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Journal of Bacteriology, February 2003, p. 703-713, Vol. 185, No. 3
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.3.703-713.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Novel Organization and Divergent Dockerin Specificities in the Cellulosome System of Ruminococcus flavefaciens{dagger}

Marco T. Rincon,1 Shi-You Ding,2,3,{ddagger} Sheila I. McCrae,1 Jennifer C. Martin,1 Vincenzo Aurilia,4 Raphael Lamed,3 Yuval Shoham,5, Edward A. Bayer,2 and Harry J. Flint1*

Gut Microbiology Group, Rowett Research Institute, Aberdeen, United Kingdom,1 Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot,2 Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv,3 Department of Food Engineering and Biotechnology,5 Institute of Catalysis Science and Technology, Technion—Israel Institute of Technology, Haifa, Israel,6 Institute of Protein Biochemistry, National Research Council, Naples, Italy4

Received 5 July 2002/ Accepted 30 September 2002

The DNA sequence coding for putative cellulosomal scaffolding protein ScaA from the rumen cellulolytic anaerobe Ruminococcus flavefaciens 17 was completed. The mature protein exhibits a calculated molecular mass of 90,198 Da and comprises three cohesin domains, a C-terminal dockerin, and a unique N-terminal X domain of unknown function. A novel feature of ScaA is the absence of an identifiable cellulose-binding module. Nevertheless, native ScaA was detected among proteins that attach to cellulose and appeared as a glycosylated band migrating at around 130 kDa. The ScaA dockerin was previously shown to interact with the cohesin-containing putative surface-anchoring protein ScaB. Here, six of the seven cohesins from ScaB were overexpressed as histidine-tagged products in E. coli; despite their considerable sequence differences, each ScaB cohesin specifically recognized the native 130-kDa ScaA protein. The binding specificities of dockerins found in R. flavefaciens plant cell wall-degrading enzymes were examined next. The dockerin sequences of the enzymes EndA, EndB, XynB, and XynD are all closely related but differ from those of XynE and CesA. A recombinant ScaA cohesin bound selectively to dockerin-containing fragments of EndB, but not to those of XynE or CesA. Furthermore, dockerin-containing EndB and XynB, but not XynE or CesA, constructs bound specifically to native ScaA. XynE- and CesA-derived probes did however bind a number of alternative R. flavefaciens bands, including an ~110-kDa supernatant protein expressed selectively in cultures grown on xylan. Our findings indicate that in addition to the ScaA dockerin-ScaB cohesin interaction, at least two distinct dockerin-binding specificities are involved in the novel organization of plant cell wall-degrading enzymes in this species and suggest that different scaffoldins and perhaps multiple enzyme complexes may exist in R. flavefaciens.

* Corresponding author. Mailing address: Rowett Research Institute, Greenburn Rd., Bucksburn, Aberdeen AB21 9SB, United Kingdom. Phone: 44-1224-716 651. Fax: 44-1224-716 687. E-mail: h.flint{at}rri.sari.ac.uk.

{dagger} For a commentary on this article, see page 701 in this issue.

{ddagger} Present address: National Renewable Energy Laboratory, Biotechnology Center for Fuels and Chemicals, Golden, CO 80401.

Journal of Bacteriology, February 2003, p. 703-713, Vol. 185, No. 3
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.3.703-713.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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