The killer toxin secreted by Kluyveromyces phaffii (KpKt) is active against spoilage yeast under winemaking conditions and thus has potential applications in the biocontrol of undesired micro-organisms in the wine industry. Biochemical characterization and N-terminal sequencing of the purified toxin show that KpKt is a glycosylated protein with a molecular mass of 33 kDa. Moreover, it shows 93 % and 80 % identity to a b-1,3-glucanase of Saccharomyces cerevisiae and a b-1,3-glucan transferase of Candida albicans, respectively, and it is active on laminarin and glucan, thus showing a b-glucanase activity. Competitive inhibition of killer activity by cell-wall polysaccharides suggests that glucan (b-1,3 and b-1,6 branched glucans) represents the first receptor site of the toxin on the envelope of the sensitive target. Flow cytometry analysis of the sensitive target after treatment with KpKt and K1 toxin of S. cerevisiae, known to cause loss of cell viability via formation of pores in the cell membrane, suggests a different mode of action for KpKt. INTRODUCTIONKiller toxins were first discovered in Saccharomyces cerevisiae by Makower & Bevan (1963) and they have since been found in numerous other yeast genera (Philliskirk & Young, 1975; Stumm et al., 1977;Rosini, 1983Rosini, , 1985Starmer et al., 1987). Through the following years, killer yeasts and their toxins found applications in several fields. For example, killer yeasts have provided an interesting model for studying the mechanisms involved in the processing and secretion of extracellular proteins, and the identification of killer toxin receptors on the envelope of sensitive targets has helped in the elucidation of the structure and function of the yeast cell wall. Killer yeasts have been used to combat contaminating wild yeasts in the food and fermentation industries, and to control wooddecay basidiomycetes and plant-pathogenic fungi (Young, 1987; Boone et al., 1990;Palpacelli et al., 1991, van Vuuren & Jacobs, 1992Walker et al., 1995). In the medical field, these yeasts have been used in the biotyping of pathogenic yeasts and yeast-like fungi (Morace et al., 1989;Boeckhout & Scorzetti, 1997;Buzzini & Martini, 2001), and in the development of novel antimycotics for the treatment of human and animal fungal infections Seguy et al., 1998; Conti et al., 2002;Schmitt & Breinig, 2002).Recently, it has been shown that Kluyveromyces phaffii (since reclassified as Tetrapisispora phaffii) (Ueda-Nishimura & Mikata, 1999) produces a killer toxin (KpKt) that is active on wine spoilage yeasts (Ciani & Fatichenti, 2001). KpKt has an extensive anti-Hansenispora/Kloeckera activity under winemaking conditions and, therefore, is of particular interest for its potential application as an antimicrobial agent in the wine industry (Ciani & Fatichenti, 2001). At present, the inhibition of wild spoilage yeast at the prefermentative stage is achieved by the addition of SO 2 to freshly pressed must. This antiseptic agent, which has been shown to have a toxic action on humans, is also r...

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The practice of exposing liquid cultures of the white-rot fungus Phanerochaete chrysosporium to a pure oxygen atmosphere under conditions of nutrient starvation has been widely adopted to induce lignin peroxidase (LiP) synthesis. Transmission electron microscopy was used to examine hyphal cells of carbonlimited cultures that had been exposed to an atmosphere of pure oxygen, and revealed evidence of a major loss in organization of cellular ultrastructure, which may be attributed to oxygen toxicity. Under some conditions (continuous agitation in air with cellulose as the carbon source) cultures will produce LiP without needing to be exposed to a pure oxygen atmosphere. A similar major loss of cellular ultrastructure was found in hyphal cells from such cultures upon examination. Investigation of the levels of H 2 O 2 , catalase and carbonyl content of intracellular proteins suggests that the latter cultures developed a hyperoxidant state because the rate of supply of carbon from cellulose hydrolysis was insufficient for oxygen homeostasis. The association of LiP with these cultures and with those exposed to an atmosphere of pure oxygen infers that LiP may be triggered in response to oxidant stress.

Phanerochaete chrysosporium maintained on glucose as the carbon source contained severely impaired mitochondria that were characterised by the loss of both succinate dehydrogenase and cytochrome oxidase activities. These cells maintained a constant value for energy charge using anaerobic metabolism. Cells with these properties express lignin peroxidase when supplied with a pure oxygen atmosphere, which may reflect a response to accumulating reactive oxygen species. Cells maintained on cellulose retained fully functional mitochondria, but expressed lignin peroxidase without being exposed to a pure oxygen atmosphere. In the cells maintained on cellulose, mitochondrial function may be limited by the supply of glucose, leading to the accumulation of reactive oxygen species. ß

Phanerochaete chrysosporium maintained on glucose as the carbon source contained severely impaired mitochondria that were characterised by the loss of both succinate dehydrogenase and cytochrome oxidase activities. These cells maintained a constant value for energy charge using anaerobic metabolism. Cells with these properties express lignin peroxidase when supplied with a pure oxygen atmosphere, which may reflect a response to accumulating reactive oxygen species. Cells maintained on cellulose retained fully functional mitochondria, but expressed lignin peroxidase without being exposed to a pure oxygen atmosphere. In the cells maintained on cellulose, mitochondrial function may be limited by the supply of glucose, leading to the accumulation of reactive oxygen species.