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Journal of Bacteriology, March 2001, p. 1945-1953, Vol. 183, No. 6
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.6.1945-1953.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Cellulosomal Scaffoldin-Like Proteins from
Ruminococcus flavefaciens
Shi-You
Ding,1,2,
Marco T.
Rincon,3
Raphael
Lamed,2
Jennifer C.
Martin,3
Sheila I.
McCrae,3
Vincenzo
Aurilia,4
Yuval
Shoham,5
Edward A.
Bayer,1,* and
Harry J.
Flint3
Department of Biological Chemistry, The Weizmann Institute
of Science, Rehovot,1 Department of
Molecular Microbiology and Biotechnology, Tel Aviv University,
Ramat Aviv,2 and Department of Food
Engineering and Biotechnology, and Institute of Catalysis Science
and Technology, Technion
Israel Institute of Technology,
Haifa,5 Israel; Gut Microbiology Group,
Rowett Research Institute, Aberdeen, United
Kingdom3; and CNR-IABBAM,
Ponticelli-Naples, Italy4
Received 5 September 2000/Accepted 14 December 2000
Two tandem cellulosome-associated genes were identified in the
cellulolytic rumen bacterium, Ruminococcus flavefaciens.
The deduced gene products represent multimodular scaffoldin-related proteins (termed ScaA and ScaB), both of which include several copies
of explicit cellulosome signature sequences. The scaB gene was completely sequenced, and its upstream neighbor scaA
was partially sequenced. The sequenced portion of scaA
contains repeating cohesin modules and a C-terminal dockerin domain.
ScaB contains seven relatively divergent cohesin modules, two extremely
long T-rich linkers, and a C-terminal domain of unknown function.
Collectively, the cohesins of ScaA and ScaB are phylogenetically
distinct from the previously described type I and type II cohesins, and
we propose that they define a new group, which we designated here type
III cohesins. Selected modules from both genes were overexpressed in
Escherichia coli, and the recombinant proteins were used as probes in affinity-blotting experiments. The results strongly indicate
that ScaA serves as a cellulosomal scaffoldin-like protein for several
R. flavefaciens enzymes. The data are supported by the
direct interaction of a recombinant ScaA cohesin with an expressed dockerin-containing enzyme construct from the same bacterium. The
evidence also demonstrates that the ScaA dockerin binds to a
specialized cohesin(s) on ScaB, suggesting that ScaB may act as an
anchoring protein, linked either directly or indirectly to the
bacterial cell surface. This study is the first direct demonstration in
a cellulolytic rumen bacterium of a cellulosome system, mediated by
distinctive cohesin-dockerin interactions.
*
Corresponding author. Mailing address: Department of
Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel. Phone: (972) 8-934-2373. Fax: (972) 8-946-8256. E-mail: bfbayer{at}wicc.weizmann.ac.il.

Present address: National Renewable Energy Laboratory,
Biotechnology Center for Fuels & Chemicals, Golden, CO
80401.
Journal of Bacteriology, March 2001, p. 1945-1953, Vol. 183, No. 6
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.6.1945-1953.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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