There is good evidence that the majority of plant cell-wall-degrading enzymes in R. flavefa­ciens are retained on the bacterial cell surface via a cellulosome-type multienzyme complex. The assembly of this complex depends on the specific interaction of dockerin and cohesin domains within the component proteins. The known structural components of this com­plex are encoded by the sca gene cluster, which was first described in R. flavefaciens 17 (10). A very similar gene cluster has now also been identified from the partial genome sequence of R. flavefaciens FD1 (11). In R. flavefaciens 17, the scaffolding protein ScaA carries three cohesins and a C-terminal dockerin that in turn binds to any one of seven cohesins present in the larger noncatalytic protein ScaB (12). Recent work has shown that the C-terminus of ScaB also contains an unusual type of dockerin domain that interacts with a single cohesin present in the small, cell-surface-anchored protein, ScaE (13). ScaE is covalently bound to the peptidoglycan of the cell surface at its C-terminus via a sortase-mediated mechanism (Figure 12.1). Meanwhile, a second small protein encoded by the sca cluster, ScaC, possesses a dockerin that binds to ScaA, but carries a single, distinctive cohesin. This has been de­scribed as an adaptor, because it recognizes a different type of dockerin from that recognized by ScaA cohesins (14). Dockerin-carrying enzymes from R. flavefaciens 17 have so far been shown to bind mainly to ScaA cohesins, although some divergent enzyme dockerins such as those from CesA (Ce3B) and XynE (Xyn11E) have distinct binding specificities for which the corresponding cohesin partners remain unknown (12, 15, 16).

The proteins encoded by the homologous sca cluster of R. flavefaciens FD1 differ in a few interesting respects from their homologues in R. flavefaciens 17. ScaAFD1 carries only two cohesins, while ScaBFDi carries nine, five homologous to those of ScaBi7, and four closer to those of ScaA17. Thus, ScaBFD1 is predicted to be able to bind four enzyme subunits directly, and 10 more via attached ScaA molecules, while ScaB17 is predicted to bind 21 enzyme subunits via ScaA molecules (11). It is not yet clear whether these differences have functional consequences. Significant strain diversity exists in R. flavefaciens both at the genetic level and at the level of plant cell wall degradation (17, 18).

Most plant cell wall hydrolases studied from R. flavefaciens 17 have a dockerin-like module located either at the C-terminus, or internally (15,19). One non-dockerin containing extra­cellular hydrolase is, however, known that carries family 11 and 10 xylanases domains at its N and C terminus, respectively, connected by an unusual NQ-rich linker (20). In addition to at least three types of enzyme dockerin, the dockerins found in the structural proteins ScaA

and ScaB are also distinct in their sequences and binding characteristics, suggesting that there are at least five different dockerin specificities in this strain (21). Meanwhile, genome sequencing of R. flavefaciens FD1 has revealed at least 180 polypeptides that carry dockerin sequences (22). The sequences of these dockerins fall into a number of branches in phy­logenetic analyses. Fewer than 50% of these carry glycoside hydrolase domains, and many carry proteinase domains or domains of unknown function. It remains unclear whether all these domains are required primarily for plant cell wall breakdown. Proteinases may well be involved in removing structural proteins in the plant cell wall, but it is also possible that dockerin-cohesin interactions mediate the assembly of protein complexes that play other roles on the bacterial cell surface.

Uniquely among known cellulosome systems, the R. flavefaciens scaffolding proteins so far identified carry no identified carbohydrate-binding modules. Carbohydrate-binding modules occur in many enzyme subunits, including a module in the abundant cellulase EndB (Cel44A) that represents a new CBM family. The gene that precedes ScaE, however, is now known to encode a protein (CttA) that is attached to the bacterial cell surface via ScaE, and binds crystalline cellulose (23). CttA is, therefore, a strong candidate for a carbohydrate-binding protein that may play a key role in positioning cellulosomal enzymes close to their substrates. The possible role played in adhesion by the yellow affinity substance of R. flavefaciens (24) remains unclear.