Ding S Y, Rincon M T, Lamed R, Martin J C, McCrae S I, Aurilia V, Shoham Y, Bayer E A, Flint H J
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel.
J Bacteriol. 2001 Mar;183(6):1945-53. doi: 10.1128/JB.183.6.1945-1953.2001.
Two tandem cellulosome-associated genes were identified in the cellulolytic rumen bacterium, Ruminococcus flavefaciens. The deduced gene products represent multimodular scaffoldin-related proteins (termed ScaA and ScaB), both of which include several copies of explicit cellulosome signature sequences. The scaB gene was completely sequenced, and its upstream neighbor scaA was partially sequenced. The sequenced portion of scaA contains repeating cohesin modules and a C-terminal dockerin domain. ScaB contains seven relatively divergent cohesin modules, two extremely long T-rich linkers, and a C-terminal 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 designated here type III cohesins. Selected modules from both genes were overexpressed in Escherichia coli, and the recombinant proteins were used as probes in affinity-blotting experiments. The results strongly indicate that ScaA serves as a cellulosomal scaffoldin-like protein for several R. flavefaciens enzymes. The data are supported by the direct interaction of a recombinant ScaA cohesin with an expressed dockerin-containing enzyme construct from the same bacterium. The evidence also demonstrates that the ScaA dockerin binds to a specialized cohesin(s) on ScaB, suggesting that ScaB may act as an anchoring protein, linked either directly or indirectly to the bacterial cell surface. This study is the first direct demonstration in a cellulolytic rumen bacterium of a cellulosome system, mediated by distinctive cohesin-dockerin interactions.
在纤维素分解瘤胃细菌黄化瘤胃球菌（Ruminococcus flavefaciens）中鉴定出了两个串联的与纤维小体相关的基因。推导的基因产物代表多模块支架蛋白相关蛋白（称为ScaA和ScaB），二者均包含多个明确的纤维小体特征序列拷贝。对scaB基因进行了全序列测定，其上游相邻基因scaA进行了部分序列测定。scaA的测序部分包含重复的粘着蛋白模块和一个C端dockerin结构域。ScaB包含七个相对不同的粘着蛋白模块、两个极长的富含T的接头以及一个功能未知的C端结构域。总体而言，ScaA和ScaB的粘着蛋白在系统发育上与先前描述的I型和II型粘着蛋白不同，我们提出它们定义了一个新的组，我们在此将其指定为III型粘着蛋白。从这两个基因中选择的模块在大肠杆菌中过表达，重组蛋白用作亲和印迹实验中的探针。结果有力地表明，ScaA作为几种黄化瘤胃球菌酶的类似纤维小体支架蛋白。来自同一细菌的重组ScaA粘着蛋白与表达的含dockerin的酶构建体的直接相互作用支持了这些数据。证据还表明，ScaA dockerin与ScaB上的一种特殊粘着蛋白结合，这表明ScaB可能作为一种锚定蛋白，直接或间接与细菌细胞表面相连。本研究首次在纤维素分解瘤胃细菌中直接证明了由独特的粘着蛋白-dockerin相互作用介导的纤维小体系统。
