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

Abstract

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.

Metaproteomics of cellulose methanisation under thermophilic conditions reveals a surprisingly high proteolytic activity

“…This taxon contains cellulolytic members, such as C. obsidiansis or C. bescii (formerly Anaerocellum thermophilum). These do not possess cellulosomes, their cellulolytic system being based on secreted multifunctional Metaproteomics of cellulose methanisation F Lü et al Metaproteomics of cellulose methanisationenzymes; they usually utilise a broad range of plant materials, including crystalline cellulose, cellulose, hemicelllose, starch and pectin, with a very high hydrogen yield (van de Werken et al, 2008;VanFossen et al, 2009;Yang et al, 2009;Hamilton-Brehm et al, 2010;Lochner et al, 2011). Among the non-redundant protein groups identified for Caldicellulosiruptor, four were probably related to hemicellulose degradation (two beta-mannanases UniRef50_Q9KWY5, one acetyl xylan esterase UniRef50_F8F4V8 and one endoxylanase UniRef50_ E4Q5G9).…”

Role of the CipA Scaffoldin Protein in Cellulose Solubilization, as Determined by Targeted Gene Deletion and Complementation in Clostridium thermocellum

“…Identified peptides were then score filtered (false-discovery rate [FDR], Ͻ2% peptide spectrum match) and assembled into protein identifications (minimum of 2 distinct peptides per protein call) by IDPicker 3 (26). Proteins were then spectrally balanced to deal with nonunique peptides and normalized by normalized spectral abundance factors (NSAF), and abundance values were adjusted to normalized spectral counts (nSpC) as previously described (27). Protein-to-protein abundance was then assessed across all samples to identify those that were differentially expressed (Student's t test).…”

Caldicellulosiruptor Core and Pangenomes Reveal Determinants for Noncellulosomal Thermophilic Deconstruction of Plant Biomass

“…A strong, potentially irreversible interaction between Caldicellulosiruptor proteins and Avicel would be ideal for proteomic screening to identify substrate-bound proteins, since their affinity for Avicel would have to withstand washing steps to remove cells. Previous proteomic screens from members of the genus focused on the cell-free extracellular and whole-cell fractions of cellulolytic Caldicellulosiruptor species (15,43,44). We previously reported on differential two-dimensional SDS-PAGE profiles of cell-free supernatant from cells grown on Avicel in an attempt to capture protein-level differences of weakly to strongly cellulolytic Caldicellulosiruptor species (9).…”

“…Analysis of genome sequence data from biomass-degrading microorganisms has helped to identify noncellulosomal bacteria that also lack identifiable cellobiohydrolases, such as Cytophaga hutchinsonii (96) and Fibrobacter succinogenes (77), both of which require close attachment to cellulose for efficient hydrolysis, and Sacharophagus degradans (95), which uses processive endocellulases (94), indicating that there is great diversity in strategies used for crystalline cellulose hydrolysis. As members of the phylum Firmicutes, Caldicellulosiruptor species are distinct from the thermophilic, anaerobic clostridia in that they secrete free and S-layer-bound cellulases and hemicellulases (9,23,24,43,44,58,60,63,75,84,89,90) that are not assembled into cellulosomes (85,89). In this respect, their strategy for crystalline cellulose deconstruction is similar to that for noncellulosomal biomass-degrading aerobic fungi, such as Trichoderma reesei, (54), the thermophilic fungi Myceliophthora thermophila and Thielavia terrestris (7), or the thermophilic aerobe Thermobifida fusca (48).…”