Skip to main content
Log in

Structure and regulation of the Erwinia carotovora subspecies carotovora SCC3193 cellulase gene celV1 and the role of cellulase in phytopathogenicity

  • Original Paper
  • Published:
Molecular and General Genetics MGG Aims and scope Submit manuscript

Abstract

The ceIV1 gene encoding a secreted cellulase (CelV1) of Erwinia carotovora subsp. carotovora SCC3193 was cloned and its nucleotide sequence determined. The gene contains an open reading frame of 1511 by and codes for an exported protein of 504 amino acids. The predicted amino acid sequence of Ce1V1 was highly similar to that of CeIV of another E. c. subsp. carotovora strain SCRI193 but completely different from the previously characterized cellulase, CelS, of the strain SCC3193. Gene fusions to the lacZ reporter were employed to characterize the regulation of celV1 and celS. Both genes are coordinately induced in a growth phase-dependent manner and are catabolite repressed. Expression of celV1 but not celS was stimulated by plant extracts. The celS gene was expressed at a much lower level than celV1 under all conditions tested. Inactivation of the celV1 gene in E. c. subsp. carotovora strain SCC3193 by marker exchange showed that celV1 encodes the major cellulase of strain SCC3193, as the resulting mutant strain SCC6001 was devoid of cellulase activity. Ce1Vl mutants exhibited reduced virulence suggesting that CelV1, although not absolutely required for pathogenicity, enhances the ability of strain SCC3193 to macerate plant tissue. Inactivation of the celS gene in the celV1 mutant did not lead to any further decrease in virulence.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  • Andro T, Chambost JP, Katonjansky A, Catteneo J, Bertheam Y, Barras F, van Gijsegem F, Colleno A (1984) Mutants of Erwinia chrysanthemi defective in secretion of pectinase and cellulase. J Bacteriol 160:1199–1203

    CAS  PubMed  PubMed Central  Google Scholar 

  • Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. (1987) Current protocols in molecular biology. John Wiley & Sons, New York

    Google Scholar 

  • Aymeric J-L, Guiseppi A, Pascal M-C, Chippaux M (1988) Mapping and regulation of the cel genes in Erwinia chrysanthemi. Mol Gen Genet 211:95–101

    Article  CAS  Google Scholar 

  • Beguin P (1990) Molecular biology of cellulose degradation. Annu Rev Microbiol 4:4219–248

    Google Scholar 

  • Biely P, Mislovicova D, Toman R (1985) Soluble chromogenic substrates for the assay of endo-1,4-β-xylanases and endo-1,4β-glycanases. Anal Biochem 144:142–146

    Article  CAS  Google Scholar 

  • Boccara M, Aymeric J-L, Camus C (1994) Role of endoglucanases in Erwinia chrysanthemi 3937 virulence on Sauntpaulia ionantha. J Bacteriol 176:1524–1526

    Article  CAS  Google Scholar 

  • Boyer M-H, Chambost JP, Magnan M, Cattaneo J (1987) Characterization of a new endoglucanase from Erwinia chrysanthemi. Eur J Biochem 162:311–316

    Article  CAS  Google Scholar 

  • Chatterjee AK, McEvoy JL, Murata H, Collmer A (1991) Regulation of the production of pectinases and other extracellular enzymes in the soft-rot Erwinia spp. In: Molecular strategies of pathogens and host plants. Patil SS, Ouchi S, Mills D, Vance C (eds) Springer, New York

    Google Scholar 

  • Collmer A, Keen NT (1986) The role of pectic enzymes in plant pathogenesis. Annu Rev Phytopathol 24:383–409

    Article  CAS  Google Scholar 

  • Cooper VJC, Salmond GPC (1993) Molecular analysis of the major cellulase (CeIV) of Erwinia carotovora: evidence for an evolutionary “mix-and-match” of enzyme domains. Mol Gen Genet 241:341–350

    CAS  PubMed  Google Scholar 

  • Gilkes NR, Henrissat B, Kilburn DG, Miller RC, Warren RAJ (1991) Domains in microbial β-1,4-glycanases: sequence conservation, function, and enzyme families. Microbiol Rev 55:303–315

    CAS  PubMed  PubMed Central  Google Scholar 

  • Gilman M (1987) In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Stuhl K (eds) Current protocols in molecular biology. John Wiley & Sons, New York, 4.4.1–4.4.4

    Google Scholar 

  • Gough CL, Dow JM, Barber CE, Daniels MJ (1988) Cloning of two endoglucanase genes of Xanthomonas campestris pv. campestris:analysis of the role of the major endoglucanase in pathogenesis. Mol Plant-Microbe Interact 1:257–281

    Article  Google Scholar 

  • Guiseppi A, Cami B, Aymeric JL, Ball Y (1988) Homology between endoglucanase Z of Erwinia chrysanthemi and endoglucanases of Bacillus subtilis and alkalophilic Bacillus. Mol Microbiol 2:1591–1604

    Article  Google Scholar 

  • Guiseppi A, Aymeric JL, Cami B, Barras F, Creuzet N (1991) Sequence analysis of the cellulase-encoding gene of Erwinia chrysanthemi: a possible case of interspecies gene transfer. Gene 106:109–114

    Article  CAS  Google Scholar 

  • von Heijne G (1983) Patterns of amino acid signal sequence cleavage sites. Eur J Biochem 133:17–21

    Article  Google Scholar 

  • Henikoff S (1984) Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351–359

    Article  CAS  Google Scholar 

  • Henrissat B, Claeyssens M, Tomme P, Lemesle L, Mornon J-P (1989) Cellulase families revealed by hydrophobic cluster analysis. Gene 81:83–95

    Article  CAS  Google Scholar 

  • Hinton JCD, Gill DR, Lalo D, Plastow GS, Salmond GPC (1990) Sequence of the peh gene of Erwinia carotovora: homology between Erwinia and plant enzymes. Mol Microbiol 4:1029–1036

    Article  CAS  Google Scholar 

  • Horikoshi K, Fukumori F (1988) Modification and expression of alkaline cellulase genes of alkalophilic Bacillus strains. In: Aubert JP, Beguin P, Millet J (eds) Biochemistry and genetics of cellulose degradation. Academic Press, New York, pp 203–217

    Google Scholar 

  • Karlsson M-B, Pirhonen M, Saarilahti HT, Palva ET (1991) Molecular cloning of ompRS, a regulatory locus controlling production of outer membrane proteins in Erwinia carotovora subsp. carotovora. Mol Gen Genet 226:353–360

    Article  CAS  Google Scholar 

  • Knowles J, Lehtovaara P, Teeri T (1987) Cellulase genes and their families. Trends Biotehnol 5:255–261

    Article  CAS  Google Scholar 

  • Kotoujansky A (1987). Molecular genetics of pathogenesis by soft-rot erwinias. Annu Rev Phytopathol 25:405–430

    Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  • Lång H, Palva ET (1993) The ompS gene of Vibrio cholerae encodes a growth phase-dependent maltoporin. Mol Microbiol 10:891–901

    Article  Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

    Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

    Google Scholar 

  • Murata H, McEvoy J, Chatterjee A, Collmer A, Chatterjee AK (1991) Molecular cloning of an aepA gene that activates production of extracellular pectolytic, cellulolytic, and proteolytic enzymes in Erwinia carotovora subsp. carotovora. Mol Plant-Microbe Interact 4:239–246

    Article  CAS  Google Scholar 

  • Palva ET (1978) Major outer membrane protein in Salmonella typhimurium induced by maltose. J Bacteriol 136:286–294

    CAS  PubMed  PubMed Central  Google Scholar 

  • Perombelon MCM, Kelman A (1980) Ecology of the soft rot erwinias. Annu Rev Phytopathol 18:361–387

    Article  Google Scholar 

  • Penttilä M, Lehtovaara P, Nevalainen H, Bhikhabhai R, Knowles J (1986) Homology between cellulase genes of Trichoderma reesei: complete nucleotide sequence of the endoglucanase gene I. Gene 45:253–263

    Article  Google Scholar 

  • Pirhonen M, Saarilahti H, Karlsson M-B, Palva ET (1991) Identification of pathogenicity determinants of Erwinia carotovora subspecies carotovora by transposon mutagenesis. Mol Plant-Microbe Interact 4:276–283

    Article  CAS  Google Scholar 

  • Pirhonen M, Flego D, Heikinheimo R, Palva ET (1993) A small diffusible signal molecule is responsible for the global control of virulence and exoenzyme production in plant pathogen Erwinia carotovora. EMBO J 12:2467–2476

    Article  CAS  Google Scholar 

  • Py B, Bortoli-German I, Haiech I, Chippaux M, Barras F (1991) Cellulase EG2 of Erwinia chysanthemi: structural organization and importance of His 98 and Glu 133 residues for catalysis. Prot Eng 4:325–333

    Article  CAS  Google Scholar 

  • Roberts DP, Denny TP, Schell MA (1988) Cloning of the egl gene of Pseudomonas solanacearum and analysis of its role in phytopathogenicity. J Bacteriol 170:1445–1451

    Article  CAS  Google Scholar 

  • Rosenberg M, Court D (1979) Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet 13:319–353

    Article  CAS  Google Scholar 

  • Saarilahti HT, Henrissat B, Palva ET (1990) Ce1S: a novel endoglucanase identified from Erwinia carotovora subsp. carotovora. Gene 90:9–14

    Article  CAS  Google Scholar 

  • Saarilahti HT, Pirhonen M, Karlsson M-J, Flego D, Palva ET (1992) Expression of pehA-bla gene fusion in Erwinia carotovora subsp. carotovora and isolation of regulatory mutants affecting polygalacturonase production. Mol Gen Genet 234:81–88

    CAS  PubMed  Google Scholar 

  • Saloheimo M, Lehtovaara P, Penttilä M., Teeri TT, Stahlberg J (1988) EGIII, a new endoglucanase from Trichoderma reesei, and the characterization of both gene and enzyme. Gene 63:11–21

    Article  CAS  Google Scholar 

  • Sanger F, Nicklen S, Caulson A (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    Article  CAS  Google Scholar 

  • Stephens GJ, Wood RKS (1975) Killing of protoplasts by soft-rot bacteria. Physiol Plant Pathol 5:165–181

    Article  CAS  Google Scholar 

  • Walker DS, Reeves PJ, Salmond GPC (1994) The major cellulase, CelV of Erwinia carotovora subsp. carotovora is an important soft rot virulence factor. Mol Plant-Microbe Interact 7:425–431

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Additional information

Communicated by A. Kondorosi

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mäe, A., Heikinheimo, R. & Palva, E.T. Structure and regulation of the Erwinia carotovora subspecies carotovora SCC3193 cellulase gene celV1 and the role of cellulase in phytopathogenicity. Molec. Gen. Genet. 247, 17–26 (1995). https://doi.org/10.1007/BF00425817

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00425817

Key words

Navigation