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Mixed cultures of different yeasts species and yeasts with filamentous fungi in the SCP production. I. Production of single cell protein by mixed cultures Candida lipolytica and Candida tropicalis.

The aim of this study was to determine the application of mixed cultures Candida lipolytica and Candida tropicalis in the SCP production. N-paraffin fraction of crude oil and individual n-alkanes C:7--C:17 and glucose were used as carbon sources. The cultures were grown on laboratory scale in shaking flasks and in a 7 1 fermentor. It was found that the mixed cultures gave about 18% higher yield of biomass than the individual cultures.

Alkanes↗

Purified yeast RNA polymerase II reads through intrinsic blocks to elongation in response to the yeast TFIIS analogue, P37.

Saccharomyces cerevisiae has a TFIIS-related transcription elongation factor, originally called P37 (Sawadogo, M., Sentenac, A., and Fromageot, P. (1979) J. Biol. Chem. 255, 12-15; Nakanishi, T., Nakano, A., Nomura, K., Sekimizu, K., and Natori, S. (1992) J. Biol. Chem. 267, 13200-13204), which binds directly to RNA polymerase II and stimulates read-through of intrinsic blocks to elongation. To elucidate functional features of this protein:protein interaction, we tested the ability of several forms of RNA polymerase II to respond to either full-length or an amino-terminal truncation of TFIIS. The variants of the polymerase differed in the structure of the carboxyl-terminal domain of the largest subunit or lacked two of the smaller subunits. No differences in ability to recognize intrinsic blocks to elongation or to read through them in response to either form of TFIIS were detected among these variants. Furthermore, ternary complexes containing each variant form of RNA polymerase cleave the 3' end of the nascent transcripts in response to TFIIS, a reaction previously reported for mammalian and Drosophila TFIIS (Kassavetis, G. A., and Geiduschek, E. P. (1993) Science 259, 944-945) and likely to be important in TFIIS function. Thus the carboxyl-terminal domain of the largest subunit and subunits four and seven of the polymerase, required in vivo, are not required in vitro for recognition of intrinsic blocks to elongation, read-through in response to TFIIS, or TFIIS-stimulated cleavage of the nascent transcript.

Base Sequence↗

Digestion of Histoplasma capsulatum yeasts by human macrophages.

The strategies used by Histoplasma capsulatum yeasts to survive and multiply within human macrophages (M phi) are unknown. To better understand these strategies we studied the intracellular fate of viable vs heat-killed (HK) yeasts in human monocyte-derived M phi. Initial studies demonstrated that phagolysosome fusion was present in M phi ingesting either viable or HK yeasts. Viable yeasts multiplied within M phi phagolysosomes, whereas M phi completely digested intracellular FITC-labeled HK yeasts within 24 h after ingestion. This observation was confirmed by electron microscopy. M phi that had ingested colloidal gold-labeled HK yeasts contained gold particles but no visible yeasts at 24 h. Digestion of HK yeasts was evident as early as 4 h after phagocytosis, and was complete by 24 h. M phi digestion of HK yeasts was blocked completely when M phi were cultured for 24 h in the presence of chloroquine. In M phi simultaneously ingesting both viable and HK yeasts, viable yeasts multiplied, but HK yeasts were digested within the same cell. M phi that had ingested viable yeasts digested them completely when M phi were cultured for 24 h in the presence of cycloheximide or amphotericin B. Coculture of infected M phi with nystatin or ketoconazole resulted in inhibition of growth, but the yeasts were not digested. These data indicate that: 1), HK Hc yeasts are easily digested by preformed M phi lysosomal hydrolases; 2), viable Hc yeasts survive and multiply within M phi phagolysosomes, but the yeasts do not secrete a factor(s) that affects the ability of other phagolysosomes within the same M phi to digest killed yeasts; and 3), inhibition of yeast protein synthesis or cell wall biosynthesis is sufficient to render viable yeasts susceptible to digestion by human M phi.

Cells, Cultured↗

Interactions between Brettanomyces bruxellensis and other yeast species during the initial stages of winemaking.

AIMS: Wine is the product of complex interactions between yeasts and bacteria in grape must. Amongst yeast populations, two groups can be distinguished. The first, named non-Saccharomyces (NS), colonizes, with many other micro-organisms, the surface of grape berries. In the past, NS yeasts were primarily considered as spoilage micro-organisms. However, recent studies have established a positive contribution of certain NS yeasts to wine quality. Amongst the group of NS yeasts, Brettanomyces bruxellensis, which is not prevalent on wine grapes, plays an important part in the evolution of wine aroma. Some of their secondary metabolites, namely volatile phenols, are responsible for wine spoilage. The other group contributing to wine aroma, which is also the main agent of alcoholic fermentation (AF), is composed of Saccharomyces species. The fermenting must is a complex microbial ecosystem where numerous yeast strains grow and die according to their adaptation to the medium. Yeast-yeast interactions occur during winemaking right from the onset of AF. The aim of this study was to describe the interactions between B. bruxellensis, other NS and Saccharomyces cerevisiae during laboratory and practical scale winemaking. METHODS AND RESULTS: Molecular methods such as internal transcribed spacer-restriction fragment length polymorphism and polymerase chain reaction and denaturing gradient gel electrophoresis were used in laboratory scale experiments and cellar observations. The influence of different oenological practices, like the level of sulphiting at harvest time, cold maceration preceding AF, addition of commercial active dry yeasts on B. bruxellensis and other yeast interactions and their evolution during the initial stages of winemaking have been studied. Brettanomyces bruxellensis was the most adapted NS yeast at the beginning of AF, and towards the end of AF it appeared to be more resistant than S. cerevisiae to the conditions of increased alcohol and sugar limitation. CONCLUSIONS: Among all NS yeast species, B. bruxellensis is better adapted than other wild yeasts to resist in must and during AF. Moreover, B. bruxellensis appeared to be more tolerant to ethanol stress than S. cerevisiae and after AF B. bruxellensis was the main yeast species in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: Brettanomyces bruxellensis interacts with other yeast species and adapts to the wine medium as the dominant yeast species at the end of AF. Contamination of B. bruxellensis might take place at the beginning of malolactic fermentation, which is a critical stage in winemaking.

DNA, Fungal↗

In vitro attachment of phylloplane yeasts to Botrytis cinerea, Rhizoctonia solani, and Sclerotinia homoeocarpa.

The ability of yeasts to attach to hyphae or conidia of phytopathogenic fungi has been speculated to contribute to biocontrol activity on plant surfaces. Attachment of phylloplane yeasts to Botrytis cinerea, Rhizoctonia solani, and Sclerotinia homoeocarpa was determined using in vitro attachment assays. Yeasts were incubated for 2 d on potato dextrose agar (PDA) prior to experimentation. A total of 292 yeasts cultured on PDA were screened for their ability to attach to conidia of B. cinerea; 260 isolates (89.1%) attached to conidia forming large aggregates of cells, and 22 isolates (7.5%) weakly attached to conidia with 1 or 2 yeast cells attached to a few conidia. Ten yeasts (3.4%), including 8 isolates of Cryptococcus laurentii, 1 isolate of Cryptococcus flavescens, and an unidentified species of Cryptococcus, failed to attach to conidia. All non-attaching yeasts produced copious extracellular polysaccharide (EPS) on PDA. Seventeen yeast isolates did not attach to hyphal fragments of B. cinerea, R. solani, and S. homoeocarpa after a 1 h incubation, but attachment was observed after 24 h. Culture medium, but not culture age, significantly affected the attachment of yeast cells to conidia of B. cinerea. The 10 yeast isolates that did not attach to conidia when grown on agar did attach to conidia (20%-57% of conidia with attached yeast cells) when cultured in liquid medium. Attachment of the biocontrol yeast Rhodotorula glutinis PM4 to conidia of B. cinerea was significantly greater at 1 x 10(7) yeast cells x mL(-1) than at lower concentrations of yeast cells. The ability of yeast cells to attach to fungal conidia or hyphae appears to be a common phenotype among phylloplane yeasts.

Ascomycota↗

Age-related acquisition of oral and nasopharyngeal yeast species and stability of colonization in young children.

The occurrence and stability of colonization of oral yeast species and strains was determined from 40 healthy children during a 22-month follow-up at the ages 2, 6, 12, 18 and 24 months. In addition, salivary samples were obtained from the mothers at baseline (2 months) to study the role of the mother as the source of yeasts for the child. Yeasts were recovered at least once from 17/40 (43%) children by the age of 2 years. Of the 40 children, 11 (28%) were yeast-positive at multiple sampling occasions. No significant differences were found in recovery frequency of yeasts at different ages. Candida parapsilosis was isolated in 18/33 (55%) yeast-positive samples, and it predominated (share of positive findings 76%) at ages 12 to 24 months. The same yeast species was rarely detected in successive follow-up samples and thus on species level yeasts were transient colonizers in the developing oral flora of the children. Of the mothers 20/40 (50%) harbored yeasts. Candida albicans was recovered from 19/20 (95%) of the yeast-positive mothers and C. parapsilosis from none. Only 7/20 (35%) of the mothers with a yeast-positive finding had a yeast-positive child. In 5/7 (71%) of these mother-child pairs, both harbored the same yeast species (C. albicans) and in 3/5 (60%) of the pairs the AP-PCR profiles of the yeast isolates were identical suggesting possible transmission. In children, significant relationships (Fisher's exact-test, P < 0.05) were found between recovery of yeasts and use of pacifier at age over 12 months, eruption of first teeth at age over 6 months, mother cooling the child's food by blowing and mother cleaning the child's pacifier in her own mouth. In mothers, a significant relationship existed between recovery of yeasts and use of antibiotics.

Adult↗

Glycolytic enzyme interactions with yeast and skeletal muscle F-actin.

Interaction of glycolytic enzymes with F-actin is suggested to be a mechanism for compartmentation of the glycolytic pathway. Earlier work demonstrates that muscle F-actin strongly binds glycolytic enzymes, allowing for the general conclusion that "actin binds enzymes", which may be a generalized phenomenon. By taking actin from a lower form, such as yeast, which is more deviant from muscle actin than other higher animal forms, the generality of glycolytic enzyme interactions with actin and the cytoskeleton can be tested and compared with higher eukaryotes, e.g., rabbit muscle. Cosedimentation of rabbit skeletal muscle and yeast F-actin with muscle fructose-1,6-bisphosphate aldolase (aldolase) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) followed by Scatchard analysis revealed a biphasic binding, indicating high- and low-affinity domains. Muscle aldolase and GAPDH showed low-affinity for binding yeast F-actin, presumably because of fewer acidic residues at the N-terminus of yeast actin; this difference in affinity is also seen in Brownian dynamics computer simulations. Yeast GAPDH and aldolase showed low-affinity binding to yeast actin, which suggests that actin-glycolytic enzyme interactions may also occur in yeast although with lower affinity than in higher eukaryotes. The cosedimentation results were supported by viscometry results that revealed significant cross-linking at lower concentrations of rabbit muscle enzymes than yeast enzymes. Brownian dynamics simulations of yeast and muscle aldolase and GAPDH with yeast and muscle actin compared the relative association free energy. Yeast aldolase did not specifically bind to either yeast or muscle actin. Yeast GAPDH did bind to yeast actin although with a much lower affinity than when binding muscle actin. The binding of yeast enzymes to yeast actin was much less site specific and showed much lower affinities than in the case with muscle enzymes and muscle actin.

Actins↗

Brewers dried yeast as a source of mannan oligosaccharides for weanling pigs.

Brewers dried yeast, a source of mannan oligosaccharides (MOS), was assessed as an alternative to an antimicrobial agent (carbadox) for young pigs in two experiments. The yeast contained 5.2% MOS. Agglutination tests confirmed adsorption of several serovars of E. coli and Salmonella spp. onto the yeast product. In Exp. 1, seven replicates (five pigs per pen) of 22-d-old pigs were fed a nonmedicated basal diet or the basal diet with carbadox (55 mg/kg), yeast (3%), or a combination of 3% yeast and 2% citric acid for 28 d. Carbadox did not improve growth performance. Growth rate and feed intake were depressed (P < 0.05) in pigs fed yeast alone or in combination with acid. Log counts of total coliforms, Escherichia coli, and Clostridium perfringens in feces were not affected by diet, but Bifidobacteria spp. counts were lower (P < 0.05) in pigs fed the yeast + acid diet and lactobacilli counts were higher (P < 0.05) in pigs fed yeast. Fecal pH and VFA concentrations and intestinal morphological traits were not consistently affected by diet. Serum IgG levels were elevated in the yeast + acid (P < 0.01) group. In Exp. 2, the effects of yeast and carbadox additions to the diet on enteric microbial populations in young pigs housed in isolation units were evaluated. Pigs (n = 24) were weaned at 11 d of age (4.1 kg BW) and placed in isolation chambers (two pigs per chamber) equipped with individual air filtering systems and excrement containers. Treatments were a nonmedicated basal diet and the basal diet with 55 mg/kg of carbadox or with 3% yeast. Diets were fed for 29 d, then each pig was orally dosed with approximately 9.5 x 10(8) CFU of E. coli K88. Daily fecal E. coli K88 counts were not different (P > 0.05) among treatments, but fecal shedding of carbadox-resistant coliforms was higher (P < 0.01) during the 9-d period in pigs fed carbadox. Total fecal coliforms were consistently lower throughout the postinoculation period in pigs fed yeast (P < 0.05). Yeast reduced colonization oftotal coliforms in the duodenum,jejunum, cecum, and colon, but it did not have a consistent effect on colonization of E. coli K88. Pigs fed yeast tended (P < 0.10) to have higher serum IgG levels than controls. In these experiments, brewers dried yeast and carbadox had minimal effects on growth, microbial populations, and intestinal health traits of early-weaned pigs, but certain serum immunological traits were enhanced by feeding yeast.

Agglutination Tests↗

Selective media for detecting and enumerating foodborne yeasts.

No one medium is satisfactory for detecting, isolating and enumerating all yeasts in all foods. Antibiotic-supplemented media such as dichloran rose Bengal chloramphenicol agar, tryptone glucose yeast extract chloramphenicol agar, oxytetracycline glucose yeast extract agar and rose Bengal chloramphenicol agar are superior to acidified potato dextrose agar and other acidified media for enumeration of the vast majority of spoilage yeasts. Dichloran glycerol (18%) agar performs well for enumerating moderately xerotolerant yeasts. Malt extract yeast extract glucose (up to 60%) can be used for detecting and enumerating moderate and extreme xerophiles. These media also support the growth of moulds. Lysine agar, Schwarz differential agar and Lin's wild yeast differential agar are used by the brewing industry to differentiate wild yeasts from brewer's strains. Lysine agar is selective for apiculate yeasts and ethanol sulfite yeast extract agar is selective for Saccharomyces. Both have application in wineries. Modified molybdate agar can be used to selectively isolate yeasts from tropical fruits. Preservative-resistant yeasts can be detected on malt acetic agar. The recommended incubation temperature is 25 degrees C, but incubation time between plating and counting colonies ranges from 5 days for determination of general populations of yeasts to 10 days for more for xerotolerant yeasts. There is need for new and improved media for selectively isolating various groups, genera, species and strains of yeasts capable of growing only under specific environmental conditions in specific types of foods and beverages.

Bacteriological Techniques↗

Beauveria bassiana yeast phase on agar medium and its pathogenicity against Diatraea saccharalis (Lepidoptera: Crambidae) and Tetranychus urticae (Acari: Tetranychidae).

Beauveria bassiana colonizes insect hosts initially through a yeast phase, which is common in some artificial liquid cultures, but not reported on artificial solid media. We describe a yeast-like phase for B. bassiana isolate 447 (ATCC 20872) on MacConkey agar and its virulence toward Diatraea saccharalis and Tetranychus urticae. The yeast-like cells of B. bassiana developed by budding from germinating conidia after 24-h incubation. Cells were typically 5-10 microm and fungal colonies were initially circular and mucoid, but later were covered with mycelia and conidia. Ability to produce yeast-like cells on MacConkey medium was relatively common among different B. bassiana isolates, but growth rate and timing of yeast-like cell production also varied. Metarhizium anisopliae and Paecilomyces spp. isolates did not grow as yeast-like cells on MacConkey medium. Yeast-like cells of B. bassiana 447 were more virulent against D. saccharalis than conidia when 10(7)cells/ml were used. At 10(8)cells/ml, the estimated mean survival time was 5.4 days for the yeast suspension and 7.7 days for the conidial suspension, perhaps due to faster germination. The LC(50) was also lower for yeast than conidial suspensions. Yeast-like cells and conidia had similar virulence against T. urticae; the average mortalities with yeast-like cells and conidia were, respectively, 42.8 and 45.0%, with 10(7)cells/ml, and 77.8 and 74.4%, with 10(8)cells/ml. The estimated mean survival times were 3.6 and 3.9 for yeast and conidial suspensions, respectively. The bioassay results demonstrate the yeast-like structures produced on MacConkey agar are effective as inoculum for B. bassiana applications against arthropod pests, and possibly superior to conidia against some species. Obtaining well-defined yeast phase cultures of entomopathogenic hyphomycetes may be an important step in studies of the biology and nutrition, pathogenesis, and the genetic manipulation of these fungi.

Agar↗