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At least 19 recordsLinked to original sources
PubMed1992

CSD2, CSD3, and CSD4, genes required for chitin synthesis in Saccharomyces cerevisiae: the CSD2 gene product is related to chitin synthases and to developmentally regulated proteins in Rhizobium species and Xenopus laevis.

In Saccharomyces cerevisiae, chitin forms the primary division septum and the bud scar in the walls of vegetative cells. Three chitin synthetic activities have been detected. Two of them, chitin synthase I and chitin synthase II, are not required for synthesis of most of the chitin present in vivo. Using a novel screen, I have identified three mutations, designated csd2, csd3, and csd4, that reduce levels of chitin in vivo by as much as 10-fold without causing any obvious perturbation of cell division. The csd2 and csd4 mutants lack chitin synthase III activity in vitro, while csd3 mutants have wild-type levels of this enzyme. In certain genetic backgrounds, these mutations cause temperature-sensitive growth on rich medium; inclusion of salts or sorbitol bypasses this phenotype. Gene disruption experiments show that CSD2 is nonessential; a small amount of chitin, about 5% of the wild-type level, is detected in the disruptants. DNA sequencing indicates that the CSD2 protein has limited, but statistically significant, similarity to chitin synthase I and chitin synthase II. Other significant similarities are to two developmental proteins: the nodC protein from Rhizobium species and the DG42 protein of Xenopus laevis. The relationship between the nodC and CSD2 proteins suggests that nodC may encode an N-acetylglucosaminyltransferase that synthesizes the oligosaccharide backbone of the nodulation factor NodRm-1.

Amino Acid Sequence↗
PubMed1991

Chitin utilization by marine bacteria. Chemotaxis to chitin oligosaccharides by Vibrio furnissii.

The adhesion/deadhesion apparatus of the marine bacterium Vibrio furnissii (Yu, C., Lee, A., Bassler, B. L., and Roseman, S. (1991) J. Biol. Chem. 266, 24260-24267) probably catalyzes the first step in colonizing chitin. Evidence is presented here for a second step, chemotaxis to chitin hydrolysis products. V. furnissii swarms toward chitin oligomers (GlcNAc)n, n = 1-6, at initial concentrations as low as 10 microM. A modified capillary assay was used for quantitation; the cells exhibit low level constitutive taxis to GlcNAc but not to the oligosaccharides. A mutant defective in the GlcNAc receptor (IINag of the phosphotransferase system) showed inducible taxis to the oligosaccharides. Two (or more) independently inducible receptors with overlapping specificities recognize (GlcNAc)n, n = 2-4. (GlcNAc)5 and (GlcNAc)6 were inactive in the capillary assay; expression of this receptor(s) apparently require special induction conditions. The (GlcNAc)n, n = 1-4, chemoreceptors of V. furnissii may be the most potent reported for bacteria. L-Amino acids were weak, constitutive attractants; glutamine, not known to be an attractant in other bacteria, was the most effective amino acid. The most potent receptor in Escherichia coli, Tar (aspartate), is not expressed in V. furnissii. The chemotactic responses were greatly affected by growth and induction conditions and the presence of nutrients in the assay media. Taxis to GlcNAc and GlcNAc oligomers was optimally induced by growth in lactate medium containing 0.6 mM sugar, while growth on the sugar per se resulted in poor taxis. Chemotaxis to the sugars increased 2- to 3-fold when the cells were starved. Nutrients in the assay medium, especially compounds that feed into or are part of the Krebs cycle, were potent inhibitors of taxis to the sugars and Gln. With the exception of isocitrate, inhibition of taxis correlated with the rate of oxidation of these compounds. The results suggest a link between catabolism and taxis in this organism, i.e. interactions or "cross-talk" between systems that are regulated by protein phosphorylation (Stock, J. A., Ninfa, A. J., and Stock, A. M. (1989) Microbiol. Rev. 53, 450-490).

Bacterial Adhesion↗
PubMed1991

Chitin utilization by marine bacteria. Degradation and catabolism of chitin oligosaccharides by Vibrio furnissii.

Chemotaxis of the marine bacterium Vibrio furnissii to chitin oligosaccharides has been described (Bassler, B. L., Gibbons, P. J., Yu, C., and Roseman, S. (1991) J. Biol. Chem. 266, 24268-24275). Some steps in catabolism of the oligosaccharides are reported here. GlcNAc, (GlcNAc)2, and (GlcNAc)3 are very rapidly consumed by intact cells, about 320 nmol of GlcNAc equivalents/min/mg of protein. (GlcNAc)4 is utilized somewhat more slowly. During these processes, there is virtually no release of hydrolysis products by the cells. The oligosaccharides enter the periplasmic space (via specific porins?) and are hydrolyzed by a unique membrane-bound endoenzyme (chitodextrinase) and an exoenzyme (N-acetyl-beta-glucosaminidase; beta-Glc-NAcidase). The genes encoding these enzymes have been cloned and expressed in Escherichia coli. The chitodextrinase cleaves soluble oligomers, but not chitin, to the di- and trisaccharides, while the periplasmic beta-GlcNAcidase hydrolyzes the GlcNAc termini from the oligomers. The end products in the periplasm, GlcNAc and (GlcNAc)2 (possibly (GlcNAc)3) are catabolized as follows. (a) Disaccharide pathway, A (GlcNAc)2 permease is apparently expressed by Vibrio furnissii. Translocated (GlcNAc)2 is rapidly hydrolyzed by a soluble, cytosolic beta-GlcNAcidase, and the GlcNAc is phosphorylated by an ATP-dependent, constitutive kinase to GlcNAc-6-P. (b) Monosaccharide pathway, Periplasmic GlcNAc is taken up by Enzyme IINag of the phosphoenolpyruvate:glycose phosphotransferase system, yielding GlcNAc-6-P, the common intermediate for both pathways. Finally, GlcNAc-6-P----Ac- + GlcNH2-6-P----Fru-6-P + NH3. (GlcNAc)2 is probably the "true" inducer of the chitin degradative enzymes described in this report and, depending on its concentration in the growth medium, differentially induces the periplasmic and cytosolic beta-GlcNAcidases. The disaccharide pathway appears to be the most important when the cells are confronted with low concentrations of the oligomers (e.g. in chemotaxis swarm plates). The relative activities of the induced enzymes suggest that the rate-limiting steps in oligosaccharide catabolism are the glycosidase activities in the periplasm.

Acetylglucosamine↗
PubMed1992

Synthesis and drug-release characteristics of the conjugates of mitomycin C with N-succinyl-chitosan and carboxymethyl-chitin.

By condensation of mitomycin C (MMC) with N-succinyl-chitosan (Suc-chitosan) and carboxymethyl-chitin (CM-chitin) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, Suc-chitosan-MMC conjugate (Suc-chitosan-MMC) and CM-chitin-MMC conjugate (CM-chitin-MMC) were prepared, respectively. The reaction conditions for 45 min at pH 5 and for 2 h at pH 5 were selected as the most appropriate for the preparations of Suc-chitosan-MMC and CM-chitin-MMC, respectively. Suc-chitosan-MMC was obtained as a water-insoluble product, while CM-chitin-MMC was partially water-soluble. When the ratio of MMC to the polymer supports changed in the conjugation reaction, the conjugates with 33% (w/w) and 23% (w/w) MMC contents were obtained as those most highly drug-loaded for Suc-chitosan-MMC and CM-chitin-MMC, respectively. At pH 7.4 at 37 degrees C, Suc-chitosan-MMC regenerated MMC very slowly, while the release of MMC from CM-chitin-MMC was relatively fast. Each drug release followed very nearly pseudo-first order kinetics, in which the apparent drug release rate constants (k(apps)) of Suc-chitosan-MMC and CM-chitin-MMC were 3.9 x 10(-3) and 1.1 x 10(-1) (h-1), respectively.

Chitin↗
PubMed1976

Proteolytic activation and inactivation of chitin synthetase from Mucor rouxii.

Crude chitin synthetase preparations from the mycelial and yeast forms of Mucor rouxii behaved differently. The mycelial preparations, incubated at 28 degrees C, lost virtually all chitin synthetase activity in a few hours; by contrast, the activity of enzyme preparations from yeast cells increased several fold during similar incubations. These spontaneous changes were probably caused by endogenous protease(s). Seemingly, the chitin synthetase in yeast preparations was present mainly in a latent, 'zymogenic', form that was activated by proteases. In the mycelial preparations, chitin synthetase was present mainly in an active state and was rapidly degraded by endogenous proteolysis. Exogenous proteases accelerated activation and destruction of chitin synthetase; an acid protease from Rhizopus chinensis was the most effective activator. The activation of chitin synthetase was inhibited by a soluble protein in the cell-free extract. Treatment with the detergent Brij 36T stabilized the chitin synthetase of crude preparations against spontaneous changes. Stabilized preparations were rapidly activated by exogenous proteases. The different behaviour of chitin synthetases in crude extracts of mycelium and yeast cells is consistent with, and perhaps partially responsible for, the differences in wall construction between mycelial and yeast forms of M. rouxii.

Chitin↗
PubMed2025

Adaptive traits for chitin utilization in the saprotrophic aquatic chytrid fungus Rhizoclosmatium globosum.

The Chytridiomycota (chytrids) are early diverging fungi, many of which function in ecosystems as saprotrophs; however, associated adaptive traits are poorly understood. We focused on chitin degradation, a common ecosystem function of aquatic chytrids, using the model chitinophilic Rhizoclosmatium globosum and comparison of other chytrid genomes. Zoospores are chemotactic to the chitin monomer N-acetylglucosamine and accelerate development when grown with chitin. The R. globosum secretome is dominated by different glycoside hydrolase (GH) family GH18 chitinases, with abundance matching reciprocal transcriptome mRNA sequences. Models of the secreted chitinases indicate a range of sizes and domain configurations. Along with R. globosum, the genomes of other chitinophilic chytrids also have expanded inventories of GH-encoding genes responsible for chitin processing. Several R. globosum GH18 chitinases have bacteria-like chitin-binding module domains, also present in the genomes of other chitinophilic chytrids yet absent in non-chitinophilic chytrids. Chemotaxis, increased abundance and diversity of secreted chitinases, complemented with the acquisition of novel chitin-binding capability, are probably adaptive traits that facilitate chitin saprotrophy. Our study reveals the underpinning mechanisms that have supported the niche expansion of some chytrids to utilize lucrative chitin-rich particles in aquatic ecosystems and is a demonstration of the adaptive ability of this successful fungal group.

Chitin↗
PubMed1991

Inhibition of tumor-induced angiogenesis by sulfated chitin derivatives.

The effect of antimetastatic sulfated chitin derivatives (SCM-chitin III) on angiogenesis induced by B16-BL6 cells was examined in syngeneic mice. SCM-chitin III caused a marked decrease of the number of vessels toward tumor mass (angiogenic response) without affecting the tumor cell growth when coinjected with tumor cells (on day 0), or injected into tumor site on day 1 or 3 after tumor inoculation. In contrast, carboxymethyl chitin as well as heparin had no effect. Invasion of endothelial cells through reconstituted basement membrane (Matrigel) toward tumor-conditioned media was significantly inhibited by SCM-chitin III in a Transwell chamber assay. SCM-chitin III also inhibited the haptotactic migration of endothelial cells to fibronectin-substrate, but did not inhibit the chemotactic activity in tumor conditioned media in vitro. SCM-chitin III did not directly affect the viability and the growth of tumor cells and endothelial cells in vitro. These results suggest that inhibition of lung tumor metastasis by SCM-chitin III may in part be due to the inhibition of tumor-associated angiogenesis.

Animals↗
PubMed1992

DIT101 (CSD2, CAL1), a cell cycle-regulated yeast gene required for synthesis of chitin in cell walls and chitosan in spore walls.

A mutant screen has been designed to isolate mutants in Saccharomyces cerevisiae deficient in spore wall dityrosine. As shown by electron microscopy, most of the mutant spores lacked only the outermost, dityrosine-rich layer of the spore wall. Mutant dit101, however, was additionally lacking the chitosan layer of the spore wall. Chemical measurements showed that this mutant does not synthesize chitosan during sporulation. The mutant spores were viable but sensitive to lytic enzymes (glusulase or zymolyase). Unlike most of the dit-mutants, dit101 did show a distinctive phenotype in vegetative cells: they grew normally but contained very little chitin and were therefore resistant to the toxic chitin-binding dye, Calcofluor White. The cells showed barely detectable staining of the walls with Calcofluor White or primulin. The decrease in the amount of chitin in vegetative cells and the absence of chitosan in spores suggested that the mutant dit101 could be defective in a chitin synthase. Indeed, a genomic yeast clone harboring the gene, CSD2, sharing significant sequence similarity with yeast chitin synthases I and II (C. E. Bulawa (1992), Mol. Cell. Biol. 12, 1764-1776), complemented our mutant and was shown to correspond to the chromosomal locus of dit101. Thus, the mutations dit101 and csd2 (and probably also call; M. H. Valdivieso et al., (1991), J. Cell Biol. 114, 101-109) were shown to be allelic. The gene was mapped to chromosome II and was located about 3 kb distal of GAL1. Using this DNA clone, a transcript of about 3500-4000 nucleotides was detected. Comparing RNA isolated from vegetative cells and from sporulating cells at different times throughout the sporulation process, no significant differences in DIT101 transcript levels could be detected indicating absence of sporulation-specific transcriptional regulation. However, the amount of DIT101 transcript changed significantly at different stages of the mitotic cell cycle, peaking after septum formation, but before cytokinesis. As most of the chitin synthesis of vegetative cells occurs at this stage of the cell division cycle, chitin synthesis mediated by DIT101 could be primarily regulated at the level of transcription in vegetatively growing cells.

Cell Cycle↗
PubMed1991

Chitin synthase 1 plays a major role in cell wall biogenesis in Neurospora crassa.

In filamentous fungi, chitin is a structural component of morphologically distinct structures assembled during various phases of growth and development. To investigate the role of chitin synthase in cell wall biogenesis in Neurospora crassa, we cloned a chitin synthase structural gene and examined the consequences of its inactivation. Using degenerate oligonucleotide mixtures designed on the basis of conserved sequences of the Saccharomyces cerevisiae CHS1 and CHS2 polypeptides, a DNA fragment encoding a similar predicted amino acid sequence was amplified from N. crassa genomic DNA. This product was used to probe N. crassa libraries for a gene homologous to one of the yeast genes. Full-length genomic and partial cDNA clones were identified, isolated, and sequenced. The amino acid sequence deduced from a cloned 3.4-kb gene [designated chitin synthase 1 (chs-1)] was very similar to that of the S. cerevisiae CHS1 and CHS2 and the Candida albicans CHS1 polypeptides. Inactivation of the N. crassa chs-1 gene by repeat-induced point mutation produced slow-growing progeny that formed hyphae with morphologic abnormalities. The chs-1RIP phenotype was correlated with a significant reduction in chitin synthase activity. Calcofluor staining of the chs-1RIP strain cross-walls, residual chitin synthase activity, and the increased sensitivity of the chs-1RIP strain to Nikkomycin Z suggest that N. crassa produces additional chitin synthase that can participate in cell wall formation.

Amino Acid Sequence↗
PubMed1975

Timing and function of chitin synthesis in yeast.

A temperature-sensitive mutant of Saccharomyces cerevisiae, L-2-42, is blocked at 37 C at a stage of the cell cycle prior to septum formation. When single cells of the mutant are allowed to bud at 37 C in a medium containing tritiated glucose, a large incorporation of radioactivity into chitin takes place. Thus, the synthesis of chitin, the major component of the primary septum, is initiated in a phase of the cell cycle which precedes septum closure. This early period of chitin synthesis is not required for emergence and growth of buds because, in the wild type, budding takes place normally in the presence of concentrations of polyoxin D that effectively and specifically prevent chitin formation. However, at a later time a majority of these cells lyse, presumably because of the inability to form a septum. Polyoxin D also prevents the appearance of enhanced fluorescence at the junction between mother cell and bud, as observed in the presence of a brightener. Therefore, the fluorescence is due to chitin and its presence at the base of very early buds indicates that chitin synthesis begins at or shortly after bud emergence. A scheme for chitin synthesis and primary septum formation which embodies these and other results is presented.

Anti-Bacterial Agents↗
PubMed1978

Localization of chitin in algal and fungal cell walls by light and electron microscopy.

Chitin was visualized in cell walls after hydrolysis with potassium hydroxide and subsequent postfixation of the deacetylated polysaccharide (chitosan) in OsO4. Areas of chitin deposition appeared dark borwn by light microscopy and electron dense in the electron microscope. With this method, the presence of chitin was demonstrated in the cell walls of the green alga Pithophora oedogonia (Montagne) Wittrock and two fungi, Ceratocystis ulmi Buism. (C. Moreau) and Blastocladiella emersonii Cantino and Hyatt. Most of the chitin in P. oedogonia ws found in the crosswall disk and small amounts occurred in the outer longitudinal walls. The septal disk of C. ulmi also contained chitin, but significant amounts were present in the inner and outer regions of longitudinal walls as well. Chitin was present throughout the walls of B. emersonii. Small amounts of chitin were not easily demonstrated by this technique, but removal of chitosan by exposure to dilute acetic acid before osmium fixation disrupted cell wall integrity, suggesting that small amounts of the structural polysaccharide had been removed.

Ascomycota↗
PubMedDate not supplied

Quantitative determination of carboxymethyl chitin in polymer-coated liposomes.

A simple turbidity method for the quantitative determination of carboxymethyl chitin (CM-chitin) in coated liposomes has been developed. Isopropanol:buffer (1:1 v/v) was selected as a good solvent system for dimyristoyl-, dipalmitoyl-, or egg phosphatidylcholines but not CM-chitin. The turbidity of CM-chitin in this solvent system was spectrophotometrically determined at 400 nm without significant interference by these phosphatidylcholines. The dependence of turbidity on the CM-chitin concentration, pH, and ionic strength were determined. It was found that analyses of the coating efficiency of dipalmitoylphosphatidylcholine liposomes by CM-chitin were reproducible within the specified ranges of formulation conditions and enabled differentiation of the effect of order of CM-chitin addition on the coating efficiency.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1992

A hemagglutinating substance in chitin.

Chitin from crustacean shells has often been used to isolate and purify plant lectins that have an affinity for poly-N-acetylglucosamine (poly-GlcNAc). When we used washed chitin from crab shells as an affinity medium to isolate a lectin from Pinus strobus L. (eastern white pine) ovules, we found that a substance having a strong capacity to agglutinate red blood cells was eluted from the chitin during a weak acid desorption step. The chitin agglutinin is a complex structure containing protein and poly-GlcNAc. Chitin samples from four biochemical suppliers were tested; all contained the elutable agglutinin. Acid (0.05 N HCl or 0.1 N acetic acid) appears to hydrolyze the material from the solid chitin. NaOH at 0.5 N does not remove the agglutinin. Since agglutination is the assay used to monitor lectin purification, care must be taken to avoid the native agglutinin if chitin is used as an affinity matrix.

Acetylglucosamine↗
PubMed1977

Properties of chitin synthetase in isolated chitosomes from yeast cells of Mucor rouxii.

Chitin synthetase was isolated and purified 120-fold from the supernatant fraction (54,500 X g) of broken yeast cells of Mucor rouxii. The purified preparations consisted mainly of chitin synthetase particles (chitosomes) with an average size larger than 7 X 10(6) daltons (by gel filtration) and an average sedimentation coefficient of 105 S. The samples also contained other enzyme complexes (fatty acid synthetase, pyruvate dehydrogenase, and, depending on method, ribosomes). Nearly all of the chitosomal chitin synthetase occurred in a zymogenic form that required proteolytic activation. In most properties, the chitosomal enzyme was similar to crude enzyme (54,000 X g sediment): kinetics, activation by proteases, response to metals, stimulation by N-acetylglucosamine, and inhibition by polyoxin or UDP. One mamor difference was the much greater stability of the chitosomal chitin synthetase zymogen against spontaneous activation and destruction. Product (chitin microfibril) and enzyme (chitin synthetase) remained associated in a complex that was readily separated by centrifugation.

Acetylglucosamine↗
PubMed1979

Chitin structures of the cell walls of synchronously grown virgin cells of Saccharomyces cerevisiae.

The ability of a lytic beta-glucanase of Arthrobacter GJM-1 to dissolve cell walls of Saccharomyces cerevisiae with exception of the chitin-containing fraction was employed for the isolation of chitin-rich residues of the cell walls of synchronously growing populations of virgin cells. Electron microscopical examination of such wall residues isolated from cells at various stages of the budding cycle showed that the first phase of chitin deposition in the wall corresponds to the formation of an annular structure found as a part of the bud scar after cell division. The annular chitin-rich structure could not be isolated at cell cycle stages preceding the bud emergence and at earliest stages of bud development. The observations confirmed that the annular structure (chitin ring) formed during bud growth represents a major part of total chitin present in the bud scar after septum closure.

Arthrobacter↗
PubMed1978

Purification and properties of an inhibitory protein of chitin synthetase from Mucor rouxii.

A soluble protein inhibitor of chitin synthetase (UDP-2-acetamindo-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxyglucosyltransferase, EC 2.4.1.16) was isolated from the cytoplasm of Mucor rouxii. By gel filtration, the molecular weight of the inhibitor was estimated to be 17 500. The inhibitor was effective against crude or purified (chitosome) preparations of chitin synthetase. Unlike the chitin synthesis inhibitor from Saccharomyces spp., the inhibitor from M. rouxii does not operate by blocking the proteolytic activation of chitin synthetase zymogen but by inhibiting the operation of activated enzyme. Presumably, the inhibitor forms part of the regulatory mechanism of chitin synthesis in the cell.

Chitin Synthase↗
PubMed1975

Chitin synthetase zymogen is attached to the yeast plasma membrane.

Pretreatment of yeast protoplasts with concanavalin A, according to the method used by G. A. Scarborough for Neurospora (J. Biol. Chem. 250, 1106-1111, 1975), reinforced the plasma membranes, and helped to maintain their integrity during subsequent lysis of the protoplasts. After purification by centrifuging on a Renografin density gradient, practically intact membranes were obtained. Previous labeling of the protoplasts with 125I or with [3H]concanavalin A resulted in recovery of the radioactivity in the membrane fraction. The bulk of the chitin synthetase (chitin synthase; UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxyglucosyltransferase; EC 2.4.1.16) recovered in the gradient was also found In this fraction; in the zymogen form. About 20% of the activity sedimented in a plasma-membrane-free fraction at lower density. Glutaraldehyde inactivated chitin synthetase when it was added to a lysate, but not when applied to intact protoplasts. It is concluded that chitin synthetase is so oriented in the membrane that it is only accessible from the inside of the cell. These results confirm our previous hypothesis that the chitin synthetase zymogen is associated with the plasma membrane, a basic assumption for the explanation of localized activation of the enzyme and initiation of septum formation.

Cell Fractionation↗