Isolated Cell Walls Research Articles

Saccharomyces cerevisiae Cell Wall Isolates are Potential Inhibitors of Bacterial Growth

Saccharomyces cerevisiae cell wall components are thought to bind and help pass pathogenic bacteria, such as E. coli, through the intestines 1. The binding between bacteria and S. cerevisiae is thought to occur between type‐1 mannose specific fimbriae, a common feature of pathogenic bacteria, and the mannoproteins that comprise 30–50% of the cell wall of S. cerevisiae by weight 2, 3, 4. With the fimbriae bound to mannoproteins, bacteria are unable to adhere to the walls of the intestine and thus, no colonization occurs. This interaction provides a potential antibiotic free method to prevent E. coli infection. The goals of this project are two‐fold. First, to develop a cell wall harvesting procedure and characterize the cell wall fractions. Second, to evaluate the binding interaction of S. cerevisiae with E. coli and its effect on E. coli proliferation. A cell wall isolation procedure has been optimized using temperature induced autolysis of a cell culture at 50 °C and pH 5.0 for 24 hours followed by purification washes with salt solutions. Cell wall fractions have been characterized using the Bradford assay, Coomassie stained SDS gels, and Western blot analysis employing the lectin Concanavalin A antibody to detect mannose. The cell wall fractions have been found to contain high levels of mannoproteins. An in‐vitro binding assay, adapted from Ganner et al. 2013, has been employed to evaluate the interaction between the cell wall fractions and E. coli. Cell wall coated microplate wells display a faster progression to log phase than controls due to the cell wall‐fimbriae binding interaction increasing retention of initial colony forming units. In‐vivo, a stronger binding interaction would inhibit bacterial infection by disrupting initial attachment of E. coli to the intestinal lining. Work is currently being performed to better understand factors influencing the binding mechanism as previous studies have indicated an unclear connection between total mannoprotein content and binding capacity. 2Support or Funding InformationSJFC Internal funds

Cellular Localization of Carbonic Anhydrase Nce103p in Candida albicans and Candida parapsilosis.

Pathogenic yeasts Candida albicans and Candida parapsilosis possess a ß-type carbonic anhydrase Nce103p, which is involved in CO2 hydration and signaling. C. albicans lacking Nce103p cannot survive in low CO2 concentrations, e.g., in atmospheric growth conditions. Candida carbonic anhydrases are orthologous to the Saccharomyces cerevisiae enzyme, which had originally been detected as a substrate of a non-classical export pathway. However, experimental evidence on localization of C. albicans and C. parapsilosis carbonic anhydrases has not been reported to date. Immunogold labeling and electron microscopy used in the present study showed that carbonic anhydrases are localized in the cell wall and plasmatic membrane of both Candida species. This localization was confirmed by Western blot and mass spectrometry analyses of isolated cell wall and plasma membrane fractions. Further analysis of C. albicans and C. parapsilosis subcellular fractions revealed presence of carbonic anhydrases also in the cytosolic and mitochondrial fractions of Candida cells cultivated in shaken liquid cultures, under the atmospheric conditions.

Wall teichoic acids govern cationic gold nanoparticle interaction with Gram-positive bacterial cell walls.

Molecular-level understanding of nanomaterial interactions with bacterial cell surfaces can facilitate design of antimicrobial and antifouling surfaces and inform assessment of potential consequences of nanomaterial release into the environment. Here, we investigate the interaction of cationic nanoparticles with the main surface components of Gram-positive bacteria: peptidoglycan and teichoic acids. We employed intact cells and isolated cell walls from wild type Bacillus subtilis and two mutant strains differing in wall teichoic acid composition to investigate interaction with gold nanoparticles functionalized with cationic, branched polyethylenimine. We quantified nanoparticle association with intact cells by flow cytometry and determined sites of interaction by solid-state 31P- and 13C-NMR spectroscopy. We find that wall teichoic acid structure and composition were important determinants for the extent of interaction with cationic gold nanoparticles. The nanoparticles interacted more with wall teichoic acids from the wild type and mutant lacking glucose in its wall teichoic acids than those from the mutant having wall teichoic acids lacking alanine and exhibiting more restricted molecular motion. Our experimental evidence supports the interpretation that electrostatic forces contributed to nanoparticle–cell interactions and that the accessibility of negatively charged moieties in teichoic acid chains influences the degree of interaction. The approaches employed in this study can be applied to engineered nanomaterials differing in core composition, shape, or surface functional groups as well as to other types of bacteria to elucidate the influence of nanoparticle and cell surface properties on interactions with Gram-positive bacteria.

Cell Wall Membrane Fraction of Chlorella sorokiniana Enhances Host Antitumor Immunity and Inhibits Colon Carcinoma Growth in Mice.

A colon cancer growth inhibitor partially purified from the isolated cell wall membrane fraction of Chlorella sorokiniana, here referred to as Chlorella membrane factor (CMF), was evaluated for its antitumor and immunomodulatory effects in cell culture and in a colon carcinoma mouse model. The CMF treatment dose- and time-dependently inhibited colon carcinoma cell growth in 2-dimensional cultures. Treatment with CMF also significantly inhibited the growth of colon carcinoma spheroids in 3-dimensional cell culture in coculture with T lymphocytes. In a mouse CT26 colon carcinoma peritoneal dissemination model, intraperitoneal injection of CMF (10 or 30 mg dry weight/kg body weight, every other day) dose-dependently and significantly attenuated the growth of tumor nodules via induction of tumor cell apoptosis. Evaluation of immune cell populations in ascites showed that CMF treatment tended to increase T lymphocytes but lower granulocyte populations. The present study suggests that the cell wall membrane fraction of Chlorella sorokiniana contains a bioactive material that inhibits colon carcinoma growth via direct cell growth inhibition and stimulation of host antitumor immunity. Hence, it is suggested that the Chlorella cell wall membrane extract or a bioactive substance in the extract is an attractive complementary medicine for cancer therapy.

Cell wall components regulate photosynthesis and leaf water relations of Vitis vinifera cv. Grenache acclimated to contrasting environmental conditions

Environmental conditions determine plants performance as they shape – among other key factors – leaf features and physiology. However, little is known regarding to the changes occurring in leaf cell wall composition during the acclimation to an environmental stress and, specially, if these changes have an impact on other leaf physiology aspects. In order to induce changes in photosynthesis, leaf water relations and cell wall main components (i.e., cellulose, hemicelluloses and pectins) and see how they co-vary, Vitis vinifera cv. Grenache was tested under four different conditions: (i) non-stress conditions (i.e., control, with high summer temperature and irradiance), (ii) growth chamber conditions, (iii) growth chamber under water stress and (iv) cold growth chamber. Plants developed in growth chambers decreased net CO2 assimilation (AN) and mesophyll conductance (gm) compared to control. Although cold did not change the bulk modulus of elasticity (ε), it decreased in growth chamber conditions and water stress. Control treatment showed the highest values for photosynthetic parameters and ε as well as for leaf structural traits such as leaf mass area (LMA) and leaf density (LD). Whereas cellulose content correlated with photosynthetic parameters, particularly AN and gm, pectins and the amount of alcohol insoluble residue (AIR) – an approximation of the isolated cell wall fraction – correlated with leaf water parameters, specifically, ε. Although preliminary, our results suggest that cell wall modifications due to environmental acclimations can play a significant role in leaf physiology by affecting distinctly photosynthesis and water relations in a manner that might depend on environmental conditions.

Nanostructure and poroviscoelasticity in cell wall materials from onion, carrot and apple: Roles of pectin

The hierarchical organisation of polysaccharides in primary plant cell walls is responsible for their unique mechanical properties, and in turn for the textural and rheological properties of plant-based foods and ingredients. It is expected that at the nano scale, the mechanical properties of cell wall materials arise from a combination of structural deformation of the polysaccharide networks and hydraulic properties of the continuous water phase, as has been shown for other cellulose-based composites. Pectin plays a key role in the load bearing properties of (bacterial) cellulose-pectin composites due to its contribution to both hydration structure and the dynamics of water movement. To investigate whether these features are also important in plant cell wall materials we have used a set of advanced characterisation techniques to elucidate cell wall structural features at different length scales (X-ray diffraction and small angle X-ray and neutron scattering) in cell walls from two dicotyledons (apple and carrot) and a non-commelinid monocotyledon (onion). The strength of isolated cell walls was measured under compression and fitted to a poroviscoelastic mechanical model, demonstrating that the mechanical properties of the isolated cell wall materials are directly linked to both polysaccharide networks and fluid flow through the networks. Our results show how pectin polysaccharides influence the viscoelastic behaviour of these materials and contribute to the texture of plant-derived food systems.

Augmenting Staphylococcal infection: the importance of timing

Staphylococcus aureus (S. aureus) acts as a commensal in the microbiome of the skin and nasopharynx. However, on gaining access to the bloodstream it can cause an array of pathogenic outcomes. S. aureus can crowdsource the microflora to assist in becoming an opportunistic pathogen as our lab has recently published findings that co-inoculation of S. aureus with commensals, acting as pro-infectious agents, leads to a much more robust, virulent infection. This benefits S. aureus at doses where it would otherwise be cleared by the immune system. Pro-infectious agents do not need to be live commensals as isolated cell wall peptidoglycan also augments infection. This work aimed to assess the effects of inoculation with pro-infectious agents before and after infection with S. aureus. It was found that pro-infectious agents needed to be co-administered in order to fully augment infection. This gives mechanistic insight where S. aureus and the pro-infectious agents need to be in the same local environment or phagocyte to augment infection.

Isolation and characterization of cereal cell walls

ABSTRACT The present study was aimed to isolate and characterize cereal cell walls with special reference to monosaccharide and total phenolic content. For the purpose, two varieties of each cereal, i.e. wheat (Ujala-16 and FSD-08), barley (Jau-87 and Haider-93), and sorghum (Sorghum-11 and JS-02) were procured from Ayub Agriculture Research Institute, Faisalabad. In phase I, endospermic cell walls were isolated from cereals through popping method. In phase II, isolated cell walls were analyzed for monosaccharides, beta-glucan, and total phenolics according to their respective methods. Higher cell wall contents were shown by both varieties of barley (8.32–8.99 g/100 g) followed by wheat (5.21–5.68 g/100 g) and sorghum (3.88–4.02 g/100 g) varieties. Results regarding monosaccharides of endospermic cell walls revealed that these cell walls had arabinose, xylose, mannose, galactose, and glucose with glucose absence in wheat and mannose and galactose absence in sorghum. Furthermore, barley varieties were higher in beta-glucan and total phenolics content followed by wheat and sorghum. Conclusively, barley cell wall was considered more nutritious as compared to wheat and sorghum.

Desiccation tolerance in plants: Structural characterization of the cell wall hemicellulosic polysaccharides in three Selaginella species

Drought-induced dehydration of vegetative tissues in lycopods affects growth and survival. Different species of Selaginella have evolved a series of specialized mechanisms to tolerate desiccation in vegetative tissues in response to water stress. In the present study, we report on the structural characterization of the leaf cell wall of the desiccation-tolerant species S. involvens and two desiccation-sensitive species, namely S. kraussiana and S. moellendorffii. Isolated cell walls from hydrated and desiccated leaves of each species were fractionated and the resulting oligosaccharide fragments were analyzed to determine their structural features. Our results demonstrate that desiccation induces substantial modifications in the cell wall composition and structure. Altogether, these data highlight the fact that structural remodeling of cell wall hemicellulosic polysaccharides including XXXG-rich xyloglucan, arabinoxylan and acetylated galactomannan is an important process in order to mitigate desiccation stress in Selaginella.

Peptidoglycan and Teichoic Acid Levels and Alterations in Staphylococcus aureus by Cell-Wall and Whole-Cell Nuclear Magnetic Resonance.

Gram-positive bacteria surround themselves with a multilayered macromolecular cell wall that is essential to cell survival and serves as a major target for antibiotics. The cell wall of Staphylococcus aureus is composed of two major structural components, peptidoglycan (PG) and wall teichoic acid (WTA), together creating a heterogeneous and insoluble matrix that poses a challenge to quantitative compositional analysis. Here, we present 13C cross polarization magic angle spinning solid-state nuclear magnetic resonance (NMR) spectra of intact cell walls, purified PG, and purified WTA. The spectra reveal the clear molecular differences in the two polymers and enable quantification of PG and WTA in isolated cell walls, an attractive alternative to estimating teichoic acid content from a phosphate analysis of completely pyrolyzed cell walls. Furthermore, we discovered that unique PG and WTA spectral signatures could be identified in whole-cell NMR spectra and used to compare PG and WTA levels among intact bacterial cell samples. The distinguishing whole-cell 13C NMR contributions associated with PG include the GlcNAc-MurNAc sugar carbons and glycyl α-carbons. WTA contributes carbons from the phosphoribitol backbone. Distinguishing 15N spectral signatures include glycyl amide nitrogens in PG and the esterified d-alanyl amine nitrogens in WTA. 13C NMR analysis was performed with samples at natural abundance and included 10 whole-cell sample comparisons. Changes consistent with altered PG and WTA content were detected in whole-cell spectra of bacteria harvested at different growth times and in cells treated with tunicamycin. This use of whole-cell NMR provides quantitative parameters of composition in the context of whole-cell activity.

On the S-layer of Thermus thermophilus and the assembling of its main protein SlpA

We have isolated and analysed the cell envelope of the thermophilic bacterium Thermus thermophilus HB8. Isolated cell walls, characterized by the dominance of the S-layer protein SlpA, are found to be constituted by several protein complexes of high molecular weights. Further isolation steps, starting from the cell wall samples, led to the selective release of the S-layer protein SlpA in solution as confirmed by mass spectrometry. Blue Native gel electrophoresis on these samples showed that SlpA is organized into a specific hierarchical order of oligomeric states that are consistent with the complexes at high molecular weight identified on the total cell wall fraction. The analysis showed that SlpA bases this peculiar organization on monomers and exceptionally stable dimers, leading to the formation of tetramers, heptamers, and decamers. Furthermore, the two elementary units of SlpA, monomers and dimers, are regulated by the presence of calcium not only for the assembling of monomers into dimers, but also for the splitting of dimers into monomers. Finally, the SlpA protein was found to be subjected to specific proteolysis leading to characteristic degradation products. Findings are discussed in terms of S-layer assembling properties as bases for understanding its structure, turn-over and organization.

Peptidoglycan Compositional Analysis of Enterococcus faecalis Biofilm by Stable Isotope Labeling by Amino Acids in a Bacterial Culture.

Peptidoglycan (PG) is a major component of the cell wall in Enterococcus faecalis. Accurate analysis of PG composition provides crucial insights into the bacterium's cellular functions and responses to external stimuli, but this analysis remains challenging because of various chemical modifications to PG-repeat subunits. We characterized changes to the PG composition of E. faecalis grown as planktonic bacteria and biofilm by developing "stable isotope labeling by amino acids in bacterial culture" (SILAB), optimized for bacterial cultures with incomplete amino acid labeling. This comparative analysis by mass spectrometry was performed by labeling E. faecalis in biofilm with heavy Lys (l-[13C6,2D9,15N2]Lys) and planktonic bacteria with natural abundance l-Lys, then mixing equal amounts of bacteria from each condition, and performing cell wall isolation and mutanolysin digestion necessary for liquid chromatography and mass spectrometry. An analytical method was developed to determine muropeptide abundances using correction factors to compensate for incomplete heavy Lys isotopic enrichment (98.33 ± 0.05%) and incorporation (83.23 ± 1.16%). Forty-seven pairs of PG fragment ions from isolated cell walls of planktonic and biofilm samples were selected for SILAB analysis. We found that the PG in biofilm showed an increased level of PG cross-linking, an increased level of N-deacetylation of GlcNAc, a decreased level of O-acetylation of MurNAc, and an increased number of stem modifications by d,d- and l,d-carboxypeptidases.

Dietary polyphenols bind to potato cells and cellular components

The ability of phenolic compounds to bind to dietary polysaccharides such as starch and plant cell wall components impacts their nutritional value. Here, we report interactions between potato cells and three different phenolic compounds (+)-catechin, phloridzin and vanillic acid. The binding interactions of the phenolic compounds with intact potato cells, as well as disrupted cells, cooked cells, isolated cell walls and starch granules was explored varying polyphenol concentration, pH and incubation time. Results showed that binding capacity depends on the type of phenolic compound ((+)-catechin>phloridzin>vanillic acid) as well as the type of substrate, to a maximum of ∼50µmol/g (dry weight). The observed differences (p<0.05) were ascribed to the amount and accessibility of potential binding sites in both the phenolic compounds and the polysaccharides. Remarkably, polyphenols could penetrate intact cells and bind the starch within them, suggesting their potential as delivery vehicles, whose loading capacity more than doubled after cooking.

Characterization of the tertiary structure of the peptidoglycan of Enterococcus faecalis

Solid-state NMR spectra of whole cells and isolated cell walls of Enterococcus faecalis grown in media containing combinations of 13C and 15N specific labels in d- and l-alanine and l-lysine (in the presence of an alanine racemase inhibitor alaphosphin) have been used to determine the composition and architecture of the cell-wall peptidoglycan. The compositional variables include the concentrations of (i) peptidoglycan stems without bridges, (ii) d-alanylated wall teichoic acid, (iii) cross-links, and (iv) uncross-linked tripeptide and tetra/pentapeptide stems. Connectivities of l-alanyl carbonyl‑carbon bridge labels to d-[3-13C]alanyl and l-[ε-15N]lysyl stem labels prove that the peptidoglycan of E. faecalis has the same hybrid short-bridge architecture (with a mix of parallel and perpendicular stems) as the FemA mutant of Staphylococcus aureus, in which the cross-linked stems are perpendicular to one another and the cross-linking is close to the ideal 50% value. This is the first determination of the cell-wall chemical and geometrical architecture of whole cells of E. faecalis, a major source of nosocomial infections worldwide.

The role of cell walls and pectins in cation exchange and surface area of plant roots

We aimed to assess role of cell walls in formation of cation exchange capacity, surface charge, surface acidity, specific surface, water adsorption energy and surface charge density of plant roots, and to find the input of the cell wall pectins to the above properties. Whole roots, isolated cell walls and the residue after the extraction of pectins from the cell walls of two Apiaceae L. species (celeriac and parsnip) were studied using potentiometric titration curves and water vapor adsorption – desorption isotherms.Total amount of surface charge, as well as the cation exchange capacity were markedly higher in roots than in their cell walls, suggesting large contribution of other cell organelles to the binding of cations by the whole root cells. Significantly lower charge of the residues after removal of pectins was noted indicating that pectins play the most important role in surface charge formation of cell walls. The specific surface was similar for all of the studied materials. For the separated cell walls it was around 10% smaller than of the whole roots, and it increased slightly after the removal of pectins. The surface charge density and water vapor adsorption energy were the highest for the whole roots and the lowest for the cell walls residues after removal of pectins.The results indicate that the cell walls and plasma membranes are jointly involved in root ion exchange and surface characteristics and their contribution depends upon the plant species.

Isolation of wheat endosperm cell walls: Effects of non-endosperm flour components on structural analyses

We report the isolation of a pure form of cell walls from wheat endosperm ‘popped’ out from the whole, enzyme deactivated and soaked grain, and compare them with cell walls isolated from milled flours of extraction rates from 45% to 75%, at physiological (37 °C) and elevated (70 °C) temperatures. Cell walls isolated from flours all contained non-endosperm walls whereas walls from popped endosperm were apparently pure. The monosaccharide composition of ‘popped’ cell walls was different to that of cell walls isolated from flour, particularly glucose and mannose contents (34 and 7% for ‘popped’ cf 29 and 3% for flour respectively) and arabinose to xylose ratios (0.45 for ‘popped’ cf 0.58 for flour). Total phenolic contents of popped endosperm cell walls were three to four times lower than for cell walls from flour. Elevated isolation temperature also had a solubilising effect, altering the cell wall composition. This study provides a novel method of isolating pure wheat endosperm cell walls, and demonstrates how contaminating (thick cell walled) non-endospermic material in milled flours can have a major influence on cell wall compositional analyses.

Quantification of the d-Ala-d-Lac-Terminated Peptidoglycan Structure in Vancomycin-Resistant Enterococcus faecalis Using a Combined Solid-State Nuclear Magnetic Resonance and Mass Spectrometry Analysis.

Induction of vancomycin resistance in vancomycin-resistant enterococci (VRE) involves replacement of the d-Ala-d-Ala terminus of peptidoglycan (PG) stems with d-Ala-d-Lac, dramatically reducing the binding affinity of vancomycin for lipid II. Effects from vancomycin resistance induction in Enterococcus faecalis (ATCC 51299) were characterized using a combined solid-state nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS) analysis. Solid-state NMR directly measured the total amounts of d-Lac and l,d-Ala metabolized from [2-13C]pyruvate, accumulated Park's nucleotide, and changes to the PG bridge-linking density during the early exponential growth phase (OD660 = 0.4) in intact whole cells of VRE. A high level of accumulation of depsipeptide-substituted Park's nucleotide consistent with the inhibition of the transglycosylation step of PG biosynthesis during the initial phase of vancomycin resistance was observed, while no changes to the PG bridge-linking density following the induction of vancomycin resistance were detected. This indicated that the attachment of the PG bridge to lipid II by the peptidyl transferases was not inhibited by the d-Ala-d-Lac-substituted PG stem structure in VRE. Compositions of mutanolysin-digested isolated cell walls of VRE grown with and without vancomycin resistance induction were determined by LC-MS. Muropeptides with PG stems terminating in d-Ala-d-Lac were found only in VRE grown in the presence of vancomycin. Percentages of muropeptides with a pentapeptide stem terminating in d-Ala-d-Lac for VRE grown in the presence of vancomycin were 26% for the midexponential phase (OD660 = 0.6) and 57% for the stationary growth phase (OD660 = 1.0). These high percentages indicate that d-Ala-d-Lac-substituted lipid II was efficiently utilized for PG biosynthesis in VRE.

Iron incorporation in biosilica of the marine diatom Stephanopyxisturris: dispersed or clustered?

Iron incorporation into diatom biosilica was investigated for the species Stephanopyxisturris. It is known that several "foreign" elements (e.g., germanium, titanium, aluminum, zinc, iron) can be incorporated into the siliceous cell walls of diatoms in addition to silicon dioxide (SiO2). In order to examine the amount and form of iron incorporation, the iron content in the growth medium was varied during cultivation. Fe:Si ratios of isolated cell walls were measured by ICP-OES. SEM studies were performed to examine of a possible influence of excess iron during diatom growth upon cell wall formation. The chemical state of biosilica-attached iron was characterized by a combination of infrared, 29Si MAS NMR, and EPR spectroscopy. For comparison, synthetic silicagels of variable iron content were studied. Our investigations show that iron incorporation in biosilica is limited. More than 95% of biosilica-attached iron is found in the form of iron clusters/nanoparticles. In contrast, iron is preferentially dispersedly incorporated within the silica framework in synthetic silicagels leading to Si-O-Fe bond formation.

Cell wall chemical characteristics of whole-crop cereal silages harvested at three maturity stages.

BackgroundIn cooler climates such as found in Scandinavian countries cereals are important feedstuffs for ruminants often ensiled as whole‐crop cereal silages (WCCS) to preserve nutrients. Animal performance varies with the type of cereal forage and stage of cereal development being ensiled. Cell wall isolation and analysis was undertaken to determine differences among cereal silages harvested at different stages of maturity.ResultsA set of 27 WCCS samples of barley, wheat and oats harvested at heading, early milk, and dough stages of maturity were analyzed for cell wall (CW) composition and compared to previous NDF analyses. Total CW concentrations of the WCCS were higher than the NDF concentration. The lignin concentration was higher (P < 0.001) in oats (111 g kg−1 DM) than in barley (88 g kg−1 DM) and wheat (91 g kg−1 DM). Ferulates (ester and ether linked) ranged from 12.2 to 14.9 g kg−1 across forage types and maturity stages. The correlation between total cell wall xylose and HC concentrations (NDF‐ADF) was lower than expected in all forages (R = 0.63).ConclusionThe more comprehensive analyses of cell walls provide detailed composition of the different WCCS that vary due to the maturity and type of cereal. © 2016 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The GCA1 gene encodes a glycosidase-like protein in the cell wall of Candida albicans.

Candida albicans Gca1p is a putative glucoamylase enzyme which contains 946 amino acids, 11 putative sites for N-glycosylation and 9 for O-glycosylation. Gca1p was identified in β-mercaptoethanol extracts from isolated cell walls of strain C. albicans SC5314 and it is involved in carbohydrate metabolism. The significance and the role of this protein within the cell wall structure were studied in the corresponding mutants. The homozygous mutant showed that GCA1 was not an essential gene for cell viability. Subsequent phenotypic analysis performed in the mutants obtained did not show significant difference in the behavior of mutant when compared with the wild strain SC5314. Zymoliase, Calcofluor White, Congo red, SDS, caffeine or inorganic compounds did not affect the integrity of the cell wall. No differences were observed when hyphal formation assays were carried out. However, an enzyme assay in the presence of substrate p-nitrophenyl-α-D-glucopyranoside enabled us to detect a significant decrease in glycosidase activity in the mutants compared with the parental strain, revealing the function of Gca1.