S-Layer Homology Domain Proteins Csac_0678 and Csac_2722 Are Implicated in Plant Polysaccharide Deconstruction by the Extremely Thermophilic Bacterium Caldicellulosiruptor saccharolyticus

Abstract

The genus Caldicellulosiruptor contains extremely thermophilic bacteria that grow on plant polysaccharides. The genomes of Caldicellulosiruptor species reveal certain surface layer homology (SLH) domain proteins that have distinguishing features, pointing to a role in lignocellulose deconstruction. Two of these proteins in Caldicellulosiruptor saccharolyticus (Csac_0678 and Csac_2722) were examined from this perspective. In addition to three contiguous SLH domains, the Csac_0678 gene encodes a glycoside hydrolase family 5 (GH5) catalytic domain and a family 28 carbohydrate-binding module (CBM); orthologs to Csac_0678 could be identified in all genome-sequenced Caldicellulosiruptor species. Recombinant Csac_0678 was optimally active at 75°C and pH 5.0, exhibiting both endoglucanase and xylanase activities. SLH domain removal did not impact Csac_0678 GH activity, but deletion of the CBM28 domain eliminated binding to crystalline cellulose and rendered the enzyme inactive on this substrate. Csac_2722 is the largest open reading frame (ORF) in the C. saccharolyticus genome (predicted molecular mass of 286,516 kDa) and contains two putative sugar-binding domains, two Big4 domains (bacterial domains with an immunoglobulin [Ig]-like fold), and a cadherin-like (Cd) domain. Recombinant Csac_2722, lacking the SLH and Cd domains, bound to cellulose and had detectable carboxymethylcellulose (CMC) hydrolytic activity. Antibodies directed against Csac_0678 and Csac_2722 confirmed that these proteins bound to the C. saccharolyticus S-layer. Their cellular localization and functional biochemical properties indicate roles for Csac_0678 and Csac_2722 in recruitment and hydrolysis of complex polysaccharides and the deconstruction of lignocellulosic biomass. Furthermore, these results suggest that related SLH domain proteins in other Caldicellulosiruptor genomes may also be important contributors to plant biomass utilization.

Comparative Analysis of Extremely Thermophilic Caldicellulosiruptor Species Reveals Common and Unique Cellular Strategies for Plant Biomass Utilization

“…Switchgrass typically contains approximately 15% water-soluble material, 25 to 50% of which is carbohydrate (27,28). After approximately 24 h, as the readily solubilized sugars were utilized by the bacteria, planktonic cells entered a second, slower growth phase, which was tracked over the next 200 h. During the slower growth phase, the Caldicellulosiruptor species can recruit carbohydrates by deploying extracellular, multidomain glycoside hydrolases (GHs), both free (29,30) and attached to the cell surface via the S-layer (15). These GHs are thought to degrade the more recalcitrant complex carbohydrate fractions, thereby releasing the soluble sugars necessary for growth.…”

“…To date, Caldicellulosiruptor species have been isolated globally from terrestrial hot springs and thermal features in locations including the United States (C. owensensis [2,3] and C. obsidiansis [4,5]), Russia (C. bescii [6,7]), C. kronotskyensis [2,8], and C. hydrothermalis [2,8]), New Zealand (C. saccharolyticus [9][10][11]), and Iceland (C. kristjanssonii [2,12] and C. lactoaceticus [2,13]). Characteristic of Caldicellulosiruptor species are multidomain extracellular and S-layer-associated glycoside hydrolases (GHs) that mediate the microbial conversion of complex carbohydrates (14)(15)(16). Sequenced genomes for Caldicellulosiruptor species (2,4,6,10) indicate that some, but not all, encode GH48-containing enzymes (17), and these appear to be essential for crystalline cellulose degradation (18).…”

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

“…Indeed, C. kristjanssonii has 11 GH domain-containing enzymes above the core Caldicellulosiruptor set, the lowest number of total GH domain-containing enzymes in the genus. Note that the minimal set of carbohydrate-active enzymes in the genus does not equip the microbe for crystalline cellulose hydrolysis, although the GH5-containing enzyme does allow for random cleavage of amorphous cellulose (63). C. lactoaceticus, a species closely related to C. kristjanssonii, is cellulolytic and possesses only 6 more CAZy-related ORFs above that of C. kristjanssonii (Table 2).…”

“…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).…”

Discrete and Structurally Unique Proteins (Tāpirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose

“…Although the mechanism by which this occurs has not been established, insights along these lines have been described. Some of the modular enzymes from the genus Caldicellulosiruptor are cell-anchored using S-layer homology domains (30) and are hypothesized to mediate cell-substrate proximity. In addition, substrate-binding proteins (in some cases, components of ATP-binding cassette transporters (24,31)) may also play a role in orienting Caldicellulosiruptor species toward carbohydrate moieties in plant biomass, possibly through positively charged amino acid residues interacting with negatively charged areas (32).…”