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

Abstract

Extremely thermophilic bacteria of the genus Caldicellulosiruptor utilize carbohydrate components of plant cell walls, including cellulose and hemicellulose, facilitated by a diverse set of glycoside hydrolases (GHs). From a biofuel perspective, this capability is crucial for deconstruction of plant biomass into fermentable sugars. While all species from the genus grow on xylan and acidpretreated switchgrass, growth on crystalline cellulose is variable. The basis for this variability was examined using microbiological, genomic, and proteomic analyses of eight globally diverse Caldicellulosiruptor species. The open Caldicellulosiruptor pangenome (4,009 open reading frames [ORFs]) encodes 106 GHs, representing 43 GH families, but only 26 GHs from 17 families are included in the core (noncellulosic) genome (1,543 ORFs). Differentiating the strongly cellulolytic Caldicellulosiruptor species from the others is a specific genomic locus that encodes multidomain cellulases from GH families 9 and 48, which are associated with cellulose-binding modules. This locus also encodes a novel adhesin associated with type IV pili, which was identified in the exoproteome bound to crystalline cellulose. Taking into account the core genomes, pangenomes, and individual genomes, the ancestral Caldicellulosiruptor was likely cellulolytic and evolved, in some cases, into species that lost the ability to degrade crystalline cellulose while maintaining the capacity to hydrolyze amorphous cellulose and hemicellulose. Interest in cellulosic biofuels (29) has sparked efforts to isolate microorganisms capable of both hydrolysis and fermentation of plant biomass, a process referred to as consolidated bioprocessing (CBP) (49, 50). Since plant biomass deconstruction could be accelerated at elevated temperatures, thermophilic microorganisms have been considered catalysts for CBP (8). Of particular note in this regard are members of the extremely thermophilic genus Caldicellulosiruptor that inhabit globally diverse, terrestrial hot springs (12,27,56,57,61,69,80,98) and thermally heated mud flats (31). Caldicellulosiruptor species are Gram-positive bacteria and typically associate with plant debris; consequently, all isolates characterized to date hydrolyze certain complex carbohydrates characteristic of plant cell walls (8, 97). As such, members of the genus Caldicellulosiruptor are excellent genetic reservoirs of enzymes for plant biomass degradation and, pending the development of functional genetics systems, are potential metabolic hosts for CBP (9).Currently, there are two main paradigms described for microbial degradation of crystalline cellulose: cellulosomal (3) and noncellulosomal (48, 54). Enzymatically, both systems require the concerted efforts of cellobiohydrolases, endocellulases, and ␤-glucosidases (49). Crystalline cellulose deconstruction via cell membrane-bound cellulosomes was first described in the thermophile Clostridium thermocellum and has since been described in other mesophilic Firmicutes, such as Clostridium cellulolyticum,...

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

“…Genome-wide homology (95.6% identity at the nucleotide level) is highest for the two Russian isolates (C. bescii and C. kronotskyensis). Homology of C. saccharolyticus to the other two species is lower but still relatively high, with 80.3% and 79.6% nucleotide identity to C. bescii and C. kronotskyensis, respectively (17). Homologous protein sequences were identified by sequence clustering using UCLUST software (drive5) (based on 50% identity and 50% coverage) (33), genes from all three species were organized into 3,223 families with 885 singletons (family with only a single gene).…”

“…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). The cellulolytic Caldicellulosiruptor species also utilize novel binding proteins (ta pirins) to adhere to plant biomass (19).…”

Genome Stability in Engineered Strains of the Extremely Thermophilic Lignocellulose-Degrading Bacterium Caldicellulosiruptor bescii

“…Upon closer inspection of the insertions and deletions in the alignment, most were located in homopolymer runs of four or more nucleotides and were on average less than one nucleotide long, so these may be attributed to sequencing or assembly artifacts. Overall, the two genome sequences generally agree but with only one notable exception, and this is at the so-called glucan degradation locus (GDL) (23). As shown in Fig.…”

“…This observation is particularly important, as it is the second instance of the loss of ISCbe4 without the duplication of the direct repeats. Interestingly, one ISCbe4 element in JWCB018 disrupted a gene previously identified as a cellulosebinding protein termed a ta pirin (Athe_1870) (23,24), and this could potentially give rise to changes in attachment to and degradation of biomass substrates (Table S5). However, the cause of the swimming motility defect seen in strain WT-Parent and JWCB005 or the clumping phenotype in JWCB018 could not be attributed to movement of the known insertion elements.…”

“…Cells from 50-ml and 500-ml cultures were harvested at 6,000 ϫ g for Southern blot and DNA sequencing analyses, respectively. A phenol-chloroform-isoamyl alcohol extraction was used, as described previously (23). Ethanol precipitation was performed to obtain highly pure DNA.…”