Abstract
InSaccharomyces cerevisiae, most of the cellular chitin is produced by chitin synthase III, which requires the product encoded by theCSD2/CAL1/DIT101/KT12 gene. We have identified, isolated and structurally characterized aCSD2/CAL1/DIT101/KT12 homologue in the filamentous ascomyceteNeurospora crassa and have used a “reverse genetics” approach to determine its role in vivo. The yeast gene was used as a heterologous probe for the isolation of aN. crassa gene (designatedchs-4) encoding a polypeptide belonging to a class of chitin synthases which we have designated class IV. The predicted polypeptide encoded by this gene is highly similar to those ofS. cerevisiae andCandida albicans. N. crassa strains in whichchs-4 had been inactivated by the Repeat-Induced Point mutation (RIP) process grew and developed in a normal manner under standard growth conditions. However, when grown in the presence of sorbose (a carbon source which induces morphological changes accompanied by elevated chitin content), chitin levels in thechs-4 RIP strain were significantly lower than those observed in the wild type. We suggest that CHS4 may serve as an auxiliary enzyme inN. crassa and that, in contrast to yeasts, it is possible that filamentous fungi may have more than one class IV chitin synthase.
Similar content being viewed by others
References
Beth Din A, Yarden O (1994) TheNeurospora crassa chs-2 gene encodes a non-essential chitin synthase. Microbiology 140:2189–2197
Bowen AR, Chen-Wu JL, Momany M, Young R, Szaniszlo PJ, Robbins PW (1992) Classification of fungal chitin synthases. Proc Natl Acad Sci USA 89:519–523
Bulawa CE (1992)CSD2, CSD3, andCSD4, genes required for chitin synthesis inSaccharomyces cerevisiae: theCSD2 gene product is related to chitin synthases and to developmentally regulated proteins inRhizobium species andXenopus laevis. Mol Cell Biol 12:1764–1776
Bulawa CE (1993) Genetics and molecular biology of chitin synthesis in fungi. Annu Rev Microbiol 47:505–534
Bulawa CE, Slater M, Cabib E, Au-Young J, Sburlati A, Adair WL, Robbins PW (1986) TheS. cerevisiae structural gene for chitin synthase in not required for chitin synthesis in vivo. Cell 46:213–225
Burnett JH (1979) Aspects of the structure and growth of hyphal walls. In: Burnett JH, Trinci APJ (eds) Fungal walls and hyphal growth. Cambridge University Press, Cambridge, pp 1–25
Cabib E, Mol PC, Shaw JA, Choi WJ (1993) Biosynthesis of cell wall and septum during yeast growth. Arch Med Res 24:301–303
Chen-Wu JL, Zwicker J, Bowen AR, Robbins PW (1992) Expression of chitin synthase genes during yeast and hyphal growth phases ofCandida albicans. Mol Microbiol 6:497–502
Choi WJ, Santos B, Duran A, Cabib E (1994) Are yeast chitin synthases regulated at the transcriptional or posttranslational level? Mol Cell Biol 14:7685–7694
Davis RH, de Serres FJ (1970) Genetic and microbiological research techniques forNeurospora crassa. Methods Enzymol 17A:79–143
Gold SE, Kronstad JW (1994) Disruption of two genes for chitin synthase in the phytopathogenic fungusUstilago maydis. Mol Microbiol 11:897–902
Hunsley D, Gooday GW (1974) The structure and development of septa inNeurospora crassa. Protoplasma 82:125–146
Kollar R, Petrakova E, Ashwell G, Robbins PW, Cabib E (1995) Architecture of the yeast cell wall — the linkage between chitin and beta (1 → 3)-glucan. J Biol Chem 270:1170–1178
Mahadevan PR, Tatum EL (1965) Relationship of the major constituents of theNeurospora crassa cell wall to wild-type and colonial morphology. J Bacteriol 90:1073–1081
Mehmann B, Brunner I, Braus GH (1994) Nucleotide sequence variation of chitin synthase genes among ectomycorrhizal fungi and its potential use in taxonomy. Appl Environ Microbiol 60:3105–3111
Mellado E, Aufauvre-Brown A, Specht CA, Robbins PW, Holden DW (1995) A multigene family related to chitin synthase genes of yeast in the opportunistic pathogenAspergillus fumigatus. Mol Gen Genet 246:353–359
Metzenberg RL, Stevens JN, Selker EU, Morzycka-Wroblewska E (1985) Identification and chromosomal distribution of 5S rRNA genes inNeurospora crassa. Proc Natl Acad Sci USA 82:2067–2071
Motoyama T, Sudoh M, Horiuchi H, Ohta A, Takagi M (1994a) Isolation and characterization of two chitin synthase gene ofRhizopus oligosporus. Biosci Biotech Biochem 58:1685–1693
Motoyama T, Kojima N, Horiuchi H, Ohta A, Takagi M (1994b) Isolation of a chitin synthase gene (chsC) ofAspergillus nidulens. Biosci Biotech Biochem 58:2254–2257
Orbach MJ, Porro EB, Yanofsky C (1986) Cloning and characterization of the gene forβ-tubulin from a benomyl-resistant mutant ofNeurospora crassa and its use as a dominant selectable marker. Mol Cell Biol 6:2452–2461
Orbach MJ, Sachs MS, Yanofsky C (1990) TheNeurospora crassa arg-2 locus: structure and expression of the gene encoding the small subunit of the arginine-specific carbamoyl phosphate synthethase. J Biol Chem 265:10981–10987
Pammer M, Briza P, Ellinger A, Schuster T, Stucka R, Feldman H, Breitenbach M (1992)DIT101 (CSD2, CAL1), a cell cycle-regulated yeast gene required for synthesis of chitin in cell walls and chitosan in spore walls. Yeast 9:1089–1099
Reissig JL, Stringer JL, Leloir LF (1955) A modified colorimetric method for the estimation of N-acetylamino sugars. J Biol Chem 217:959–966
Robbins PW, Albright C, Benfield B (1988) Cloning and expression of aStreptomyces plicatus chitinase (chitinase-63) inEscherichia coli. J Biol Chem 263:443–447
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Schmidt JC, Edson CM, Brody S (1975) Changes in glucosamine and galactoseamine levels during conidial germination inNeurospora crassa. J Bacteriol 122:1062–1070
Sedmak JJ, Grossbery SE (1977) A rapid sensitive and versatile assay for protein using Coomassie brilliant blue (G-250). Anal Biochem 79:544–552
Selker EU (1990) Premeiotic instability of repeated sequences inNeurospora crassa. Annu Rev Genet 24:579–613
Selker EU, Garrett PW (1988) DNA sequence duplications trigger gene inactivation inNeurospora crassa. Proc Natl Acad Sci USA 85:6870–6874
Shaw JA, Mol PC, Bowers B, Silverman SJ, Valdivieso MH, Duran A, Cabib E (1991) The function of chitin synthases 2 and 3 in theSaccharomyces cerevisiae cell cycle. J Cell Biol 114:111–123
Staben C, Jensen B, Singer M, Pollock J, Schectman M, Kinsey J, Selker E (1989) Use of a bacterial hygromycin B resistance gene as a dominant selectable marker inNeurospora crassa transformation. Fungal Genet Newslett 36:79–81
Sudoh M, Nagahasi S, Doi M, Ohta A, Takagi M, Arisawa M (1993) Cloning of the chitin synthase 3 gene fromCandida albicans and its expression during the yeast-hyphal transition. Mol Gen Genet 241:351–358
Valdivieso MH, Mol PC, Shaw JA, Cabib E, Duran A (1991)CAL1, a gene required for activity of chitin synthase 3 inSaccharomyces cerevisiae. J Cell Biol 114:101–109
Vogel HJ (1956) A convenient growth medium forNeurospora crassa (medium N). Microbiol Genet Bull 13:42–43
Yanai K, Kojima N, Takaya N, Horiuchi H, Ohta A, Takagi M (1994) Isolation and characterization of two chitin synthase genes fromAspergillus nidulans. Biosci Biotech Biochem 58:1828–1835
Yarden O, Yanofsky C (1991) Chitin synthase 1 plays a major role in cell wall biogenesis ofNeurospora crassa. Genes Dev 5:2420–2430
Author information
Authors and Affiliations
Additional information
Communicated by C. A. M. J. J. van den Hondel
A. Beth Din and C. A. Specht contributed equally to this work
Rights and permissions
About this article
Cite this article
Din, A.B., Specht, C.A., Robbins, P.W. et al. chs-4, a class IV chitin synthase gene fromNeurospora crassa . Molec. Gen. Genet. 250, 214–222 (1996). https://doi.org/10.1007/BF02174181
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02174181