FLO Genes Research Articles

Amino acid starvation and Gcn4p regulate adhesive growth and FLO11 gene expression in Saccharomyces cerevisiae.

In baker's yeast Saccharomyces cerevisiae, cell-cell and cell-surface adhesion are required for haploid invasive growth and diploid pseudohyphal development. These morphogenetic events are induced by starvation for glucose or nitrogen and require the cell surface protein Flo11p. We show that amino acid starvation is a nutritional signal that activates adhesive growth and expression of FLO11 in both haploid and diploid strains in the presence of glucose and ammonium, known suppressors of adhesion. Starvation-induced adhesive growth requires Flo11p and is under control of Gcn2p and Gcn4p, elements of the general amino acid control system. Tpk2p and Flo8p, elements of the cAMP pathway, are also required for activation but not Ste12p and Tec1p, known targets of the mitogen-activated protein kinase cascade. Promoter analysis of FLO11 identifies one upstream activation sequence (UASR) and one repression site (URS) that confer regulation by amino acid starvation. Gcn4p is not required for regulation of the UASR by amino acid starvation, but seems to be indirectly required to overcome the negative effects of the URS on FLO11 transcription. In addition, Gcn4p controls expression of FLO11 by affecting two basal upstream activation sequences (UASB). In summary, our study suggests that amino acid starvation is a nutritional signal that triggers a Gcn4p-controlled signaling pathway, which relieves repression of FLO11 gene expression and induces adhesive growth.

Construction of yeast strains with high cell surface lipase activity by using novel display systems based on the Flo1p flocculation functional domain.

We constructed a novel cell-surface display system, using as a new type of cell-wall anchor 3,297 or 4,341 bp of the 3' region of the FLO1 gene (FS or FL gene, respectively), which encodes the flocculation functional domain of Flo1p. In this system, the N terminus of the target protein was fused to the FS or FL protein and the fusion proteins were expressed under the control of the inducible promoter UPR-ICL (5' upstream region of the isocitrate lyase of Candida tropicalis). Using this new system, recombinant lipase with a pro sequence from Rhizopus oryzae (rProROL), which has its active site near the C terminus, was displayed on the cell surface. Cell-surface display of the FSProROL and FLProROL fusion proteins was confirmed by immunofluorescence microscopy and immunoblotting. Lipase activity reached 145 IU/liter (61.3 IU/g [dry cell weight]) on the surface of the yeast cells, which successfully catalyzed the methanolysis reaction. Using these whole-cell biocatalysts, methylesters synthesized from triglyceride and methanol reached 78.3% after 72 h of reaction. To our knowledge, this is the first example of cell-surface display of lipase with high activity. Interestingly, the yeast cells displaying the FLProROL protein showed strong flocculation, even though the glycosylphosphatidylinositol anchor attachment signal and cell-membrane-anchoring region of Flo1p had been deleted from this gene. The cell-surface display system based on FL thus endows the yeast strain with both novel enzyme display and strong flocculation ability.

Analysis of an inactivated Lg- FLO1 gene present in bottom-fermenting yeast

During the course of investigating a flocculation-related gene of a bottom-fermenting yeast, we identified a new Lg- FLO1 homologue which contains the N-terminal domain of the Lg- FLO1 gene. The results of the partial DNA sequence analysis of the amplified product obtained by inverse-PCR suggested that the homologue contains a sequence present in the YIL169c (chr. IX of Saccharomyces cerevisiae). Southern blot analyses using the VTH1, HXT12, SDL1 and UBP7 genes as probes for chr. IX strongly indicated that an approximately 20-kb region from the YIL169c ORF to the left telomere in chr. IX translocated to the Lg- FLO1 ORF region in chr. VIII of bottom-fermenting yeast. This translocation might convert a flocculent cell to a non-flocculent one.

Use of Saccharomyces cerevisiae for Cu2+ removal from solution: the advantages of using a flocculent strain

A flocculent strain of Saccharomyces cerevisiae S646-1B accumulated more Cu2+ (81 nmol mg−1 dry wt) than the isogenic (except for the marker genes ade1 and trp1 and the gene FLO1) non-flocculent strain S646-8D (30 nmol mg−1 dry wt), in the first 10 min of contact of the cells with Cu2+. Additionally, this strain flocculated in solutions of 0.2 mM Cu2+, Ni2+, Zn2+ and Cd2+. The potential of using flocculent strains in the bioremediation of heavy metals contaminated waste waters is discussed.

Antagonistic remodelling by Swi-Snf and Tup1-Ssn6 of an extensive chromatin region forms the background for FLO1 gene regulation.

Novel yeast histone mutations that confer Swi-Snf independence (Sin(-)) were used to investigate the mechanisms by which transcription coactivator complexes relieve chromatin repression in vivo. Derepression of the flocculation gene FLO1, which is normally repressed by the Tup1-Ssn6 corepressor, leads to its identification as a constitutive Swi-Snf-dependent gene. We demonstrate that Tup1-Ssn6 is a chromatin remodelling complex that rearranges and also orders nucleosomal arrays on the promoter and over 5 kb of upstream intergenic region. Our results confirm that the Swi-Snf complex disrupts nucleosome positioning on promoters, but reveal that it can also rearrange nucleosomes several kilobases upstream from the transcription start site. The antagonistic chromatin remodelling activities of Swi-Snf and Tup1-Ssn6 detected in an array of 32 nucleosomes upstream of FLO1 extend far beyond the scale of promoter-based models of chromatin-mediated gene regulation. The Swi-Snf coactivator and Tup1-Ssn6 corepressor control an extensive chromatin domain in which regulation of the FLO1 gene takes place.

Late Fermentation Expression of FLO1 in Saccharomyces Cerevisiae

A strategy to alter the flocculation properties of nonflocculent yeast in such a way that flocculation occurred toward the end of fermentation was developed. In a double cross-over event, the wild-type FLO1 promoter of the haploid, nonflocculent S. cerevisiae FY23 strain was replaced by a construct consisting of the SMRI-410 marker gene and the HSP30 promoter. In this way, the genomic copy of the wild-type FLO1 open reading frame was brought under transcriptional control of the HSP30 promoter. The transformants showed strong flocculation toward the end of fermentation, resulting in a distinctly clearer beer than the beer obtained with wild-type cells. The other properties of the wild-type strain were conserved. Moreover, it was shown that the transformants were extremely stable and that flocculation could be induced earlier during fermentation by a heat-shock treatment or the addition of ethanol to the medium. These results suggest that the flocculation properties of weakly flocculent brewer's yeast strains can be improved using controlled expression of the FLO1 gene.

Different domains of the essential GTPase Cdc42p required for growth and development of Saccharomyces cerevisiae.

In budding yeast, the Rho-type GTPase Cdc42p is essential for cell division and regulates pseudohyphal development and invasive growth. Here, we isolated novel Cdc42p mutant proteins with single-amino-acid substitutions that are sufficient to uncouple functions of Cdc42p essential for cell division from regulatory functions required for pseudohyphal development and invasive growth. In haploid cells, Cdc42p is able to regulate invasive growth dependent on and independent of FLO11 gene expression. In diploid cells, Cdc42p regulates pseudohyphal development by controlling pseudohyphal cell (PH cell) morphogenesis and invasive growth. Several of the Cdc42p mutants isolated here block PH cell morphogenesis in response to nitrogen starvation without affecting morphology or polarity of yeast form cells in nutrient-rich conditions, indicating that these proteins are impaired for certain signaling functions. Interaction studies between development-specific Cdc42p mutants and known effector proteins indicate that in addition to the p21-activated (PAK)-like protein kinase Ste20p, the Cdc42p/Rac-interactive-binding domain containing Gic1p and Gic2p proteins and the PAK-like protein kinase Skm1p might be further effectors of Cdc42p that regulate pseudohyphal and invasive growth.

A mannose-binding protein from the cell surface of flocculent Saccharomyces cerevisiae (NCIM 3528): its role in flocculation.

A cell surface lectin was isolated and purified to homogeneity from the cell walls of a highly flocculent strain of Saccharomyces cerevisiae (NCIM 3528) by chromatography on DEAE-cellulose, phenyl Sepharose and Sephacryl S-300. It showed a molecular mass of 40 kDa on SDS-PAGE. It is an acidic protein with a pI of 4.0 and contains 44% hydrophobic amino acids. The N-terminal sequence up to 10 amino acid residues showed at least 70% homology with the predicted N-terminal sequence of the putative FLO1 as well as FLO5 gene products. The mannose-binding nature of the lectin was indicated by its high affinity and specificity towards the branched trisaccharide of mannose, a ligand which also inhibits the flocculation of yeast cells. Immunofluorescence studies confirmed the presence of lectin on the yeast cell surface and lectin-specific IgGs prevented flocculation of the cells. This cell surface mannose-specific lectin probably plays an important role in flocculation, with the branched trimannoside on the cell wall being the apparent carbohydrate receptor. The N-terminal sequence data gives a primary indication that the lectin could be a product of one of the FLO genes.

Detection of multidrug-resistant Salmonella typhimurium DT104 by multiplex polymerase chain reaction.

Salmonella typhimurium definitive type 104 (DT104) is a virulent pathogen for humans and animals with many strains having multiple drug resistance characteristics. The organism typically carries resistance to ampicillin, chloramphenicol, florfenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT-resistant). A multiplex PCR method was developed to simultaneously amplify four genes, florfenicol (flo(st)), virulence (spvC), invasion (invA), and integron (int) from S. typhimurium DT104 (ACSSuT-type). Twenty-two ACSSuT-resistant DT104 isolates in our collection gave 100% positive reactions to this PCR assay by amplifying 584-, 392-, 321- and 265-bp PCR products, using primers specific to the respective target genes. One Salmonella strain DT23, ACSSuT-resistant, phage type 711 failed to amplify the 584-bp fragment, indicating that this method is specific for DT104-type ACSSuT-resistant S. typhimurium strains. One clinical and one bovine ASSuT-resistant strains that were sensitive to chloramphenicol and florfenicol did not yield a 584-bp fragment, indicating the absence of the flo(st) gene. This method will be useful for rapid identification of ACSSuT-type DT104 strains from clinical, food and environmental samples.

Analysis of the genes activated by the FLO8 gene in Saccharomyces cerevisiae.

It is thought that the FLO8 gene encodes a transcriptional activator of the dominant flocculation gene FLO1 in Saccharomyces cerevisiae. To determine other genes which are regulated by FLO8, a detailed comparison of the transcripts from the FLO8 and Deltaflo8 strains was carried out. In addition to the FLO1 gene, it was found that transcription of the FLO11 and STA1 genes is positively regulated by FLO8. In flo8 strains, not only transcripts of the FLO11, STA1, and FLO1 genes but also invasive growth, extracellular glucoamylase production, and flocculation were undetected. From these results, it is suggested that FLO8 regulates these characteristics via the transcriptional regulation of the FLO11, STA1, and FLO1 genes.

MAP kinase and cAMP filamentation signaling pathways converge on the unusually large promoter of the yeast FLO11 gene.

In Saccharomyces cerevisiae, two major signal transduction pathways, the Kss1 MAPK pathway and the cAMP-regulated pathway, are critical for the differentiation of round yeast form cells to multicellular, invasive pseudohyphae. Here we report that these parallel pathways converge on the promoter of a gene, FLO11, which encodes a cell surface protein required for pseudohyphal formation. The FLO11 promoter is unusually large, containing at least four upstream activation sequences (UASs) and nine repression elements which together span at least 2.8 kb. Several lines of evidence indicate that the MAPK and cAMP signals are received by distinct transcription factors and promoter elements. First, regulation via the MAPK pathway requires the transcription factors Ste12p/Tec1p, whereas cAMP-mediated activation requires a distinct factor, Flo8p. Secondly, mutations in either pathway block FLO11 transcription. Overexpression of STE12 can suppress the loss of FLO8, and overexpression of FLO8 can suppress the loss of STE12. Finally, multiple distinct promoter regions of the FLO11 promoter are required for its activation by either Flo8p or Ste12p/ Tec1p. Thus, like the promoters of the key developmental genes, HO and IME1, the FLO11 promoter is large and complex, endowing it with the ability to integrate multiple inputs.

Distribution of the flocculation protein, flop, at the cell surface during yeast growth: the availability of flop determines the flocculation level.

The yeast FLO genes encode cell surface proteins which are expected to play a major role in the control of flocculation. We have assessed the availability of the Flo proteins at the cell surface during the growth of two flocculent strains, ABXL-1D (FLO1) and STX347-1D (FLO5) using immunological approaches, enzyme-linked immunosorbent assays and immunofluorescence. Our data show that they are not permanently present at the cell surface but that their amount increases during growth. With both strains the flocculation level is tightly correlated to the amount of Flop antigen detected, suggesting that it is the availability of the Flo proteins at the cell surface which determines the flocculation level. Our data are consistent with the idea that the Flo proteins correspond to the flocculation lectins. The differences of flocculation pattern among strains could originate from variations in the regulation of the expression of the FLO genes. Monitoring of the distribution of the Flo proteins during cellular development revealed that they are incorporated essentially in the cell wall of growing buds. Incorporation of the Flo proteins in the cell wall displays a highly polarized aspect, at the bud tip and at the mother-daughter neck junction, which can persist in mature cells. Such a localization could be relevant to constraints of the cell wall incorporation of the mannoproteins. Depending on the regulation of Flop expression and on the incorporation of the proteins in the cell wall, a yeast population can be highly heterogeneous in Flo protein equipment.

Breeding of flocculent industrial alcohol yeast strains by self-cloning of the flocculation gene FLO1 and repeated-batch fermentation by transformants.

A nonflocculent industrial polyploid yeast strain, Saccharomyces cerevisiae 396-9-6V, was converted to a flocculent one by introducing a functional FLO1 gene at the URA3 locus. The flocculent strain FSC27 obtained was a so-called self-cloned strain, having no bacterial DNA. FSC27 cells could be easily recovered for reuse from fermentation mash without any physical energy. The strain produced a concentration of alcohol as high as 396-9-6V, although the fermentation rate of FSC27 was slightly lower than that of 396-9-6V. When uracil was added to the medium or when URA3 was reintroduced into FSC27 (named FSCU-L18), the fermentation rate and the growth rate increased, and the ethanol concentration produced was higher than that produced by the parent strain. The stable flocculation and high ethanol productivity were observed by using FSCU-L18 during 10 cycles of repeated-batch fermentation test.

Localization and cell surface anchoring of the Saccharomyces cerevisiae flocculation protein Flo1p.

The Saccharomyces cerevisiae FLO1 gene encodes a large 1,536-amino-acid serine- and threonine-rich protein involved in flocculation. We have assessed the localization of Flo1p by immunoelectron microscopy, and in this study we show that this protein is located in the external mannoprotein layer of the cell wall, at the plasma membrane level and in the periplasm. The protein was also visualized in the endoplasmic reticulum and in the nuclear envelope, indicating that it was secreted through the secretory pathway. The protein was detected by Western blotting in cell wall extracts as a high-molecular-mass (>200 kDa) polydisperse material obviously as a result of extensive N and probably O glycosylation. Flo1p was extracted from cell walls in large amounts by boiling in sodium dodecyl sulfate, suggesting that it is noncovalently anchored to the cell wall network. The membranous forms of Flo1p were shown to be solubilized by phosphatidylinositol-phospholipase C treatment, suggesting that Flo1p is glycosyl phosphatidylinositol (GPI) anchored to this organelle. The expression of truncated forms with the hydrophobic C-terminal domain deleted led to the secretion of the protein in the culture medium. The hydrophobic C terminus, which is a putative GPI anchoring domain, is therefore necessary for the attachment of Flo1p in the cell wall. Deletion analysis also revealed that the N-terminal domain of Flo1p was essential for cellular aggregation. On the whole, our data indicate that Flo1p is a true cell wall protein which plays a direct role in cell-cell interaction.

Constitutive flocculation in Saccharomyces cerevisiae through overexpression of the GTS1 gene, coding for a 'Glo'-type Zn-finger-containing protein.

The product of the cloned GTS1 gene is characterized by structural features found in transcription factors. It contains one Zn-finger motif (CXXCX16CXXC) situated in the N-terminal end with a high degree of homology to the newly identified 'Glo' family of Zn-finger proteins (Ireland et al., 1994, EMBO J. 13, 3812-3821). The C-terminal end of the protein is characterized by poly (Ala-Gln) and poly-Gln stretches. Poly-Gln are part of trans-acting motifs in known transcription factors. Overexpression of the GTS1 gene results in constitutive flocculation. Whole cell electrophoretic mobility and hydrophobicity of GTS1 overexpressing cells was respectively lower and higher relative to control cells. GTS1-induced flocculation is hardly sensitive to mannose in contrast to FLO1-determined flocculation. Overexpression of the GTS1 gene in a flo1 background does not abolish flocculation, suggesting that the FLO1 gene is not linked with the GTS1 gene in a 'flocculation pathway'.

Sequence analysis of a 37.6 kbp cosmid clone from the right arm of Saccharomyces cerevisiae chromosome XII, carrying YAP3, HOG1, SNR6, tRNA-Arg3 and 23 new open reading frames, among which several homologies to proteins involved in cell division control and to mammalian growth factors and other animal proteins are found.

The nucleotide sequence of 37,639 bp of the right arm of chromosome XII has been determined. Twenty-five open reading frames (ORFs) longer than 300 bp were detected, two of which extend into the flanking cosmids. Only two (L2931 and L2961) of the 25 ORFs correspond to previously sequenced genes (HOG1 and YAP3, respectively). Another ORF is distinct from YAP3 but shows pronounced similarity to it. About half of the remaining ORFs show similarity to other genes or display characteristic protein signatures. In particular, ORF L2952 has striking homology with the probable cell cycle control protein crn of Drosophila melanogaster. L2949 has significant similarity to the human ZFM1 (related to a potential suppressor oncogene) and mouse CW17R genes, though it lacks the carboxy-terminal oligoproline and oligoglutamine stretches encoded by these mammalian genes. The small ORF L2922 is similar to part of the much larger yeast flocculation gene FLO1. Other sequences found in the 37639 bp fragment are one delta and one solo-sigma element, the tRNA-Arg3 gene, the small nuclear RNA gene SNR6 and three ARS consensus sequences.

Cloning of a new FLO gene from the flocculating Saccharomyces cerevisiae IM1-8b strain

A flocculation conferring gene was cloned from a genomic library of the flocculating strain Saccharomyces cerevisiae IM1-8b as a 5 kb DNA fragment. The shortest DNA fragment ( XbaI -XbaI) able to confer the flocculating phenotype was 3.1 kb. Southern analysis revealed that this gene was not homologous to the already reported FLO1 gene since strong hybridization signals were obtained when chromosomes IV and XII were probed with a digoxygenin-labelled fragment and no signal at all was detected for chromosome I. Partial sequencing data unequivocally ascribed the cloned fragment to chromosome XII. The gene was detected in a variety of S. cerevisiae strains regardless of their being phenotypically flocculating. This gene which, we propose as FLO2, is able to complement the flo1 mutation and is suppressed by suppressors ( fsu3) that do not affect other FLO genes.

FLO11, a yeast gene related to the STA genes, encodes a novel cell surface flocculin.

We report the characterization of a gene encoding a novel flocculin related to the STA genes of yeast, which encode secreted glucoamylase. The STA genes comprise sequences that are homologous to the sporulation-specific glucoamylase SGA and to two other sequences, S2 and S1. We find that S2 and S1 are part of a single gene which we have named FLO11. The sequence of FLO11 reveals a 4,104-bp open reading frame on chromosome IX whose predicted product is similar in overall structure to the class of yeast serine/threonine-rich GPI-anchored cell wall proteins. An amino-terminal domain containing a signal sequence and a carboxy-terminal domain with homology to GPI (glycosyl-phosphatidyl-inositol) anchor-containing proteins are separated by a central domain containing a highly repeated threonine- and serine-rich sequence. Yeast cells that express FLO11 aggregate in the calcium-dependent process of flocculation. Flocculation is abolished when FLO11 is disrupted. The product of STA1 also is shown to have flocculating activity. When a green fluorescent protein fusion of FLO11 was expressed from the FLO11 promoter on a single-copy plasmid, fluorescence was observed in vivo at the periphery of cells. We propose that FLO11 encodes a flocculin because of its demonstrated role in flocculation, its structural similarity to other members of the FLO gene family, and the cell surface location of its product. FLO11 gene sequences are present in all yeast strains tested, including all standard laboratory strains, unlike the STA genes which are present only in the variant strain Saccharomyces cerevisiae var. diastaticus. FLO11 differs from all other yeast flocculins in that it is located near a centromere rather than a telomere, and its expression is regulated by mating type. Repression of FLO11-dependent flocculation in diploids is conferred by the mating-type repressor al/alpha2.

Molecular cloning and analysis of the dominant flocculation geneFLO8 fromSaccharomyces cerevisiae

A flocculation gene was cloned from a Saccharomyces cerevisiae ATCC60715 genomic library, known to contain the FLO8 gene, on the basis of its ability to confer a flocculation phenotype on a nonflocculent strain. From a total of 11 130 clones, four clones sharing the several restriction fragments were isolated, suggesting that these were derived from the same locus. The results of integration mapping and disruption of the cloned gene indicated that this gene was the FLO8 gene. After disruption of the FLO8 gene, the strain lost its ability to flocculate. The DNA sequence of the FLO8 gene was determined. This gene includes a 2187-bp open reading frame that encodes a 729-amino acid protein. Computer analysis indicated that the FLO8 gene has a significant degree of homology with a S. cerevisiae chromosome V DNA sequence, but no homology with the FLO1 gene. The hydrophobicity profile of the putative FLO8 gene product did not indicate the presence of any significantly hydrophobic regions. Southern analysis of the FLO8 gene present in various yeast strains indicated that the FLO8 gene is highly conserved in yeast strains having a variety of flocculation phenotypes and genotypes. Northern analysis revealed that the level of FLO1 gene transcription is dependent on the rate of transcription of the FLO8 gene. These results suggest that the FLO8 gene mediates flocculation via transcriptional activation of the FLO1 gene.

The Saccharomyces cerevisiae FLO1 flocculation gene encodes for a cell surface protein.

The sequencing of a 6619 bp region encoding for a flocculation gene previously cloned from a strain defined as FLO5 (Bidard et al., 1994) has revealed that it was a FLO1 gene. The FLO1 gene product has been localized at the cell surface of the yeast cell by immunofluorescent microscopy. The Flo1 protein contains four regions with repeated sequences which account for about 70% of the amino acids of this protein. A functional analysis of the major repeated region has revealed that it plays an important role in determining the flocculation level. A gene disruption experiment has shown that FLO5 strain STX 347-1D contains at least two flocculation genes of the FLO1 type but that they are supposed to be inactive and do not contribute to its flocculation. However, enzyme-linked immunosorbent assays performed on intact cells have revealed that a protein expressed at the cell surface of the FLO5 strain STX 347-1D is antigenically related to Flo1p. A deletion analysis of the 5' region of the FLO1 gene has shown that the expression is submitted to controls which depend on the genetic background of the strain.