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Applied and Environmental Microbiology, March 2009, p. 1667-1673, Vol. 75, No. 6
0099-2240/09/$08.00+0     doi:10.1128/AEM.01838-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Isolation and Identification of Cellulose-Binding Proteins from Sheep Rumen Contents{triangledown}

Atsushi Toyoda,1,2* Wataru Iio,1 Makoto Mitsumori,3 and Hajime Minato1

College of Agriculture, Ibaraki University, Ami, Ibaraki 300-0393, Japan,1 United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchu City, Tokyo 183-8509, Japan,2 National Institute of Livestock and Grassland Sciences, Tsukuba, Ibaraki 305-0901, Japan3

Received 8 August 2008/ Accepted 6 January 2009

To extend our understanding of the mechanisms of plant cell wall degradation in the rumen, cellulose-binding proteins (CBPs) from the contents of a sheep rumen were directly isolated and identified using a metaproteomics approach. The rumen CBPs were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and some CBPs revealed endoglucanase activities toward carboxymethyl cellulose. Using mass spectrometry analyses, four CBPs were identified and annotated as known proteins from the predominant rumen cellulolytic bacterium Fibrobacter succinogenes: tetratricopeptide repeat domain protein, OmpA family protein, fibro-slime domain protein, and cellulose-binding endoglucanase F (EGF). Another CBP was identified as the cellulosomal glycosyl hydrolase family 6 exoglucanase, Cel6A, of Piromyces equi. F. succinogenes cells expressing EGF were found to be major members of the bacterial community on the surface or at the inner surface of hay stems by immunohistochemical analyses using anti-EGF antibody. The finding that four of the five CBPs isolated and identified from sheep rumen contents were from F. succinogenes indicates that F. succinogenes is significantly involved in cellulose degradation in the rumen.

* Corresponding author. Mailing address: College of Agriculture, Ibaraki University, Ami, Ibaraki 300-0393, Japan. Phone and fax: 81 29 888 8584. E-mail: atoyoda{at}mx.ibaraki.ac.jp

{triangledown} Published ahead of print on 16 January 2009.

Applied and Environmental Microbiology, March 2009, p. 1667-1673, Vol. 75, No. 6
0099-2240/09/$08.00+0     doi:10.1128/AEM.01838-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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