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At least 19 recordsLinked to original sources

PubMed1994

The Neurospora crassa chs-2 gene encodes a non-essential chitin synthase.

Chitin is a structural component of morphologically distinct structures assembled during various phases of growth and development in filamentous fungi. In Neurospora crassa, at least three different DNA fragments related to chitin synthase have been identified. In this study we cloned, sequenced and characterized the chitin synthase 2 structural gene (designated chs-2). The amino acid sequence deduced from the cloned chs-2 genomic DNA fragments is very similar to that of chitin synthase genes isolated from other fungi. Inactivation of the N. crassa chs-2 gene by repeat-induced point (RIP) mutation produced progeny which under standard growth conditions were indistinguishable from the wild-type. However, a significant reduction in chitin synthase activity and increased sensitivity to the phosphatidylcholine biosynthesis inhibitor edifenphos are characteristic of the chs-2RIP strain.

Amino Acid Sequence↗

PubMed1990

Chitin synthase I and chitin synthase II are not required for chitin synthesis in vivo in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the polysaccharide chitin forms the primary division septum between mother cell and bud. Two related enzymes, chitin synthase I and chitin synthase II (UDP-acetamido-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxyglucosyltransferase, EC 2.4.1.16), have been identified and their structural genes, CHS1 and CHS2, respectively, have been cloned and sequenced. Gene disruption experiments led to the conclusion that CHS2 is essential for cell division [Silverman, S.J., Sburlati, A., Slater, M.L. & Cabib, E. (1988) Proc. Natl. Acad. Sci. USA 85, 4735-4739], whereas CHS1 is not. We repeated the disruption of CHS2 and determined that it is not essential for vegetative growth. The viability of chs1::HIS3 chs2::TRP1 spores is influenced by strain background and germination conditions. The double disruption mutant has no detectable chitin deficiency in vivo, as judged by quantitative assay and by staining cells with Calcofluor. Assay of membrane preparations from the double disruption mutant indicates the presence of chitin synthetic activity. Unlike the CHS gene products, this third activity is not stimulated by trypsin. Characterization of the double disruption mutant revealed abnormalities in morphology and nuclear migration.

Blotting, Southern↗

PubMed1986

The S. cerevisiae structural gene for chitin synthase is not required for chitin synthesis in vivo.

The chitin synthase of Saccharomyces is a plasma membrane-bound zymogen. Following proteolytic activation, the enzyme synthesizes insoluble chitin that has chain length and other physical properties similar to chitin found in bud scars. We isolated mutants lacking chitin synthase activity (chs1) and used these to clone CHS1. The gene has an open reading frame of 3400 bases and encodes a protein of 130 kd. The fission yeast S. pombe lacks chitin synthase and chitin. When a plasmid encoding a CHS1-lacZ fusion protein is introduced into S. pombe, both enzymatic activities are expressed in the same ratio as in S. cerevisiae, demonstrating that CHS1 encodes the structural gene of chitin synthase. Three CHS1 gene disruption experiments were performed. In all cases, strains with the disrupted gene have a recognizable phenotype, lack measurable chitin synthase activity in vitro but are viable, contain normal levels of chitin in vivo, and mate and sporulate efficiently.

Amino Acid Sequence↗

PubMed1993

Identification of a Saccharomyces cerevisiae mutation that allows cells to grow without chitin synthase 1 or 2.

Chitin is a component of the yeast cell wall which is localized to the septum between mother and daughter cells. Previous work in Saccharomyces cerevisiae has shown that this organism possesses three chitin synthases, 1, 2, and 3. Disruption experiments have shown that loss of chitin synthase 2 has a more profound effect on cell viability than loss of either of the other two and is lethal in complete media. We report here the finding of an S. cerevisiae strain which does not require the chitin synthase 2 structural gene for viability. We present evidence that there is a gene in this strain which suppresses the lethality of disruption of the chitin synthase 2 structural gene and is genetically distinct from the structural genes for chitin synthase 1 and chitin synthase 2. We show that an S. cerevisiae mutant containing the suppressor and lacking both structural genes for chitin synthase 1 and 2 has normal amounts of chitin in its cell wall. We hypothesize that the suppressor gene encodes or controls the expression of chitin synthase 3.

Antifungal Agents↗

PubMed1987

Chitin synthase activity and the rate of chitin formation in cell-division cycle mutant Saccharomyces cerevisiae cdc 24.

At the nonpermissive temperature (37 degrees C) the cells of the temperature-sensitive mutant Saccharomyces cerevisiae cdc 24 accumulated chitin 10 times faster than at 22 degrees C. In situ determinations of the activity of chitin synthase revealed that in the cells grown at 37 degrees C more than 37% of the total chitin synthase were in the active state whereas in cells grown at 22 degrees C only 7% of the potential enzyme activity were expressed. When the enzyme activity was calculated per cell number unit, there was 10 times more of the active chitin synthase per cell in the cells grown at 37 degrees C than in the cells grown at 22 degrees C, a value which correlated well with the observed difference in the rates of chitin accumulation at different temperatures.

Chitin↗

PubMed1994

Characterization of chitin synthase from Botrytis cinerea.

Chitin synthase in a microsomal preparation from Botrytis cinerea had an apparent Km for UDP-N-acetylglucosamine of 2.0 mM while nikkomycin Z and polyoxin D inhibited enzyme activity competitively with apparent Ki values of approximately 0.1 microM and 6 microM respectively. The organophosphorus fungicide edifenphos was a non-competitive inhibitor (Ki(app) 54 microM). Preincubation of microsomes for 2 h at 25 degrees C resulted in a maximum twofold stimulation of chitin synthase activity while preincubation with trypsin (25 micrograms ml-1) or cytosol (350 micrograms cytosolic protein ml-1) for 10 min at 25 degrees C resulted in approximately fourfold and 20-fold increases in chitin synthase activity, respectively. A range of protease inhibitors reduced the degree of activation of microsomal chitin synthase by cytosol. Most potent were phenylmethanesulphonyl fluoride and chymostatin; these compounds completely inhibited activation of enzyme activity. Two fragments (approx. 600 bp; CHS1 and CHS2) were amplified from B. cinerea genomic DNA using degenerate PCR primers based on regions of complete amino acid homology between previously published chitin synthase gene sequences. When the DNA and predicted amino acid sequences of CHS1 were used to probe computer databases for related sequences, B. cinerea CHS1 was found to be most similar to CHS1 from Neurospora crassa.

Amino Acid Sequence↗

PubMed1989

Chitin synthase 1, an auxiliary enzyme for chitin synthesis in Saccharomyces cerevisiae.

Previously, we showed that chitin synthase 2 (Chs2) is required for septum formation in Saccharomyces cerevisiae, whereas chitin synthase 1 (Chs1) does not appear to be an essential enzyme. However, in strains carrying a disrupted CHS1 gene, frequent lysis of buds is observed. Lysis occurs after nuclear separation and appears to result from damage to the cell wall, as indicated by osmotic stabilization and by a approximately 50-nm orifice at the center of the birth scar. Lysis occurs at a low pH and is prevented by buffering the medium above pH 5. A likely candidate for the lytic system is a previously described chitinase that is probably involved in cell separation. The chitinase has a very acidic pH optimum and a location in the periplasmic space that exposes it to external pH. Accordingly, allosamidin, a specific chitinase inhibitor, substantially reduced the number of lysed cells. Because the presence of Chs1 in the cell abolishes lysis, it is concluded that damage to the cell wall is caused by excessive chitinase activity at acidic pH, which can normally be repaired through chitin synthesis by Chs1. The latter emerges as an auxiliary or emergency enzyme. Other experiments suggest that both Chs1 and Chs2 collaborate in the repair synthesis of chitin, whereas Chs1 cannot substitute for Chs2 in septum formation.

Acetylglucosamine↗

PubMed1995

Cloning of two chitin synthase gene fragments from a protoplastic entomophthorale.

Chitin synthase expression was studied in spontaneously produced protoplasts and in hyphal bodies of the Entomophthorale species Entomophaga aulicae. The absence of wall in protoplasts was correlated to an absence of chitin synthase. Two chitin synthase activities with different biochemical characteristics have been detected in the hyphal bodies. Two chitin synthase gene fragments EaCHS1 and EaCHS2 of 600 bp were obtained using PCR amplification of genomic DNA. Their amino acid sequences showed 75% identity. Compared with other fungal chitin synthases, they belong to class II. EaCHS1 and EaCHS2 were used to probe total RNA from E. aulicae hyphal bodies and protoplasts. A single transcript of 2.4 kb hybridized only with EaCHS1 in protoplasts and hyphal bodies.

Amino Acid Sequence↗

PubMed1987

Two chitin synthases in Saccharomyces cerevisiae.

Disruption of the yeast CHS1 gene, which encodes trypsin-activable chitin synthase I, yielded strains that apparently lacked chitin synthase activity in vitro, yet contained normal levels of chitin (Bulawa, C. E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, W. L., and Robbins, P. W. (1986) Cell 46, 213-225). It is shown here that disrupted (chs1 :: URA3) strains have a particulate chitin synthetic activity, chitin synthase II, and that wild type strains, in addition to chitin synthase I, have this second activity. Chitin synthase II is measured in wild type strains without preincubation with trypsin, the condition under which highest chitin synthase II activities are obtained in extracts from the chs1 :: URA3 strain. Chitin synthase II, like chitin synthase I, uses UDP-GlcNAc as substrate and synthesizes alkali-insoluble chitin (with a chain length of about 170 residues). The enzymes are equally sensitive to the competitive inhibitor Polyoxin D. The two chitin synthases are distinct in their pH and temperature optima, and in their responses to trypsin, digitonin, N-acetyl-D-glucosamine, and Co2+. In contrast to the report by Sburlati and Cabib (Sburlati, A., and Cabib, E. (1986) Fed. Proc. 45, 1909), chitin synthase II activity in vitro is usually lowered on treatment with trypsin, indicating that chitin synthase II is not activated by proteolysis. Chitin synthase II shows highest specific activities in extracts from logarithmically growing cultures, whereas chitin synthase I, whether from growing or stationary phase cultures, is only measurable after trypsin treatment, and levels of the zymogen do not change. Chitin synthase I is not required for alpha-mating pheromone-induced chitin synthesis in MATa cells, yet levels of chitin synthase I zymogen double in alpha factor-treated cultures. Specific chitin synthase II activities do not change in pheromone-treated cultures. It is proposed that of yeast's two chitin synthases, chitin synthase II is responsible for chitin synthesis in vivo, whereas nonessential chitin synthase I, detectable in vitro only after trypsin treatment, may not normally be active in vivo.

Cations, Divalent↗

PubMed1992

Classification of fungal chitin synthases.

Comparison of the chitin synthase genes of Saccharomyces cerevisiae CHS1 and CHS2 with the Candida albicans CHS1 gene (UDP-N-acetyl-D-glucosamine:chitin 4-beta-N-acetylglucosaminyltransferase, EC 2.4.1.16) revealed two small regions of complete amino acid sequence conservation that were used to design PCR primers. Fragments homologous to chitin synthase (approximately 600 base pairs) were amplified from the genomic DNA of 14 fungal species. These fragments were sequenced, and their deduced amino acid sequences were aligned. With the exception of S. cerevisiae CHS1, the sequences fell into three distinct classes, which could represent separate functional groups. Within each class phylogenetic analysis was performed. Although not the major purpose of the investigation, this analysis tends to confirm some relationships consistent with current taxonomic groupings.

Amino Acid Sequence↗

PubMed2026

Cell wall-forming chitin synthases in a chytrid fungus.

Chitin is a critical structural component of fungal cell walls, yet our understanding of its synthesis across the kingdom Fungi remains limited. Here, we investigate chitin synthase diversity, transcription and localisation in the aquatic saprotrophic chytrid Rhizoclosmatium globosum, expanding insights into fungal cell wall biology beyond Dikaryan models. We identified 20 chitin synthase genes in the R. globosum genome, mostly canonical Division I and II types with conserved functional motifs. Transcriptomic analysis through zoospore, germling and immature thallus developmental stages revealed stage-specific expression patterns, with active gene diversity correlating with increasing morphological complexity. Using electroporation-based transformation and fluorescent fusion constructs, we indicate expression and localisation of two chitin synthases during cell development. Localisation patterns suggest dynamic redistribution from cytoplasmic dispersion in early encysted cells to concentrated signals at the sporangium wall. Expression in and around the apophysis indicates the importance of these structures in cell maintenance. Our findings highlight functional specialisation among chitin synthases and underscore the importance of cell wall integrity in chytrid development. This work establishes R. globosum as a genetically tractable model for studying chytrid cell biology and contributes to a broader understanding of fungal evolution and cell wall dynamics.

Biotechnology↗

PubMed1994

Isolation and characterization of two chitin synthase genes of Rhizopus oligosporus.

Two chitin synthase genes (chs1 and chs2) were isolated from Rhizopus oligosporus by plaque hybridization probed with the chitin synthase 2 gene of Saccharomyces cerevisiae. From their deduced amino acid sequences, they were both class II chitin synthases according to the classification proposed by Bowen et al. The expression of these genes was controlled differently in each stage of differentiation. It was suggested that the gene products of chs1 and chs2 function mainly in the hyphae growing stage but not in the late stage of spore formation. When each of these genes was expressed in S. cerevisiae, elevation of chitin synthase activity was observed in both cases.

Amino Acid Sequence↗

PubMed1994

Isolation and characterization of two chitin synthase genes from Aspergillus nidulans.

Two chitin synthase genes, designated chsA and chsB, were isolated from Aspergillus nidulans with the Saccharomyces cerevisiae CHS2 gene as the hybridization probe. Nucleotide sequencing showed that chsA and chsB encoded polypeptides consisting of 1013 and 916 amino acid residues, respectively; the hydropathy profiles of the enzymes were similar to those of other fungal chitin synthases. Northern analysis indicated that both genes were transcribed, suggesting that cellular chitin in A. nidulans is synthesized by at least two chitin synthases. For examination of the roles of the chitin synthase genes in cell growth, gene disruption experiments were done. The chsA disruptant grew as well as the wild-type strain, but the chsB disruptant had severe growth defects that could not be overcome by the addition of 1.2M sorbitol as an osmotic stabilizer. These findings suggested that chsB but not chsA is essential for hyphal growth.

Amino Acid Sequence↗

PubMed1993

Purification and characterization of membrane-bound chitin synthase.

The membrane-bound chitin synthase, a key enzyme of chitin biosynthesis, was purified, for the first time to homogeneity as a zymogen form. Digitonin could solubilize the enzyme from microsomal fraction of the filamentous fungus Absidia glauca, with 60-70% of the enzyme activity. The solubilized form of the enzyme was effectively purified by a sequence of chelating Sepharose, concanavalin A-Sepharose, and Mono Q column. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme gave a single band with a molecular weight of 30,000. IgG prepared against this 30-kDa species on SDS-polyacrylamide gel electrophoresis immunoprecipitated chitin synthase. The purified enzyme existed as a zymogen, was converted into active form by treatment with trypsin, and the limited digestion with trypsin produced a little smaller polypeptide (28.5 kDa) of which the amino-terminal sequence was identical to the zymogen. The purified enzyme was the glycoprotein and showed a requirement for Mg2+. N-Acetylglucosamine stimulated the enzyme activity approximately 5-fold and polyoxin D, an analogue of substrate, and UDP, a byproduct of enzyme reaction, strongly inhibited the enzyme activity.

Amino Acid Sequence↗

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↗

PubMed1990

Isolation of a chitin synthase gene (CHS1) from Candida albicans by expression in Saccharomyces cerevisiae.

Chitin synthase activity was studied in yeast and hyphal forms of Candida albicans. pH-activity profiles showed that yeast and hyphae contain a protease-dependent activity that has an optimum at pH 6.8. In addition, there is an activity that is not activated by proteolysis in vitro and which shows a peak at pH 8.0. This suggests there are two distinct chitin synthases in C. albicans. A gene for chitin synthase from C. albicans (CHS1) was cloned by heterologous expression in a Saccharomyces cerevisiae chs1 mutant. Proof that the cloned chitin synthase is a C. albicans membrane-bound zymogen capable of chitin biosynthesis in vitro was based on several criteria. (i) the CHS1 gene complemented the S. cerevisiae chs1 mutation and encoded enzymatic activity which was stimulated by partial proteolysis; (ii) the enzyme catalyses incorporation of [14C]-GlcNAc from the substrate, UDP[U-14C]-GlcNAc, into alkali-insoluble chitin; (iii) Southern analysis showed hybridization of a C. albicans CHS1 probe only with C. albicans DNA and not with S. cerevisiae DNA; (iv) pH profiles of the cloned enzyme showed an optimum at pH 6.8. This overlaps with the pH-activity profiles for chitin synthase measured in yeast and hyphal forms of C. albicans. Thus, CHS1 encodes only part of the chitin synthase activity in C. albicans. A gene for a second chitin synthase in C. albicans with a pH optimum at 8.0 is proposed. DNA sequencing revealed an open reading frame of 2328 nucleotides which predicts a polypeptide of Mr 88,281 with 776 amino acids. The alignment of derived amino acid sequences revealed that the CHS1 gene from C. albicans (canCHS1) is homologous (37% amino acid identity) to the CHS1 gene from S. cerevisiae (sacCHS1).

Amino Acid Sequence↗

PubMed1981

Effects of amphotericin B, nystatin, and other polyene antibiotics on chitin synthase.

The effects of amphotericin B (AmB), nystatin, filipin, and pimaricin were tested chitin synthase (EC 2.4.1.16) (chitosomes from yeast cells of Mucor rouxii). AmB and nystatin inhibited the enzyme at concentrations greater than or equal to 10 micrograms/ml, filipin was weakly inhibitory, and pimaricin had no effect. The inhibition of chitin synthase by AmB appears to be noncompetitive, with a Ki value of about 0.13 mM. the effect of nystatin was more complex and included a sharp stimulation of chitin synthase activity at approximately 50 micrograms/ml. Our findings suggest the existence of binding sites (sterols?) on the chitosome that are selective for certain polyenes and that play a role in the operation of chitin synthase. Because the minimal growth inhibitory concentrations of AmB or nystatin are lower than the concentrations that inhibit chitin synthase in vitro, the possibility of chitosomal chitin synthase being a primary target for the antifungal action of these polyenes seems unlikely.

Amphotericin B↗

PubMed1994

Nikkomycin Z is a specific inhibitor of Saccharomyces cerevisiae chitin synthase isozyme Chs3 in vitro and in vivo.

Nikkomycin Z inhibits chitin synthase in vitro but does not exhibit antifungal activity against many pathogens. Assays of chitin synthase isozymes and growth assays with isozyme mutants were used to demonstrate that nikkomycin Z is a selective inhibitor of chitin synthase 3. The resistance of chitin synthase 2 to nikkomycin Z in vitro is likely responsible for the poor activity of this antibiotic against Saccharomyces cerevisiae.

Aminoglycosides↗