agitated at 30oC for 24 h in the dark (aluminium foil was wrapped around the culture flask to avoid light). The culture was then streaked onto YPDG-agar medium for single colonies. Colonies were confirmed petite by the absence of growth on YPG-agar medium ÍI7o (wlv) yeast extract, 27o (wlv) peptone, 37o (vlv) glycerol, 2Vo (wlv) agarl.

6.2.3 Selection of spontaneous petites

The strain of interest was streaked for single colonies on YPD-agar medium, and incubated at 30oC for 48 h. A single colony was then selected to inoculate the entire surface of a single YPD-agar plate, which was incubated at 30t for 24h, after which the culture was retrieved from the plate using 1.5 ml of sterile water. This culture was diluted 104, 10-5 and 10-6, and 0.2 ml of each dilution was spread onto YPDG-agar medium. Colonies were confirmed petite by the absence of growth on YPG-agar medium.

6.2.4 Killer assays

The solid agar streak-test assay is described in Section3.2.2, and the well test killer assay is described in Section 5.2.2. The method for undertaking killer assays in an anaerobic environment is essentially that of Polonelli et al. (1992), using a Model B Coy Anaerobic Cha:rrber (Coy Laboratory Products Inc., MI, USA). YPD-agar plates to be anaerobically assayed were prepared, before the inoculum of the sensitive strain, by placing them in the anaerobic hood for 4 hours. Killer activity was expressed in terms of minimal activity (appearance of a dark blue halo), weak (0.1 - 1.5 mm), medium (1.5 - 2.5 mm) or strong killer activity (2.5+ mm).

6.2.5 Forming diploids

Crosses were undertaken by co-inoculation on SD drop out media, with the diploids selected based on their growth in the absence of particular growth requirements, as outlined in Adams et at. (1997). Diploid isolates were identified as having a wild type (p+) or petite (p-) phenotype by their ability to grow on YPG-agar medium.

6.2.6 Concentration of tlrre Píchía membranífacíens CBS 7374 killer toxin supernatant 59

The toxin supernatant of Pichia membranifaci¿ns CBS 7374, was conoentrated using the method outlined in Section 5.2.10.

6.2.7 mtDNA isolation and restriction analysis

The procedure for the isolation of mtDNA and its subsequent restriction enzyme analysis is essentially that of Querol et aI. (1992). Yeast cells were grown in an overnight culture of 5 rnl of YEPD. Cells were spun down in a microcentrifuge and resuspended in 0.5 ml of a spheroplast buffer (1 M sorbitol, 0.1 M EDTA, pH 7.5), to which 0.02 ml of Zymolyase 60 (Sigma) (2.5 mgtml) was added. The cells were incubated at 37oC for 60 minutes, pelleted by centrifugation, then resuspended in 0.5 rnl of a spheroplast-lysing buffer (50 mM Tris-

IJCI, 20 mM EDTA, pH 7 .4). Spheroplasts were lysed with the addition of I07o SDS, incubated at 65oC for 30 minutes. Immediately thereafter, O.2 ml of 5 M potassium acetate was added, and then placed on ice for 30 minutes prior to centrifugation for 5 minutes at 13,000 rpm. The supernatant was retained and the DNA precipitated with the addition of 1 volume of isopropanol, incubating at RT for 5 minutes. The precipitated DNA was pelleted by centrifugation for 10 minutes at 13,000 rpm, washed with TOVI (vlv) ethanol, vacuum dried in a speedi-vac, and dissolved in 50 pl of TE (10 mM Tris-HCl, 1 mM EDTA' pH 7.5). A volume of 2 ¡tl of DNA was digested with the restriction endonucleases Rs¿I (Boehringer Mannheim) and Hinfl @oehringer Mannheim) as outlined by the supplier. Restriction fragments were separated in 27o (wtv) agarose gel electrophoresis and visualised under a UV transilluminator after ethidium bromide staining.

6.3 RESULTS

6.3.1 Sacchøromyces cerevisiae AWRI t36L, derived from Saccharomyces cerevísiae A\ryRI 1360, harbours deletions in its mitochondrial genome

As reported in Chapter Four, the petite strain of the sensitive Saccharomyces cerevisiae AWRI 1360, strain AIVRI l3íl,displayed resistance to Pichi.a membranifaciens CBS 7374 and CBS 7373. Due to the significance of this result there was a need to confirm the relatedness of these two Saccharomyces cerevisiae strains, both of which were sourced from Dr Ian Dawes of the University of New South Wales, Australia. This first involved verifying the auxotrophic markers of these strains (ie. ade- his- leu- ura-) by plating them to drop out media (Table 6.3).

The relatedness of these strains were further verified by checking the mutant gene (ie. his4) echoing the auxotrophic marker. This was done by crossing these strains with strains which Table 6.3

SD drop-out media

AWRI strain Auxotrophic markers adenine histidine - leucine uracil tryptophan

1360 adel his4leu2 ura3 x x x x {

1361 adel his4leu2 ura3 x x x x {

1363 his3leu2 ura3 trpl { x x x x

15d adel hisl leu2 uraj trpl x x x x x

1360 x 1363 adel/ ADEI his3/HIS3 his4/HIS4 ./ { x x { leu2/leu2 ura3/ura3 TRP I /trp I

L36l x 1363 adel/ ADEL his3/HIS3 his4/HIS4 ./ ./ x x ./ leu2/leu2 ura3/ura3 TRP I /trp I

,V 1360 x 15d ade ]/ade I his4/HIS4 HIS I /his4 x { x x Ieu2/leu2 ura3/ura3 TRP I hrp I ,l 1361 x 15d ade I /ade I his4/HIS4 HIS 1/his4 x x x { leu2/leu2 ura3/ura3 TRP I hrp I

Table 6.3: Haploid and diploid strains, and their growth on various SD drop-out media. The tick ({) indicates growth on that particular medium, whereas the cross (x) indicates no growth. Table 6.4

S. cerevisia¿ AWRI tester strain Killer Yeast 1360 1361 1360 t362 1375 1316 t377 i. 1363 t364 1363 1365 1366 - 0r* - 0rj

S. cerevisia¿ CBS 6505 +++ +++ +++ n.d. n.d. n.d. +++ +++ +++ ++ +

W. saturnus var. mraktt CBS 1707 +++ +++ +++ n.d. n.d. n.d. +++ +++ +++ +++ ++

K. lactis var. Iactis CBS 2359 + + n.d. n.d. n.d. + + +

P. anomnla CBS 1982 ++ +l- n.d. n.d. n.d. + + ++

P. anom"ala NCYC 434 ++ n.d. n.d. n.d. n.d. n.d. ++ +l- n.d. +++ +l-

D. vanrijia¿ CBS 40721 + n.d. n.d. n.d. n.d. n.d. + +l- n.d. + +l-

P. membranifaciens CBS 7374 +++ +++ +++ +++ +++ +++ ++ +l- n.d ++ +l-

P. membranifaciens CBS 7373 ++ ++ ++ ++ ++ ++ ++ +l- n.d + +l-

P. membranifaciens CBS 638 + n.d n.d. n.d. n.d. n.d. + +l- n.d + +l-

P. membranifaciens CBS 107 + n d n.d. n.d. n.d. n.d. + +l- n.d + +l-

growth mediumT +l-

Table 6.4: Respiratory competent (p*) and incompetent (p-) strains of Sacchnromyces cerevisiae, assayed for their level of sensitivity to killer yeasts. * killer assays were undertaken in an anaerobic environment.

T the well-test killer assay was used instead of the solid-agar diffusion assay. Petite strains are highlighted in blue. 60 share different auxotrophic markers (Table 6.3). When assayed for their growth on various SD drop-out media, the diploids arising fuom Saccharomyces cerevisiae AWRI 1360 and AWRI 1361 crossed with either Sacchnromyces cerevist¿¿ A\MRI 1363 or AWRI 1377 shared the same growth requirements. These results strongly suggest fhat Saccharomyces cerevisiae AWRI 1360 is the wild type (wt) of the petite AWRI 1360 as they both share the same auxotrophic markers. Saccharomyces cerevisiae AWRI 1360 was further confirmed as the wt of strain AWRI 1361 using the molecular technique amplified fragment length polymorphism. Of more than 300 amplified fragments, all were monomorphic between the two strains (data not shown).

Mtochondrial DNA (mtDNA) restriction analysis of the three strains Saccharomyces cerevisiaz AWRI 1360, AWRI 1361 and 15d was undertaken, as depicted in Figure 6.1. This technique was employed initially in an attempt to differentiate these strains based on their mtDNA amplihed restriction patterns. The gel of Figure 6.1 demonstrates that Saccharomyces cerevis¡¿¿ AWRI 1360 (Lane 2) has a mitochondria similar to the respiratory competent Sacclnromyces cerevisíae I5d (Lane 3). However, Sacchnromyces cerevisiae AWRI 1361 (Lane 1) shows a very different RFLP pattern indicating that either no mtDNA exists, or at least a large portion of it has been deleted.

6.3.2 The petite Sa.ccharomyces cerevísiae A\ryRI 1361 displays killer toxin resistance

To investigate the association between functional mitochondria and cell sensitivity to yeast killer toxins the petite strains, Saccharomyces cerevisia¿ AWRI L362, AWRI 1375, AWRI L376 and AWRI L377, were derived from the killer-sensitive (kS), respiratory compotent (p+) Saccluromyces cerevisiae strain AIVRI 1360 (Iable 6.1). These petites were assayed for their sensitivity to a number of different killer yeasts, starting with different killer strains of Pichiamembranifaciens (Table 6.2). Also assayed for its sensitivity to these killer yeasts was the petite Sacchnromyces cerevisiae AWRI 1361 (Table 6.1), which was derived from Saccharomyces cerevisiae AWRI 1360 as described by Collinson and Dawes (1992).

Results from the solid agar well-test assay showed that four of the five petites, Saccharomyces cerevis,ø¿ AWRI 1362 and AWRI 1375-1377, displayed the same level of sensitivity as the respiratory competent parent to the killer yeasts Pichin membranifaciens CBS 7374 and CBS 7373 (Table 6.4 and Figure 6.2). This is unlike the petite Saccharomyces cerevß¡¿¿ AWRI 136I, which displayed resistance (kR) to these two killer yeasts, and resistance to a further two killer strains of Pichia membraniþciens, CBS 638 and CBS 107 (Figure 6.2). Figure 6.1

123 rú^ r.l a

Figure 6.1: The mitochondrial DNA restriction analysis of: Lane 1: Saccharomyces cerevisía¿ AWRI 1361 (petite) Lane 2: Saccharomyces cerevisiø¿ AV/RI 1360 (respiratory competent) Lane 3: Saccharomyces cerevisiae ISd (respiratory competent)

(Photograph kindly supplied by J .Bellon) Figure 6.2: The parental Saccharomyces cerevisiae AWRI 1360 (p*ks), and the petites Saccharomyces cerevis¿ø¿ AWRI 1361 (p-lë) and Saccharomyces cerevisiae AWRI 1362 (p- ks), were assayed for their level of sensitivity to the toxins of Pichia membranifaciens klller strains. Saccharomyces cerevisí¿¿ AWRI 1360 was assayed both in an aerobic (+0r) and an anaerobic (-0r) environment.

Ã. Pichia membranifaciens CBS 7374 B. Pichia membranifaciens CBS 7373 C. Pichia membranifaci¿ns CBS 638 D. Pichia membranifaciens CBS I07

The killer activity was scaled based on the radius of the killer halo (mm)

- : no visible sign of killer activity +/- : minimal activity (the appearance of a dark blue halo) + : weak killer activity (0.1 - 1.5 mm) ++ : medium killer activity (1.5 - 2.5 mm) +++: strong killer activity (2.5+ mm) Figure 6.2

Saccharomyc e s c erevísiae tester strains

AWRr 1360 AWRr 1361 AWRr 1362 ++++ ++++

A.

A\ryRr 1360 A\ryRI 1361 AWRr 1362 ++ ++

B.

Sac charomy c e s c erevisiae testq strains

A\ryRr 1360 AWRI 1361 +

C.

AWRr 1360 AWRI 1361 +

D. Figure 6.3: The parental Saccharomyces cerevisiae AWRI 1360 (p*ks), and the petites Saccharomyces cerevisiø¿ AWRI 1361 (p-lð) and Saccharomyces cerevisine AWRI 1362 (p ks), were assayed for their level of sensitivity to the killer yeasts: A.. Saccharomyces cerevisíae CBS 6505 B.Williopsis saturnus var. mrakü CBS 1707 C. Kluyveromyces lactis var. lactis CBS 2359 D. Pichia anomala CBS 1982 Saccharomyces cerevisi¿¿ AWRI 1360 was assayed both in an aerobic (+0r) and an anaerobic (-0r) environment. Figure 6.3 S ac charo my c e s c e rev i s iae tester strains

AWRr Ur69 1+02) AV/RI 1361 AWRI 1362 AwRI tzí} (02) +++ +++ +++

A" :1 rFEl,

+++ +++

B

. ;j.' "' - ...., '':' :'-.i : :

+

C. -'5¡Fr-Ër

++

tt t D. '.t '4 ,' . ..'., . .":+ ..r: Figure 6.4

Saccharomy ces c erevisiae tester strains

AWRr 1360 AWRr 1361

++

A.

+

B.

Figure 6.4: The parental SaccharomTtces cerevisiae AWRI 1360 (p+ks) and Saccharomyces cerevisiae AWRI 136I (p-kR), were assayed for their level of sensitivity to the killer yeasts Ã. Pichia anomala NCYC 434 and B. Debarornyces vanrijiae CBS 4072. Table 6.5

Killer yeast Genetic basis Toxin structure Primary receptor Killing action Resistant mutant

K. lactis var.lactis two linearDNA heterotrimeric protein Possibly chitin a subunit has chitinase actiYity Chitin deificient strains. plasmids CBS 2359 a subunit (99 kDa) g subunit induces cell a¡rest at Genes encoding tRNA3Glu pGKLI (8.9 kb) Gl phase and an unidentied 35 kDa b subunit (30 kDa) polypetide pGKL2 (13.4 kb) Unknown intracellulaler target(s) g subunit (27.5 Y.Da)

S. cerevßiae two dsRNA virus- heterodimeric protein A b-glucan Osmolysis by pore formation in None like particles (38.7 kDa) component of the cell cell wall CBS 6505 wall, possibly p-1,6- L-A virus (4.6 kb) a subunit (I72 aa) glucan M2 virus (1.5 kb) b subunit (140 aa)

W. saturnus var. mrakü nuclea¡ single polypetide A Sglucan Osmolysis by inhibiting p-1,3- KNR4 gene which is CBS 1707 component of the cell glucan synthetase actiYity at the associated with þ l,3-glucan wall budding point synthetase

Table 6.5: The killer yeast systems of Kluyveromyces lactis var. Iactis CBS 2359, Saccharomyces cerevisiae CBS 6505 and Wílliopsis saturnus var- mrakü CBS 1707. Figure 6.5

Saccharomyces cerevisiae tester strains

A\ /RI 1363 AWRI 1364 ++ +l-

A.

+ +l-

B.

+ +l-

C

+ +l-

D

Figure 6.5: The petite Saccharomyces cerevisiø¿ AWRI 1364 (p-knS¡ and its parental

Saccharomyces cerevisla¿ AWRI 1363 (p+ts), were assayed for their sensitivity to the toxins of Pichia membranifaciens þJller strains.

t¡. Pichia membraniþciens CBS 7374 B. Pichia membranifaciens CBS 7373 C. Pichia membranifaci¿ns CBS 638 D. Pichia membranifaciens CBS I07 Figure 6.6: The petite Saccharomyces cerevisr'a¿ AIWRI 136a þ-Ës) and its parental Sacch.aromyces cerevisia¿ AWRI 1363 (p.ks), were assayed for their level of sensitivity to the killer yeasts: Ã. Sacchnromyces cerevisiae CBS 6505 B.Williopsis saturnus var. mrakü CBS 1707 C. Kluyverornyces lactis var. lactis CBS 2359 D. Pichia anomala CBS 1982 Saccharomyces cerevisiø¿ AWRI 1363 was assayed both in an aerobic (+0r) and an anaerobic (-0r) environment. Figure 6.6

S ac c haromy c e s c e r ev i s iae tester strains

(02) AWRr tr63 $0) AWRI 1364 AwRI n$

+++ +++ +++

A

+++ +++ +++

B '--4L,----+

+

C.

' ..''.; :,' . ,

D -I:¡¿ilf-.fltfl1: Figure 6.7

S ac charomy c e s c e rev isiae testet strains

AWRI 1363 AWRr 1364

++ +l-

A

+ +l-

B.

Figure 6.7: The petite Saccharomyces cerevisi¿¿ AWRI 1364 (p-kRS) and its parental

Saccharomyces cerevisi¿¿ AWRI 1363 (p+ks), were assayed for their level of sensitivity to the killer yeasts d,. Pichia anomala NCYC 434 and B. Debaromyces vanrijiae CBS 4072. 6r

To determine whether this resistance was confined to killer strains of Pichit membranifaciens, Saccharomyces cerevisia¿ AIWRI 1361 was assayed for it's sensitivity to six different killer yeasts that the parental yeast showed sensitivity to (fable 6.4). The solid agar streak test assay was employed for all six killer yeasts with the exception of Debaromycesvanrijiae CBS 4072, whose killer activity is more readily identified using the well test killer assay. Results from these killer assays revealed that Sacclwromyces cerevisiae AWRI 1361 displayed resistance to all six killer yeasts (Figures 6.3 and 6.4). This included resistance to Saccharomyces cerevisiae CBS 6505, Kluyveromyces lactis var. /¿crls CBS 2359 andWiltiopsis saturnusvar. mrakit CBS 1707, where information on structure and mode of action for the toxins of these killer yeasts is known (Table 6.5).

To determine what effect the lack of respiration alone would have on the sensitivity of Saccharomyces cerevisi¿¿ AWRI 13601p+¡s), this strain was assayed to six different killer yeasts in an anaerobic environment (fable 6.4). There was no change in the sensitivity of Saccharomyces cerevisia¿ AWRI 1360 to five of the six killer yeasts when assayed in an anaerobic environment (Figures 6.2 and 6.3). However, the sensitivity of this strain to Pichia anomala CBS 1982 decreased (Figure 6.3).

6.3.3 Reduction in yeast killer toxin sensitivity displayed by petite Saccharomyces cerevísíøe strains

Apetite of sacclnromycescerevisiae AWRI 1360 (p+kS), strain AWRI 1362, was assayed for its sensitivity to the killer yeasts Saccharornyces cerevisiae CBS 6505 , Kluyveromyces I.actís var. tnÊtß CBS 2359, Williopsis saturnus var. mrakü CBS 1707 and Píchia arcmala CBS 1982 (table 6.4). This petite strain displayed the same level of sensitivity to Sacclnromyces cerevisr.d¿ CBS 6505 and Williopsis saturnus var. mrakit CBS l7O7 as compared to its parent strain @igure 6.3). However, sensitivity of Saccharomyces cerevísiae AIVRI 1362 to both Kluyveromyces l.actis var. lactis CBS 2359 and Pichia arcmala CBS 1982 was not detectable (Figure 6.3).

Employing the ethidium bromide method, petite strains were derived from the sensitive parent strains Saccharomyces cerevisia¿ AWRI 1363 and Sacchnromyces cerevis¡¿¿ AWRI 1365, and their sensitivity to ten different killer yeasts assayed (Iable 6.4). The petite of Saccharomyces cerevisr.c¿ AWRI 1363, strain AWRI 1364, displayed a reduction in sensitivity (kns¡ to all four killer strains of Píchia membranifaciens @igure 6.5), and to four of the six non-Pichin membranifaciens killer yeasts @igures 6.6 and 6.7). Like Saccharomyces cerevisr.d¿ AWRI 1362, Saccharomyces cerevisra¿ AIVRI 1364 shared the same level of sensitivity to Saccharomyces cerevisiae CBS 6505 andWilliopsis saturnus var. mrakä CBS 1707 as its parent strain. This differs to a petite of Saccharomyces ceratßiae 62

AWRI 1365, strain AWRI 1366, which displayed a reduction in sensitivity to all ten killer yeasts (Table 6.4 and Figures 6.8-6.10).

To determine whether the lack of respiration alone has an effect on the sensitivity of Saccharomyces cerevisr.d¿ AWRI 1363 (p+ks) to yeast killer toxins, this respiratory competent strain was assayed for its sensitivity to four killer yeasts in an anaerobic environment Clable 6.4). When assayed, however, Saccharomyces cerevisi¿¿ AWRI 1363 displayed the same level of sensitivity to all four killer yeasts in both an anaerobic and aerobic environment (Figure 6.6).

6.3.4 The killer resistant phenotype of Saccharomyces cerevísíae AWRI 1361 is partially attributable to a nuclear mutation

Of thefivepetites of SacchnrornycescerevisiaeAwRl 1360, only Sacchnromyces cerevisia¿ AWRI 1361 displayed a complete resistant phenotype to all killer yeasts tested (kR). To determine whether this resistant phenotype was atftibutable to a nuclear or a mitochondrial mutation (or both), two different crosses were undertaken, and the resultant diploids assayed for their sensitivity (Figure 6.11).

Saccharomyces cerevisia¿ AWRI 1361 (p-kn) was crossed with the sensitive, respiratory competent Saccharomyces cerevisía¿ AWRI 1363 (p+kS) (Cross One, Figure 6.11). The resultant diploid, Saccharomyces cerevisrd¿ AWRI L37L, was found to be respiratory competent and thus, homoplasmic for the mitochondria of Sacclwromyces cerevisiae AWRI 1361. When assayed Saccharomyces cerevisia¿ AWRI 1371 (p+) displayed sensitivity to all sixkilleryeasts assayed (Figures 6.12 and 6.13), with the level of sensitivity being similar to the respiratory competent parental strains AWRI 1360 and AWRI 1363.

The sensitivity level of Saccharomyces cerevisia¿ AWRI 1371 (p+kS¡ remained the same in an anaerobic environment to all but one of the killer yeasts assayed, Pichin atnmaln CBS 1982 (Figure 6.13). This result was consistent with the observation that the haploid parcnt Saccharomyces cerevisr¿¿ AWRI 1360 displayed a reduced level of sensitivity to Pichia anomala CBS 1982 in an anaerobic environment (Table 6.4).

'When two petites of the diploid strain Saccharomyces cerevisld¿ AWRI 1371 (p+kS¡ were assayed for their sensitivity, both petites displayed resistance to Saccharornyces cerevisiae CBS 6505, Kluyveromyces l.actß var. /¿cr¿s CBS 2359, Pichia arcmala CBS 1982, ild Pichiamembranifací¿¿s CBS 7373,with only minimal sensitivity to the remaining two killer yeasts assayed @igures 6.12 and6.13). Therefore, the diploid arising from the cross of the sensitive Sacclaromyces cerevisu¿ AWRI 1363 (p+ks) with the resistant Saccharomyces Figure 6.8

Saccharomy ce s cerevisiae testet strains

AWRr 1365 AWRI 1366 ++

A.

+ +l-

B.

+ +l-

C

+ +l-

D.

Figure 6.8: The petite Saccharomyces cerevisiae AWRI 1366 and its parental Saccharomyces cerevisiø¿ AWRI 1365, were assayed for their sensitivity to the toxins of Pichia membraniføciens kJller strains.

A¡. Pichia membranifaciens CBS 7374 B. Pichia membraniþciens CBS 7373 C. Pichia membranifaci¿ns CBS 638 D. Pichiamembraniþciens CBS lO7 Figure 6.9

S ac charomy c e s c e revisiae tester strains

AlvRr 1365 AWRI 1366

++ +

A.

+++ ++

B

+

C. f-rì,.++--.-+L\ ''...-._ __-._,,.i

++

D.

Figure 6.9: The petite Saccharomyces cerevisiae AWRI 1366 and its parental Saccharomyces cerevisia¿ AÌWRI 1365, were assayed for their sensitivity to the killer yeasts: !¡. Saccharomyces cerevisiae CBS 6505 B.Williopsis saturnus var. mrakü CBS 1707 C. Kluyveromyces lactis var.IactisCBS 2359 D. Pichia anomalaCBs 1982 Figure 6.10

S accharomyc es cerevisiae testet strains

AWRr 1365 AWRr 1366

+++ +l-

A.

+ +l-

B

Figure 6.10: The petite Saccharomyces cerevisl¿¿ AWRI 1366 and its parental Saccharomyces cerevisia¿ AWRI 1363, were assayed for their level of sensitivity to the killer yeasts A,. Pichia anomala NCYC 434 andB. Debaromyces vanrijiae CBS 4072. Figure 6.11

Cross One

AWRI 1361 (p-kn) x AWRI1363 (p+ks) resistant sensitive phenotype phenotype

AWRI 1371 (p+ts; sensitive phenotype

EtBr-induced petite

AWRI 1372 (p-kNn) AWRI 1373 (p-kun¡ near-resistant near-resistant phenotype* phenotype*

Cross Two

AWRI 1361 (p-kn) x AWRIt364 (t-tns¡ resistant reduced-sensitivity phenotype pehnotype

AV/RI 137a (P-kxn; near-resistant phenotypex

Figure 6.11: Crosses One and Two were undertaken to gain further insight into the resistant mutation of Saccharomyces cerevisiae AWRI L361. *The two petites of Saccharomyces cerevisiae AWRI I37I, and the petite diploid Saccharomyces cerevisiae AWRI 1374, displayed what was essentially a resistant phenotype. These results suggest the resistant mutation is nuclear and partially dominant, but is suppressed in the presence of functional mitochondria. Figure 6.122 The diploid strains arising from Cross One, Saccharomyces cerevisiae AWRI 1371 (p*ks), and Cross Two, ,Søccharomyces cerevisiae AIWRI 1374 (p-kN*), were assayed for their level of sensitivity to the killer toxins of A. Pichin membranifaciens CBS 7374 and$. Pichiamembranifoci¿r¿s CBS 7373. The petites of Sacchnromyces cerevisine AWRI 1371 (p.ks), Søccharomyces cerevisine AIVRI 1372 (p-lCR) and AWRI 1374 (p-kNR) were assayed for their sensitivity to these toxins also. Sacch.aromyces cerevisiae AWR^I L37l was assayed to the Pichin membranifaci¿ns CBS 7374 in both an aerobic (+Or) and an anaerobic (-Or) environment. Figure 6.12

Saccharomyce s cerevisiae tester strains

AlvRr tztt (úz) A\MRI 1374 AWRI 1372 AWRI I37L (-02) ++ +l- +l- ++

A.

AWRI 1371 AWRI 1374 A\ryRI 1372 AWRI 1373 + +l- +l- +l-

B Figure 6.13: The diploid strains arising from Cross One, Saccharomyces cerevisiae AWRI 1371 (p.ks), and Cross Two, Saccharomyces cerevisiae AIVRI 1374 (p-kNR), and the petite diploids Saccharomyces cerevisra¿ A\ilRI 1372 (p-kM) and AWRI 1373 (p-kN*), were assayed for their level of sensitivity to the killer toxins: ì¡. Saccharomyces cerevisiae CBS 6505 B.Williopsis saturnus var. mrakü CBS 1707 C. Kluyveromyces lactis var. Iactis CBS 2359 D. Pichia anomala CBS 1982 Sacch.aromyces cerevista¿ AWRI 1371 was assayed to these killer yeasts in both an aerobic (+Or) and an anaerobic (-Or) environment. Figure 6.L3 Saccharomyces cerevisiae tester strains

AlvRr tztt (úù AWRI tZtt G0ù AWRI 1372 AWRI 1373 AWRI 1374

++ ++ A.

+l-

B.

+ +

C

+ +l-

D. 63 cerevßiaeA\ryRl 1361 (p-kn), displayed a near-resistant ftNR) phenotype, but only when petite.

The diploid arising from the cross of Saccharomyces cerevisi¿¿ AWRI 1361 (p-kn) and Sacchnromyces cerevis¡'a¿ AWRI 1364 (p-kns) (Cross Two, Figure 6.11), Saccharomyces cerevisiae AWRI 1374, displayed the same near-resistant GNR) phenotype as the two petites of Saccharomyces cerevisiae AWRI 1371 (Figures6.12 andFigure 6.13).

6.3.5 Estimating the level of resistance of Sacchøromyces cerevísíae AWRI 1361

To quantify the level of resistance shown by the petite Saccharomyces cerevis¿'a¿ AWRI 1361 relative to its sensitive parent, the two strains were assayed for their sensitivity to various concentrations of the Pichia membranifaci¿ns CBS 7374 ktller toxin supernatant (Table 6.6). Also assayed for their sensitivity were the two petites of the diploid arising from Cross One, Sacchnromyces cerevisínc A\ryRI 1372 (p-kNn) and AWRI 1373 (p-kt'lR), -d the petite diploid arising from Cross Two, Saccharomyces cerevisiae AWRI 1374 (p-kNn¡.

Saccharomyces cerevisia¿ AWRI 1360 (p+ks) displayed sensitivity to the Pichia membranifacr¿zs CBS 7474 tonn supernatant to up to a 1 in 6 dilution (Figure 6.14). In comparison, Saccharomyces cerevisiø¿ AWRI 1361 (p-kn) displayed resistance to at least a 2 fold concentration. This would infer that Sacchøromyces cerevisia¿ AWRI 1361 is at least twelve times more resistant to the toxin supernatant as compared to the parent strain. The two petites Saccharomyces cerevislo¿ AWRI 1372 (p-kxn) and AWRI 1373 (p-kNn), and the petite Sacclnromyces cerevisia¿ AWRI 1374 (p-kM), displayed a minimal level of sensitivity to a stock concentration, and resistance to a 1 in 2 dilution (Figure 6.14). Therefore, it would appear that these near-resistant petites are 3 times more resistant to the Pichia membranifaciens CBS 7474 toxin supematant as compared to Saccløromyces cerevisiae AWRI 1360.

6.4 DISCUSSION

The association of killer toxin sensitivity and mitochondria was investigated using five petiæ strains of Saccharomyces cerevisiae AWRI 1360 (p+ks). These were assayed for their sensitivity to the killer yeasts Pichiamembranifaciens CB,S7474 andCBS 7373. Four of the five petite strains were constructed in this study, whilst the fifth petite, Saccharomyces cerevisi.ae AWRI 1361 (p-kn), was provided by Dr Ian Dawes of the University of New South Wales. Table 6.6

Concentration S. cerevisia¿ ArWRI tester strain P. membranifuciens 1360 r36L 1374 1372 r373 CBS 7374 toxin 2x ++++ + + +

Stock +++ +l- +l- +l- I in2 ++

1in5 + lin6 +l- I in7 1in8 1in9 1in10

Table 6.6: Respiratory competent (p+) and incompetent (p-) strains of Saccharomyces cerervisiae assayed for their level of sensitivity to various concentrations of the Pichía me mb r anifaci¿n s CB S 7 37 4 ktller toxin supernatant. Figure 6.142 Strains of Sacchnromyces cerevisi.ae were assayed for their level of sensitivity to varying concentrations of the Pichin membranifaciens CBS 7374 killer toxin supernatant. A. and l. Saccharomyces cerevisiae AWRI 1360 (p.ks) B. Sacch,a.romyces cerevisiae AWRI 1361 (p-kR) C. Saccharomyces cerevisiae AWRI 1374 (p-lCR) D. Saccharomyces cerevisiae AWRI 1372 (p-kM) E. Saccharomyces cerevisiae AIVRI 1373 (p-lCR) Figure 6.14 Concentration 2x 1x I in2 ++++ +++ ++

A.

-

B.

+ +l- -

C.

+ +l-

D.

+ +l-

E.

1in5 1in6 I in7 + +l-

F. 64

Saccharomyces cerevis¡¿¿ AWRI 1360 was validated as the wt of Saccharomyces cerevßiae AWRI 1361, by crossing these two haploids with strains harbouring different genetic markers (Table 6.3). The growth requirements of these diploids verified that Saccharomyces cerevisiae AWRI 1360 and AWRI 1361 shared the same auxotrophic markers. A\ryRI 1360 was further confirmed as the parent of AWRI L36l by amplified fragment length polymorphism analysis (de Barros Lopes, personal communication). The petite AWRI 1361 was also shown to carry deletions in its mitochondrial genome (Figure 6.1).

Assaying the killer sensitivity of petites showed that the four petite strains constructed in this study displayed the same level of sensitivity as the parent strain. The fifth petite AWRI 136I, however, displayed resistance to all ten killer yeasts. The sensitivity of Saccharomyces cerevisiae AWRI 1360 (p+¡s) to five of the six killer yeasts assayed was the same in both an anaerobic and aerobic environment (Figures 6.2 and 6.3). This result suggests that the resistance phenotype of AWRI 136I (p-kR) is not due to the lack of oxidative- phosphorylation.Instead, it appears that a nuclear or a mitochondrial mutation, independent of oxidative-phosphorylation, confers the resistance. To further investigate the genetics of this mutation, two different crosses were undertaken, and the resultant diploids assayed for their sensitivity (Figure 6.11).

Saccharomyces cerevisrø¿ AWRI 1361 (p-kn) was crossed with AWRI 1363 (p+ks¡ lcross One), with the resultant diploid, AWRI 137I, found to be respiratory proficient. As described in Chapter 2 (2.6.2 Resistant mutants and functional mitochondria), the resultant diploid from the cross of two haploid strains with different mitochondria, will bear only one mitochondrial type ie., the resultant diploid will be homoplasmic for the mitochondria of one of the haploids @ujon, 1981; Gingold, 1988). The observation that AWRI 1371 is respiratory competent implies that this diploid is homoplasmic for the p+ mitochondria of AWRI 1363.

Saccharomyces cerevisi¿¿ AWRI 1371 displayed a kS phenotype in both an aerobic and an anaerobic environment (Figures 6.12 and 6.13). However, petites of AWRI 1371 (p+ks), AWRI 1372 (p-km) and AWRI 1373 (p-kNR), displayed what was a near-resistant phenotype. These results suggest that, because AWRI 1371 is devoid of mtDNA from AWRI 136I, the mutation conferring killer-resistance is nuclear encoded and partially dominant. However, this partially dominant, nuclear mutation was found to confer resistance to the cell, but only when the mitochondria are non-functional.

If in fact the resistant phenotype can be attributed to a partially dominant, nuclear mutation, visible only in the presence of non-functional mitochondria, then a diploid arising from the cross of Saccharomyces cerevßrd¿ AWRI 1361 and a sensitive, petite strain, would give a resistant phenotype. Consistent with this model, the diploid arising from the cross of AWRI 65

1361 and AIWRI 1364 (Cross 2, Figure 6.11), displayed the same'near-resistant' phenotype as the petites of AWRI 1371 @gures 6.12 and 6.13).

Included in the panel of ten killer yeasts that Saccharomyces cerevisiae AWRI 1361 displayed resistance to, were Kluyveromyces lactis var. lactis CBS 2359, Saccharomyces cerevisine CBS 6505 andWilliopsis saturnu.r var. mrakü CBS 1707. The primary rcceptor and mode of action differs for each of these three killer yeasts (Iable 6.5). Killer toxin resistant mutants have been isolated for only two of these killer yeasts, revealing genes specific for their toxin receptors; chitin for Kluyveromyces Inctß var loctß CBS 2359, and p-glucan for Williopsis saturnus var mrakü CBS I7O7. This is the first known report of a killer resistant mutant displaying resistance to more than one killer type.

It is possible that the nuclear mutation of Saccharomyces cerevisiac AWRI L36I, in the presence of non-functional mitochondria, resulted in many cell wall components being affected. This would reflect the resistance to killer toxins with different primary cell wall receptors.

Evans et aI. (1980) have shown that defective mitochondria can affect yeast cell surface characteristics such as concanavalin A agglutinability, cell movement in a biphasic polymer system, and cell adhesion. Furthermore,Iung et aI. (1999) found that yeast cell flocculation is decreased for petite cells. It is not known, however, whether functional mitochondria play a direct role in cell wall synthesis. As speculated by Iung et al. (L999), the mitochondrial genome can influence the expression of nuclear genes encoding cell wall components, as is known with the expression of the nuclear genes encoding for the citrate synthetase enzyme (Liao et aI.,I99l). Cell wall stains such as Calcofluor White (CFW) or Congo Red could be employed to test for gross cell changes in Sacclnrornyces cerevisiae AIVRI 1361 (p-kn). Mutants found resistant to CFÌW have been shown to be deficient in cell wall chitin and resistant also to the Kluyveromyces lactis var. Iactis CBS 2359 toxin, whilst mutants hypersensitive to CFW are deficient in cell wall p-glucan, and subsequently, resistant to eittrer the HM-l or the Kl toxin Clakita and Castilho-Valavicius, 1993; Ram ef al., 1994; Ram ¿t a1.,1995).

A second possibility is that resistance of Sacchnromyces cerevistd¿ AWRI L36l is attributable to a single molecule interacting with, and subsequently preventing, yeast killer toxins binding to their primary cell wall receptors. Recently, Suzuki and Shimma (1999) reported the mutation of the SPFI gene conferred a sensitive yeast resistance to the Pichin farfuwsa SMKT toxin. The SPF/ gene belongs to the P-type ATPase family. However, its function remains unclear. Spfl- cells were found to have a glycosylation-defective phenotype, and appeared to be defective in some aspect of cell wall synthesis. Moreover, characterisation by indirect immunofluorescence microscopy and FACS analysis revealed 66 thatthe PichiafarinosøSMKTtoxin interacted with the cell surface of the Spfl- resistant cells. Therefore, the Spfl- mutation appears to provide a preferential target for the Pichia farinosa SMKT toxin, akin to sequestering the toxin away from it's native receptor. t Like the Spfl- mutation, the partially dominant, nuclear mutation of AWRI 1361 may sequester or block the killer toxins from binding their primary receptors, by providing a non- specific, preferential target. To prevent chitin and p-glucan binding toxins finding their target, this alternative receptor would need to be positioned on the surface of the cell wall, extending out into the extracellular environment. Such yeast cell wall proteins do exist, like the flocculation protein Flol, whose heavily N- and O-glycosylated repeated sequences stiffens and extends the protein, thus exposing it's reacting domain to the extracellular environment (Bony et aI.,1997).

An alternative model for explaining the killer resistant phenotype, is that induction of cell death by yeast killer toxins may function in a similar fashion as cell apoptosis. Recent developments in the research of mammalian mitochondria has revealed that, besides their role in respiration and energy conservation, mitochondria supply other functions essential for cell survival (Grivell, 1995; Kiberstis, 1999).In particular, mitochondria play a cental role in the process of mammalian programmed cell death, otherwise known as apoptosis (reviewed by Adams and Cory, 1998; Green and Reed, 1998).

The intracellular expression of the mammalian pro-apoptotic protein Bax, induces yeasts such as Saccharomyces cerevisia¿ (Zha et al, !996; Ligr et al, 1998), Sacclwromycodes pombe (Ink et al., 1997; Juergensmeier et aI., 1997) and Pichía farirnsa (Martinet et al., 1999), to undergo an apoptotic-like cell death. It is thought that some of the mechanisms employed in mammalian apoptosis, have been conserved in yeasts (Green and Reed, 1998; Martinent et al., L999).

The pro-apoptotic Bax protein has structural similarities to channel-forming proteins such as colicins and the diphtheria toxin, and can form ion channels when added to synthetic membranes (Adams and Cory, 1998; Green and Reed, 1993). When expressed in Saccharomyces cerevisiae,Baxcreates a channel in the outer mitochondrial membrane, while leaving the inner mitochondrial membrane intact (Priault et al., 1999). It has been postulaÛed that as Bax forms this channel, there would be an outflux of H+ ions from the intermembrane space. As a consequence, the FgFl-ATPase would run in reverse, consuming ATP and alkalising the mitochondrial matrix. A deletion of the nuclear gene ATP4, which results in a non-functional FsFl-ATPase, renders Saccharomyces cerevislø¿ resistant to Bax. This resistance is independent of oxidative phosphorylation (Matsuyama et al., 1998). Alkalisation of the mitochondrial matrix induces opening of the yeast permeability transition pore (Jung et aI.,1997;Manon et a1.,1998), which results in the outflux of cytochrome c to 67 the cell's cytoplasm (Manon et aI., 1997; Matsuyama et al., 1998). In mammalian cells, the migration of cytochrome c from the mitochondria to the cytosol triggers the activation of the apoptotic pathway (Reed, 1997; Green and Reed, 1998).

It may be the case that yeast killer toxins can target the mitochondrial membrane in a similar fashion as the pro-apoptotic Bax protein. By forming a pore or a channel in the outer mitochondrial membrane, these killer toxins could trigger the release of cytochrome c from the mitochondria, resulting in an apoptoticJike cell death. The killer-resistant nuclear mutation, seen in AWRI L361, when complemented with a deletion of mtDNA, ñây in fact be inhibiting this killer-toxin triggered apoptoticJike cell death.

This may hold true for killer yeasts such as Kluyveromyces lactis var. lactis CBS 2359, whose toxin has an (unidentified) innacellular target (Butler et al., 1994; Magliani et al., 1997). However, it is more difficult to underctand how this could affect other toxins such as those of Saccharomyces cerevisiae CBS 6505 and Williopsis saturnus var. mrakü CBS 1707, which appear to induce cell death by osmolysis (Yamamoto et a1.,1986a; Hong et al., L994; Takasuka et al., L995; Komiyama et aI., 1996; Franken et al., 1998). Therefore, it would be of interest to determine if AWRI 1361 is resistance to killer toxin at an intacellular level. To determine so,c AWRI 1361 could be transformed to internally expresses the y subunit of the Kluyveromyces lactís var. lactis CBS 2359 toxin. Arrest at the Gl phase of the cell cycle would suggest the killer-resistant mutation does not confer resistance at an intracellula¡ level, and consequently, ruling out the killer-toxin triggered apoptosis hypothesis.

The observation that the killer-resistant, nuclear mutation of AWRI 1361 appears to be partially dominant, suggests a possible gain of function. To determine if only a single gene has been mutated, respiratory competent diploids arising from the cross of AWRI 1361 and its parent strain, would be sporulated. The subsequent haploids would be re-cultured for a petite phenotype (petite diploids can not sporulate), and their sensitivity assayed. It would be hoped that a 2;2 ratio of sensitive:resistant would prevail, suggesting a single mutation only. A subsequent cloning strategy could be employed, to identify the mutation responsible for the killer-resistant mutation.

In addition to the killer-resistant mutation of Saccharomyces cerevisiae AWRI 1360, this investigation revealed that respiratory profîcient strains showed no change in their sensitivity to killer yeasts in an anaerobic environment, yet petites of these strains displayed a reduction in sensitivity. This was observed for Saccharomyces cerevß,¿¿ AWRI 1360, AWRI 1363 and AWRI 1365, whose respective petites showed a reduction in sensitivity, yet they themselves showed no change when assayed in an anaerobic environment. Therefore, being petiæ, and not the lack of respirati on per se, confers the cell a kRS phenotype. 68

There was however, one example of a respiratory competent strain displaying a reduction in sensitivity to a killer yeast in an anaerobic environment. The sensitivity of AWRI 1360 (p+ks) to the killer yeast Pichia anomala CBS t1982 was reduced in an anaerrcbic environment (Figure 6.3). The sensitivity of AWRI 1363 (p+k\ to Pichia arcmaln CBS 1982 remained unchanged in the same anaerobic environment @igure 6.6), which suggests there was no change in the exogenous expression of killer toxin by Pißhin arcmala CBS 1982 in the absence of respiration.

The reduction in sensitivity due to petiteness was found to be both sensitive strain and killer yeast dependent. This is seen with the petite AWRI 1366 displaying a reduced sensitive phenotype to all ten killer yeasts @igures 6.8-6.10), yet the petites AWRI 1364 (Figure 6.5- 6.7) and AWRI 1362 @gures 6.2-6.3) showed the same level of sensitivity to two and four killer yeasts respectively when compared to their parent strains. Furthermore, the quantitative reduction in sensitivity to a killer yeast differed from strain to strain (Table 6.4).

Polonelli et aI. (1992) found that some strains of Pichia sp. showed a reduction in sensitivity to killer yeasts in an anaerobic environment, and that this reduction in sensitivity was both killer yeast and sensitive strain dependent. Polonelli et aI. (L992) suggests the reduction in sensitivity to killer yeasts may be due to the change in the sensitive cell's metabolic rate in the absence of respiration. It is worth noting that although the sensitivity level of AWRI 1360 to Pichia anomalacBs 1982 was reduced to a minimum in an anaerobic environment, the petite of AWRI 1360, AWRI 1362, displayed no sensitivity at all. Therefore, having a petiæ phenotype confers the cell a grcater reduction in sensitivity, than just the lack of respiration alone.

It may be that a petite phenotype results in alterations in the cell wall, thus effecting the toxin- cell wall receptor interaction. As mentioned previously, mitochondrial defects results in changes in the cell wall structure @vans et aI. 1980; Iung et aI., 1999), and that CFW or Congor Red could be employed to possible to detect any cell wall defects.

Baganz et at. (1998) have shown that under certain physiological conditions, nuclear petite strains can out+ompete the wild type for growth in continuous culture. A strain carrying a PET191 deletion (completely respiratory deficient) and a strain carrying a COX4 deletion (partially respiratory deficient) displayed a competitive advantage over the wild type under nitrogenous, phosphorus and sulphur limiøtions in an aerobic environment. Only under glucose limitations or on a non-fermentable carbon source did the wild type out{ompete both deletants. Perhaps the reduction in sensitivity due to petiteness, could be a mechanism by which some sensitive yeast strains can improve their fitness in an environment 69 comprising of killer yeasts. To date, there have been no reports on the incidence of petiteness in the ecological studies of killer yeasts in natural environments.

Like the killer-resistant mutation of AWRI 1361, the reduction in sensitivity due to petiteness is independent of respiration. Whether the two observations share some common mechanism is unknown.In recent times mammalian mitochondrial research has revealed functions of ttre mitochondria, independent of respiration, which are essential for cell survival. Further research into the association of functional mitochondria and killer toxin sensitivity, may also provide further insight into this organelle, akin to the wealth of information killer-resistant mutants have provided on yeast cell wall biosynthesis. 70

Chapter 7

GnNBn¿.L DISCUSSION AND CONCLUSIONS \

An investigation was undertaken to identify a yeast with broad spectrum killer activity towards indigenous non-Saccharomyces yeasts of the wine ferment. The growth of these indigenous yeasts during wine fermentation may result in inappropriate sensory properties to the wine. To suppress indigenous yeast growth, sulphur dioxide is added to the grape juice or must, in conjunction with a highly active Saccharomyces cerevisiae startet culture. However, the introduction of a starter culture expressing a killer protein toxic to a range of indigenous yeast species, could reduce or replace the use of sulphur dioxide in winemaking.

The sensitivity of 26 tester strains characteristic of the wine ferment microflora to 14 killer yeasts were assayed at pH 4.5, revealing a total of I47 killer-sensitive reactions. At a pH comparable to a wine ferment (pH 3.5), 28Vo of these 147 killer-sensitive reactions were observed. Intraspecific differences in killer susceptibility were identified for strains of a number of yeast species, whilst intraspecific differences in killer activity was identified for strains of Pichia anomala, Kluyveromyces lactis and Pichia membranifaciens.

To generate a wine yeast with effective killer capacity it may be necessary to express more than one killer toxin. This investigation revealed that the combination of the killer yeasts Pichinanomala NCYC 434andWilliopsis saturnus var. mrakit CBS 1707 would see a combined killing range of over 907o at pH 4.5. However, due to the ineffectiveness of the killer yeasts at a reduced pH, this combined killing range would be reduced to less than 607o at pH 3.5. Other factors of the wine ferment, such as high tifratable acidity, proteolytic activity, and binding to grape derived phenolic compounds may further reduce killer activity. The Williopsis saturnus var. mrakü toxins HY1 and HM-l have been noted for their exceptional temperature and pH stability, features which make them attractive for use as new food preservatives (Yamamoto et at. !986b; Kimura et al. 1993; Komiyama et al. 1995; Lowes et a1.,2000). With the increased understanding of the molecular interactions that maintain protein structure at low pH (Mortensen and Breddam, 1994), it may be possible to engineer killer proteins which maintain activity at wine pH. Combining these strategies may provide a yeast with a broad killer range at an acidic pH.

To gain further insight into the little known killer phenotype of Pichin membraniþciens, the killer activity of the ten Pichiø membranifaciens strains was assayed towards 15 æster strains. Prior to this investigation, intron primer PCR confirmed that all these strains were related but were different yeasts. An outcome of this investigation was that each Pichia mcmbranifaci¿ns strain was allocated one of a possible four killer types based on their killer 7T activity. This classification of killer type was undertaken in a similar fashion as the classification of strains of Saccharomyces cerevisiae as either Kl, K2 or K28 @oger and Bevan, 1978). Subsequent investigations revealed that the Kl, K2 and K28 killer types each expressed a unique toxin, which accounted for the innaspecific differences in killer activity. The killer phenotype of all three killer types were encoded by extrachromosomal dsRNAs.

The two killer strains of the Class C killer type, Píchia membranifaci¿ns CBS 638 and the type strain CBS 107, had no extrachromosomal elements. Therefore, the killer phenotype of at least these two strains is encoded by nuclear genes. In contrast, the killer strains of the Class B and D killer types harboured an extrachromosomal element of the same molecular weight. For Pichia membranífaci¿ns CBS 7374 of Class D this extrachromosomal element (pPM01) is dsRNA in nature. Pichiamembranifaciens CBS7374 was unable to be cured of pPM01, however, when fteated with various curing techniques. Consequently, its not known whether pPM01 is associated with the killer phenotype.

Other curing techniques could be employed to cure Pichiø membranifaci¿ns CBS 7374 of pPM01. Curing treatments other than those methods mentioned previously , have had some limited success in curing yeast species of their killer phenotype. Incubating cultures at elevated temperatures (37-40oC) has been proven successful in curing Saccharomyces cerevisiae strains of their Kl killer type, and some Saccharomyces cerevisiae strains of their K2 killer type, but failed to cure strains of Hanseniaspora uvarum, Zygosacchnromyces bailíi and Saccharomyces cerevisiae K28 (Zorg et al., 1988; Petering et al., L99I; Radler et al., 1993). Treating cultures with S-fluorouracil or acridine orange have proven successful also, in curing killer strains of Sacchørotnyces cerevßiae (Mitchell et al., 1973; Cansado et aI., 1939). It may be the case that one of these less successful methods may cure Pbhia membranifaciens CBS 7374 ofpPMOl, if the extrachromosomal element is not essential for its survival.

Of the ten Pichia membranifaciens struns investigated, strain CBS 7374 displayed the broadest killing range. Prior to this investigation the killer toxin of Pichin membranifaciens CBS 7374 had not been characterised, consequently, a method for its purification was developed. Like all killer toxins characterised to date, the toxin of Pichia membranifaciens CBS 7374 is proteinaceous in nature. Like most characterised killer toxins, this toxin was found to be heat liable and its pI acidic in nature. The activity of this toxin in a liquid culture of Pichin membranifaci¿ns CBS 7374 was optimal at late exponential phase of growth, but declined rapidly in activity during stationary phase. Using the purification protocol developed in this study, a protein of 20.5 kDa was identified as a candidate for the Pichia membranifacl¿ns CBS 7374 ktller toxin. Further experiments are required, however, to confirm this 20.5 kDa protein harbours killer activity. 72

To confirm the 20.5 kDa protein as the killer toxin, the protein band in question would need to be assayed for killer activity, using a method similar to that of McCracken et al. (1994). This would first involve visualising the protein by native gel electrophoresis. The lane from this electrophoretic gel would then be cut into thin stçips, placed on a YEPD-agar plate that had been pre-seeded with a sensitive strain, and assayed for killer activity. Visualising the toxin by native gel electrophoresis would also provide information on its structure. The SDS PAGE of the 22nd fraction suggests the toxin is a monomeric protein of 20.5 kDa. Alternatively, the Pichin membranifaciens CBS 7374 Y¡ller toxin may consist of more than one subunit of, or approximately,20.5 kDa in size. Size exclusion chromatography could also be employed, in identifying more clearly the toxin's structure and molecular weight.

Whilst investigating the sensitivity of tester strains to Pichia membranifacl¿ns CBS 7374, a petite of the sensitive parent Saccharomyces cerevisØ¿ AWRI 1360 (p+kS), strain AWRI 1361 (p-kn), was found to be resistant. Subsequent studies showed this petiûe to be at least twelve times more resistant to the Pichia membranifaci¿ns CBS 7374 toxtn as compared to its parent strain. Furthermore, this petite displayed resistance to ten killer yeasts to which the parent was sensitive. This included resistance to the killer yeasts Sacclwrornyces cerevisiae K2, Kluyveromyces lactis var.lactis andWilliopsis saturnus var. mrakü, where the primary receptor and mode of action differs for each killer protein. This is the first known report of a mutant displaying resistance to more than one killer type.

Characterisation of this petite revealed that its resistance to these killer toxins is attributed to a partially dominant, nuclear mutation. This mutation is independent of oxidative- phosphorylation and yet, confers resistance only in the presence of non-functional mitochondria. It is possible this nuclear mutation, in the presence of defective mitochondria, results in change(s) in the yeast cell wall, thus preventing killer toxins binding to their primary cell wall receptors. Previous studies have shown that defective mitochondria can affect yeast cell wall characteristics @vans et al., 1980, lung et aI., 1999). This study also revealed that for some strains, petites of sensitive parents show a reduction in sensitivity to killer yeasts, and that this reduction in sensitivity is independent of oxidative- phosphorylation. Further characterisation of the mutation of Saccharomyces cerevßin¿ AWRI 1361 may provide further insight into the association of functional mitochondria and cell wall synthesis.

Further characterisation of the mutation of Saccharomyces cerevisi.ae AWRI 1361 may provide insight into the association of functional mitochondria and cell wall synthesis. Cell wall stains such as Calcofluor rWhiæ (CFW) or Congo Red could be employed to test for gross cell changes in Saccharomyces cerevisíae AWRI 1361 (p-kR). Mutants found resistant to CFW have been shown to be deficient in cell wall chitin and resistant also to the Kluyveromyces lactis var.lactis CBS 2359 toxin, whilst mutants hypersensitive to CFW are 73 deficient in cell wall glucan, and subsequently, resistant to either the HM-l or the Kl toxin (Takita and Castilho-Valavicius, 1993; Ram ¿f a1.,1994; Ram ¿r al., L995).

Yeast genetics, and a subsequent cloning strategy, could be employed to identify the mutated gene responsible for conferring AWRI 1361 resistance. The observation that the killer- resistant, nuclear mutation of AWRI 1361 appears to be partially dominant, suggests a possible gain of function. To determine if only a single gene has been mutated, respiratory competent diploids arising from the cross of AWRI 1361 and its parent strain, would be sporulated. The subsequent haploids would be re-cultured for a petite phenotype (petite diploids can not sporulate), and their sensitivity assayed. It would be hoped that a 2:2 ratio of sensitive:resistant would prevail, suggesting a single mutation only.

A cloning strategy to complement the finding that the killer-resistant mutation is conferred by a single, dominant mutation, would be the transformation of the sensitive wt with a Saccharomyces cerevisia¿ AWRI 1361 cDNA expression library. The sensitive wt would first require a petite phenotype prior to transformation, as the killer-resistant mutation is suppressed by functional mitochondria. Resistant transformants would be selecæd in the presence of an extracellular killer toxin, and subsequently assayed to confirm their resistance to more than one killer yeast. Further analysis of the resistant transformants, such as sequence analysis of the cDNA insert, would provide further insight into this gain of function mutation. Identifying where the cDNA product resides in the cell, say intracellularly, or on the surface of the cell wall, would also provide information on how resistance is conferred to the cell. Experiments such as green fluorescent protein tagging or immunoelectron microscopy would provide such information. Together, this information may reveal what aspect of functional mitochondria suppresses the resistant phenotype. 74 BIBLIOGRAPHY

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