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PubMed2002

Immunolocalization of chitin synthase in the tobacco hornworm.

To start investigation of chitin synthesis and peritrophic membrane formation in the midgut of Manduca sexta, we have cloned a cDNA fragment encoding chitin synthase. Northern blots with a corresponding RNA probe revealed a single transcript of 4.7 kb, which was most prominent in poly(A) RNA isolated from the anterior and median midgut as well as from tracheal cells. In situ hybridization showed that the amount of chitin synthase transcripts in the cytoplasm of columnar cells decreased from the anterior to the posterior midgut. Moreover, in the anterior midgut they were localized in the apical region of columnar cells. Southern blots suggested more than one gene locus for chitin synthase in the Manducagenome. To analyze the distribution of chitin synthases on the protein level, we expressed a polymerase chain reaction (PCR) fragment of 119 amino acids in Escherichia coli and generated polyclonal antibodies to the purified recombinant protein. In immunoblots of crude extracts derived from the anterior midgut as well as from partially purified brush border membranes of columnar cells the affinity-purified anti-chitin synthase antiserum labeled a single protein with an apparent molecular mass of 150-200 kDa. Immunohistochemistry showed intense labeling in midgut brush border membranes. Immunofluorescence was restricted to the apical ends of microvilli. Apical membranes of salivary glands and tracheal cells were labeled as well, but not those of Malpighian tubules. This is the first time that chitin synthase expression has been visualized in insect tissues on the level of proteins.

Amino Acid Sequence↗
PubMed1998

Identification of four chitin synthase genes in the rice blast disease agent Magnaporthe grisea.

Four chitin synthase gene fragments were isolated from Magnaporthe grisea by use of polymerase chain reaction. A pair of degenerate primers based on conserved zymogen-type chitin synthase sequences amplified a approximately 600-bp product containing three chitin synthase gene fragments. A second pair of degenerate primers, based on conserved sequences between the chitin synthases 3 of Saccharomyces cerevisiae and Candida albicans, amplified a single 770-bp fragment. The four corresponding amino acid sequences each fell into one of the chitin synthase classes I-IV, as deduced from sequence analysis. Northern analysis demonstrated that the class II gene was expressed transiently in early phases of growth, whereas the class I and III genes as well as the class IV gene were expressed throughout the entire life cycle. Furthermore, the class III gene was the most expressed.

Amino Acid Sequence↗
PubMed2004

Role of chitin synthase genes in Fusarium oxysporum.

Three structural chitin synthase genes, chs1, chs2 and chs3, were identified in the genome of Fusarium oxysporum f. sp. lycopersici, a soilborne pathogen causing vascular wilt disease in tomato plants. Based on amino acid identities with related fungal species, chs1, chs2 and chs3 encode structural chitin synthases (CSs) of class I, class II and class III, respectively. A gene (chs7) encoding a chaperone-like protein was identified by comparison of the deduced protein with Chs7p from Saccharomyces cerevisiae, an endoplasmic reticulum (ER) protein required for the export of ScChs3p (class IV) from the ER. So far no CS gene belonging to class IV has been isolated from F. oxysporum, although it probably contains more than one gene of this class, based on the genome data of the closely related species Fusarium graminearum. F. oxysporum chs1-, chs2- and chs7-deficient mutants were constructed through targeted gene disruption by homologous recombination. No compensatory mechanism seems to exist between the CS genes studied, since chitin content determination and expression analysis of the chs genes showed no differences between the disruption mutants and the wild-type strain. By fluorescence microscopy using Calcofluor white and DAPI staining, the wild-type strain and Deltachs2 and Deltachs7 mutants showed similar septation and even nuclear distribution, with each hyphal compartment containing only one nucleus, whereas the Deltachs1 mutant showed compartments containing up to four nuclei. Pathogenicity assays on tomato plants indicated reduced virulence of Deltachs2 and Deltachs7 null mutants. Stress conditions affected normal development in Deltachs2 but not in Deltachs1 or Deltachs7 disruptants, and the three chs-deficient mutants showed increased hyphal hydrophobicity compared to the wild-type strain when grown in sorbitol-containing medium. The chitin synthase mutants will be useful for elucidating cell wall biogenesis in F. oxysporum and the relationship between fungal cell wall integrity and pathogenicity.

Amino Acid Sequence↗
PubMed1996

The localization of chitin synthase in membranous vesicles (chitosomes) in Neurospora crassa.

Polyclonal anti-chitin synthase antibodies raised against the Saccharomyces cerevisiae CHS2 gene product were used to identify and localize chitin synthase in the filamentous ascomycete Neurospora crassa. A single band of approximately 110 kDa was observed in Western blots of total protein extracts of N. crassa, probed with these antibodies. However, several additional bands were labelled when membrane fraction proteins (microsomes) were probed. Histo-immunochemical localization of chitin synthase confirmed that the polypeptide is compartmentalized in membranous vesicles (chitosomes), which are abundant in the vicinity of the hyphal tip. TEM analysis did not reveal chitin synthase in the plasma membrane. However, dense labelling of membrane-associated chitin synthase was observed by light-microscopic analysis of N. crassa protoplasts and at young hyphal tips.

Antibodies, Fungal↗
PubMed2004

Independent regulation of chitin synthase and chitinase activity in Candida albicans and Saccharomyces cerevisiae.

Chitin is an essential structural polysaccharide in fungi that is required for cell shape and morphogenesis. One model for wall synthesis at the growing cell surface suggests that the compliance that is necessary for turgor-driven expansion of the cell wall involves a delicate balance of wall synthesis and lysis. Accordingly, de novo chitin synthesis may involve coordinated regulation of members of the CHS chitin synthase and CHT chitinase gene families. To test this hypothesis, the chitin synthase and chitinase activities of cell-free extracts were measured, as well as the chitin content of cell walls isolated from isogenic mutant strains that contained single or multiple knock-outs in members of these two gene families, in both Candida albicans and Saccharomyces cerevisiae. However, deletion of chitinase genes did not markedly affect specific chitin synthase activity, and deletion of single CHS genes had little effect on in vitro specific chitinase activity in either fungus. Chitin synthesis and chitinase production was, however, regulated in C. albicans during yeast-hypha morphogenesis. In C. albicans, the total specific activities of both chitin synthase and chitinase were higher in the hyphal form, which was attributable mainly to the activities of Chs2 and Cht3, respectively. It appeared, therefore, that chitin synthesis and hydrolysis were not coupled, but that both were regulated during yeast-hypha morphogenesis in C. albicans.

Benzenesulfonates↗
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↗
PubMed2006

Survey and expression analysis of five new chitin synthase genes in the biotrophic rust fungus Puccinia graminis.

We have isolated and characterised the first set of chitin synthase genes from a rust fungus, a large group of economically highly important, obligately biotrophic plant pathogens. Puccinia graminis was used as a model organism for the rust fungi which are not well investigated on the molecular level today. One of the major structural components of most fungal cell walls is the chitin polymer which is synthesised by a family of enzymes called chitin synthases. In P. graminis, we have isolated five new chitin synthase genes from four different classes, chsII, chsIIIa, chsIIIb, chsIV, and chsV. The genes contain a high number of introns, unusual for other known fungal chitin synthases. The dinucleic stage of the fungus seems to contain two slightly different genes or alleles for four isoforms. One isoform, chsIIIa, seems to be expressed only in the youngest stages of fungal growth. Analysis of the derived proteins shows that together with other basidiomycete CHS, the pgtCHS form separate subgroups in the phylogenetic tree. This set of five rust chitin synthase genes, with some unusual features compared to known fungal chitin synthases, allows new insights into chitin synthase classification, and may help in the development of novel functional fungicides.

Basidiomycota↗
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↗
PubMed1979

Chitin synthase activity from the slime variant of Neurospora crassa.

Chitin synthase (UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxy-D-glucosyltransferase, EC 2.4.1.16) activity from the wall-less variant of Neurospora crassa (slime) was partially characterized. The slime enzyme activity was found to be similar to that reported for slime-like and wild-type chitin synthase activities with respect to the following: specific activity, particulate cell-fraction localization, activation by N-acetylglucosamine, apparent Km with respect to substrate, pH optimum and ion requirement. It appears that the phenotype of slime cannot be solely accounted for by the absence of chitin synthase enzyme activity.

Acetylglucosamine↗
PubMed2000

WdCHS3, a gene that encodes a class III chitin synthase in Wangiella (Exophiala) dermatitidis, is expressed differentially under stress conditions.

Class III chitin synthases are important for hyphal growth in some filamentous fungi but are not found in yeasts. Using a specific PCR product that encodes a portion of the class III chitin synthase of W. dermatitidis as a probe, we isolated the chitin synthase gene, WdCHS3, from this polymorphic melanized pathogen of humans. Northern blotting showed that WdCHS3 was highly expressed under stress conditions, such as the shift of cells to temperatures commensurate with infection, or to conditions that induce cellular morphogenesis in this fungus. Analysis of the 5' upstream sequence of WdCHS3 provided evidence for a negative regulatory element at between -780 and -1600 bp. Western blotting indicated that the production of the WdChs3p was temperature dependent and temporally regulated. Disruption of WdCHS3 in a wild-type strain and in two temperature-sensitive morphological mutants resulted in significantly reduced chitin synthase activities but did not obviously affect their morphologies, growth rates, chitin contents, or virulence. This paradox suggested that the contributions of the high levels of WdCHS3 gene expression and WdChs3p production in strains subjected to stress reside in unknown or unexamined parts of the life cycle of this ecologically poorly known member of the Fungi Imperfecti. Nonetheless, this report presents the first evidence that transcription of a chitin synthase gene is regulated by a negative regulatory element in its 5' upstream sequence.

Amino Acid Sequence↗
PubMed2006

Immunolocalization of chitin synthases in the phytopathogenic dimorphic fungus Ustilago maydis.

Conserved polypeptides of the chitin synthase genes UmCHS3 and UmCHS6 from the phytopathogenic fungus Ustilago maydis were utilized as immunogens to obtain polyclonal antibodies that were purified by affinity procedures. Because of their similarities at the regions encoded by either polypeptide, it was concluded that anti-Chs3 antibodies recognized both Chs3 and Chs4 chitin synthases, whereas anti-Chs6 antibodies recognized Chs6 and Chs8 polypeptides. These antibodies were used to analyze the localization of the corresponding chitin synthases in U. maydis cells, using both indirect immunofluorescence microscopy and immunoelectron microscopy with colloidal-gold-labeled secondary antibodies. It was observed that chitin synthase proteins were accumulated both in the surface and in the cytoplasm of the fungal cells. Electron microscopy images revealed the accumulation of clusters of gold particles in vesicles, providing evidence for the possible origin and destination of chitin synthases in the fungal cells.

Centrifugation, Density Gradient↗
PubMed2002

The zygomycetous fungus, Benjaminiella poitrasii contains a large family of differentially regulated chitin synthase genes.

Benjaminiella poitrasii is a zygomycetous, non-pathogenic dimorphic fungus. Chitin synthases are the membrane bound enzymes involved in the synthesis of chitin and are key enzymes in the cell wall metabolism. Multiplicity of these enzymes is a common occurrence. Here, we identify eight distinct CHS genes in B. poitrasii as confirmed through DNA sequence and Southern analysis. These genes are related to other fungal CHS genes. BpCHS1-4 are class I-III chitin synthases while BpCHS5-8 are class IV-V chitin synthases. These eight genes are differentially expressed during morphogenesis and under different growth conditions. Two of these genes viz. BpCHS2 and BpCHS3 appear to be specific to the mycelial growth form. These are the first B. poitrasii sequences to be reported. Based on CHS gene sequences, B. poitrasii chitin synthase genes place it with other zygomycetes on a fungal phylogenetic tree.

Amino Acid Sequence↗
PubMed1997

A putative role for calmodulin in the activation of Neurospora crassa chitin synthase.

The possible role of calmodulin in cell wall formation and chitin synthesis was studied in Neurospora crassa by examining the effects of anti-calmodulin agents on protoplast regeneration and possible associations between chitin synthase and calmodulin related proteins in microsomal isolates. Protoplast regeneration was inhibited by trifluoperazine (> 20 microM), an anticalmodulin agent. Chitin synthase activity in microsomes was associated with that of calmodulin-dependent protein kinase and inhibited by trifluoperazine (100 microM). In vitro activity of chitin synthase was enhanced upon inclusion of calmodulin (300 ng) in the assay mix, 63% over and above the stimulation brought about by trypsin, an activator of the enzyme. Autoradiography studies on microsomal proteins revealed calmodulin-dependent phosphorylation of two microsomal calmodulin-binding proteins (106 and 89 kDa). The results indicate that calmodulin-mediated phosphorylation of specific microsomal proteins may be important in the in vivo activation of chitin synthase.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed2002

Altering the expression of two chitin synthase genes differentially affects the growth and morphology of Aspergillus oryzae.

In Aspergillus oryzae, one full-length chitin synthase (chsB) and fragments of two other chitin synthases (csmA and chsC) were identified. The deduced amino acid sequence of chsB was similar (87% identity) to chsB from Aspergillus nidulans, which encodes a class III chitin synthase. The sequence obtained for csmA indicated that it had high similarity to class V chitin synthases. chsB and csmA disruption strains and a strain in which chsB transcription was controlled were constructed using the nitrite reductase (niiA) promoter. The strains were examined during hyphal growth by Northern analysis, analysis of the cell-wall composition and growth in the presence of Calcofluor white (CFW). The chsB disrupted strain and the uninduced p(niiA)-chsB strain exhibited hyperbranching, they had a lower level of conidiation than the wild-type and were sensitive to CFW at 50 mg l(-1). When chsB transcription was induced in the strain containing the p(niiA)-chsB construct, the strain displayed wild-type morphology on solid medium and at sub-maximum growth rates but the wild-type morphology was not fully restored during rapid growth in batch cultivation. The csmA disruption strain displayed morphological abnormalities, such as ballooning cells, intrahyphal hyphae and conidial scars. The growth was severely inhibited in the presence of 10 mg CFW l(-1). In none of the constructed strains did the cell-wall composition differ from the wild-type. Northern analysis indicated no change in the transcription of the chitin synthase genes csmA and chsC when chsB expression was altered, and there was no change in the transcription of chsB and chsC when csmA was disrupted.

Aspergillus oryzae↗
PubMed1988

Chitin synthase 2 is essential for septum formation and cell division in Saccharomyces cerevisiae.

Previous work led to the puzzling conclusion that chitin synthase 1, the major chitin synthase activity in Saccharomyces cerevisiae, is not required for synthesis of the chitinous primary septum. The mechanism of in vivo synthesis of chitin has now been clarified by cloning the structural gene for the newly found chitin synthase 2, a relatively minor activity in yeast. Disruption of the chitin synthase 2 gene results in the loss of well-defined septa and in growth arrest, establishing that the gene product is essential for both septum formation and cell division.

Cell Division↗
PubMed2001

Inhibitory activity for chitin synthase II from Saccharomyces cerevisiae by tannins and related compounds.

In the course of search for potent inhibitors of chitin synthase II from natural resources, seven tannins and related compounds were isolated from the aerial part of Euphorbia pekinensis and identified as gallic acid (1), methyl gallate (2), 3-O-galloyl-(-)-shikimic acid (3), corilagin (4), geraniin (5), quercetin-3-O-(2"-O-galloyl)-beta-D-glucoside (6), and kaempferol-3-O-(2"-O-galloyl)-beta-D-glucoside (7). These and nine related compounds, (-)-quinic acid (8), (-)-shikimic acid (9), ellagic acid (10), kaempferol (11), quercetin (12), quercitrin (13), rutin (14), quercetin-3-O-(2"-O-galloyl)-beta-D-rutinoside (15) and 1,3,4,6-tetra-O-galloyl-beta-D-glucose (16), were evaluated for the inhibitory activity against chitin synthase II and III. They inhibited chitin synthase II with IC(50) values of 18-206 microM, except for two organic acids, (-)-quinic acid (8) and (-)-shikimic acid (9). Among them, 3-O-galloyl-(-)-shikimic acid (3) was the most potent inhibitor against chitin synthase II of Saccharomyces cerevisiae with an IC(50) value of 18 microM. The inhibition appears to be selective for chitin synthase II, as they did not appreciably inhibit chitin synthase III.

Antifungal Agents↗