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

1999 ◽  
Vol 181 (10) ◽  
pp. 3076-3086 ◽  
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.

Identification and study of a Candida albicans protein homologous to Saccharomyces cerevisiae Ssr1p, an internal cell-wall protein

Microbiology ◽  
2003 ◽  
Vol 149 (8) ◽  
pp. 2137-2145 ◽  
Author(s):  
Ana Garcerá ◽  
Ana Isabel Martínez ◽  
Luis Castillo ◽  
M. Victoria Elorza ◽  
Rafael Sentandreu ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Candida Albicans ◽  
Cell Walls ◽  
Cell Wall Protein ◽  
Calcofluor White ◽  
Genome Database ◽  
Internal Cell ◽  
Increased Sensitivity ◽  
Covalently Linked

After screening of a Candida albicans genome database, the product of an ORF (IPF 3054) that has 62 % homology with Saccharomyces cerevisiae Ssr1p, an internal cell-wall protein, was identified and named CaSsr1p. The deduced amino acid sequence shows that CaSsr1p contains an N-terminal hydrophobic signal peptide, is rich in Ser and Thr amino acids and has a potential glycosylphosphatidylinositol-attachment signal. CaSsr1p is released following degradation of isolated cell walls by zymolyase (mainly a 1,3-β-glucanase) and therefore seems to be covalently linked to the β-glucan of the cell walls. Both disruption and overexpression of the CaSSR1 gene caused an increased sensitivity to calcofluor white, Congo red and zymolyase digestion. These results suggest that CaSsr1p has a structural role associated with the cell-wall β-glucan.

Cell wall associations of some antigenic proteins of slime-forming, encapsulated Streptococcus cremoris from the fermented milk product "viili"

1986 ◽  
Vol 32 (2) ◽  
pp. 176-178 ◽  
Author(s):  
Raili Forsén ◽  
Teuvo Hentunen ◽  
Kaua Valkonen ◽  
Sirpa Kontusaari
Keyword(s):  
Cell Wall ◽  
Molecular Mass ◽  
Cell Walls ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fermented Milk ◽  
Milk Product ◽  
Streptococcus Cremoris ◽  
Protein Components

Cell walls were isolated from mechanically disrupted cells of the slime-forming, encapsulated Streptococcus cremoris strains T5 and MLS96 by using sucrose gradient centrifugation as the last purification step. This cell wall isolation procedure was developed to obtain cell wall associated protein components. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis revealed several polypeptide bands; the 50 kiloDalton band was major in strain T5 cell walls and the 26 and 30 kiloDalton bands were major in strain MLS96 cell walls. Both strains contained five antigenic polypeptides with molecular radius (Mr) values of 40, 47, 50, 54, and 70 kiloDaltons as analysed by immunoblotting and autoradiography. The polypeptides of strain MLS96 with molecular mass of 40 and 70 kiloDaltons reacted most strongly with homologous anti-whole cell serum. In addition, antigenic polypeptides with molecular mass of 100 and 160 kiloDaltons were also detected in strain T5.

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

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.

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

2006 ◽  
Vol 5 (2) ◽  
pp. 313-320 ◽  
Author(s):  
Matthew G. Slattery ◽  
Dritan Liko ◽  
Warren Heideman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Quaternary Structure ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Carbon Sources ◽  
Extraction Procedure ◽  
Growth Conditions ◽  
Growth Defect ◽  
Calcofluor White

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.

Detection of bacterial cell wall hydrolases after denaturing polyacrylamide gel electrophoresis

1989 ◽  
Vol 35 (8) ◽  
pp. 749-753 ◽  
Author(s):  
Denis Leclerc ◽  
Alain Asselin
Keyword(s):  
Cell Wall ◽  
Bacterial Cell ◽  
Gel Electrophoresis ◽  
Cell Walls ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Cell Wall Hydrolases

Cell walls from various Gram-positive bacteria were incorporated at a concentration of 0.2% (w/v) into polyacrylamide gels as a substrate for detection of cell wall hydrolases. Bacterial extracts from crude cell wall preparations were denatured with sodium dodecyl sulfate and 2-mercaptoethanol and subjected to denaturing polyacrylamide gel electrophoresis in gels containing bacterial cell walls. After renaturation in the presence of purified and buffered 1% (v/v) Triton X-100, cell wall hydrolases were visualized as clear lytic zones against the opaque cell wall background. One to fifteen bands with lytic activity could be detected, depending on bacterial extracts and on the nature of the cell walls incorporated into gels. Crude cell wall extracts were the best source of cell wall hydrolases from various Gram-positive bacteria such as Clostridium perfringens (15 bands), Micrococcus luteus (1 band), Bacillus megaterium (4 bands), Bacillus sp. (6 bands), B. cereus (3 bands), B. subtilis (7 bands), Staphylococcus aureus (13 bands), Streptococcus faecalis (3 bands), and Strep. pyogenes (5 bands). Molecular masses of cell wall hydrolases ranged from 17 to 114.6 kDa. Lytic activities against cell walls of Corynebacterium sepedonicum (Clavibacter michiganense pv. sepedonicum) could be shown with the cell wall extracts of Strep. pyogenes (45.7 kDa), Strep. faecalis (67 kDa), B. megaterium (67 kDa), and Staph. aureus (67 kDa).Key words: autolysins, electrophoresis, hydrolases, muramidases, peptidoglycan.

scholarly journals Sed1p Is a Major Cell Wall Protein ofSaccharomyces cerevisiae in the Stationary Phase and Is Involved in Lytic Enzyme Resistance

1998 ◽  
Vol 180 (13) ◽  
pp. 3381-3387 ◽  
Author(s):  
Hitoshi Shimoi ◽  
Hiroshi Kitagaki ◽  
Hisanobu Ohmori ◽  
Yuzuru Iimura ◽  
Kiyoshi Ito
Keyword(s):  
Cell Wall ◽  
Stationary Phase ◽  
Cell Walls ◽  
Secretory Pathway ◽  
Signal Sequence ◽  
Sodium Dodecyl ◽  
Cell Wall Protein ◽  
Lytic Enzyme ◽  
Cell Wall Proteins ◽  
Structural Cell

ABSTRACT A 260-kDa structural cell wall protein was purified from sodium dodecyl sulfate-treated cell walls of Saccharomyces cerevisiae by incubation with Rarobacter faecitabidusprotease I, which is a yeast-lytic enzyme. Amino acid sequence analysis revealed that this protein is the product of the SED1 gene.SED1 was formerly identified as a multicopy suppressor oferd2, which encodes a protein involved in retrieval of luminal endoplasmic reticulum proteins from the secretory pathway. Sed1p is very rich in threonine and serine and, like other structural cell wall proteins, contains a putative signal sequence for the addition of a glycosylphosphatidylinositol anchor. However, the fact that Sed1p, unlike other cell wall proteins, has six cysteines and seven putative N-glycosylation sites suggests that Sed1p belongs to a new family of cell wall proteins. Epitope-tagged Sed1p was detected in a β-1,3-glucanase extract of cell walls by immunoblot analysis, suggesting that Sed1p is a glucanase-extractable cell wall protein. The expression of Sed1p mRNA increased in the stationary phase and was accompanied by an increase in the Sed1p content of cell walls. Disruption of SED1 had no effect on exponentially growing cells but made stationary-phase cells sensitive to Zymolyase. These results indicate that Sed1p is a major structural cell wall protein in stationary-phase cells and is required for lytic enzyme resistance.

scholarly journals Conservation, Surface Exposure, and In Vivo Expression of the Frp Family of Iron-Regulated Cell Wall Proteins in Staphylococcus aureus

2002 ◽  
Vol 70 (5) ◽  
pp. 2399-2407 ◽  
Author(s):  
Julie A. Morrissey ◽  
Alan Cockayne ◽  
Jane Hammacott ◽  
Keith Bishop ◽  
Amy Denman-Johnson ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Growth Conditions ◽  
Open Reading Frames ◽  
Cell Wall Proteins ◽  
Phenotypic Analysis

ABSTRACT Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis identified two conserved, immunogenic Staphylococcus aureus cell wall proteins, of 40 and 87 kDa, expressed under iron-restricted growth conditions in vitro and in vivo. N-terminal sequencing and subsequent genome analysis showed that these proteins are encoded by adjacent monocistronic open reading frames designated frpA and frpB, respectively. Studies with an S. aureus fur mutant confirmed that expression of FrpA and FrpB is regulated by Fur but that there also appears to be differential expression of these proteins in different iron-restricted media in vitro. FrpA and FrpB share some amino acid sequence homology with each other and with a putative S. aureus membrane protein, FrpC. frpC is the first gene of a Fur-regulated operon encoding four proteins of unknown function (FrpC, -D, -G, and -H) and the binding protein (FrpE) and permease (FrpF) of a putative iron transporter. Antisense mutagenesis and bioassays showed that FrpA and FrpB are not required for growth of S. aureus under iron-restricted conditions in vitro and do not appear to be involved in the transport of iron from siderophores or in binding of hemin. Further phenotypic analysis suggested that FrpA may be involved in adhesion of S. aureus to plastic in vitro. Binding of S. aureus to microtiter wells was found to be iron regulated, and iron-restricted S. aureus containing antisense frpA or frpAB but not frpB constructs showed reduced binding compared to vector construct controls.

scholarly journals Preliminary characterization of the transcriptional and translational products of the Saccharomyces cerevisiae cell division cycle gene CDC28.

1982 ◽  
Vol 2 (4) ◽  
pp. 412-425 ◽  
Author(s):  
S I Reed ◽  
J Ferguson ◽  
J C Groppe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
In Vitro Mutagenesis ◽  
Coding Region ◽  
Loop Analysis ◽  
Partial Digestion ◽  
Polyadenylic Acid

The CDC28 gene was subcloned from a plasmid containing a 6.5-kilobase-pair segment of Saccharomyces cerevisiae DNA YRp7(CDC28-3) by partial digestion with Sau3A and insertion of the resulting fragments into the BamHI sites of YRp7 and pRC1. Recombinant plasmids were obtained containing inserts of 4.4 and 3.1 kilobase pairs which were capable of complementing a cdc28(ts) mutation. R-loop analysis indicated that each yeast insert contained two RNA coding regions of about 0.8 and 1.0 kilobase pairs, respectively. In vitro mutagenesis experiments suggested that the smaller coding region corresponded to the CDC28 gene. When cellular polyadenylic acid-containing RNA, separated by agarose gel electrophoresis after denaturation with glyoxal and transferred to nitrocellulose membrane, was reacted with labeled DNA from the smaller coding region, and RNA species of about 1 kilobase in length was detected. Presumably, the discrepancy in size between the R-loop and electrophoretic determinations is due to a segment of polyadenylic acid which is excluded from the R-loops. By using hybridization of the histone H2B mRNAs to an appropriate probe as a previously determined standards, it was possible to estimate the number of CDC28 mRNA copies per haploid cell as between 6 and 12 molecules. Hybrid release translation performed on the CDC29 mRNA directed the synthesis of a polypeptide of 27,000 daltons, as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This polypeptide was not synthesized when mRNA prepared from a cdc28 nonsense mutant was translated in a parallel fashion. However, if the RNA from a cell containing the CDC28 gene on a plasmid maintained at a high copy number was translated, the amount of in vitro product was amplified fivefold.

scholarly journals Requirement of Autolytic Activity for Bacteriocin-Induced Lysis

2000 ◽  
Vol 66 (8) ◽  
pp. 3174-3179 ◽  
Author(s):  
M. Carmen Martínez-Cuesta ◽  
Jan Kok ◽  
Elisabet Herranz ◽  
Carmen Peláez ◽  
Teresa Requena ◽  
...  
Keyword(s):  
Cell Wall ◽  
Sodium Dodecyl Sulfate ◽  
Growth Phase ◽  
Cell Walls ◽  
Cell Damage ◽  
Sodium Dodecyl ◽  
Exponential Growth Phase ◽  
Cell Wall Degradation ◽  
Intracellular Proteins ◽  
Autolytic Activity

ABSTRACT The bacteriocin produced by Lactococcus lactis IFPL105 is bactericidal against several Lactococcus andLactobacillus strains. Addition of the bacteriocin to exponential-growth-phase cells resulted in all cases in bacteriolysis. The bacteriolytic response of the strains was not related to differences in sensitivity to the bacteriocin and was strongly reduced in the presence of autolysin inhibitors (Co2+ and sodium dodecyl sulfate). When L. lactis MG1363 and its derivative deficient in the production of the major autolysin AcmA (MG1363acmAΔ1) were incubated with the bacteriocin, the latter did not lyse and no intracellular proteins were released into the medium. Incubation of cell wall fragments of L. lactisMG1363, or of L. lactis MG1363acmAΔ1 to which extracellular AcmA was added, in the presence or absence of the bacteriocin had no effect on the speed of cell wall degradation. This result indicates that the bacteriocin does not degrade cell walls, nor does it directly activate the autolysin AcmA. The autolysin was also responsible for the observed lysis of L. lactis MG1363 cells during incubation with nisin or the mixture of lactococcins A, B, and M. The results presented here show that lysis of L. lactis after addition of the bacteriocins is caused by the resulting cell damage, which promotes uncontrolled degradation of the cell walls by AcmA.

Extraction and properties of hemagglutinin from cell wall fragments of Fusobacterium nucleatum

1982 ◽  
Vol 152 (1) ◽  
pp. 298-305
Author(s):  
P Dehazya ◽  
R S Coles
Keyword(s):  
Cell Wall ◽  
Sodium Dodecyl Sulfate ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fusobacterium Nucleatum ◽  
Blue Staining ◽  
Sephadex Column ◽  
Dodecyl Sulfate ◽  
Triton X

To study the hemagglutinin of Fusobacterium nucleatum, methods were sought to solubilize and purify this component. When cells of F. nucleatum were ruptured by passage through a French press, the fragments lost virtually all ability to agglutinate human erythrocytes. Extraction of the fragments with 2% Triton X-100 for 30 min at 22 degrees C restored hemagglutinating activity (HA). Hemagglutination by these fragments could be inhibited by arginine, as can hemagglutination by intact bacteria. Treatment of active cell wall fragments with pronase and 2% Triton X-100-EDTA at 37 degrees C or with pronase and 0.1% Triton X-100-EDTA at pH 10.0 allowed recovery of solubilized HA. The former HA was inhibited by arginine (arg+) whereas the latter was not (arg-). Fractionation of the arg+ extract by preparative isoelectric focusing showed that HA was recovered from the gel sections having a pH between 4.5 and 5.5. Hemagglutination by this preparation was still arg+. Chromatography of this hemagglutinin on DEAE-Sephadex increased the specific activity to high levels with a loss of inhibition by arginine. A fraction from the DEAE-Sephadex column containing 10,700 HA units per mg of protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Solubilization at 22 degrees C before electrophoresis revealed three Coomassie blue-staining bands which migrated with apparent molecular weights of about 21,000, 38,000 and 60,000. When the same DEAE fraction was boiled in sodium dodecyl sulfate, electrophoresis revealed only one band with an apparent molecular weight of 21,000.

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