Structure of Secondary Cell Wall Polymers in Cell Surfaces of Anaerobes and Interaction between Them and SLH Domains

1.从3种梭菌中制备次级细胞壁聚合物（SCWP）和肽聚糖2，并在大肠杆菌中制备并纯化了来自3种梭菌的SLH模块多肽：Josui梭菌木聚糖酶Xyn 10A、C.stercorarium梭菌木聚糖酶Xyn 10 B和C.thermocellum梭菌细胞表面蛋白SdbA。因此，在本发明中，发现来自C.josui Xyn 10A和C.stercorarium Xyn 10 B的SLH模块多肽与SCWP结合，但来自C.thermocellum SdbA的SLH模块多肽出乎意料地与肽聚糖结合。4.使用从大肠杆菌制备的肽聚糖级分的结合测定证实，来自C.thermocellum SdbA的SLH模块多肽对肽聚糖具有亲和力，尽管通常认为SLH模块与肽聚糖结合。SCWP。热纤梭菌SdbA的SLH模块多肽可用于SCWP缺失细菌的细胞表面工程。5.采用盐酸水解短梗梭菌、粪生梭菌和热纤梭菌的SCWP，并对SCWP的氨基糖组分进行了定性分析。N-乙酰葡萄糖胺和N-乙酰甘露糖胺被检测为粪梭菌SCWP的成分，N-乙酰半乳糖胺以及这些氨基糖被鉴定为柔井梭菌和热纤梭菌SCWP的成分，表明这两种细菌的SCWP包括未鉴定的结构。

1.Secondary cell wall polymers(SCWP) and peptidoglycans were prepared from 3 clostridia, C.josui, C.stercorarium and C.thermocellum2.SLH module polypeptides from C.josui xylanase Xyn10A, C.stercorarium xylanase Xyn10B and C.thermocellum cell surface protein SdbA were produced in Escherichia coli and purified.3.Interactions between these polypeptides and various cell wall fractions were investigated. As a result, it was found that the SLH module polypeptides from C.josui Xyn10A and C.stercorarium Xyn10B bound to SCWP but that the SLH module polypeptide from C.thermocellum SdbA unexpectedly bound to peptidoglycan.4.Binding assay using peptidoglycan fraction prepared from E.coli confirmed that the SLH module polypeptide from C.thermocellum SdbA had an affinity for peptidoglycan although it is generally believed that SLH modules bind to SCWP. The SLH module polypeptide from C.thermocellum SdbA should be applicable for cell surface engineering in bacteria lacking SCWP.5.SCWP of C.josui, C.stercorarium and C.thermocellum were hydrolyzed with HC1 and constituent amino sugars were qualitatively analyzed. N-Acetylglucosamine and N-acetylmannosamine were detected as constituents for C.stercorarium SCWP and N-acetylgalactosamine as well as these amino sugars were identified for C.josui and C.thermocellum SCWP, suggesting that SCWP of these two bacteria include an unidentified structure.