Jun Hyun-Sik, Qi Meng, Gong Joshua, Egbosimba Emmanuel E, Forsberg Cecil W
Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1, Canada.
J Bacteriol. 2007 Oct;189(19):6806-15. doi: 10.1128/JB.00560-07. Epub 2007 Jul 20.
Comparative analysis of binding of intact glucose-grown Fibrobacter succinogenes strain S85 cells and adhesion-defective mutants AD1 and AD4 to crystalline and acid-swollen (amorphous) cellulose showed that strain S85 bound efficiently to both forms of cellulose while mutant Ad1 bound to acid-swollen cellulose, but not to crystalline cellulose, and mutant Ad4 did not bind to either. One- and two-dimensional electrophoresis (2-DE) of outer membrane cellulose binding proteins and of outer membranes, respectively, of strain S85 and adhesion-defective mutant strains in conjunction with mass spectrometry analysis of tryptic peptides was used to identify proteins with roles in adhesion to and digestion of cellulose. Examination of the binding to cellulose of detergent-solubilized outer membrane proteins from S85 and mutant strains revealed six proteins in S85 that bound to crystalline cellulose that were absent from the mutants and five proteins in Ad1 that bound to acid-swollen cellulose that were absent from Ad4. Twenty-five proteins from the outer membrane fraction of cellulose-grown F. succinogenes were identified by 2-DE, and 16 of these were up-regulated by growth on cellulose compared to results with growth on glucose. A protein identified as a Cl-stimulated cellobiosidase was repressed in S85 cells growing on glucose and further repressed in the mutants, while a cellulose-binding protein identified as pilin was unchanged in S85 grown on glucose but was not produced by the mutants. The candidate differential cellulose binding proteins of S85 and the mutants and the proteins induced by growth of S85 on cellulose provide the basis for dissecting essential components of the cellulase system of F. succinogenes.
对完整的以葡萄糖为生长底物的琥珀酸纤维杆菌菌株S85细胞以及黏附缺陷突变体AD1和AD4与结晶态和酸溶胀（无定形）纤维素的结合进行比较分析，结果表明，菌株S85能有效地结合两种形式的纤维素；而突变体Ad1能结合酸溶胀纤维素，但不能结合结晶态纤维素；突变体Ad4则两种纤维素都不能结合。分别对菌株S85和黏附缺陷突变体菌株的外膜纤维素结合蛋白和外膜进行一维和二维电泳（2-DE），并结合胰蛋白酶肽段的质谱分析，以鉴定在纤维素黏附和消化过程中起作用的蛋白质。对来自S85和突变体菌株的经去污剂溶解的外膜蛋白与纤维素的结合情况进行检测，结果显示，S85中有6种与结晶态纤维素结合的蛋白在突变体中不存在，Ad1中有5种与酸溶胀纤维素结合的蛋白在Ad4中不存在。通过2-DE鉴定出25种来自以纤维素为生长底物的琥珀酸纤维杆菌外膜部分的蛋白质，其中16种在以纤维素为生长底物时相对于以葡萄糖为生长底物时表达上调。一种被鉴定为Cl刺激的纤维二糖酶的蛋白质在以葡萄糖为生长底物的S85细胞中受到抑制，在突变体中进一步受到抑制；而一种被鉴定为菌毛蛋白的纤维素结合蛋白在以葡萄糖为生长底物的S85中未发生变化，但在突变体中不产生。S85和突变体的候选差异纤维素结合蛋白以及S85在纤维素上生长所诱导的蛋白质为剖析琥珀酸纤维杆菌纤维素酶系统的关键成分提供了基础。
