Use of RAPD and killer toxin sensitivity in Saccharomyces cerevisiae strain typing.

AIMS: Two different strain characterization techniques, random amplified polymorphic DNA (RAPD) and killer toxin sensitivity (KTS), were compared to assess their typing performance using a set of 30 certified Saccharomyces cerevisiae strains. METHODS AND RESULTS: A sequential random resampling procedure was employed to subdivide the 32 descriptors in eight sets, in order to compare the differential performances of the two techniques with diverse number of characters. Results showed that RAPD performs better than killer, although the complete differentiation of the strains under study could be obtained only by combining profiles from the two techniques. CONCLUSIONS: The combination of different typing techniques was useful when discriminating similar organisms. In such cases, the introduction of a second typing technique can be more advantageous than increasing the number of characters obtained with a single method. SIGNIFICANCE AND IMPACT OF THE STUDY: The distribution of among-strains pairwise distances and the relative performance of the two techniques has implications for the study of biodiversity, taxonomy and microbial ecology.

Killer toxin of Kluyveromyces phaffii DBVPG 6076 as a biopreservative agent to control apiculate wine Yeasts.

The use of Kluyveromyces phaffii DBVPG 6076 killer toxin against apiculate wine yeasts has been investigated. The killer toxin of K. phaffii DBVPG 6076 showed extensive anti-Hanseniaspora activity against strains isolated from grape samples. The proteinaceous killer toxin was found to be active in the pH range of 3 to 5 and at temperatures lower than 40 degrees C. These biochemical properties would allow the use of K. phaffii killer toxin in wine making. Fungicidal or fungistatic effects depend on the toxin concentration. Toxin concentrations present in the supernatant during optimal conditions of production (14.3 arbitrary units) exerted a fungicidal effect on a sensitive strain of Hanseniaspora uvarum. At subcritical concentrations (fungistatic effect) the saturation kinetics observed with the increased ratio of killer toxin to H. uvarum cells suggest the presence of a toxin receptor. The inhibitory activity exerted by the killer toxin present in grape juice was comparable to that of sulfur dioxide. The findings presented suggest that the K. phaffii DBVPG 6076 killer toxin has potential as a biopreservative agent in wine making.

Influence of glycerol production on the aerobic and anaerobic growth of the wine yeast Candida stellata.

Candida stellata is frequently found in wine fermentations and may be used as a yeast starter in beverage production. In order to acquire additional knowledge on the physiology of C. stellata, a study on sugar metabolism in aerobic and anaerobic conditions was carried out. We found that under anaerobic conditions the low growth rate and biomass yield of C. stellata were due to the diversion of carbon flux from ethanol to glycerol. C. stellata had lower ADHI (alcohol dehydrogenase) activity (3-4 fold) and higher GPDH (glycerol-3-phosphate dehydrogenase) activity (40 and 15 times higher in anaerobiosis and aerobiosis respectively) than that of a Saccharomyces cerevisiae control strain. In aerobic sugar-limited chemostat culture C. stellata exhibited lower maximum biomass concentration [5.23 gl(-1) (dry weight)] than other respirofermentative yeasts at very low dilution rates (up to D = 0.042 h(-1)). While glycerol was constantly produced, ethanol and sugar residue appeared at D = 0.042 h(-1) and D = 0.065 h(-1) respectively. The tendency of C. stellata to form glycerol is probably the main cause of its very low growth and fermentation rates.

Vanadate and copper induce overlapping oxidative stress responses in the vanadate-tolerant yeast Hansenula polymorpha.

The mechanisms by which vanadate exerts a toxic effect on living organisms are not completely understood. This is principally due to the variety of intracellular targets of the metal and to the changes in the chemical form and oxidation states that vanadate can undergo, both in the external environment and intracellularly. In order to further elucidate the reasons for vanadate toxicity, and assuming that common detoxification mechanisms can be evoked by a general heavy metal response, we have compared some aspects of the cellular responses to vanadate and copper in the yeast Hansenula polymorpha. By means of 2D electrophoresis we show the existence of common determinants in the responses to vanadate- and copper-induced stresses. Moreover, we demonstrate that both metals induce significant increases in antioxidant enzyme levels, and that there are significant overlaps in the heavy metal and oxidative stress responses. Interestingly, vanadate induces an increase in catalase activity that is much higher than that seen with copper and, unlike copper, does not cause lipid peroxidation of cellular membranes. This suggests that H. polymorpha cells activate a further specific detoxification pathway against vanadate-induced oxidative insults.

Selective sugar consumption by apiculate yeasts.

Selective consumption of glucose and fructose among apiculate yeasts was evaluated. Results showed that Hanseniaspora guilliermondii and H. uvarum type strains were fructosophilic, unlike the other type strains. The difference in glucose and fructose use was confirmed in different media and throughout sugar consumption. Selective consumption of fructose is widely diffused among apiculate wine yeasts and could positively interfere with fermentation behaviour of Saccharomyces cerevisiae.

Secondary products formation as a tool for discriminating non-Saccharomyces wine strains. Strain diversity in non-Saccharomyces wine yeasts.

A total of 78 strains of non-Saccharomyces yeasts were isolated: 30 strains of Kloeckera apiculata, 20 of Candida stellata, 8 of Candida valida and 20 of Zygosaccharomyces fermentati. The diversity of yeast species and strains was monitored by determining the formation of secondary products of fermentation, such as acetaldehyde, ethyl acetate and higher alcohols. Within each species, the strains were distinguishable in phenotypes through the production of different amounts of by-products. In particular, a great variability was found in C. stellata, where six different phenotypes were identified by means of the production of acetaldehyde, ethyl acetate, isobutanol and isoamyl alcohol. At different stages of the spontaneous fermentation different phenotypes of the non-Saccharomyces yeasts were represented, characterized by consistent differences in some by-products involved in the wine bouquet, such as acetaldehyde.

Vanadium affects vacuolation and phosphate metabolism in Hansenula polymorpha.

The yeast Hansenula polymorpha is able to grow on vanadate concentrations that are toxic to other organisms. Transmission electron microscopy analysis showed that H. polymorpha cells growing on a vanadate-containing medium undergo a significant increase in cell vacuolation and a thickening of the cell wall; the presence of small cytoplasmic vesicles and an increase in cristae at the level of the plasma membrane were also observed. These ultrastructural modifications were accompanied by a change in the intracellular polyphosphate level, as shown by in vivo 31P-NMR. The involvement of these observed changes in vanadium detoxification is discussed.

Kluyveromyces fragilis SS-437: an associatively-profiled thermotolerant yeast.

The lactose-utilizing Kluyveromyces fragilis SS-437 was found to have an associative temperature profile, but a thermotolerant growth yield behaviour. Cardinal growth temperatures were: 3 degrees C minimum for growth; 41.5 degrees C optimum; 44.5 degrees C final maximum (growth and death rates equalize); 46.1 degrees C initial maximum (maximum limit for growth).

Antagonistic activity of Debaryomyces hansenii towards Clostridium tyrobutyricum and Cl. butyricum.

A study of the inhibitory action of Debaryomyces hansenii (31 strains) on Clostridium tyrobutyricum (5 strains) and Cl. butyricum (2 strains) on laboratory media showed that Deb. hansenii inhibited the growth of these organisms, and that this effect was due not only to competition for nutrients but also to the production of both extra- and intracellular antimicrobial metabolites. The inhibitory effect varied with strain and occurred whether the yeasts were grown aerobically or under reduced O2 tension.