Genomic Analyses of Anaerobically Induced Genes in Saccharomyces cerevisiae: Functional Roles of Rox1 and Other Factors in Mediating the Anoxic Response

ABSTRACT DNA arrays were used to investigate the functional role of Rox1 in mediating acclimatization to anaerobic conditions in Saccharomyces cerevisiae. Multiple growth conditions for wild-type and rox1 null strains were used to identify open reading frames with a statistically robust response to this repressor. These results were compared to those obtained for a wild-type strain in response to oxygen availability. Transcripts of nearly one-sixth of the genome were differentially expressed (P < 0.05) with respect to oxygen availability, the majority (>65%) being down-regulated under anoxia. Of the anaerobically induced genes, about one-third (106) contain putative Rox1-binding sites in their promoters and were significantly (P < 0.05) up-regulated in the rox1 null strains under aerobiosis. Additional promoter searches revealed that nearly one-third of the anaerobically induced genes contain an AR1 site(s) for the Upc2 transcription factor, suggesting that Upc2 and Rox1 regulate the majority of anaerobically induced genes in S. cerevisiae. Functional analyses indicate that a large fraction of the anaerobically induced genes are involved in cell stress (∼1/3), cell wall maintenance (∼1/8), carbohydrate metabolism (∼1/10), and lipid metabolism (∼1/12), with both Rox1 and Upc2 predominating in the regulation of this latter group and Upc2 predominating in cell wall maintenance. Mapping the changes in expression of functional regulons onto metabolic pathways has provided novel insight into the role of Rox1 and other trans-acting factors in mediating the physiological response of S. cerevisiae to anaerobic conditions.

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Role of Saccharomyces cerevisiae Oxidoreductases Bdh1p and Ara1p in the Metabolism of Acetoin and 2,3-Butanediol

Applied and Environmental Microbiology ◽

10.1128/aem.01521-09 ◽

2009 ◽

Vol 76 (3) ◽

pp. 670-679 ◽

Cited By ~ 26

Author(s):

Eva González ◽

M. Rosario Fernández ◽

Didac Marco ◽

Eduard Calam ◽

Lauro Sumoy ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Type Strain ◽

Wild Type Strain ◽

Growth Conditions ◽

Wild Type ◽

Genome Expression ◽

Whole Genome Expression ◽

Histidine Tag ◽

Butanediol Dehydrogenase

ABSTRACT NAD-dependent butanediol dehydrogenase (Bdh1p) from Saccharomyces cerevisiae reversibly transforms acetoin to 2,3-butanediol in a stereospecific manner. Deletion of BDH1 resulted in an accumulation of acetoin and a diminution of 2,3-butanediol in two S. cerevisiae strains under two different growth conditions. The concentrations of (2R,3R)-2,3-butanediol are mostly dependent on Bdh1p activity, while those of (meso)-2,3-butanediol are also influenced by the activity of NADP(H)-dependent oxidoreductases. One of them has been purified and shown to be d-arabinose dehydrogenase (Ara1p), which converts (R/S)-acetoin to meso-2,3-butanediol and (2S,3S)-2,3-butanediol. Deletion of BDH2, a gene adjacent to BDH1, whose encoded protein is 51% identical to Bdh1p, does not significantly alter the levels of acetoin or 2,3-butanediol in comparison to the wild-type strain. Furthermore, we have expressed Bdh2p with a histidine tag and have shown it to be inactive toward 2,3-butanediol. A whole-genome expression analysis with microarrays demonstrates that BDH1 and BDH2 are reciprocally regulated.

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A Crucial Role of Mitochondrial Dynamics in Dehydration Resistance in Saccharomyces cerevisiae

International Journal of Molecular Sciences ◽

10.3390/ijms22094607 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4607

Author(s):

Chang-Lin Chen ◽

Ying-Chieh Chen ◽

Wei-Ling Huang ◽

Steven Lin ◽

Rimantas Daugelavičius ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Survival Rate ◽

Mitochondrial Dynamics ◽

Growth Conditions ◽

Stationary Growth Phase ◽

Yeast Cells ◽

Mitochondrial Activity ◽

Wild Type ◽

Stationary Growth

Mitochondria are dynamic organelles as they continuously undergo fission and fusion. These dynamic processes conduct not only mitochondrial network morphology but also activity regulation and quality control. Saccharomyces cerevisiae has a remarkable capacity to resist stress from dehydration/rehydration. Although mitochondria are noted for their role in desiccation tolerance, the mechanisms underlying these processes remains obscure. Here, we report that yeast cells that went through stationary growth phase have a better survival rate after dehydration/rehydration. Dynamic defective yeast cells with reduced mitochondrial genome cannot maintain the mitochondrial activity and survival rate of wild type cells. Our results demonstrate that yeast cells balance mitochondrial fusion and fission according to growth conditions, and the ability to adjust dynamic behavior aids the dehydration resistance by preserving mitochondria.

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Mapping the genetic landscape of biomineralization in Magnetospirillum magneticum AMB-1 with RB-Tnseq

10.1101/2021.08.13.456315 ◽

2021 ◽

Author(s):

Hayley C McCausland ◽

Kelly M Wetmore ◽

Adam P. Arkin ◽

Arash Komeili

Keyword(s):

Essential Gene ◽

Growth Conditions ◽

Wild Type ◽

Anaerobic Conditions ◽

High Iron ◽

Genetic Landscape ◽

Transposon Insertions ◽

Magnetospirillum Magneticum ◽

Markerless Deletion

Magnetotactic bacteria (MTB) are a phylogenetically diverse group of bacteria remarkable for their ability to biomineralize magnetite (Fe3O4) or greigite (Fe3S4) in organelles called magnetosomes. The majority of genes required for magnetosome formation are encoded by a magnetosome gene island (MAI). Here, we conducted random barcoded transposon mutagenesis (RB-TnSeq) in Magnetospirillum magneticum AMB-1 to identify the global genetic requirements for magnetosome formation under different growth conditions. We generated a library of 184,710 unique strains in a wild-type background, generating ~34 mutant strains for each gene. RB-TnSeq also allowed us to determine the essential gene set of AMB-1 under standard laboratory growth conditions. To pinpoint novel genes that are important for magnetosome formation, we subjected the library to magnetic selection screens in varied growth conditions. We compared biomineralization in standard growth conditions to biomineralization in high iron and anaerobic conditions, respectively. Strains with transposon insertions in the MAI gene mamT had an exacerbated biomineralization defect under both high iron and anerobic conditions compared to standard conditions, adding to our knowledge of the role of MamT in magnetosome formation. Mutants in amb4151, a gene outside of the MAI, are more magnetic than wild-type cells under anaerobic conditions. All three of these phenotypes were validated by creating a markerless deletion strain of the gene and evaluating with TEM imaging. Overall, our results indicate that growth conditions affect which genes are required for biomineralization and that some MAI genes may have more nuanced functions than was previously understood.

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Loss of Ubp3 increases silencing, decreases unequal recombination in rDNA, and shortens the replicative life span in Saccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.e13-10-0591 ◽

2014 ◽

Vol 25 (12) ◽

pp. 1916-1924 ◽

Cited By ~ 3

Author(s):

David Öling ◽

Rehan Masoom ◽

Kristian Kvint

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Life Span ◽

Rna Polymerase Ii ◽

Ribosomal Dna ◽

Genetic Evidence ◽

Wild Type ◽

Replicative Life Span ◽

Unequal Recombination

Ubp3 is a conserved ubiquitin protease that acts as an antisilencing factor in MAT and telomeric regions. Here we show that ubp3∆ mutants also display increased silencing in ribosomal DNA (rDNA). Consistent with this, RNA polymerase II occupancy is lower in cells lacking Ubp3 than in wild-type cells in all heterochromatic regions. Moreover, in a ubp3∆ mutant, unequal recombination in rDNA is highly suppressed. We present genetic evidence that this effect on rDNA recombination, but not silencing, is entirely dependent on the silencing factor Sir2. Further, ubp3∆ sir2∆ mutants age prematurely at the same rate as sir2∆ mutants. Thus our data suggest that recombination negatively influences replicative life span more so than silencing. However, in ubp3∆ mutants, recombination is not a prerequisite for aging, since cells lacking Ubp3 have a shorter life span than isogenic wild-type cells. We discuss the data in view of different models on how silencing and unequal recombination affect replicative life span and the role of Ubp3 in these processes.

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Significance of C-terminal cysteine modifications to the biological activity of the Saccharomyces cerevisiae a-factor mating pheromone

Molecular and Cellular Biology ◽

10.1128/mcb.11.7.3603-3612.1991 ◽

1991 ◽

Vol 11 (7) ◽

pp. 3603-3612

Author(s):

S Marcus ◽

G A Caldwell ◽

D Miller ◽

C B Xue ◽

F Naider ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Biological Activity ◽

Methyl Ester ◽

Total Synthesis ◽

Biologically Active ◽

Structural Genes ◽

Wild Type ◽

Mating Pheromone ◽

Carboxy Terminal

We have undertaken total synthesis of the Saccharomyces cerevisiae a-factor (NH2-YIIKGVFWDPAC[S-farnesyl]-COOCH3) and several Cys-12 analogs to determine the significance of S-farnesylation and carboxy-terminal methyl esterification to the biological activity of this lipopeptide mating pheromone. Replacement of either the farnesyl group or the carboxy-terminal methyl ester by a hydrogen atom resulted in marked reduction but not total loss of bioactivity as measured by a variety of assays. Moreover, both the farnesyl and methyl ester groups could be replaced by other substituents to produce biologically active analogs. The bioactivity of a-factor decreased as the number of prenyl units on the cysteine sulfur decreased from three to one, and an a-factor analog having the S-farnesyl group replaced by an S-hexadecanyl group was more active than an S-methyl a-factor analog. Thus, with two types of modifications, a-factor activity increased as the S-alkyl group became bulkier and more hydrophobic. MATa cells having deletions of the a-factor structural genes (mfal1 mfa2 mutants) were capable of mating with either sst2 or wild-type MAT alpha cells in the presence of exogenous a-factor, indicating that it is not absolutely essential for MATa cells to actively produce a-factor in order to mate. Various a-factor analogs were found to partially restore mating to these strains as well, and their relative activities in the mating restoration assay were similar to their activities in the other assays used in this study. Mating was not restored by addition of exogenous a-factor to a cross of a wild-type MAT alpha strain and a MATaste6 mutant, indicating a role of the STE6 gene product in mating in addition to its secretion of a-factor.

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Significance of C-terminal cysteine modifications to the biological activity of the Saccharomyces cerevisiae a-factor mating pheromone.

Molecular and Cellular Biology ◽

10.1128/mcb.11.7.3603 ◽

1991 ◽

Vol 11 (7) ◽

pp. 3603-3612 ◽

Cited By ~ 74

Author(s):

S Marcus ◽

G A Caldwell ◽

D Miller ◽

C B Xue ◽

F Naider ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Biological Activity ◽

Methyl Ester ◽

Total Synthesis ◽

Biologically Active ◽

Structural Genes ◽

Wild Type ◽

Mating Pheromone ◽

Carboxy Terminal

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Hypolignification: A Decisive Factor in the Development of Hyperhydricity

Plants ◽

10.3390/plants10122625 ◽

2021 ◽

Vol 10 (12) ◽

pp. 2625

Author(s):

Nurashikin Kemat ◽

Richard G. F. Visser ◽

Frans A. Krens

Keyword(s):

Cell Wall ◽

Lignin Content ◽

Coumaric Acid ◽

Wild Type ◽

Higher Plant ◽

Gene Coding ◽

Phenylpropanoid Biosynthesis ◽

Total Lignin ◽

Total Lignin Content

One of the characteristics of hyperhydric plants is the reduction of cell wall lignification (hypolignification), but how this is related to the observed abnormalities of hyperhydricity (HH), is still unclear. Lignin is hydrophobic, and we speculate that a reduction in lignin levels leads to more capillary action of the cell wall and consequently to more water in the apoplast. p-coumaric acid is the hydroxyl derivative of cinnamic acid and a precursor for lignin and flavonoids in higher plant. In the present study, we examined the role of lignin in the development of HH in Arabidopsis thaliana by checking the wild-types (Ler and Col-0) and mutants affected in phenylpropanoid biosynthesis, in the gene coding for cinnamate 4-hydroxylase, C4H (ref3-1 and ref3-3). Exogenously applied p-coumaric acid decreased the symptoms of HH in both wild-type and less-lignin mutants. Moreover, the results revealed that exogenously applied p-coumaric acid inhibited root growth and increased the total lignin content in both wild-type and less-lignin mutants. These effects appeared to diminish the symptoms of HH and suggest an important role for lignin in HH.

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Protein Glycosylation inAspergillus fumigatusIs Essential for Cell Wall Synthesis and Serves as a Promising Model of Multicellular Eukaryotic Development

International Journal of Microbiology ◽

10.1155/2012/654251 ◽

2012 ◽

Vol 2012 ◽

pp. 1-21 ◽

Cited By ~ 14

Author(s):

Cheng Jin

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Posttranslational Modification ◽

Mammalian Cells ◽

Protein Glycosylation ◽

Cell Wall Synthesis ◽

Fungal Cell ◽

Multiple Functions ◽

Significant Attention

Glycosylation is a conserved posttranslational modification that is found in all eukaryotes, which helps generate proteins with multiple functions. Our knowledge of glycosylation mainly comes from the investigation of the yeastSaccharomyces cerevisiaeand mammalian cells. However, during the last decade, glycosylation in the human pathogenic moldAspergillus fumigatushas drawn significant attention. It has been revealed that glycosylation inA. fumigatusis crucial for its growth, cell wall synthesis, and development and that the process is more complicated than that found in the budding yeastS. cerevisiae. The present paper implies that the investigation of glycosylation inA. fumigatusis not only vital for elucidating the mechanism of fungal cell wall synthesis, which will benefit the design of new antifungal therapies, but also helps to understand the role of protein glycosylation in the development of multicellular eukaryotes. This paper describes the advances in functional analysis of protein glycosylation inA. fumigatus.

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Role of CpALS4790 and CpALS0660 in Candida parapsilosis Virulence: Evidence from a Murine Model of Vaginal Candidiasis

Journal of Fungi ◽

10.3390/jof6020086 ◽

2020 ◽

Vol 6 (2) ◽

pp. 86

Author(s):

Marina Zoppo ◽

Fabrizio Fiorentini ◽

Cosmeri Rizzato ◽

Mariagrazia Di Luca ◽

Antonella Lupetti ◽

...

Keyword(s):

Cell Wall ◽

Murine Model ◽

Type Strain ◽

Wild Type Strain ◽

Candida Parapsilosis ◽

Vaginal Candidiasis ◽

Phenotypic Characterization ◽

Wild Type ◽

Mutant Strains

The Candida parapsilosis genome encodes for five agglutinin-like sequence (Als) cell-wall glycoproteins involved in adhesion to biotic and abiotic surfaces. The work presented here is aimed at analyzing the role of the two still uncharacterized ALS genes in C. parapsilosis, CpALS4790 and CpALS0660, by the generation and characterization of CpALS4790 and CpALS066 single mutant strains. Phenotypic characterization showed that both mutant strains behaved as the parental wild type strain regarding growth rate in liquid/solid media supplemented with cell-wall perturbing agents, and in the ability to produce pseudohyphae. Interestingly, the ability of the CpALS0660 null mutant to adhere to human buccal epithelial cells (HBECs) was not altered when compared with the wild-type strain, whereas deletion of CpALS4790 led to a significant loss of the adhesion capability. RT-qPCR analysis performed on the mutant strains in co-incubation with HBECs did not highlight significant changes in the expression levels of others ALS genes. In vivo experiments in a murine model of vaginal candidiasis indicated a significant reduction in CFUs recovered from BALB/C mice infected with each mutant strain in comparison to those infected with the wild type strain, confirming the involvement of CpAls4790 and CpAls5600 proteins in C. parapsilosis vaginal candidiasis in mice.

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Expression of the Schwanniomyces occidentalis SWA2 amylase in Saccharomyces cerevisiae: role of N-glycosylation on activity, stability and secretion

Biochemical Journal ◽

10.1042/bj3290065 ◽

1998 ◽

Vol 329 (1) ◽

pp. 65-71 ◽

Cited By ~ 36

Author(s):

Esther YÁÑEZ ◽

A. Teresa CARMONA ◽

Mercedes TIEMBLO ◽

Antonio JIMÉNEZ ◽

María FERNÁNDEZ-LOBATO

Keyword(s):

Saccharomyces Cerevisiae ◽

Catalytic Efficiency ◽

Extracellular Enzyme ◽

Site Directed Mutagenesis ◽

Activity Levels ◽

Wild Type ◽

Schwanniomyces Occidentalis ◽

Type Protein ◽

Wild Type Protein

The role of N-linked glycosylation on the biological activity of Schwanniomyces occidentalis SWA2 α-amylase, as expressed in Saccharomyces cerevisiae, was analysed by site-directed mutagenesis of the two potential N-glycosylation sites, Asn-134 and Asn-229. These residues were replaced by Ala or Gly individually or in various combinations and the effects on the activity, secretion and thermal stability of the enzyme were studied. Any Asn-229 substitution caused a drastic decrease in activity levels of the extracellular enzyme. In contrast, substitutions of Asn-134 had little or no effect. The use of antibodies showed that α-amylase was secreted in all the mutants tested, although those containing substitutions at Asn-229 seemed to have a lower rate of synthesis and/or higher degradation than the wild-type strain. α-Amylases with substitution at Asn-229 had a 2 kDa lower molecular mass than the wild-type protein, as did the wild-type protein itself after treatment with endoglycosidase F. These findings indicate that Asn-229 is the single glycosylated residue in SWA2. Thermostability analysis of both purified wild-type (T50 = 50 °C, where T50 is the temperature resulting in 50% loss of activity) and mutant enzymes indicated that removal of carbohydrate from the 229 position results in a decrease of approx. 3 °C in the T50 of the enzyme. The Gly-229 mutation does not change the apparent affinity of the enzyme for starch (Km) but decreases to 1/22 its apparent catalytic efficiency (kcat/Km). These results therefore indicate that glycosylation at the 229 position has an important role in the extracellular activity levels, kinetics and stability of the Sw. occidentalis SWA2 α-amylase in both its wild-type and mutant forms.

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