Current Microbiology

, Volume 53, Issue 3, pp 217–221 | Cite as

Isolation, Characterization, and Transcriptional Analysis of the Chitinase chi2 Gene (DQ011663) from the Biocontrol Fungus Metarhizium anisopliae var. anisopliae

  • César Milton Baratto
  • Valéria Dutra
  • Juliano Tomazzoni Boldo
  • Leonardo Barbosa Leiria
  • Marilene Henning Vainstein
  • Augusto Schrank
Article

Abstract

Metarhizium anisopliae infects arthropods via a combination of specialized structures and cuticle degradation. Hydrolytic enzymes are accepted as key factors for the host penetration step and include chitinases. The characterization of the chi2 chitinase gene from M. anisopliae var. anisopliae is reported. The chi2 gene is interrupted by two short introns and is 1,542-bp long, coding a predicted protein of 419 amino acids with a stretch of 19 amino acid residues displaying characteristics of signal peptide. The predicted chitinase molecular mass is 44 kDa with a mature protein of 42 kDa and a theoretical pI of 4.8. The comparison of the CHI2 predicted protein to fungal orthologues revealed similarity to the glycohydrolase family 18 and a phylogenetic analysis was conducted. The chi2 gene is up-regulated by chitin as a carbon source and in conditions of fungus autolysis, and is down-regulated by glucose. This regulation is consistent with the presence of putative CreA/Crel/Crr1 carbon catabolic repressor binding domains on the regulatory sequence.

Notes

Acknowledgments

This work was supported by FAPERGS (Fundação de Amparo a Pesquisa do Estado do Rio Grande do Sul), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), PADCT (Programa de Apoio ao Desenvolvimento Científico e Tecnológico), and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior). We thank Irene Schrank for a critical reading of the manuscript and Giancarlo Pasqualli for the use of sequencing facilities.

Literature Cited

  1. 1.
    Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedCrossRefGoogle Scholar
  2. 2.
    Baratto CM, da Silva MV, Santi L, Passaglia L, Schrank IS, Vainstein MH, Schrank A (2003) Expression and characterization of the 42 kDa chitinase of the biocontrol fungus Metarhizium anisopliae in Escherichia coli. Can J Microbiol 49:723–726PubMedCrossRefGoogle Scholar
  3. 3.
    Barreto CC, Staats CC, Schrank A, Vainstein MH (2004) Distribution of chitinases in the entomopathogen Metarhizium anisopliae and effect of N-acetylglucosamine in protein secretion. Curr Microbiol 48:102–107PubMedCrossRefGoogle Scholar
  4. 4.
    Bogo MR, Queiroz MV, Silva DM, Gimenez Pecci MP, Azevedo JL, Schrank A (1996) Double-stranded RNA and isometric virus-like particles in the entomopathogenic fungus Metarhizium anisopliae. Mycol Res 100:1468–1472CrossRefGoogle Scholar
  5. 5.
    Bogo MR, Rota CA, Pinto H Jr., Ocampos M, Correa CT, Vainstein MH, Schrank A (1998) A chitinase encoding gene (chit1 gene) from the entomopathogen Metarhizium anisopliae: isolation and characterization of genomic and full-length cDNA. Curr Microbiol 37:221–225PubMedCrossRefGoogle Scholar
  6. 6.
    Carsolio C, Gutierrez A, Jimenez B, Van MM, Herrera-Estrella A (1994) Characterization of ech-42, a Trichoderma harzianum endochitinase gene expressed during mycoparasitism. Proc Natl Acad Sci USA 91:10903–10907PubMedCrossRefGoogle Scholar
  7. 7.
    Cubero B, Scazzocchio C (1994) Two different, adjacent and divergent zinc finger binding sites are necessary for CREA-mediated carbon catabolite repression in the proline gene cluster of Aspergillus nidulans. EMBO J 13:407–415PubMedGoogle Scholar
  8. 8.
    da Silva MV, Santi L, Staats CC, da Costa AM, Colodel EM, Driemeier D, Vainstein MH, Schrank A (2005) Cuticle-induced endo/exoacting chitinase CHIT30 from Metarhizium anisopliae is encoded by an ortholog of the chi3 gene. Res Microbiol 156:382–392PubMedCrossRefGoogle Scholar
  9. 9.
    Dutra V, Nakazato L, Broetto L, Silveira SI, Henning VM, Schrank A (2004) Application of representational difference analysis to identify sequence tags expressed by Metarhizium anisopliae during the infection process of the tick Boophilus microplus cuticle. Res Microbiol 155:245–251PubMedCrossRefGoogle Scholar
  10. 10.
    Eberle J, Russo VE (1994) Neurospora crassa blue light-inducible gene bli-3. Biochem Mol Biol Int 34:737–744PubMedGoogle Scholar
  11. 11.
    Falquet L, Pagni M, Bucher P, Hulo N, Sigrist CJ, Hofmann K, Bairoch A (2002) The PROSITE database, its status in 2002. Nucleic Acids Res 30:235–238PubMedCrossRefGoogle Scholar
  12. 12.
    Fang W, Leng B, Xiao Y, Jin K, Ma J, Fan Y, Feng J, Yang X, Zhang Y, Pei Y (2005) Cloning of Beauveria bassiana chitinase gene Bbchit1 and its application to improve fungal strain virulence. Appl Environ Microbiol 71(1):363–370PubMedCrossRefGoogle Scholar
  13. 13.
    Frazzon AP, da Silva VJI, Masuda A, Schrank A, Vainstein MH (2000) In vitro assessment of Metarhizium anisopliae isolates to control the cattle tick Boophilus microplus. Vet Parasitol 94:117–125PubMedCrossRefGoogle Scholar
  14. 14.
    Gillespie JP, Bateman R, Charnley AK (1998) Role of cuticle-degrading proteases in the virulence of Metarhizium spp. for the desert locust, Schistocerca gregaria. J Invertebr Pathol 71:128–137PubMedCrossRefGoogle Scholar
  15. 15.
    Kang SC, Park S, Lee DG (1999) Purification and characterization of a novel chitinase from the entomopathogenic fungus, Metarhizium anisopliae. J Invertebr Pathol 73:276–281PubMedCrossRefGoogle Scholar
  16. 16.
    Kanzok SM, Jacobs-Lorena M (2006) Entomopathogenic fungi as biological insecticides to control malaria. Trends Parasitol 22:49–51PubMedCrossRefGoogle Scholar
  17. 17.
    Kim DJ, Baek JM, Uribe P, Kenerley CM, Cook DR (2002) Cloning and characterization of multiple glycosyl hydrolase genes from Trichoderma virens. Curr Genet 40:374–384PubMedCrossRefGoogle Scholar
  18. 18.
    Krieger de MC, Schrank A, Vainstein MH (2003) Regulation of extracellular chitinases and proteases in the entomopathogen and acaricide Metarhizium anisopliae. Curr Microbiol 46:205–210CrossRefGoogle Scholar
  19. 19.
    Kumar S, Tamura K, Jakobsen IB, Nei M (2001) MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17:1244–1245PubMedCrossRefGoogle Scholar
  20. 20.
    Limon MC, Chacon MR, Mejias R, gado-Jarana J, Rincon AM, Codon AC, Benitez T (2004) Increased antifungal and chitinase specific activities of Trichoderma harzianum CECT 2413 by addition of a cellulose binding domain. Appl Microbiol Biotechnol 64:675–685PubMedCrossRefGoogle Scholar
  21. 21.
    Nakazato L, Dutra V, Broetto L, Staats CC, Vainstein MH, Schrank A (2006) Development of an expression vector for Metarhizium anisopliae based on the tef-1alpha homologous promoter. Appl Microbiol Biotechnol 10:1–8CrossRefGoogle Scholar
  22. 22.
    Orikoshi H, Baba N, Nakayama S, Kashu H, Miyamoto K, Yasuda M, Inamori Y, Tsujibo H (2003) Molecular analysis of the gene encoding a novel cold-adapted chitinase (ChiB) from a marine bacterium, Alteromonas sp. strain O-7. J Bacteriol 185:1153–1160PubMedCrossRefGoogle Scholar
  23. 23.
    Pinto AS, Barreto CC, Schrank A, Ulhoa CJ, Vainstein MH (1997) Purification and characterization of an extracellular chitinase from the entomopathogenic Metarhizium anisopliae. Can J Microbiol 43:322–327CrossRefGoogle Scholar
  24. 24.
    Sambrook J, Russel DW (2001) Molecular cloning: A laboratory manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor LaboratoryGoogle Scholar
  25. 25.
    Screen S, Bailey A, Charnley K, Cooper R, Clarkson J (1997) Carbon regulation of the cuticle-degrading enzyme PR1 from Metarhizium anisopliae may involve a trans-acting DNA-binding protein CRR1, a functional equivalent of the Aspergillus nidulans CREA protein. Curr Genet 31:511–518PubMedCrossRefGoogle Scholar
  26. 26.
    Screen SE, Hu G, St. Leger RJ (2001) Transformants of Metarhizium anisopliae sf. anisopliae overexpressing chitinase from Metarhizium anisopliae sf. acridum show early induction of native chitinase but are not altered in pathogenicity to Manduca sexta. J Invert Pathol 78:260–266CrossRefGoogle Scholar
  27. 27.
    Seidl V, Huemer B, Seiboth B, Kubicek CP (2005) A complete survey of Trichoderma chitinases reveals three distinct subgroups of family 18 chitinases. FEBS J 272:5923–5939PubMedCrossRefGoogle Scholar
  28. 28.
    St. Leger RJ, Charnley AK, Cooper RM (1986) Cuticle-degrading enzymes of entomopathogenic fungi: Mechanisms of interaction between pathogen enzymes and insect cuticle. J Invert Pathol 47:295–302CrossRefGoogle Scholar
  29. 29.
    St. Leger RJ, Cooper RM, Charnley AK (1991) Characterization of chitinase and chitobiase produced by the entomopathogenic fungus Metarhizium anisopliae. J Invert Pathol 58:415–426CrossRefGoogle Scholar
  30. 30.
    Takaya N, Yamazaki D, Horiuchi H, Ohta A, Takagi M (1998) Cloning and characterization of a chitinase-encoding gene (chiA) from Aspergillus nidulans, disruption of which decreases germination frequency and hyphal growth. Biosci Biotechnol Biochem 62:60–65PubMedCrossRefGoogle Scholar
  31. 31.
    Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882PubMedCrossRefGoogle Scholar
  32. 32.
    Viterbo A, Haran S, Friesem D, Ramot O, Chet I (2001) Antifungal activity of a novel endochitinase gene (chit36) from Trichoderma harzianum Rifai TM. FEMS Microbiol Lett 200:169–174PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • César Milton Baratto
    • 1
  • Valéria Dutra
    • 1
  • Juliano Tomazzoni Boldo
    • 1
  • Leonardo Barbosa Leiria
    • 1
  • Marilene Henning Vainstein
    • 1
    • 2
  • Augusto Schrank
    • 1
    • 3
  1. 1.Centro de BiotecnologiaPrograma de Pós-graduação em Biologia Celular e Molecular Universidade Federal do Rio Grande do SulPorto AlegreBrazil
  2. 2.Departamento de Microbiologia, Instituto de Ciências Básicas da SaúdeUniversidade Federal do Rio Grande do SulPorto AlegreBrazil
  3. 3.Departamento de Biologia Molecular e Biotecnologia. Instituto de BiociênciasUniversidade Federal do Rio Grande do SulPorto AlegreBrazil

Personalised recommendations