The Function and Properties of the Azf1 Transcriptional Regulator Change with Growth Conditions in Saccharomyces cerevisiae

ABSTRACT Azf1 activates CLN3 transcription in Saccharomyces cerevisiae cells growing in glucose. Paradoxically, other studies have shown Azf1 to be almost undetectable in glucose-grown cells. Microarray experiments showed that Azf1 activates nonoverlapping gene sets in different carbon sources: in glucose, Azf1 activates carbon and energy metabolism genes, and in glycerol-lactate, Azf1 activates genes needed for cell wall maintenance. Consistent with the decreased expression of cell wall maintenance genes observed with azf1Δ mutants, we observed a marked growth defect in the azf1Δ cells at 37°C in nonfermentable medium. Cell wall integrity assays, such as sensitivity to calcofluor white, sodium dodecyl sulfate, or caffeine, confirmed cell wall defects in azf1Δ mutants in nonfermentable medium. Gel shift experiments show that Azf1 binds to DNA elements with the sequence AAAAGAAA (A4GA3), a motif enriched in the promoters of Azf1-sensitive genes and predicted by whole-genome studies. This suggests that many of the Azf1-dependent transcripts may be regulated directly by Azf1 binding. We found that the levels of Azf1 protein in glucose-grown cells were comparable to Azf1 levels in cells grown in glycerol-lactate; however, this could only be demonstrated with a cell extraction procedure that minimizes proteolysis. Glucose produces conditions that destabilize the Azf1 protein, a finding that may reflect a glucose-induced change in Azf1 tertiary or quaternary structure.

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Deletion of YLR358C Contributes to Reduce Cell Wall Integrity in Saccharomyces Cerevisiae

10.21203/rs.3.rs-1235133/v1 ◽

2022 ◽

Author(s):

Yu Zhang ◽

Mengyan Li ◽

Hanying Wang ◽

Juqing Deng ◽

Jianxing Liu ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Sodium Dodecyl ◽

Wall Stress ◽

Pathogenic Fungi ◽

Cell Wall Integrity ◽

Calcofluor White ◽

Fungal Cell ◽

Reading Frame ◽

Stress Signals

Abstract The mechanism of fungal cell wall synthesis and assembly is still unclear. Saccharomyces cerevisiae (S. cerevisiae) and pathogenic fungi are conserved in cell wall construction and response to stress signals, and often respond to cell wall stress through activated cell wall integrity (CWI) pathways. Whether the YLR358C open reading frame regulates CWI remains unclear. This study found that the growth of S. cerevisiae with YLR358C knockout was significantly inhibited on the medium containing different concentrations of cell wall interfering agents Calcofluor White (CFW), Congo Red (CR) and sodium dodecyl sulfate (SDS). CFW staining showed that the cell wall chitin was down-regulated, and transmission electron microscopy also observed a decrease in cell wall thickness. Transcriptome sequencing and analysis showed that YLR358C gene may be involved in the regulation of CWI signaling pathway. It was found by qRT-PCR that WSC3, SWI4 and HSP12 were differentially expressed after YLR358C was knocked out. The above results suggest that YLR358C may regulate the integrity of the yeast cell walls and has some potential for application in fermentation.

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Deletion of New Covalently Linked Cell Wall Glycoproteins Alters the Electrophoretic Mobility of Phosphorylated Wall Components of Saccharomyces cerevisiae

Journal of Bacteriology ◽

10.1128/jb.181.10.3076-3086.1999 ◽

1999 ◽

Vol 181 (10) ◽

pp. 3076-3086 ◽

Cited By ~ 57

Author(s):

Vladimir Mrsa ◽

Margit Ecker ◽

Sabine Strahl-Bolsinger ◽

Manfred Nimtz ◽

Ludwig Lehle ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Cell Walls ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Radioactive Material ◽

Cell Wall Proteins ◽

Triple Mutant ◽

Calcofluor White ◽

Covalently Linked

ABSTRACT The incorporation of radioactive orthophosphate into the cell walls of Saccharomyces cerevisiae was studied.33P-labeled cell walls were extensively extracted with hot sodium dodecyl sulfate (SDS). Of the remaining insoluble radioactivity more than 90% could be released by laminarinase. This radioactive material stayed in the stacking gel during SDS-polyacrylamide gel electrophoresis but entered the separating gel upon treatment with N -glycosidase F, indicating that phosphate was linked directly or indirectly to N-mannosylated glycoproteins. The phosphate was bound to covalently linked cell wall proteins as mannose-6-phosphate, the same type of linkage shown previously for soluble mannoproteins (L. Ballou, L. M. Hernandez, E. Alvarado, and C. E. Ballou, Proc. Natl. Acad. Sci. USA 87:3368–3372, 1990). From the phosphate-labeled glycoprotein fraction released by laminarinase, three cell wall mannoproteins, Ccw12p, Ccw13p and Ccw14p, were isolated and identified by N-terminal sequencing. For Ccw13p (encoded by DAN1 [also called TIR3]) and Ccw12p the association with the cell wall has not been described before; Ccw14p is identical with cell wall protein Icwp (I. Moukadiri, J. Armero, A. Abad, R. Sentandreu, and J. Zueco, J. Bacteriol. 179:2154–2162, 1997). In ccw12, ccw13, orccw14 single or double mutants neither the amount of radioactive phosphate incorporated into cell wall proteins nor its position in the stacking gel was changed. However, the triple mutant brought about a shift of the 33P-labeled glycoprotein components from the stacking gel into the separating gel. The disruption of CCW12 results in a pronounced sensitivity of the cells to calcofluor white and Congo red. In addition, theccw12 mutant shows a decrease in mating efficiency and a defect in agglutination.

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The Putative α-1,2-Mannosyltransferase AfMnt1 of the Opportunistic Fungal Pathogen Aspergillus fumigatus Is Required for Cell Wall Stability and Full Virulence

Eukaryotic Cell ◽

10.1128/ec.00221-08 ◽

2008 ◽

Vol 7 (10) ◽

pp. 1661-1673 ◽

Cited By ~ 57

Author(s):

Johannes Wagener ◽

Bernd Echtenacher ◽

Manfred Rohde ◽

Andrea Kotz ◽

Sven Krappmann ◽

...

Keyword(s):

Cell Wall ◽

Aspergillus Fumigatus ◽

Secretory Pathway ◽

Sodium Dodecyl ◽

Brefeldin A ◽

Antifungal Drugs ◽

Growth Defect ◽

Calcofluor White ◽

Necrosis Factor Alpha ◽

Opportunistic Fungal Pathogen

ABSTRACT Proteins entering the eukaryotic secretory pathway commonly are glycosylated. Important steps in this posttranslational modification are carried out by mannosyltransferases. In this study, we investigated the putative α-1,2-mannosyltransferase AfMnt1 of the human pathogenic mold Aspergillus fumigatus. AfMnt1 belongs to a family of enzymes that comprises nine members in Saccharomyces cerevisiae but only three in A. fumigatus. A Δafmnt1 mutant is viable and grows normally at 37°C, but its hyphal cell wall appears to be thinner than that of the parental strain. The lack of AfMnt1 leads to a higher sensitivity to calcofluor white and Congo red but not to sodium dodecyl sulfate. The growth of the mutant is abrogated at 48°C but can be restored by osmotic stabilization. The resulting colonies remain white due to a defect in the formation of conidia. Electron and immunofluorescence microscopy further revealed that the observed growth defect of the mutant at 48°C can be attributed to cell wall instability resulting in leakage at the hyphal tips. Using a red fluorescence fusion protein, we localized AfMnt1 in compact, brefeldin A-sensitive organelles that most likely represent fungal Golgi equivalents. The tumor necrosis factor alpha response of murine macrophages to hyphae was not affected by the lack of the afmnt1 gene, but the corresponding mutant was attenuated in a mouse model of infection. This and the increased sensitivity of the Δafmnt1 mutant to azoles, antifungal agents that currently are used to treat Aspergillus infections, suggest that α-1,2-mannosyltransferases are interesting targets for novel antifungal drugs.

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EslB is required for cell wall biosynthesis and modification in Listeria monocytogenes

Journal of Bacteriology ◽

10.1128/jb.00553-20 ◽

2020 ◽

Author(s):

Jeanine Rismondo ◽

Lisa M. Schulz ◽

Maria Yacoub ◽

Ashima Wadhawan ◽

Michael Hoppert ◽

...

Keyword(s):

Cell Wall ◽

Cell Division ◽

Listeria Monocytogenes ◽

Abc Transporter ◽

Cell Lysis ◽

Growth Conditions ◽

Cell Wall Integrity ◽

Growth Defect ◽

Cell Wall Biosynthesis ◽

High Concentrations

Lysozyme is an important component of the innate immune system. It functions by hydrolysing the peptidoglycan (PG) layer of bacteria. The human pathogen Listeria monocytogenes is intrinsically lysozyme resistant. The peptidoglycan N-deacetylase PgdA and O-acetyltransferase OatA are two known factors contributing to its lysozyme resistance. Furthermore, it was shown that the absence of components of an ABC transporter, here referred to as EslABC, leads to reduced lysozyme resistance. How its activity is linked to lysozyme resistance is still unknown. To investigate this further, a strain with a deletion in eslB, coding for a membrane component of the ABC transporter, was constructed in L. monocytogenes strain 10403S. The eslB mutant showed a 40-fold reduction in the minimal inhibitory concentration to lysozyme. Analysis of the PG structure revealed that the eslB mutant produced PG with reduced levels of O-acetylation. Using growth and autolysis assays, we show that the absence of EslB manifests in a growth defect in media containing high concentrations of sugars and increased endogenous cell lysis. A thinner PG layer produced by the eslB mutant under these growth conditions might explain these phenotypes. Furthermore, the eslB mutant had a noticeable cell division defect and formed elongated cells. Microscopy analysis revealed that an early cell division protein still localized in the eslB mutant indicating that a downstream process is perturbed. Based on our results, we hypothesize that EslB affects the biosynthesis and modification of the cell wall in L. monocytogenes and is thus important for the maintenance of cell wall integrity. IMPORTANCE The ABC transporter EslABC is associated with the intrinsic lysozyme resistance of Listeria monocytogenes. However, the exact role of the transporter in this process and in the physiology of L. monocytogenes is unknown. Using different assays to characterize an eslB deletion strain, we found that the absence of EslB not only affects lysozyme resistance, but also endogenous cell lysis, cell wall biosynthesis, cell division and the ability of the bacterium to grow in media containing high concentrations of sugars. Our results indicate that EslB is by a yet unknown mechanism an important determinant for cell wall integrity in L. monocytogenes.

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Saccharomyces cerevisiae Mid2p Is a Potential Cell Wall Stress Sensor and Upstream Activator of thePKC1-MPK1 Cell Integrity Pathway

Journal of Bacteriology ◽

10.1128/jb.181.11.3330-3340.1999 ◽

1999 ◽

Vol 181 (11) ◽

pp. 3330-3340 ◽

Cited By ~ 194

Author(s):

Troy Ketela ◽

Robin Green ◽

Howard Bussey

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Wall Stress ◽

Structural Features ◽

Mitogen Activated Protein Kinase ◽

Cell Integrity ◽

Chitin Synthesis ◽

Calcofluor White ◽

Reading Frame ◽

Cell Wall Stress

ABSTRACT The MID2 gene of Saccharomyces cerevisiaeencodes a protein with structural features indicative of a plasma membrane-associated cell wall sensor. MID2 was isolated as a multicopy activator of the Skn7p transcription factor. Deletion ofMID2 causes resistance to calcofluor white, diminished production of stress-induced cell wall chitin under a variety of conditions, and changes in growth rate and viability in a number of different cell wall biosynthesis mutants. Overexpression ofMID2 causes hyperaccumulation of chitin and increased sensitivity to calcofluor white. α-Factor hypersensitivity ofmid2Δ mutants can be suppressed by overexpression of upstream elements of the cell integrity pathway, includingPKC1, RHO1, WSC1, andWSC2. Mid2p and Wsc1p appear to have overlapping roles in maintaining cell integrity since mid2Δ wsc1Δ mutants are inviable on medium that does not contain osmotic support. A role for MID2 in the cell integrity pathway is further supported by the finding that MID2 is required for induction of Mpk1p tyrosine phosphorylation during exposure to α-factor, calcofluor white, or high temperature. Our data are consistent with a role for Mid2p in sensing cell wall stress and in activation of a response that includes both increased chitin synthesis and the Mpk1p mitogen-activated protein kinase cell integrity pathway. In addition, we have identified an open reading frame, MTL1, which encodes a protein with both structural and functional similarity to Mid2p.

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Saccharomyces cerevisiae HOC1, a Suppressor of pkc1, Encodes a Putative Glycosyltransferase

Genetics ◽

10.1093/genetics/145.3.637 ◽

1997 ◽

Vol 145 (3) ◽

pp. 637-645 ◽

Cited By ~ 12

Author(s):

Aaron M Neiman ◽

Vijay Mhaiskar ◽

Vladimir Manus ◽

Francis Galibert ◽

Neta Dean

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Cell Lysis ◽

Integral Membrane Protein ◽

Protein Glycosylation ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Calcofluor White ◽

Kinase C

The Saccharomyces cerevisiae gene PKC1 encodes a protein kinase C isozyme that regulates cell wall synthesis. Here we describe the characterization of HOC1, a gene identified by its ability to suppress the cell lysis phenotype of pkc1-371 cells. The HOC1 gene (Homologous to OCH1) is predicted to encode a type II integral membrane protein that strongly resembles Och1p, an α-1,6-mannosyltransferase. Immunofluorescence studies localized Hoc1p to the Golgi apparatus. While overexpression of HOC1 rescued the pkc1-371 temperature-sensitive cell lysis phenotype, disruption of HOC1 lowered the restrictive temperature of the pkc1-371 allele. Disruption of HOC1 also resulted in hypersensitivity to Calcofluor White and hygromycin B, phenotypes characteristic of defects in cell wall integrity and protein glycosylation, respectively. The function of HOC1 appears to be distinct from that of OCH1. Taken together, these results suggest that HOC1 encodes a Golgi-localized putative mannosyltransferase required for the proper construction of the cell wall.

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Physiological and genetic analysis of the carbon regulation of the NAD-dependent glutamate dehydrogenase of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4455-4465.1991 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4455-4465

Author(s):

P W Coschigano ◽

S M Miller ◽

B Magasanik

Keyword(s):

Saccharomyces Cerevisiae ◽

Nitrogen Source ◽

Glutamate Dehydrogenase ◽

Carbon Sources ◽

Growth Conditions ◽

Neighboring Element ◽

Carbon Regulation ◽

Activation Of Transcription ◽

Regulated Expression ◽

Nonfermentable Carbon Source

We found that cells of Saccharomyces cerevisiae have an elevated level of the NAD-dependent glutamate dehydrogenase (NAD-GDH; encoded by the GDH2 gene) when grown with a nonfermentable carbon source or with limiting amounts of glucose, even in the presence of the repressing nitrogen source glutamine. This regulation was found to be transcriptional, and an upstream activation site (GDH2 UASc) sufficient for activation of transcription during respiratory growth conditions was identified. This UAS was found to be separable from a neighboring element which is necessary for the nitrogen source regulation of the gene, and strains deficient for the GLN3 gene product, required for expression of NAD-GDH during growth with the activating nitrogen source glutamate, were unaffected for the expression of NAD-GDH during growth with activating carbon sources. Two classes of mutations which prevented the normal activation of NAD-GDH in response to growth with nonfermentable carbon sources, but which did not affect the nitrogen-regulated expression of NAD-GDH, were found and characterized. Carbon regulation of GDH2 was found to be normal in hxk2, hap3, and hap4 strains and to be only slightly altered in a ssn6 strain; thus, in comparison with the regulation of previously identified glucose-repressed genes, a new pathway appears to be involved in the regulation of GDH2.

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ABF1 is a phosphoprotein and plays a role in carbon source control of COX6 transcription in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.4197-4208.1992 ◽

1992 ◽

Vol 12 (9) ◽

pp. 4197-4208

Author(s):

S Silve ◽

P R Rhode ◽

B Coll ◽

J Campbell ◽

R O Poyton

Keyword(s):

Saccharomyces Cerevisiae ◽

Carbon Source ◽

Glucose Repression ◽

Carbon Sources ◽

Growth Conditions ◽

Source Control ◽

Specific Target Gene ◽

Phosphorylation Pattern ◽

Nonfermentable Carbon Source ◽

In Cells

Previously, we have shown that the Saccharomyces cerevisiae DNA-binding protein ABF1 exists in at least two different electrophoretic forms (K. S. Sweder, P. R. Rhode, and J. L. Campbell, J. Biol. Chem. 263: 17270-17277, 1988). In this report, we show that these forms represent different states of phosphorylation of ABF1 and that at least four different phosphorylation states can be resolved electrophoretically. The ratios of these states to one another differ according to growth conditions and carbon source. Phosphorylation of ABF1 is therefore a regulated process. In nitrogen-starved cells or in cells grown on nonfermentable carbon sources (e.g., lactate), phosphorylated forms predominate, while in cells grown on fermentable carbon sources (e.g., glucose), dephosphorylated forms are enriched. The phosphorylation pattern is affected by mutations in the SNF1-SSN6 pathway, which is involved in glucose repression-depression. Whereas a functional SNF1 gene, which encodes a protein kinase, is not required for the phosphorylation of ABF1, a functional SSN6 gene is required for itsd ephosphorylation. The phosphorylation patterns that we have observed correlate with the regulation of a specific target gene, COX6, which encodes subunit VI of cytochrome c oxidase. Transcription of COX6 is repressed by growth in medium containing a fermentable carbon source and is derepressed by growth in medium containing a nonfermentable carbon source. COX6 repression-derepression is under the control of the SNF1-SSN6 pathway. This carbon source regulation is exerted through domain 1, a region of the upstream activation sequence UAS6 that binds ABF1 (J. D. Trawick, N. Kraut, F. Simon, and R. O. Poyton, Mol. Cell Biol. 12:2302-2314, 1992). We show that the greater the phosphorylation of ABF1, the greater the transcription of COX6. Furthermore, the ABF1-containing protein-DNA complexes formed at domain 1 differ according to the phosphorylation state of ABF1 and the carbon source on which the cells were grown. From these findings, we propose that the phosphorylation of ABF1 is involved in glucose repression-derepression of COX6 transcription.

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SUPPRESSORS OF THE ras2 MUTATION OF SACCHAROMYCES CEREVISIAE

Genetics ◽

10.1093/genetics/113.2.247 ◽

1986 ◽

Vol 113 (2) ◽

pp. 247-264 ◽

Cited By ~ 3

Author(s):

John F Cannon ◽

Jackson B Gibbs ◽

Kelly Tatchell

Keyword(s):

Saccharomyces Cerevisiae ◽

Carbon Sources ◽

Gene Mutations ◽

Yeast Cells ◽

Growth Defect ◽

Ras Gene ◽

Respiratory Capacity ◽

Glycogen Accumulation ◽

Suppressor Mutations ◽

Specific Locus

ABSTRACT Saccharomyces cerevisiae contains two members of the ras gene family. Strains with disruptions of the RAS2 gene fail to grow efficiently on nonfermentable carbon sources. This growth defect can be suppressed by extragenic mutations called sra. We have isolated 79 independent suppressor mutations, 68 of which have been assigned to one of five loci. Eleven additional dominant mutations have not been assigned to a specific locus. Some sra1 and SRA4 and all SRA3 mutations were RAS independent, allowing growth of yeast cells that lack a functional RAS gene. Mutations in sra1, SRA3, SRA4 and sra6 are linked to his6, ino1, met3 and ade6, respectively. Some sra mutants have pleitropic phenotypes that affect glycogen accumulation, sporulation, viability, respiratory capacity and suppression of two cell-division-cycle mutations, cdc25 and cdc35. The proposed functions of many of the suppressor genes are consistent with the model in which RAS activates adenylate cyclase.

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EslB is required for cell wall biosynthesis and modification in Listeria monocytogenes

10.1101/2020.02.03.932061 ◽

2020 ◽

Author(s):

Jeanine Rismondo ◽

Lisa M. Schulz ◽

Maria Yacoub ◽

Ashima Wadhawan ◽

Michael Hoppert ◽

...

Keyword(s):

Cell Wall ◽

Cell Division ◽

Listeria Monocytogenes ◽

Abc Transporter ◽

Cell Lysis ◽

Growth Conditions ◽

Cell Wall Integrity ◽

Growth Defect ◽

Cell Wall Biosynthesis ◽

High Concentrations

ABSTRACTLysozyme is an important component of the innate immune system. It functions by hydrolysing the peptidoglycan (PG) layer of bacteria. The human pathogen Listeria monocytogenes is intrinsically lysozyme resistant. The peptidoglycan N-deacetylase PgdA and O-acetyltransferase OatA are two known factors contributing to its lysozyme resistance. Furthermore, it was shown that the absence of components of an ABC transporter, here referred to as EslABC, leads to reduced lysozyme resistance. How its activity is linked to lysozyme resistance is still unknown. To investigate this further, a strain with a deletion in eslB, coding for a membrane component of the ABC transporter, was constructed in L. monocytogenes strain 10403S. The eslB mutant showed a 40-fold reduction in the minimal inhibitory concentration to lysozyme. Analysis of the PG structure revealed that the eslB mutant produced PG with reduced levels of O-acetylation. Using growth and autolysis assays, we show that the absence of EslB manifests in a growth defect in media containing high concentrations of sugars and increased endogenous cell lysis. A thinner PG layer produced by the eslB mutant under these growth conditions might explain these phenotypes. Furthermore, the eslB mutant had a noticeable cell division defect and formed elongated cells. Microscopy analysis revealed that an early cell division protein still localized in the eslB mutant indicating that a downstream process is perturbed. Based on our results, we hypothesize that EslB affects the biosynthesis and modification of the cell wall in L. monocytogenes and is thus important for the maintenance of cell wall integrity.IMPORTANCEThe ABC transporter EslABC is associated with the intrinsic lysozyme resistance of Listeria monocytogenes. However, the exact role of the transporter in this process and in the physiology of L. monocytogenes is unknown. Using different assays to characterize an eslB deletion strain, we found that the absence of EslB not only affects lysozyme resistance, but also endogenous cell lysis, cell wall biosynthesis, cell division and the ability of the bacterium to grow in media containing high concentrations of sugars. Our results indicate that EslB is by a yet unknown mechanism an important determinant for cell wall integrity in L. monocytogenes.

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