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Journal of Bacteriology, September 2001, p. 5325-5333, Vol. 183, No. 18
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.18.5325-5333.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Noncatalytic Docking Domains of Cellulosomes of Anaerobic Fungi

Peter J. M. Steenbakkers,1 Xin-Liang Li,2 Eduardo A. Ximenes,3 Jorik G. Arts,1 Huizhong Chen,2 Lars G. Ljungdahl,2 and Huub J. M. Op den Camp1,*

Department of Microbiology, Faculty of Science, University of Nijmegen, NL-6525 ED Nijmegen, The Netherlands1; Department of Biochemistry and Molecular Biology and Center for Biological Resource Recovery, University of Georgia, Athens, Georgia 30602-72292; and Laboratorio De Enzimologia, Departamento De Biologia Celular, Universidade De Brasilia, Asa Norte, Brasilia-DF, Brazil 70910-9003

Received 10 April 2001/Accepted 22 June 2001

A method is presented for the specific isolation of genes encoding cellulosome components from anaerobic fungi. The catalytic components of the cellulosome of anaerobic fungi typically contain, besides the catalytic domain, mostly two copies of a 40-amino-acid cysteine-rich, noncatalytic docking domain (NCDD) interspaced by short linkers. Degenerate primers were designed to anneal to the highly conserved region within the NCDDs of the monocentric fungus Piromyces sp. strain E2 and the polycentric fungus Orpinomyces sp. strain PC-2. Through PCR using cDNA from Orpinomyces sp. and genomic DNA from Piromyces sp. as templates, respectively, 9 and 19 PCR products were isolated encoding novel NCDD linker sequences. Screening of an Orpinomyces sp. cDNA library with four of these PCR products resulted in the isolation of new genes encoding cellulosome components. An alignment of the partial NCDD sequence information obtained and an alignment of database-accessible NCDD sequences, focusing on the number and position of cysteine residues, indicated the presence of three structural subfamilies within fungal NCDDs. Furthermore, evidence is presented that the NCDDs in CelC from the polycentric fungus Orpinomyces sp. strain PC-2 specifically recognize four proteins in a cellulosome preparation, indicating the presence of multiple scaffoldins.

* Corresponding author. Mailing address: Department of Microbiology, Faculty of Science, University of Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands. Phone: 31-(0)243652657. Fax: 31-(0)243652830. E-mail: huubcamp{at}sci.kun.nl.

Journal of Bacteriology, September 2001, p. 5325-5333, Vol. 183, No. 18
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.18.5325-5333.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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