CHARACTERIZATION OF 4 OUTER-MEMBRANE PROTEINS THAT PLAY A ROLE IN UTILIZATION OF STARCH BY BACTEROIDES-THETAIOTAOMICRON

Results of earlier work had suggested that utilization of polysacchari des by Bacteroides spp. did not proceed via breakdown by extracellular polysaccharide-degrading enzymes. Rather, it appeared that the polysa ccharide was first bound to a putative outer membrane receptor complex and then translocated into the periplasm, where the degradative enzym es were located, In a recent article, we reported the cloning and sequ encing of susC, a gene from Bacteroides thetaiotaomicron that encoded a 115-kDa outer membrane protein. SusC protein proved to be essential for utilization not only of starch but also of intermediate-sized malt ooligosaccharides (maltose to maltoheptaose). In this paper, we report the sequencing of a 7-kbp region of the B. thetaiotaomicron chromosom e that lies immediately downstream of susC. We found four genes in thi s region (susD, susE, susF, and susG). Transcription of these genes wa s maltose inducible, and the genes appeared to be part of the same ope ron as susC. Western blot (immunoblot) analysis using antisera raised against proteins encoded by each of the four genes showed that all fou r were outer membrane proteins. Protein database searches revealed tha t SusE had limited similarity to a glucanohydrolase from Clostridium a cetobutylicum and SusG had high similarity to amylases from a variety of sources. SusD and SusF had no significant similarity to any protein s in the databases. Results of C-14-starch binding assays suggested th at SusD makes a major contribution to binding. SusE and SusF also appe ar to contribute to binding but not to the same extent as SusD. SusG i s essential for growth on starch but appears to contribute little to s tarch binding. Our results demonstrate that the binding of starch to t he B. thetaiotaomicron surface involves at least four outer membrane p roteins (SusC, SusD, SusE, and SusF), which may form a surface recepto r complex, The role of SusG in binding is still unclear.