title: Novel organization and divergent dockerin specificities in the cellulosome system of Ruminococcus flavefaciens.
abstract: 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 approximately 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.
authors.affiliation: Gut Microbiology Group, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, Scotland, United Kingdom.
authors.forename: Marco T
authors.initials: MT
authors.lastname: Rincon
authors.forename: Shi-You
authors.initials: SY
authors.lastname: Ding
authors.forename: Sheila I
authors.initials: SI
authors.lastname: McCrae
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: Martin
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Aurilia
authors.forename: Raphael
authors.initials: R
authors.lastname: Lamed
authors.forename: Yuval
authors.initials: Y
authors.lastname: Shoham
authors.forename: Edward A
authors.initials: EA
authors.lastname: Bayer
authors.forename: Harry J
authors.initials: HJ
authors.lastname: Flint
mesh_terms: D000818:Animals; D001421:Bacteria, Anaerobic / Q000201:enzymology*; D001426:Bacterial Proteins / Q000502:physiology; D001483:Base Sequence; D002473:Cell Wall / Q000378:metabolism; D002480:Cellulase / Q000502:physiology*; D002482:Cellulose / Q000378:metabolism; D006095:Gram-Positive Cocci / Q000201:enzymology*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000502:physiology*; D012417:Rumen / Q000382:microbiology*
chemical_list: D001426:Bacterial Proteins; D009097:Multienzyme Complexes; C422913:ScaA protein, Ruminococcus; C422914:ScaB protein, Ruminococcus; D002482:Cellulose; D002480:Cellulase