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Journal of Bacteriology, November 2000, p. 6440-6450, Vol. 182, No. 22
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Characterization of the Collagen-Binding S-Layer Protein CbsA of Lactobacillus crispatus

Jouko Sillanpää,1 Beatriz Martínez,2 Jenni Antikainen,1 Takahiro Toba,1,3 Nisse Kalkkinen,4 Sanna Tankka,1 Kari Lounatmaa,5 Jaakko Keränen,6 Magnus Höök,7 Benita Westerlund-Wikström,1 Peter H. Pouwels,2 and Timo K. Korhonen1,*

Division of General Microbiology, Department of Biosciences,1 and Institute of Biotechnology,4 FIN-00014 University of Helsinki, Laboratory of Electronics Production Technology, Helsinki University of Technology, FIN-02015 HUT,5 and Centre for Electron Microscopy, Tampere University of Technology, FIN-33101 Tampere,6 Finland; TNO Nutrition and Food Research Institute, 3700 AJ Zeist, The Netherlands2; Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Japan3; and Center for Extracellular Matrix Research, Institute for Biosciences and Technology, Texas A&M University, Houston, Texas7

Received 10 May 2000/Accepted 25 August 2000

The cbsA gene of Lactobacillus crispatus strain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in Escherichia coli. The cbsA open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein. The cbsA gene product was expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens. Three other Lactobacillus S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in cbsA where deletions and mutations were introduced. In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncated peptides from CbsA were constructed by gene fusion. Analysis of these molecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule. The mutated or hybrid CbsA molecules and peptides that failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA. Strain JCM 5810 was found to contain another S-layer gene termed cbsB that was 44% identical in sequence to cbsA. RNA analysis showed that cbsA, but not cbsB, was transcribed under laboratory conditions. S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsA-mediated collagen binding represents a true tissue adherence property of L. crispatus.

* Corresponding author. Mailing address: Division of General Microbiology, Department of Biosciences, P.O. Box 56, FIN 00014 University of Helsinki, Finland. Phone: 358-9-19159260. Fax: 358-9-19159262. E-mail: timo.korhonen{at}helsinki.fi.

Journal of Bacteriology, November 2000, p. 6440-6450, Vol. 182, No. 22
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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