Cellulosomal scaffoldin-like proteins from Ruminococcus flavefaciens

Two tandem cellulosome-associated genes were identified in the cellulolytic rumen bacterium, Ruminococcus flavefaciens. The deduced gene products repr esent multimodular scaffoldin-related proteins (termed ScaA and ScaB), both of which include several copies of explicit cellulosome signature sequence s. The scaB gene was completely sequenced, and its upstream neighbor scaA w as partially sequenced. The sequenced portion of scaA contains repeating co hesin modules and a C-terminal dockerin domain. ScaB contains seven relativ ely divergent cohesin modules, two extremely long T-rich linkers, and a C-t erminal 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 de signated here type III cohesins, Selected modules from both genes were over expressed in Escherichia coli, and the recombinant proteins were used as pr obes in affinity-blotting experiments. The results strongly indicate that S caA serves as a cellulosomal scaffoldin-like protein for several R. flavefa ciens enzymes. The data are supported by the direct interaction of a recomb inant ScaA cohesin with an expressed dockerin-containing enzyme construct f rom the same bacterium. The evidence also demonstrates that the ScaA docker in binds to a specialized cohesin(s) on ScaB, suggesting that ScaB may act as an anchoring protein, linked either directly or indirectly to the bacter ial cell surface. This study is the first direct demonstration in a cellulo lytic rumen bacterium of a cellulosome system, mediated by distinctive cohe sin-dockerin interactions.