Thiamine secretion in yeast.

To isolate thiamine excretors and (or) overproducers, 188 cultures belonging to nine yeast and three fungal genera were screened. Nine excreted thiamine as determined by both the presence of cross-feeding zones on a thiamine-free agar medium seeded with a thiamine-requiring yeast strain and by the direct detection of excreted thiamine on agar plates. Several of these cultures produced several-fold more intracellular thiamine than the general culture population. Thiamine-requiring strains of Saccharomyces cerevisiae and S. uvarum (carlsbergensis) were identified and were tentatively assigned to 10 complementation groups.

Applicability of yeast extracellular proteinases in brewing: physiological and biochemical aspects.

A general screening survey for expression of extracellular acid proteinase production was performed on over 100 cultures belonging to the genus Saccharomyces. Although two strains of Saccharomyces cerevisiae showed positive extracellular proteinase phenotypes in plate tests, it was not possible to demonstrate proteolytic activities in cell-free culture supernatants in assays performed at beer pH values. Of several yeasts from other genera examined, Saccharomycopsis fibuligera and Torulopsis magnoliae produced extracellular proteinases with desirable properties. Proteolytic activities were detected in assays performed at beer pH values and at lower temperature. Brewer's wort served as a highly inducing medium for extracellular proteinase production, with T. magnoliae yielding enzyme of highest specific activity. In fact, commencement of enzyme production was detected shortly after the onset of exponential growth in brewer's wort. Inclusion of crude enzyme preparations in brewer's wort inoculated simultaneously with brewer's yeast reduced final ethanol yields slightly and was found to be effective in reducing chill haze formation in bottled beer.

Cell division age dependency of meiosis in an apomictic variant of Saccharomyces cerevisiae.

The cell division age dependency of sporulation was investigated in a diploid strain of Saccharomyces cerevisiae (19el) which undergoes a single equational nuclear division during sporulation with consequent formation of asci containing two uninucleate diploid spores (apomictic dyads). Under modified nutritional conditions which partially restore meiosis and hence normal tetrad formation, newly formed (age 0) daughter cells were observed to be capable of formation of apomictic dyads but not of meiotic tetrads. Even under conditions in which only apomictic dyads developed, approximately 20% of the asci resulted from differentiation of newborn 'inexperienced' cells. Thus, the data indicated production of at least one bud to be a prerequisite for meiosis but not for apomixis; however, occurrence of at least one complete mitotic cell division cycle was evidently insufficient for the morphogenetic switch from diploid to haploid spore formation, since older cells bearing several bud scars often underwent apomictic dyad development, and some produced no spores.

Identification and characterization of antimicrobial activity in two yeast genera.

A general screening test for the expression of antibacterial activity was performed on over 400 cultures belonging to 31 yeast genera. Of these cultures, only two, Kluyveromyces thermotolerans and Kloeckera apiculata, were found to produce zones of inhibition of bacterial growth on Diagnostic Sensitivity Test Agar medium supplemented with 0.002% methylene blue. Of nine bacteria used as test organisms, only Lactobacillus plantarum and Bacillus megaterium were inhibited. No antibacterial activity was evident against four gram-negative bacteria used in this study. Optimal activities were found to be expressed after yeasts were grown at pH 6. A requirement for cultivation in the presence of methylene blue added to culture media for the expression of apparent antibacterial activity was demonstrated.

Partial restoration of meiosis in an apomictic strain of Saccharomyces cerevisiae: a model system for investigation of nucleomitochondrial interactions during sporulation.

In an apomictic strain of Saccharomyces cerevisiae (ATCC 4117-H2) which undergoes a single nuclear division during sporulation and consequently forms asci containing two uninucleate diploid spores, a study was undertaken to investigate the effects of cultivation in three presporulation media (YPA; YNB; SMM) on nuclear division and ascosporogenesis in sporulation medium. Comparison of effects of presporulation culture in these media on the number of spores formed per ascus showed that a marked induction (30 +/- 4.3 per cent) of three- and four-spored asci could occur in sporulation medium following cultivation in a defined YNB medium supplemented with a 1 per cent solution of vitamins and containing decreased ammonium sulphate and increased glucose levels. Experiments in which the concentrations of glucose and of ammonium sulphate were varied simultaneously indicated that the initial presporulation carbon to nitrogen source ratio is an important factor in determining tetrad formation in sporulation medium. Nuclear staining demonstrated two classes of asci: binucleate (one- and two-spored) and tetranucleate (three- and four-spored). Genetic evidence and data concerning effects of inclusion in sporulation medium of a meiotic inhibitor (glucose) indicated spores in tetrads were haploid rather than diploid. This ability to condition a significant number of cells for meiotic rather than apomictic differentiation made possible investigation of effects of mitochondrial inhibitors on both developmental processes simultaneously. It was found possible to selectively inhibit meiotic development by inclusion in sporulation medium of appropriate concentrations of specific inhibitors. Moreover, the data suggest meiotic sporulation is more strictly dependent than apomictic sporulation on mitochondrial function.

Morphological and genetic effects of benomyl on polyploid brewing yeasts: isolation of auxotrophic mutants.

An enrichment procedure after ethyl methanesulfonate mutagenesis and exposure to the fungicide benomyl yielded mutants auxotrophic for several amino acids from two polyploid Saccharomyces spp. Benomyl treatment was found to have a marked morphological effect on polyploid Saccharomyces cerevisiae, causing cells to adopt a characteristic doublet cell morphology in which buds are nearly as large as the parent cells. Experiments in which nuclear division was monitored in benomyl-induced doublet cells by Giemsa nuclear staining demonstrated an unusual sequence of cytological events which culminated in the formation of binucleate parental and mononucleate bud components. The frequency of formation of doublet and binucleate parent cells was found to depend on the strain employed and the benomyl concentration administered.

Translocation of zinc from vacuole to nucleus during yeast meiosis.

A novel staining procedure employing the UV fluorochrome DAPI (4',6-diamidino-2-phenylindole X 2HCl) and dithizone (diphenylthiocarbazone) was developed for microcytochemical determination of sites of zinc localization in Saccharomyces cerevisiae Hansen. In vegetative cells vacuolar polyphosphate bodies stained with dithizone, whereas in sporulating cells nucleoli and centriolar plaques were dithizone-positive. Hence, dithizone not only permitted localization of zinc but also indicated zinc translocation from vacuolar to nuclear compartments during differentiation from the vegetative to sporulated state.

Events associated with restoration by zinc of meiosis in apomictic Saccharomyces cerevisiae.

The effects of nutritional alterations (carbon source and zinc) on nuclear division and protein synthesis during apomictic and meiotic development in Saccharomyces cerevisiae 19e1 were investigated. Unlike cells cultivated under meiosis-promoting conditions, cells cultured under apomixis-promoting conditions exhibited extensive protein synthesis during the first 3 h of incubation in sporulation medium, and nuclear divisions were evident during this time. Cycloheximide treatment of the latter cells induced meiosis, and maximum yields of meiotic asci resulted when this treatment was given for the first 3 h in sporulation medium. The results indicate that the decision concerning which developmental route cells will follow is made shortly after transfer to sporulation medium. Electrophoretic analysis of labeled proteins synthesized during sporulation revealed bands unique to both developmental routes.

Induction of normal ascosporogenesis in two-spored Saccharomyces cerevisiae by glucose, acetate, and zinc.

In an apomictic strain of Saccharomyces cerevisiae that normally produces two-spored asci, production of three and four-spored asci was restored by adjusting zinc and carbon source levels in presporulation and sporulation media. Sporogenic capability was compared after growth in defined and undefined presporulation environments. Cultivation in defined presporulation medium containing 6% glucose followed by sporulation in 3.5% acetate yielded 16 +/- 3% three- and four-spored asci. Inclusion of 25 mug of zinc sulfate per ml in presporulation medium or 25 mug of zinc acetate per ml in sporulation medium gave a further increase in yield of three- and four-spored asci; it reached 48 +/- 1% when zinc was added to both media. Cells grown in undefined presporulation medium were incapable of three- and four-spored ascus development in acetate sporulation medium unless zinc was included in the latter medium. Cultivation in defined presporulation medium amended with yeast extract, peptone, or casein hydrolysate greatly reduced three- and four-spored ascus yields. Inclusion of a meiotic inhibitor (glucose) in sporulation medium inhibited three- and four-spored ascus production but not two-spored ascus production. Nuclear staining demonstrated two classes of asci: binucleate (one and two spored) and tetranucleate (three and four spored). Hence, some control over apomictic versus normal ascus development has been gained by nutritional manipulation.