Novel organization and divergent dockerin specificities in the cellulosome system of Ruminococcus flavefaciens.
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.
Rincon
Marco T
MT
Gut Microbiology Group, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, Scotland, United Kingdom.
Ding
Shi-You
SY
McCrae
Sheila I
SI
Martin
Jennifer C
JC
Aurilia
Vincenzo
V
Lamed
Raphael
R
Shoham
Yuval
Y
Bayer
Edward A
EA
Flint
Harry J
HJ
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*
D001426:Bacterial Proteins; D009097:Multienzyme Complexes; C422913:ScaA protein, Ruminococcus; C422914:ScaB protein, Ruminococcus; D002482:Cellulose; D002480:Cellulase