The separation of beta-glucanases produced by Cytophaga johnsonii and their role in the lysis of yeast cell walls.
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ABSTRACT:
SUBMITTER: Bacon JS
PROVIDER: S-EPMC1179570 | biostudies-other | 1970 Nov
REPOSITORIES: biostudies-other
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Project description:BackgroundThe fungal cell wall is an essential and robust external structure that protects the cell from the environment. It is mainly composed of polysaccharides with different functions, some of which are necessary for cell integrity. Thus, the process of fractionation and analysis of cell wall polysaccharides is useful for studying the function and relevance of each polysaccharide, as well as for developing a variety of practical and commercial applications. This method can be used to study the mechanisms that regulate cell morphogenesis and integrity, giving rise to information that could be applied in the design of new antifungal drugs. Nonetheless, for this method to be reliable, the availability of trustworthy commercial recombinant cell wall degrading enzymes with non-contaminating activities is vital.ResultsHere we examined the efficiency and reproducibility of 12 recombinant endo-β(1,3)-D-glucanases for specifically degrading the cell wall β(1,3)-D-glucan by using a fast and reliable protocol of fractionation and analysis of the fission yeast cell wall. This protocol combines enzymatic and chemical degradation to fractionate the cell wall into the four main polymers: galactomannoproteins, α-glucan, β(1,3)-D-glucan and β(1,6)-D-glucan. We found that the GH16 endo-β(1,3)-D-glucanase PfLam16A from Pyrococcus furiosus was able to completely and reproducibly degrade β(1,3)-D-glucan without causing the release of other polymers. The cell wall degradation caused by PfLam16A was similar to that of Quantazyme, a recombinant endo-β(1,3)-D-glucanase no longer commercially available. Moreover, other recombinant β(1,3)-D-glucanases caused either incomplete or excessive degradation, suggesting deficient access to the substrate or release of other polysaccharides.ConclusionsThe discovery of a reliable and efficient recombinant endo-β(1,3)-D-glucanase, capable of replacing the previously mentioned enzyme, will be useful for carrying out studies requiring the digestion of the fungal cell wall β(1,3)-D-glucan. This new commercial endo-β(1,3)-D-glucanase will allow the study of the cell wall composition under different conditions, along the cell cycle, in response to environmental changes or in cell wall mutants. Furthermore, this enzyme will also be greatly valuable for other practical and commercial applications such as genome research, chromosomes extraction, cell transformation, protoplast formation, cell fusion, cell disruption, industrial processes and studies of new antifungals that specifically target cell wall synthesis.
| S-EPMC8254974 | biostudies-literature
Project description:1. A number of yeast species were examined for the presence of beta-glucanases. Extracts obtained by cell disruption of Saccharomyces cerevisiae, Fabospora fragilis and Hansenula anomala hydrolysed laminarin and pustulan with the production of glucose. Enzymic activities were also detected in the culture fluids of F. fragilis and H. anomala grown aerobically in buffered mineral medium with glucose as the carbon source. 2. F. fragilis and H. anomala possessed approximately sevenfold higher beta-(1-->3)-glucanase activity than S. cerevisiae. 3. Intracellular exo-beta-glucanase from baker's yeast was purified 344-fold from the dialysed cell extract. 4. Exo-beta-glucanase from F. fragilis was purified 114-fold from the dialysed culture fluid and 423-fold from the dialysed intracellular extract. The purified extracellular and intracellular enzymes had similar properties and essentially the same specific activity, 79 enzyme units/mg. of protein. 5. Extracellular exo-beta-glucanase of H. anomala was purified 600-fold. 6. The optimum pH of the enzymes from F. fragilis, S. cerevisiae and H. anomala was 5.5 in each case. Chromatographic evidence indicated that the three enzymes remove glucosyl units sequentially from laminarin as well as pustulan. 7. The ratio of activities towards laminarin and pustulan remained constant during purification of the exo-beta-glucanase obtained from the three species, suggesting a single enzyme. Additional evidence for its unienzymic nature are: (i) the two activities were destroyed at exactly the same rate on heating of the purified enzyme from F. fragilis at three different temperatures; (ii) the competitive inhibitor glucono-delta-lactone gave the same value of K(i) when tested with either substrate; (iii) quantitative application of the ;mixed-substrate' method with the purified enzyme of S. cerevisiae gave data that were in excellent agreement with those calculated on the assumption of a single enzyme. 8. The purified exo-beta-glucanases of the different species of yeast had different kinetic constants. The ratios of maximal velocities and K(m) values with laminarin and pustulan differed markedly. Comparison of V(max.) and K(m) values suggests that the rapid release of spores from asci in F. fragilis might be explained in terms of an enzyme with higher maximal velocity and higher affinity to the ascus wall than that present in baker's yeast. 9. The estimated molecular weights for exo-beta-glucanases from F. fragilis, S. cerevisiae and H. anomala were 22000, 40000 and 30000 respectively.
| S-EPMC1186829 | biostudies-other
Project description:Cellulosimicrobium cellulans (also known with the synonyms Cellulomonas cellulans, Oerskovia xanthineolytica, and Arthrobacter luteus) is an actinomycete that excretes yeast cell wall lytic enzyme complexes containing endo-beta-1,3-glucanases [EC 3.2.1.39 and 3.2.1.6] as key constituents. Three genes encoding endo-beta-1,3-glucanases from two C. cellulans strains have been cloned and characterised over the past years. The betaglII and betaglIIA genes from strain DSM 10297 (also known as O. xanthineolytica LL G109) encoded proteins of 40.8 and 28.6 kDa, respectively, whereas the beta-1,3-glucanase gene from strain ATCC 21606 (also known as A. luteus 73-14) encoded a 54.5 kDa protein. Alignment of their deduced amino acid sequences reveal that betaglII and betaglIIA have catalytic domains assigned to family 16 of glycosyl hydrolases, whereas the catalytic domain from the 54.5 kDa glucanase belongs to family 64. Notably, both betaglII and the 54.5 kDa beta-1,3-glucanase are multidomain proteins, having a lectin-like C-terminal domain that has been assigned to family 13 of carbohydrate binding modules, and that confers to beta-1,3-glucanases the ability to lyse viable yeast cells. Furthermore, betaglII may also undergo posttranslational proteolytic processing of its C-terminal domain, resulting in a truncated enzyme retaining its glucanase activity but with very low yeast-lytic activity. In this review, the diversity in terms of structural and functional characteristics of the C. cellulans beta-1,3-glucanases has been compiled and compared.
| S-EPMC1458353 | biostudies-literature
Project description:Cell division in the fission yeast Schizosaccharomyces pombe yields two equal-sized daughter cells. Medial fission is achieved by deposition of a primary septum flanked by two secondary septa within the dividing cell. During the final step of cell division, cell separation, the primary septum is hydrolyzed by an endo-(1,3)-beta-glucanase, Eng1p. We reasoned that the cell wall material surrounding the septum, referred to here as the septum edging, also must be hydrolyzed before full separation of the daughter cells can occur. Because the septum edging contains (1,3)-alpha-glucan, we investigated the cellular functions of the putative (1,3)-alpha-glucanases Agn1p and Agn2p. Whereas agn2 deletion results in a defect in endolysis of the ascus wall, deletion of agn1 leads to clumped cells that remained attached to each other by septum-edging material. Purified Agn1p hydrolyzes (1,3)-alpha-glucan predominantly into pentasaccharides, indicating an endo-catalytic mode of hydrolysis. Furthermore, we show that the transcription factors Sep1p and Ace2p regulate both eng1 and agn1 expression in a cell cycle-dependent manner. We propose that Agn1p acts in concert with Eng1p to achieve efficient cell separation, thereby exposing the secondary septa as the new ends of the daughter cells.
| S-EPMC491845 | biostudies-literature
Project description:By selective enzymolysis, or chemical fractionation, a minor polysaccharide component has been isolated from yeast (Saccharomyces cerevisiae) glucan. This minor component has a degree of polymerization of about 130-140, a highly branched structure, and a high proportion of beta-(1-->6)-glucosidic linkages. The molecules also contain a smaller proportion of beta-(1-->3)-glucosidic linkages that serve mainly as interchain linkages, but some may also be inter-residue linkages.
| S-EPMC1165785 | biostudies-other
Project description:Yeast glucan as normally prepared by various treatments of yeast (Saccharomyces cerevisiae) cell walls to remove mannan and glycogen is still heterogeneous. The major component (about 85%) is a branched beta-(1-->3)-glucan of high molecular weight (about 240000) containing 3% of beta-(1-->6)-glucosidic interchain linkages. The minor component is a branched beta-(1-->6)-glucan. A comparison of our results with those of other workers suggests that different glucan preparations may differ in the degree of heterogeneity and that the major beta-(1-->3)-glucan component may vary considerably in degree of branching.
| S-EPMC1165784 | biostudies-other
Project description:RNA polymerase II is a complex of 12 subunits, Rpb1 to Rpb12, whose specific roles are only partly understood. Rpb4 is essential in mammals and fission yeast, but not in budding yeast. To learn more about the roles of Rpb4, we expressed the rpb4 gene under the control of regulatable promoters of different strength in fission yeast. We demonstrate that below a critical level of transcription, Rpb4 affects cellular growth proportional to its expression levels: cells expressing lower levels of rpb4 grew slower compared to cells expressing higher levels. Lowered rpb4 expression did not affect cell survival under several stress conditions, but it caused specific defects in cell separation similar to sep mutants. Microarray analysis revealed that lowered rpb4 expression causes a global reduction in gene expression, but the transcript levels of a distinct subset of genes were particularly responsive to changes in rpb4 expression. These genes show some overlap with those regulated by the Sep1-Ace2 transcriptional cascade required for cell separation. Most notably, the gene expression signature of cells with lowered rpb4 expression was highly similar to those of mcs6, pmh1, sep10 and sep15 mutants. Mcs6 and Pmh1 encode orthologs of metazoan TFIIH-associated cyclin-dependent kinase (CDK)-activating kinase (Cdk7-cyclin H-Mat1), while Sep10 and Sep15 encode mediator components. Our results suggest that Rpb4, along with some other general transcription factors, plays a specialized role in a transcriptional pathway that controls the cell cycle-regulated transcription of a specific subset of genes involved in cell division.
| S-EPMC1705487 | biostudies-literature
Project description:Mechanical rupture, or lysis, of the cytoplasmic membrane is a common cell death pathway in bacteria occurring in response to β-lactam antibiotics. A better understanding of the cellular design principles governing the susceptibility and response of individual cells to lysis could indicate methods of potentiating β-lactam antibiotics and clarify relevant aspects of cellular physiology. Here, we take a single-cell approach to bacterial cell lysis to examine three cellular features-turgor pressure, mechanosensitive channels, and cell shape changes-that are expected to modulate lysis. We develop a mechanical model of bacterial cell lysis and experimentally analyze the dynamics of lysis in hundreds of single Escherichia coli cells. We find that turgor pressure is the only factor, of these three cellular features, which robustly modulates lysis. We show that mechanosensitive channels do not modulate lysis due to insufficiently fast solute outflow, and that cell shape changes result in more severe cellular lesions but do not influence the dynamics of lysis. These results inform a single-cell view of bacterial cell lysis and underscore approaches of combatting antibiotic tolerance to β-lactams aimed at targeting cellular turgor.
| S-EPMC8372035 | biostudies-literature
Project description:Plant β-glucanases are enzymes involved in the synthesis, remodelling and turnover of cell wall components during multiple physiological processes. Based on the type of the glycoside bond they cleave, plant β-glucanases have been grouped into three categories: (i) β-1,4-glucanases degrade cellulose and other polysaccharides containing 1,4-glycosidic bonds to remodel and disassemble the wall during cell growth. (ii) β-1,3-glucanases are responsible for the mobilization of callose, governing the symplastic trafficking through plasmodesmata. (iii) β-1,3-1,4-glucanases degrade mixed linkage glucan, a transient wall polysaccharide found in cereals, which is broken down to obtain energy during rapid seedling growth. In addition to their roles in the turnover of self-glucan structures, plant β-glucanases are crucial in regulating the outcome in symbiotic and hostile plant-microbe interactions by degrading non-self glucan structures. Plants use these enzymes to hydrolyse β-glucans found in the walls of microbes, not only by contributing to a local antimicrobial defence barrier, but also by generating signalling glucans triggering the activation of global responses. As a counterpart, microbes developed strategies to hijack plant β-glucanases to their advantage to successfully colonize plant tissues. This review outlines our current understanding on plant β-glucanases, with a particular focus on the latest advances on their roles in adaptative responses.
| S-EPMC9099982 | biostudies-literature
Project description:Eukaryotic cells assemble actomyosin rings during cytokinesis to function as force-generating machines to drive membrane invagination and to counteract the intracellular pressure and the cell surface tension. How the extracellular matrix affects actomyosin ring contraction has not been fully explored. While studying the Schizosaccharomyces pombe 1,3-β-glucan-synthase mutant cps1-191, which is defective in division septum synthesis and arrests with a stable actomyosin ring, we found that weakening of the extracellular glycan matrix caused the generated spheroplasts to divide under the nonpermissive condition. This nonmedial slow division was dependent on a functional actomyosin ring and vesicular trafficking, but independent of normal septum synthesis. Interestingly, the high intracellular turgor pressure appears to play a minimal role in inhibiting ring contraction in the absence of cell wall remodeling in cps1-191 mutants, as decreasing the turgor pressure alone did not enable spheroplast division. We propose that during cytokinesis, the extracellular glycan matrix restricts actomyosin ring contraction and membrane ingression, and remodeling of the extracellular components through division septum synthesis relieves the inhibition and facilitates actomyosin ring contraction.
| S-EPMC7851958 | biostudies-literature