Label-free Quantitative Proteomics for the Extremely Thermophilic Bacterium <i>Caldicellulosiruptor obsidiansis</i> Reveal Distinct Abundance Patterns upon Growth on Cellobiose, Crystalline Cellulose, and Switchgrass

Mass spectrometric analysis of Caldicellulosiruptor obsidiansis cultures grown on four different carbon sources identified 65% of the cells’ predicted proteins in cell lysates and supernatants. Biological and technical replication together with sophisticated statistical analysis were used to reliably quantify protein abundances and their changes as a function of carbon source. Extracellular, multifunctional glycosidases were significantly more abundant on cellobiose than on the crystalline cellulose substrates Avicel and filter paper, indicating either disaccharide induction or constitutive protein expression. Highly abundant flagellar, chemotaxis, and pilus proteins were detected during growth on insoluble substrates, suggesting motility or specific substrate attachment. The highly abundant extracellular binding protein COB47_0549 together with the COB47_1616 ATPase might comprise the primary ABC-transport system for cellooligosaccharides, while COB47_0096 and COB47_0097 could facilitate monosaccharide uptake. Oligosaccharide degradation can occur either via extracellular hydrolysis by a GH1 β-glycosidase or by intracellular phosphorolysis using two GH94 enzymes. When C. obsidiansis was grown on switchgrass, the abundance of hemicellulases (including GH3, GH5, GH51, and GH67 enzymes) and certain sugar transporters increased significantly. Cultivation on biomass also caused a concerted increase in cytosolic enzymes for xylose and arabinose fermentation.通过对四种不同碳源上培养的嗜热纤维素分解菌（Caldicellulosiruptor obsidiansis）培养液进行质谱分析，在细胞裂解液与上清液中鉴定出该菌株预测蛋白组的65%。本研究通过生物学重复与技术重复结合严谨的统计分析，实现了蛋白丰度及其随碳源变化情况的可靠定量。胞外多功能糖苷酶在纤维二糖上的丰度显著高于微晶纤维素底物Avicel与滤纸，提示存在二糖诱导或组成型蛋白表达两种调控模式。在不溶性底物上培养时，可检测到大量高丰度的鞭毛、趋化性及菌毛蛋白，这表明菌株可能具有运动能力或对底物的特异性附着能力。高丰度的胞外结合蛋白COB47_0549与COB47_1616 ATP酶（ATPase）可能构成纤维寡糖的主要ABC转运系统（ABC-transport system），而COB47_0096与COB47_0097则可能协助单糖的摄取。寡糖的降解可通过两种途径实现：一是由GH1家族β-糖苷酶进行胞外水解，二是借助两种GH94家族酶完成胞内磷酸解。当该菌株在柳枝稷上培养时，半纤维素酶（包含GH3、GH5、GH51及GH67家族酶类）与部分糖转运蛋白的丰度显著升高。在生物质原料上培养还会协同上调参与木糖与阿拉伯糖发酵的胞内酶的表达。