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Biochemical and genetic characterization of ChiA, the major enzyme component for the solubilization of chitin by Cellulomonas uda

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Abstract

Cellulomonas uda efficiently solubilized chitinous substrates with a simple chitinase system composed of an endochitinase, designated ChiA, which hydrolyzed insoluble substrates into long-chain chitooligosaccharides, and an as yet uncharacterized exochitinase activity. ChiA, isolated from culture supernatant fluids, was found to be a glycosylated endochitinase with an apparent molecular mass of approximately 70 kDa and pI of 8.5. The gene encoding ChiA was cloned in Escherichia coli and sequenced, revealing an open reading frame of 1,716 bp encoding a 571-amino-acid protein with a predicted molecular mass of 59.2 kDa. The region upstream of chiA included a conserved −35 hexamer flanked by two direct repeats analogous to those found in many Streptomyces chitinase promoters, and thought to function as binding sequences for regulatory proteins. Analysis of the deduced amino acid sequence showed a modular protein consisting of a signal peptide at its N terminus, a family 2 carbohydrate-binding module (CBM2) that was closely related to the substrate-binding domains of glycosyl hydrolases from distantly related bacteria, and a family 18 glycosyl hydrolase catalytic module related to Streptomyces chitinases. In contrast to the fibronectin type III domains of Streptomyces chitinases, the linker region between modules in ChiA consisted of a long proline- and threonine-rich module, thought to contribute to the glycosylation and flexibility of the mature protein.

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Fig. 1a–c
Fig. 2a, b
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Abbreviations

CBM :

Carbohydrate-binding module

P-T :

Proline- and threonine-rich domain

Fn3 :

Type III repetitive sequences of fibronectin domain

PKD :

Polycystic kidney disease I domain

References

  • Brurger MB, Nes IF, Eijsink VGH (1996) Comparative studies of chitinases A and B from Serratia marcescens. Microbiology 142:1581–1589

    CAS  PubMed  Google Scholar 

  • Cavedon K, Leschine SB, Canale-Parola E (1990) Cellulase system of a free-living, mesophilic clostridium (strain C7). J Bacteriol 172:4222–4230

    CAS  PubMed  Google Scholar 

  • Cohen-Kupiec R, Chet I (1998) The molecular biology of chitin digestion. Curr Op Biotechnol 9:270–277

    CAS  Google Scholar 

  • Edman P, Begg G (1967) A protein sequenator. Eur J Biochem 244:80–91

    Google Scholar 

  • Felse PA, Panda T (1999) Regulation and cloning of microbial chitinase genes. Appl Microbiol Biotechnol 51:141–151

    CAS  PubMed  Google Scholar 

  • Fujii T, Miyashita K (1993) Multiple domain structure in a chitinase gene (chiC) of Streptomyces lividans. J Gen Microbiol 4:677–686

    Google Scholar 

  • Gilkes NR, Warren RAJ, Miller RC, Kilburn DG (1988) Precise excision of the cellulose binding domains from two Cellulomonas fimi cellulases by a homologous protease and the effect on catalysis. J Biol Chem 263:10401–10407

    CAS  PubMed  Google Scholar 

  • Gomori G (1955) Preparation of buffers for use in enzyme studies. In: Colowick SP, Kaplan NO (eds) Methods in enzymology. Academic Press, New York, pp 138–146

  • Henrissat B (1999) Classification of chitinase modules. In: Jolles P, Muzzarelli RAA (eds) Chitin and chitinases. Birkhauser Verlag, Basel, Switzerland, pp 137–156

  • Henrissat B, Bairoch A (1993) New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 293:781–788

    CAS  PubMed  Google Scholar 

  • Heukeshoven J, Dernick R (1985) Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 6:103–112

    CAS  Google Scholar 

  • Hsu SC, Lockwood JL (1974) Powdered chitin as a selective medium for enumeration of actinomycetes in water and soil. Appl Microbiol 29:422–426

    Google Scholar 

  • Jeuniaux C (1966) Chitinases. In: Neufeld EF, Ginsburg V (eds) Methods in enzymology. Academic, New York, pp 644–650

  • Keyhani NO, Roseman S (1997) Wild-type Escherichia coli grows on the chitin disaccharide, N,N’-diacetylchitobiose, by expressing the cel operon. Proc Natl Acad Sci USA 94:14367–14371

    CAS  PubMed  Google Scholar 

  • Keyhani ND, Wang L, Lee YC, Roseman S (1996) The chitin catabolic cascade in the marine bacterium Vibrio furnissii. Characterization of an N, N’-diacetyl-chitobiose transport system. J Biol Chem 271:33409–33439

    CAS  PubMed  Google Scholar 

  • Koga D, Mitsutomi M, Kono M, Matsumiya M (1999) Biochemistry of chitinases. In: Jolles P, Muzzarelli RAA (eds) Chitin and chitinases. Birkhauser Verlag, Basel, Switzerland, pp 111–123

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

    Google Scholar 

  • Lee HS, Han DS, Choi SJ, Choi SW, Kim DS, Bai DH, Yu JH (2000) Purification, characterization, and primary structure of a chitinase from Pseudomonas sp. YHS-A2. Appl Microbiol Biotechnol 54:397–405

    CAS  PubMed  Google Scholar 

  • Leschine S, Canale-Parola E (1983) Mesophilic cellulolytic clostridia from freshwater environments. Appl Environ Microbiol 46:728–737

    CAS  Google Scholar 

  • Metcalf AC, Krsek M, Gooday GW, Prosser JI, Wellington EMH (2002) Molecular analysis of a bacterial chitinolytic community in an upland pasture. Appl Environ Microbiol 68:5042–5050

    Article  CAS  PubMed  Google Scholar 

  • Muzzarelli RAA (1977) Chitin. Pergamon Press, Oxford

  • O’Brien M, Colwell RR (1987) A rapid test for chitinase activity that uses 4-methylumbelliferyl-N-Acetyl-b-D-Glucosaminide. Appl Environ Microbiol 53:1718–1720

    CAS  PubMed  Google Scholar 

  • Ohno T, Armand S, Hata T, Nikaidou N, Henrissat B, Mitsutomi M, Watanabe T (1996) A modular family 19 chitinase found in the prokaryotic organism Streptomyces griseus HUT 6037. J Bacteriol 178:5065–5070

    CAS  PubMed  Google Scholar 

  • Ong E, Kilburn DG, Miller Jr. RC, Warren AJ (1994) Streptomyces lividans glycosylates the linker region of a beta-1,4-glycanase from Cellulomonas fimi. J Bacteriol 176:999–1008

    CAS  PubMed  Google Scholar 

  • Ponting CP, Aravind L, Schultz JB, P., Koonin EV (1999) Eukaryotic signalling domain homologues in archaea and bacteria. Ancient ancestry and horizontal gene transfer. J Mol Biol 289:729–745

    Article  CAS  PubMed  Google Scholar 

  • Reguera G, Leschine SB (2001) Chitin degradation by cellulolytic anaerobes and facultative aerobes from soils and sediments. FEMS Microbiol Lett 204:367–374

    CAS  PubMed  Google Scholar 

  • Robbins PW, Albright C, Benfield B (1988) Cloning and expression of a Streptomyces plicatus chitinase (chitinase-63) in Escherichia coli. J Biol Chem 263:443–447

    CAS  PubMed  Google Scholar 

  • Saito A, Fujii T, Yoneyama T, Redenbach M, Ohno T, Watanabe T, Miyashita K (1999) High-multiplicity of chitinase genes in Streptomyces coelicolor A3(2). Biosci Biotechnol Biochem 63:710–718

    CAS  PubMed  Google Scholar 

  • Saito A, Ishizaka M, Francisco Jr. PB, Fujii T, Miyashita K (2000) Transcriptional co-regulation of five chitinase genes scattered on the Streptomyces coelicolor A3(2) chromosome. Microbiology 146:2937–2946

    CAS  PubMed  Google Scholar 

  • Saito A, Miyashita K, Biukovic G, Schrepf H (2001) Characteristics of Streptomyces coelicolor A3(2) extracellular protein targeting chitin and chitosan. Appl Environ Microbiol 67:1268–1273

    CAS  PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Shiro M, Ueda M, Kawaguchi T, Arai M (1996) Cloning of a cluster of chitinase genes from Aeromonas sp. No. 10S-24. Biochim Biophys Acta 1305:44–48

    CAS  PubMed  Google Scholar 

  • Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85

    PubMed  Google Scholar 

  • Stackebrandt E, Kandler O (1979) Taxonomy of the genus Cellulomonas, based on phenotypic characters and deoxyribonucleic acid- deoxyribonucleic acid homology, and proposal of seven neotype strains. Int J Syst Bacteriol 29:273–282

    Google Scholar 

  • Stackebrandt E, Prauser H (1992) The family Cellulomonadaceae. In: Balows A, Truper HG, Dworkin M, Harder W, Schleifer KH (eds) The prokaryotes, 2nd edn. Springer, Berlin Heidelberg New York

  • Svitil AL, Ni Chadhain SM, Moore JA, Kirchman DL (1997) Chitin degradation proteins produced by the marine bacterium Vibrio harveyi growing on different forms of chitin. Appl Environ Microbiol 63:408–413

    CAS  Google Scholar 

  • Thompson JD, Higgins DG, Gilson TJ (1994) Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalies and weight matrix. Nucleic Acids Res 22:4674–4680

    Google Scholar 

  • Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354

    CAS  PubMed  Google Scholar 

  • Trudel J, Asselin A (1989) Detection of chitinase activity after polyacrylamide gel electrophoresis. Anal Biochem 178:362–366

    CAS  PubMed  Google Scholar 

  • Ueda M, Kojima M, Yoshikawa T, Mitsuda N, Araki K, Kawaguchi T, Miyatake K, Arai M, Fukamizo T (2003) A novel type of family 19 chitinase from Aeromonas sp. No. 10S-24. Eur J Biochem 270:2513–2520

    CAS  PubMed  Google Scholar 

  • Watanabe T, Ito Y, Hashimoto M, Yamada T, Alam MM, Tanaka H (1994) The role of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation. J Bacteriol 176:4465–4472

    CAS  PubMed  Google Scholar 

  • Williams J, McGrath WJ, Mangel WF (2000) Sensitive method to identify and characterize proteinases in situ after SDS-PAGE. Biotechniques 29:1108–1113

    CAS  PubMed  Google Scholar 

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Acknowledgements

We would like to express our appreciation to Dr. Elizabeth Stuart and Dr. Thomas Lessie for technical advice and helpful suggestions during different phases of this work. This work was supported by U.S. Department of Energy grants DE-FG02–88ER13898 and DE-FG02–02ER15330.

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  1. Department of Microbiology, University of Massachusetts, Amherst, Massachusetts , 01003, USA

    Gemma Reguera & Susan B. Leschine

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  1. Gemma Reguera
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  2. Susan B. Leschine
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Correspondence to Susan B. Leschine.

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Reguera, G., Leschine, S.B. Biochemical and genetic characterization of ChiA, the major enzyme component for the solubilization of chitin by Cellulomonas uda . Arch Microbiol 180, 434–443 (2003). https://doi.org/10.1007/s00203-003-0611-y

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  • DOI: https://doi.org/10.1007/s00203-003-0611-y

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