Genus-Wide Assessment of Lignocellulose Utilization in the Extremely Thermophilic Genus Caldicellulosiruptor by Genomic, Pangenomic, and Metagenomic Analyses

Abstract

Metagenomic data from Obsidian Pool (Yellowstone National Park, USA) and 13 genome sequences were used to reassess genus-wide biodiversity for the extremely thermophilic Caldicellulosiruptor The updated core genome contains 1,401 ortholog groups (average genome size for 13 species = 2,516 genes). The pangenome, which remains open with a revised total of 3,493 ortholog groups, encodes a variety of multidomain glycoside hydrolases (GHs). These include three cellulases with GH48 domains that are colocated in the glucan degradation locus (GDL) and are specific determinants for microcrystalline cellulose utilization. Three recently sequenced species, Caldicellulosiruptor sp. strain Rt8.B8 (renamed here Caldicellulosiruptor morganii), Thermoanaerobacter cellulolyticus strain NA10 (renamed here Caldicellulosiruptor naganoensis), and Caldicellulosiruptor sp. strain Wai35.B1 (renamed here Caldicellulosiruptor danielii), degraded Avicel and lignocellulose (switchgrass). C. morganii was more efficient than Caldicellulosiruptor bescii in this regard and differed from the other 12 species examined, both based on genome content and organization and in the specific domain features of conserved GHs. Metagenomic analysis of lignocellulose-enriched samples from Obsidian Pool revealed limited new information on genus biodiversity. Enrichments yielded genomic signatures closely related to that of Caldicellulosiruptor obsidiansis, but there was also evidence for other thermophilic fermentative anaerobes (Caldanaerobacter, Fervidobacterium, Caloramator, and Clostridium). One enrichment, containing 89.8% Caldicellulosiruptor and 9.7% Caloramator, had a capacity for switchgrass solubilization comparable to that of C. bescii These results refine the known biodiversity of Caldicellulosiruptor and indicate that microcrystalline cellulose degradation at temperatures above 70°C, based on current information, is limited to certain members of this genus that produce GH48 domain-containing enzymes.IMPORTANCE The genus Caldicellulosiruptor contains the most thermophilic bacteria capable of lignocellulose deconstruction, which are promising candidates for consolidated bioprocessing for the production of biofuels and bio-based chemicals. The focus here is on the extant capability of this genus for plant biomass degradation and the extent to which this can be inferred from the core and pangenomes, based on analysis of 13 species and metagenomic sequence information from environmental samples. Key to microcrystalline hydrolysis is the content of the glucan degradation locus (GDL), a set of genes encoding glycoside hydrolases (GHs), several of which have GH48 and family 3 carbohydrate binding module domains, that function as primary cellulases. Resolving the relationship between the GDL and lignocellulose degradation will inform efforts to identify more prolific members of the genus and to develop metabolic engineering strategies to improve this characteristic.

Keywords: Caldicellulosiruptor; extreme thermophile; pangenome.

FIG 1

Geographical biodiversity of genome-sequenced Caldicellulosiruptor species, phylogenetic relationships, and genome characteristics. Shown is the global distribution of the 14 isolated Caldicellulosiruptor species. The phylogenetic heat plot illustrates relatedness of species (along with outliers Tma and Ctherm) based on ANI. White to blue indicates distant species, while red indicates more closely related species. Genome characteristics for each sequenced species (recently sequenced highlighted in gray) are listed. Abbreviations are as follows: Cace, C. acetigenus; Cbesc, C. bescii; Calhy, C. hydrothermalis; Calkr, C. kristjanssonii; Calkro, C. kronotskyensis; Calla, C. lactoaceticus; COB47, C. obsidiansis; Calow, C. owensensis; Csac, C. saccharolyticus; F32, Caldicellulosiruptor sp. strain F32; Cmorg, C. morganii; Cdani, C. danielii; Cnaga, C. naganoensis; Tma, Thermotoga maritima MSB8; and Ctherm, Clostridium thermocellum ATCC 27405. GH, glycoside hydrolase; CBM, carbohydrate binding module; PL, polysaccharide lyase; CE, carbohydrate esterase; GT, glycosyltransferase. Numbers indicate the total number of open reading frames that contain either GH, CBM, PL, CE, or GT domains. Numbers of carbohydrate-active protein domains were retrieved from the CAZy database (http://www.cazy.org). *, no genome sequenced. See references , , , , , , , and .

FIG 2

Core genome and pangenome of the Caldicellulosiruptor genus. The number of ortholog groups present in the pangenome (red line) increases with each species sequenced, reaching a total of 3,493 ortholog groups with 13 Caldicellulosiruptor genomes. The number of core ortholog groups (black line) decreases with additional genomes; the current total is 1,401 ortholog groups.

FIG 3

Growth of Caldicellulosiruptor species and environmental enrichments on microcrystalline cellulose and switchgrass. Caldicellulosiruptor isolates (C. bescii, C. obsidiansis [COB47], C. morganii [C. morg], C. danielii [C. dani], and C. naganoensis [C. naga]) and metagenomic cultures (B6, B7, B9, and AVI) were grown on 5 g/liter of soluble and insoluble substrates. Growth was monitored for isolates on cellobiose (A), Avicel (B), and switchgrass (C), and a representative growth curve from one biological replicate is shown. Doubling times (T_d) were calculated from growth of biological triplicates; for growth on switchgrass (C), only the first phase of exponential (up to 12 h) was analyzed for T_d (59). Solubilization studies were completed by isolates on Avicel (D) and switchgrass (E) and by environmental communities on switchgrass (F). Error bars represent the SDs of cell counts for one sample per species (A to C) or triplicate solubilizations (D to F).

FIG 4

Maximum likelihood phylogenetic tree for all 13 Caldicellulosiruptor genomes, 9 large contigs from the Yellowstone National Park (YNP) metagenome sequences, and outgroups. The tree was built from concatenated nucleic acid sequences constructed from 14 conserved ribosomal genes. Percentages of times species clustered together over 500 bootstraps are indicated by black (>90%), gray (80 to 89%), and white (75 to 79%) circles on the branch. Branch lengths indicate the number of substitutions over 7,230 nucleotide sites. Font colors indicate common geographical areas of isolation, including the United States (black), New Zealand (violet), Iceland (light blue), Russia (red), Japan (green), China (maize), and Central Europe (dark blue). Evolutionary analysis used MEGA7 (54), and alignments were concatenated using SequenceMatrix (53). Abbreviations for YNP contigs represent IMG scaffold identities (IDs), the full names of which can be found in Table S2 in the supplemental material.

FIG 5

Glucan degradation locus (GDL) enzymes in Caldicellulosiruptor isolates and YNP metagenomes. Genomic locations and protein domain arrangements of CAZymes in the GDL are shown. Species abbreviations are as follows: Cbesc, C. bescii; Calkr, C. kristjanssonii; Calkro, C. kronotskyensis; Calla, C. lactoaceticus; COB47, C. obsidiansis; Csac, C. saccharolyticus; F32, Caldicellulosiruptor sp. strain F32; Cmorg, C. morganii; Cdani, C. danielii; and Cnaga, C. naganoensis. YNP sites are AVI, B6, B7, and B9. Domains numbers refer to CAZY protein families (http://www.cazy.org/), with squares, ovals, and hexagons representing glycoside hydrolases, carbohydrate binding modules, and polysaccharide lysases, respectively. “T,” tāpirins, Caldicellulosiruptor binding proteins.

FIG 6

GDL comparison in Caldicellulosiruptor genus. An unrooted phylogenetic tree derived from concatenated nucleotide sequences of GDL CAZyme homologs in the Caldicellulosiruptor isolates and YNP metagenomic enrichments, alongside grid of possible CAZyme content in each genome, is shown. YNP samples are listed by abbreviated IMG scaffold IDs (see Table S2 in the supplemental material for full IMG IDs and GDL tags). Font colors indicate isolation locations, including the United States (black), New Zealand (violet), Iceland (light blue), Russia (red), Japan (green), and China (maize). Table shading indicates similarity to the domain arrangement at the top of column and is explained by the color key. The phylogenetic tree highlights how similar common areas of isolation also map with similar GDL organization (e.g., Icelandic species group together), as well as shared domain patterns between species. Percentages of times species clustered together over 500 bootstraps are indicated by black (>90%), gray (55 to 69%), and white (<55%) circles on the branch.

FIG 7

Hydrolysis of crystalline cellulose by Caldicellulosiruptor CAZymes. CelA (Athe_1867) from C. bescii and Wai35_2053 from C. danielii were expressed recombinantly in C. bescii and tested for activity on 10 mg/ml of Avicel at 70°C, 75°C, and 80°C. Fifteen milligrams per gram of protein/glucan was added to triplicate samples (error bars signify SDs between replicates).

FIG 8

Homologous protein group conservation based on binary panmatrix for Caldicellulosiruptor and Thermotoga species. Sequence homology-based grouping of proteins among 42 species is shown as two-way clustering. In the vertical axis, protein clusters are grouped according to their presence/absence, showing more conserved families grouping at the top and moderate to low conservation in the center and the bottom. The horizontal axis shows clustering of species based on the core, accessory, and unique sets of ortholog groups. Black bars signify the presence of protein homologs. (A) Thermotoga and Caldicellulosiruptor joint core. Genes are present in both genera. (B) Thermotoga and Caldicellulosiruptor partial core. A large portion of genes are shared, except for major white areas, which signify missing genes in certain species. (C) Thermotoga core, primarily genes that are shared between most Thermotoga species. (D) Caldicellulosiruptor core, primarily genes that are shared between most Caldicellulosiruptor species. (E) Thermotoga group 2 partial core, with genes specific to a more terrestrial subset of Thermotoga. (F) Unique and accessory genes of both genera. Similar patterns between species are indicative of shared gene homologs. Abbreviations for Caldicellulosiruptor species names are as follows: CSTR8, Caldicellulosiruptor sp. strain Rt8.B8 (C. morganii); TCELL, “Thermoanaerobactor cellulolyticus” NA10 (C. naganoensis); CLACT, C. lactoaceticus; CKRIS, C. kristjanssonii; COWEN, C. owensensis; COBSI, C. obsidiansis; CKRON, C. kronotskyensis; CBESC, C. bescii; CSF32, Caldicellulosiruptor sp. strain F32; CSACC, C. saccharolyticus; CACET, C. acetigenus; CHYDR, C. hydrothermalis; and CSWAI, Caldicellulosiruptor sp. strain Wai35.B1 (C. danielii). Abbreviations for Thermotoga species names are as follows: Ga0115064, unclassified Thermotogales bacterium Bin 5 Ga0115064; Ga0115076, unclassified Thermotogales bacterium Bin 13 Ga0115076; TMO, T. lettingae strain TMO; TPROF, T. profunda; 50_64, Thermotoga sp. strain 50_64; 50_1627, Thermotoga sp. strain 50_1627; TCALDI, T. caldifontis; DSM_11164, T. hypogea DSM 11164; NBRC_106472, T. hypogea NBRC 106472; A7A, Thermotoga sp. strain A7A; RQ7, Thermotoga sp. strain RQ7; LA10, T. neapolitana sp. strain LA10; DSM 4359, T. neapolitana DSM 4359; RKU_1, T. petrophila strain RKU-1; RKU_10, T. naphthophila strain RKU-10; Xyl54, Thermotoga sp. strain Xyl54; Cell2, Thermotoga sp. strain Cell2; TBGT1765, Thermotoga sp. strain TBGT1765; TBGT1766, Thermotoga sp. strain TBGT1766; EMP, Thermotoga sp. strain EMP; 2812B, Thermotoga sp. strain 2812B; Mc24, Thermotoga sp. strain Mc24; RQ2, Thermotoga sp. strain RQ2; Tma200, T. maritima strain Tma200; MSB8_1, MSB8_2, MSB8_3, T. maritima strain MSB8; Tma100, T. maritima strain Tma100; and MSB8_DSM_3109, T. maritima strain MSB8, DSM 3109.