Discrimination by heat and proteinase treatments between flocculent phenotypes conferred on Saccharomyces cerevisiae by the genes FLO1 and FLO5

The effects of elevated temperature and of digestion with a variety of proteinases on the flocforming ability of flocculent strains of Saccharomyces cerevisiae, both genetically defined (FLO1 and FLO5) laboratory and genetically undefined brewing strains, have been determined. This has permitted classification of the flocculent phenotypes of these strains according to criteria other than quantitative grading of flocculence. The flocculent phenotypes conferred by both the FLO1 and the FLO5 gene were irreversibly lost upon treatment with pronase, proteinase K, trypsin or 2-mercaptoethanol treatments. However, the floc-forming ability of cells of the FLO1 strain ABXL-1D was destroyed by chymotrypsin digestion and was stable to incubation at 70 degrees C, whereas the floc-forming ability of cells of the FLO5 strain ABXR-11A was resistant to the action of chymotrypsin and was heat labile. Tetrad analysis of a cross of these FLO1 and FLO5 strains indicated that the chymotrypsin and heat sensitivity phenotypes were FLO-gene determined. It appears that expression of the FLO1 and FLO5 genes leads to the production of different and characteristic cell-wall proteins underlying their respective flocculent phenotypes.     

Genetic basis of flocculation phenotype conversion in Saccharomyces cerevisiae

Two NewFlo-type flocculent transformants Saccharomyces cerevisiae YTS-S and YTS-L were obtained from a partial yeast genomic library. Even though both of the transformants displayed the same flocculation phenotype, they represented different physiological characteristics during detailed investigation. Analysis of the two transformants YTS-L and YTS-S confirmed the presence of FLONL and FLONS genes, respectively. The 3396-bp ORF of FLONS encoded a protein of 1132 amino acids. Meanwhile, the presence of a 1686-bp ORF encoding a 562-amino acid protein was revealed in FLONL. Both FLONL and FLONS showed high identity to FLO1 gene. Aligned with the intact FLO1 gene, FLONS lost two internal repeated regions, whereas one repeated sequence was inserted into the middle of the FLONL gene. All of the altered regions could be found in the middle repetitive sequence of the FLO1 gene. The results indicate that FLONL and FLONS are both derived forms of the FLO1 gene. Genetic variability triggered by tandem repeats in FLO1 gene is believed to be responsible for the differential phenotypic properties of the yeast strains YTS-S and YTS-L.     

Temperature-sensitive and circadian oscillators of Neurospora crassa share components

In Neurospora crassa, the interactions between products of the frequency (frq), frequency-interacting RNA helicase (frh), white collar-1 (wc-1), and white collar-2 (wc-2) genes establish a molecular circadian clockwork, called the FRQ-WC-Oscillator (FWO), which is required for the generation of molecular and overt circadian rhythmicity. In strains carrying nonfunctional frq alleles, circadian rhythms in asexual spore development (conidiation) are abolished in constant conditions, yet conidiation remains rhythmic in temperature cycles. Certain characteristics of these temperature-synchronized rhythms have been attributed to the activity of a FRQ-less oscillator (FLO). The molecular components of this FLO are as yet unknown. To test whether the FLO depends on other circadian clock components, we created a strain that carries deletions in the frq, wc-1, wc-2, and vivid (vvd) genes. Conidiation in this ΔFWO strain was still synchronized to cyclic temperature programs, but temperature-induced rhythmicity was distinct from that seen in single frq knockout strains. These results and other evidence presented indicate that components of the FWO are part of the temperature-induced FLO.     

Pulsed-field gel electrophoresis indicates genotypic heterogeneity among Campylobacter upsaliensis strains

Abstract A non-flocculent strain of Saccharomyces cerevisiae was selected after EMS mutation of a flocculent and heterozygous FLO1 locus diploid. The analysis of 25 asci from this diploid showed in all cases segregation 0F:4NF, thus confirming that it was probably affected in the desired gene. After sporulation and dissection of asci, three haploid strains were chosen, which were altered in the locus FLO1 . Crossing these three strains with two other ones having markers for ADE1 and pho11::LEU2 , we could map the mutation at ca. 4.3 cM and ca. 37.7 cM from the PHO11 and ADE1 loci respectively.     

衔接重复序列对酵母菌絮凝蛋白功能的调控作用

【目的】了解絮凝基因FLO1中重复DNA序列B和D对絮凝蛋白Flo1p功能的影响,为构建遗传稳定的最小絮凝功能基因奠定理论基础。【方法】通过PCR和融合PCR方法分别克隆到完整的絮凝基因FLO1、重复DNA序列B和D分别缺失的衍生基因FLO1b和FLO1d,分析这些基因在非絮凝酵母中表达对细胞絮凝特性的影响。【结果】与完整絮凝基因相比,重复DNA序列B和D分别缺失后对酵母细胞絮凝强度没有明显影响,但不同基因在酵母菌中表达产生的絮凝特性受环境因素,如甘露糖浓度和pH等的影响有明显差异。FLO1中重复DNA序列B和D缺失后,细胞絮凝特性受甘露糖抑制的敏感性降低;同时对环境pH的改变具有更广泛的适应性。【结论】重复DNA序列B和D对絮凝蛋白Flo1p结构和功能具有调控作用,二者缺失后,特别是D缺失后会使絮凝蛋白在极端酸碱环境下更稳定。     

The identification, characterization, and mapping of a gene for flocculation in Saccharomyces sp.

Genetic studies of a flocculent haploid strain of Saccharomyces have revealed the flocculation to be dominant and controlled at a single gene locus. The flocculation character of both hybrids and haploids derived from such hybrids appears to be influenced by the repression or derepression status of the culture. Mapping studies of this flocculation gene have revealed that it is linked to ade 1 and therefore located on Chromosome I. Consequently, this is a different gene to the three flocculation genes studied by other laboratories because they have found such genes to be unlinked to ade 1. The flocculation gene being discussed in this paper has been designated FLO4. FLO4 has been located 32-33 cM from the Chromosome I centromere and 37 cM from ade 1 (i.e., FLO4 is on the opposite side of the centromere to ade 1).     

Possible mechanism for flocculation interactions governed by gene FLO1 in Saccharomyces cerevisiae.

A model is proposed for the mechanism of flocculation interactions in yeasts in which flocculent cells have a recognition factor which attaches to alpha-mannan sites on other cells. This factor may be governed by the expression of the single, dominant gene FLO1. Isogenic strains of Saccharomyces cerevisiae, differing only at FLO1 and the marker genes ade1 and trp1, were developed to examine the components involved in flocculene. Electron microscopy and concanavalin Aferritin labeling of aggregated cells showed that extensive and intense interactions between cell wall mannan layers mediated cell aggregation. The components of the mannan layer essential for flocculence were Ca2+ ions, alpha-mannan carbohydrates, and proteins. By studying the divalent cation dependence at various pH values and in the presence of competing monovalent cations, flocculation was found to be Ca2+ dependent; however, Mg2+ and Mn2+ ions substituted for Ca2+ under certain conditions. Reversible inhibition of flocculation by concanavalin A and succinylated concanavalin A implicated alpha-branched mannan carbohydrates as one essential component which alone did not determine the strain specificity of flocculence, since nonflocculent strains interacted with and competed for binding sites on flocculent cells. FLO1 may govern the expression of a proteinaceous, lectin-like activity, firmly associated with the cell walls of flocculent cells, which bind to the alpha-mannan carbohydrates of adjoining cells. It was selectively and irreversibly inhibited by proteolysis and reduction of disulfide bonds. The potential of this system as a model for the genetic and biochemical control of cell-cell interactions is discussed.     

Identification of novel activation mechanisms for FLO11 regulation in Saccharomyces cerevisiae

Adhesins play a central role in the cellular response of eukaryotic microorganisms to their host environment. In pathogens such as Candida spp. and other fungi, adhesins are responsible for adherence to mammalian tissues, and in Saccharomyces spp. yeasts also confer adherence to solid surfaces and to other yeast cells. The analysis of FLO11, the main adhesin identified in Saccharomyces cerevisiae, has revealed complex mechanisms, involving both genetic and epigenetic regulation, governing the expression of this critical gene. We designed a genomewide screen to identify new regulators of this pivotal adhesin in budding yeasts. We took advantage of a specific FLO11 allele that confers very high levels of FLO11 expression to wild "flor" strains of S. cerevisiae. We screened for mutants that abrogated the increased FLO11 expression of this allele using the loss of the characteristic fluffy-colony phenotype and a reporter plasmid containing GFP controlled by the same FLO11 promoter. Using this approach, we isolated several genes whose function was essential to maintain the expression of FLO11. In addition to previously characterized activators, we identified a number of novel FLO11 activators, which reveal the pH response pathway and chromatin-remodeling complexes as central elements involved in FLO11 activation.     

Saccharomyces Cerevisiae S288c Has a Mutation in Flo8, a Gene Required for Filamentous Growth

Diploid strains of baker's yeast Saccharomyces cerevisiae can grow in a cellular yeast form or in filaments called pseudohyphae. This dimorphic transition from yeast to pseudohyphae is induced by starvation for nitrogen. Not all laboratory strains are capable of this dimorphic switch; many grow only in the yeast form and fail to form pseudohyphae when starved for nitrogen. Analysis of the standard laboratory strain S288C shows that this defect in dimorphism results from a nonsense mutation in the FLO8 gene. This defect in FLO8 blocks pseudohyphal growth in diploids, haploid invasive growth, and flocculation. Since feral strains of S. cerevisiae are dimorphic and have a functional FLO8 gene, we suggest that the flo8 mutation was selected during laboratory cultivation.     

Chromosomal location of Lg-FLO1 in bottom-fermenting yeast and the FLO5 locus of industrial yeast

Aims: To determine the chromosomal location and entire sequence of Lg-FLO1, the expression of which causes the flocculation of bottom-fermenting yeast. Methods and Results: Two cosmid clones carrying DNA from a bottom-fermenting yeast chromosome VIII right-arm end were selected by colony hybridization. Sequencing revealed that the clones contained DNA derived from a Saccharomyces cerevisiae type chromosome VIII and a Saccharomyces bayanus type chromosome VIII, both from bottom-fermenting yeast. Conclusions: Lg-FLO1 is located on the S. cerevisiae type chromosome VIII at the same position as the FLO5 gene of the laboratory yeast S. cerevisiae S288c. The unique chromosome VIII structure of bottom-fermenting yeast is conserved among other related strains. FLO5 and Lg-FLO1 promoter sequences are identical except for the presence of three 42 bp repeats in the latter, which are associated with gene activity. Flocculin genes might have been generated by chromosomal recombination at these repeats. Significance and Impact of the Study: This is the first report of the exact chromosomal location and entire sequence of Lg-FLO1. This information will be useful in the brewing industry for the identification of normal bottom-fermenting yeast. Moreover, variations in the FLO5 locus among strains are thought to reflect yeast evolution.     

FLO11 expression in clinical and non-clinical Saccharomyces cerevisiae strains and its association with virulence

In Saccharomyces cerevisiae, FLO11 encodes a protein associated with phenotypic traits considered important for virulence. Here, we report the analysis of FLO11 gene expression using RT-LightCycler PCR in several S. cerevisiae strains of different origin (clinical and non-clinical) and with different degrees of in vivo virulence. An association between in vivo virulence and FLO11 expression was observed for the majority of strains when cells were grown at 37 °C in brain heart infusion (BHI) broth to mimic conditions encountered during brain colonization. However, there was a lack of correlation for two of the strains and this was probably due to the loss of a repression sequence in the FLO11 promoter and/or to changes in repetitive sequences in the ORF. The results indicate that the method proposed here, in conjunction with determination of other virulence factors, could usefully predict which S. cerevisiae strains are better suited to colonize in vivo systems.     

Exploration of a natural reservoir of flocculating genes from various Saccharomyces cerevisiae strains and improved ethanol fermentation using stable genetically engineered flocculating yeast strains

Flocculating yeast strains with good fermentation ability are desirable for brewing industry as well as for fuel ethanol production, however, the genetic diversity of the flocculating genes from natural yeast strains is largely unexplored. In this study, FLO1, FLO5, FLO9, FLO10 and FLO11 PCR products were obtained from 16 yeast strains from various sources, and the PCR product amplified from FLO1 of the self-flocculating yeast strain SPSC01 was used for the construction of expression cassette flanked by homologous fragments of the endonuclease gene HO for chromosome integration. A genetically engineered flocculating yeast BHL01 with good fermentation performance was obtained by transforming an industrial strain Saccharomyces cerevisiae 4126 with the expression cassette. The fermentation performances of SPSC01 and BHL01 in flask fermentation were compared using 208 g/L glucose. BHL01 completed the fermentation 8 h earlier than SPSC01, while no significant difference between BHL01 and S. cerevisiae 4126 was observed. In very high gravity repeated batch ethanol fermentation using 255 g/L glucose, BHL01 maintained stable flocculation for at least over 24 batches, while SPSC01 displayed severe deflocculation under the same conditions. The natural reservoir of flocculating genes from yeast strains may represent an unexplored gene source for the construction of new flocculating yeast strains for improved ethanol production.     

Region of Flo1 Proteins Responsible for Sugar Recognition

Yeast flocculation is a phenomenon which is believed to result from an interaction between a lectin-like protein and a mannose chain located on the yeast cell surface. The FLO1 gene, which encodes a cell wall protein, is considered to play an important role in yeast flocculation, which is inhibited by mannose but not by glucose (mannose-specific flocculation). A new homologue of FLO1, named Lg-FLO1, was isolated from a flocculent bottom-fermenting yeast strain in which flocculation is inhibited by both mannose and glucose (mannose/glucose-specific flocculation). In order to confirm that both FLO1 and Lg-FLO1 are involved in the yeast flocculation phenomenon, the FLO1 gene in the mannose-specific flocculation strain was replaced by the Lg-FLO1 gene. The transformant in which the Lg-FLO1 gene was incorporated showed the same flocculation phenotype as the mannose/glucose-specific flocculation strain, suggesting that the FLO1 and Lg-FLO1 genes encode mannose-specific and mannose/glucose-specific lectin-like proteins, respectively. Moreover, the sugar recognition sites for these sugars were identified by expressing chimeric FLO1 and Lg-FLO1 genes. It was found that the region from amino acid 196 to amino acid 240 of both gene products is important for flocculation phenotypes. Further mutational analysis of this region suggested that Thr-202 in the Lg-Flo1 protein and Trp-228 in the Flo1 protein are involved in sugar recognition.     

FLO11 is essential for flor formation caused by the C-terminal deletion of NRG1 in Saccharomyces cerevisiae

The flor strains of Saccharomyces cerevisiae form a flor on the surface of wine after alcoholic fermentation. High hydrophobicity of the cell surface is suggested to be important for flor formation by the flor wine yeasts. However, the molecular mechanism of flor formation is not clear. We found that expression of C-terminal deleted NRG1 lacking its two C2H2 zinc finger motifs (NRG1(1-470)) on the multicopy plasmid conferred the ability to form a flor to a non-flor laboratory strain. The cell surface hydrophobicity of NRG1(1-470) was higher than of the non-flor strain. Disruption of the Nrg1p-repressed gene FLO11, which encodes a cell surface glycoprotein that functions as a flocculin or an adhesin, abolished flor formation. Moreover, expression of FLO11 on a multicopy plasmid could also cause flor formation. These results indicate that FLO11 is essential for flor formation by NRG1(1-470). In addition, the results suggest that the C-terminal truncated form of Nrg1p exerts a dominant negative effect on FLO11 repression, resulting in FLO11 expression and, thus, flor formation.