TY - JOUR
AU - Blumer-Schuette, Sara E
AB - Abstract The genus Caldicellulosiruptor is comprised of extremely thermophilic, heterotrophic anaerobes that degrade plant biomass using modular, multifunctional enzymes. Prior pangenome analyses determined that this genus is genetically diverse, with the current pangenome remaining open, meaning that new genes are expected with each additional genome sequence added. Given the high biodiversity observed among the genus Caldicellulosiruptor, we have sequenced and added a 14th species, Caldicellulosiruptor changbaiensis, to the pangenome. The pangenome now includes 3791 ortholog clusters, 120 of which are unique to C. changbaiensis and may be involved in plant biomass degradation. Comparisons between C. changbaiensis and Caldicellulosiruptor bescii on the basis of growth kinetics, cellulose solubilization and cell attachment to polysaccharides highlighted physiological differences between the two species which are supported by their respective gene inventories. Most significantly, these comparisons indicated that C. changbaiensis possesses uncommon cellulose attachment mechanisms not observed among the other strongly cellulolytic members of the genus Caldicellulosiruptor. Electronic supplementary material The online version of this article (10.1007/s10295-019-02222-1) contains supplementary material, which is available to authorized users. Asma M. A. M. Khan and Carl Mendoza contributed equally to this manuscript. Introduction The genus Caldicellulosiruptor is comprised of extremely thermophilic, fermentative heterotrophs whose members have been isolated worldwide from terrestrial geothermal springs or other thermal environments [47]. The original isolates from the genus Caldicellulosiruptor were identified on the basis of their ability to grow on cellulose at elevated temperatures [71, 73], especially temperatures beyond the optimal growth temperature of Hungateiclostridium thermocellum [61]. Interest in thermostable enzymes produced by this genus continues, as the initial discovery of their multifunctional, modular enzymes [34, 65, 74, 88] represented an alternate paradigm to cellulosomes [3, 69]. Further discoveries on the capabilities of these thermostable enzymes include the unique mode of action used by the central cellulase, CelA, [9], synergistic activity in ionic liquid-optimized enzyme mixtures [58, 59] and the creation of designer cellulosomes from Caldicellulosiruptor catalytic domains [37]. Development of a genetics system for Caldicellulosiruptor bescii [18, 20] has also expanded the scope of work with this genus, including metabolic engineering [13, 16, 17, 64] and catalytic improvement [23, 39, 41–44]. The availability of genome sequences has precipitated deeper insights into the genus Caldicellulosiruptor, including comparative studies which have identified biomarkers for plant biomass deconstruction [6, 7, 29], novel insertion elements [19], genetic tractability [15], diverse mechanisms involved in biomass solubilization [47, 87], unique cellulose adhesins (tāpirins) [6, 47] and the identification of new combinations of catalytic domains [6, 29, 46]. Perhaps owing to the unique thermal environments that this genus inhabits, their genetic diversity is dynamic, as the first-described Caldicellulosiruptor pangenome was predicted to be open [6], and remained open after the addition of five additional genome sequences [46]. Here, we have analyzed the genome sequence of Caldicellulosiruptor changbaiensis, isolated from a hot spring in the Changbai Mountains [4], representing the 14th and most recent addition to the Caldicellulosiruptor pangenome. Past Caldicellulosiruptor pangenome analyses were comprised of multiple species from most countries of origin, which allowed for analysis on the basis of biogeography [6], with the exception of China and Japan [26]. Now with the addition of the C. changbaiensis genome sequence, insights into the biogeography of isolates from China and how they compare to the global Caldicellulosiruptor pangenome are possible. Furthermore, on the basis of the open Caldicellulosiruptor pangenome [6, 26], we hypothesize that the C. changbaiensis genome may encode for novel substrate-binding proteins and/or plant biomass-degrading enzymes. In addition to updating the Caldicellulosiruptor pangenome, we also present differences in the growth physiology of C. changbaiensis versus Caldicellulosiruptor bescii, currently the benchmark species against which most Caldicellulosiruptor are compared for their plant biomass-degrading capabilities. Materials and methods Microbial strains and medium Freeze-dried stocks of C. changbaiensis strain CBS-Z were obtained from the Leibniz Institute DSMZ—German Collection of Microorganisms and Cell Cultures (DSMZ). Glycerol stocks of C. bescii DSM-6725 were obtained from the laboratory of Robert M. Kelly, North Carolina State University (Raleigh, NC, USA). Both species were cultured at 75 °C on low osmolarity-defined (LOD) medium [32] under a nitrogen headspace to maintain anaerobic conditions and supplemented with carbohydrates as a carbon source. Carbohydrates used as a carbon source included cellobiose (≥ 99%, Chem-Impex Int’l, Inc.), pectin (Sigma-Aldrich), xylan (Sigma-Aldrich), glucomannan (NOW foods), and microcrystalline cellulose (20 µm Sigmacell, Sigma-Aldrich). For genomic DNA isolation, C. changbaiensis was cultured anaerobically at 75 °C on low osmolarity complex (LOC) medium [32] with cellobiose as a carbon source. Genomic DNA isolation Genomic DNA was isolated using the Joint Genome Institute’s CTAB-based protocol (https://jgi.doe.gov/user-programs/pmo-overview/protocols-sample-preparation-information/jgi-bacterial-dna-isolation-ctab-protocol-2012/), with modifications. 500 ml of overnight C. changbaiensis culture was harvested by centrifugation at 5000×g, 4 °C for 20 min and resuspending the cell pellet in 14.8 ml of TE buffer, prior to lysis. Gel electrophoresis in 0.7% agarose was used to assess the quality of genomic DNA and the concentration and purity of the sample for sequencing was quantified using a NanoDrop spectrophotometer, and Qubit fluorometric assay (dsDNA HS assay, Thermo Fisher). Prior to genome sequencing, a 16S rRNA gene fragment was amplified from isolated genomic DNA using oligonucleotide primers (Eton Bioscience) previously designed for the identification of C. changbaiensis [4], to confirm that the gDNA was from C. changbaiensis. Oligonucleotide primer pair 8F-207 (5′-AGAGTTTGATCCTGGCTCAG-3′) and Caldi-R-208 (5′-GTACGGCTACCTTGTTACG-3′) were used. Amplicons were sent for Sanger sequencing (Eton Bioscience), using the same oligonucleotide primers. C. changbaiensis genome sequencing, assembly and annotation The genome sequence for C. changbaiensis [52] was assembled to 60-fold coverage from long-read Oxford NanoPore (MinION) data generated in house, and short-read Illumina data generated by Molecular Research, LP (MR DNA). Hybrid assembly of the complete C. changbaiensis genome used Unicycler v0.4.7 [78], and annotation of the genome used the Prokaryotic Genome Annotation Pipeline v4.7 [72] provided by the National Center for Biotechnology Information (NCBI). The assembled genome and reads used for assembly of the C. changbaiensis genome are available through NCBI BioProject accession PRJNA511150. Phylogenomic analysis of amino acid sequences Fourteen genome sequences from the genus Caldicellulosiruptor were included in the phylogenomic analyses (see Table 1 for genome sequence accession numbers). Orthologous protein groups were classified using the GET_HOMOLOGUES v20092018 software package [25], running OrthoMCL v1.4 [50], COGtriangles v2.1 [45], or bidirectional best hits (BDBH) as determined by BLASTP [1, 12]. Orthologous protein clusters were determined using the OrthoMCL parameters: 75% pairwise coverage, maximum BLASTP E value of 1e-5, and MCL inflation of 1.5. GET_HOMOLOGUES was also used to parse the pangenome matrices comparing the C. changbaiensis genome inventory against the recent 13 Caldicellulosiruptor pangenome [47], the C. saccharolyticus genome [76], or the revised C. bescii genome [28]. PhyloPhlAn v0.99 was used to construct an unrooted core protein phylogenetic tree using default settings [67] and the resulting tree was visualized in iTOL v4.4.2 [49]. Digital measures of DNA–DNA hybridization were calculated using the online GGDC tool (https://ggdc.dsmz.de) [51]. Caldicellulosiruptor genome sequences included in the updated pangenome analysis Species name . NCBI RefSeq accession . References . C. acetigenus GCF_000421725.1 [55] C. bescii GCF_000022325.1 [28] C. changbaiensis GCF_003999255.1 [52] C. danielii GCF_000955725.1 [46, 48] C. hydrothermalis GCF_000166355.1 [6, 8] C. kristjanssonii GCF_000166695.1 [6, 8] C. kronotskyensis GCF_000166775.1 [6, 8] C. lactoaceticus GCF_000193435.2 [6, 8] C. morganii GCF_000955745.1 [46, 48] C. naganoensis GCF_000955735.1 [46, 48] C. obsidiansis GCF_000145215.1 [31] C. owensensis GCF_000166335.1 [6, 8] C. saccharolyticus GCF_000016545.1 [76] C. sp. F32 GCF_000404025.1 [84] Species name . NCBI RefSeq accession . References . C. acetigenus GCF_000421725.1 [55] C. bescii GCF_000022325.1 [28] C. changbaiensis GCF_003999255.1 [52] C. danielii GCF_000955725.1 [46, 48] C. hydrothermalis GCF_000166355.1 [6, 8] C. kristjanssonii GCF_000166695.1 [6, 8] C. kronotskyensis GCF_000166775.1 [6, 8] C. lactoaceticus GCF_000193435.2 [6, 8] C. morganii GCF_000955745.1 [46, 48] C. naganoensis GCF_000955735.1 [46, 48] C. obsidiansis GCF_000145215.1 [31] C. owensensis GCF_000166335.1 [6, 8] C. saccharolyticus GCF_000016545.1 [76] C. sp. F32 GCF_000404025.1 [84] Open in new tab Caldicellulosiruptor genome sequences included in the updated pangenome analysis Species name . NCBI RefSeq accession . References . C. acetigenus GCF_000421725.1 [55] C. bescii GCF_000022325.1 [28] C. changbaiensis GCF_003999255.1 [52] C. danielii GCF_000955725.1 [46, 48] C. hydrothermalis GCF_000166355.1 [6, 8] C. kristjanssonii GCF_000166695.1 [6, 8] C. kronotskyensis GCF_000166775.1 [6, 8] C. lactoaceticus GCF_000193435.2 [6, 8] C. morganii GCF_000955745.1 [46, 48] C. naganoensis GCF_000955735.1 [46, 48] C. obsidiansis GCF_000145215.1 [31] C. owensensis GCF_000166335.1 [6, 8] C. saccharolyticus GCF_000016545.1 [76] C. sp. F32 GCF_000404025.1 [84] Species name . NCBI RefSeq accession . References . C. acetigenus GCF_000421725.1 [55] C. bescii GCF_000022325.1 [28] C. changbaiensis GCF_003999255.1 [52] C. danielii GCF_000955725.1 [46, 48] C. hydrothermalis GCF_000166355.1 [6, 8] C. kristjanssonii GCF_000166695.1 [6, 8] C. kronotskyensis GCF_000166775.1 [6, 8] C. lactoaceticus GCF_000193435.2 [6, 8] C. morganii GCF_000955745.1 [46, 48] C. naganoensis GCF_000955735.1 [46, 48] C. obsidiansis GCF_000145215.1 [31] C. owensensis GCF_000166335.1 [6, 8] C. saccharolyticus GCF_000016545.1 [76] C. sp. F32 GCF_000404025.1 [84] Open in new tab Phylogenomic analysis of nucleotide sequences Genome-level similarity was quantified as average nucleotide identity (ANIb) from the BLASTN+ alignment of 1020 nt fragments from the 14 Caldicellulosiruptor genomes [35, 62]. ANIb were calculated by Pyani v0.2.7, (https://github.com/widdowquinn/pyani) and percent identities were plotted as a heatmap by the software package. A core-genome alignment across all 14 species was generated following the method detailed by Chung et al. [24]. Briefly, Mugsy v1.2.3 [2] was used to align the Caldicellulosiruptor core genome, and processed using mothur v1.42.1 [66]. A core-genome maximum-likelihood phylogenetic tree was constructed from the processed Mugsy output using IQ-Tree v1.6.11 [56], with optimal models selected by ModelFinder [38] and tested with 1,000 bootstraps using UFBoot [54]. The core-genome phylogenetic tree was visualized by iTOL v4.4.2 [49]. Local collinear blocks (LCBs) were visualized using progressiveMauve v2015-02-25 [30] after rearrangement of Caldicellulosiruptor sp. F32 contigs [63] using the C. changbaiensis genome as a reference. Growth kinetics on polysaccharides Caldicellulosiruptor bescii or C. changbaiensis were revived from − 80 °C glycerol stocks for growth curve analysis on microcrystalline cellulose, xylan, pectin, glucomannan, fucose, rhamnose, arabinogalactan and polygalacturonate. Glycerol stocks (1 ml) were subcultured into 50 ml LOD medium for three consecutive subcultures using 2% (v/v) inoculum at each passage. Revived cultures were then transferred (2% [v/v] inoculum) to LOD medium containing a 1:1 ratio of maltose (C. bescii) or cellobiose (C. changbaiensis) to polysaccharide. The 1:1 mixture was then passaged (2% [v/v] inoculum) three times successively in LOD medium with polysaccharide, only. Cultures for growth curves were inoculated at a starting cell density of 1 × 106 cells ml−1 in 200 ml LOD plus the respective polysaccharide. Biological replicates were used for each growth phase experiment. Cell counting used epifluorescence microscopy at 1000× total magnification and a counting reticle as described previously [36]. Cells were fixed in a final volume of 1.1 ml glutaraldehyde (2.5% [v/v] in water) prior to incubation with acridine orange (1 g l−1) and approximately 5 ml sterilized water and thoroughly mixed. Stained cells were then vacuum filtered through a polycarbonate 0.22-µm filter (GE). Samples were counted using a 10 × 10 reticle a total of ten times. Cell counts were averaged for calculation of cell density (cells ml−1). Doubling times are described as the number of hours per generation during exponential growth, calculated as Δtime divided by the number of generations. Microcrystalline cellulose solubilization Solubilization of microcrystalline cellulose followed protocols established by Zurawski et al., [87] with modifications. C. bescii or C. changbaiensis were cultured in serum bottles with 50 ml of LOD medium supplemented with 0.6 g of microcrystalline cellulose (20 µm Sigmacell) at a starting cell density of 106 cells ml−1. Cultures were then incubated without shaking at 75 °C for 7 days, after which the remaining microcrystalline cellulose was harvested by centrifugation at 6000×g, 4 °C for 15 min in a swing bucket rotor. The cellulose pellet was washed four times in sterile, deionized water and air dried at 75 °C until the weight of the microcrystalline cellulose did not change. Uninoculated LOD served as an abiotic control. Percent solubilization is reported as the difference in substrate weight divided by the starting weight multiplied by 100. All experimental conditions were measured in triplicate and significance was determined by a t test (p value < 0.05). Cell attachment assays Caldicellulosiruptor bescii and C. changbaiensis cell cultures were grown to early stationary phase on either xylan or cellulose (1 g l−1) as the carbon source, and cell densities were calculated before harvesting at 5000×g for 10 min at room temperature. Cells were resuspended and concentrated tenfold in the binding buffer (50 mM sodium phosphate, pH 7.2) to a tenfold density of approximately 1–2 × 109 cells ml−1 for cells cultured on xylan or 1 × 108 cells ml−1 for cells cultured on cellulose. For each treatment condition, 1.2 ml of C. bescii or C. changbaiensis planktonic cells in binding buffer were added to a 1.5-ml microcentrifuge tube, and supplemented with 10 mg of washed substrate (experimental condition: xylan or cellulose), or no substrate for the negative control. All assay tubes were incubated at room temperature for 1 h with gentle rotary shaking at 100 rpm. After incubation, planktonic cells were enumerated as described above for the growth curves. Each binding assay was repeated six times. Two-sample t tests were used to analyze the data using the R studio statistics package v3.3.3 [60]. Results and discussion Expansion of the Caldicellulosiruptor pangenome With the addition of the fourteenth Caldicellulosiruptor genome [52], we sought to define an updated core- and pangenome. Three different algorithms: OrthoMCL [50], bidirectional best hit and COGtriangles [45] were used to classify orthologous clusters for pangenome analysis (Table S1). Of the three, the clusters formed by OrthoMCL resulted in an estimated core- and pangenome with the lowest residual standard errors, and are reported here (Fig. 1). Overall, there are 120 new protein clusters not previously observed in the Caldicellulosiruptor pangenome that were identified in the C. changbaiensis genome [47], 75 of which are annotated as hypothetical proteins. Further transcriptomic and proteomic studies may aid in the identification of the function of these unique hypothetical proteins. By adding a 14th genome, the Caldicellulosiruptor core-genome was reduced to 1,367 orthologous clusters; however, the pangenome (3,791 genes) continues to expand at an estimated rate of 63.2 genes per additional genome (Fig. 1) highlighting the plasticity of the Caldicellulosiruptor pangenome. Fig. 1 Open in new tabDownload slide Core- and pangenome size estimates calculated from random sampling of 14 Caldicellulosiruptor genomes. a Fitted curve of the estimated Caldicellulosiruptor core genome from 10 random samples of genomes up to n = 14. The current size of the core genome is 1367 orthologous clusters. b Fitted curve of the estimated Caldicellulosiruptor pangenome from 10 random samples of genomes up to n = 14. The Caldicellulosiruptor pangenome remains open and has increased to 3791 genes. The rate of growth for the pangenome is 63.2 new genes per genome sequenced. Core- and pangenome estimates were calculated from the equations reported by Tettelin et al. [75] using GET_HOMOLOGUES software [25] C. changbaiensis shares similarity with other genomes based on biogeography In contrast to the diversity of previously released genome sequences from New Zealand [46], C. changbaiensis exhibits a similar pattern of biogeography based on average nucleotide identity (ANIb). As expected, Caldicellulosiruptor sp. F32, isolated from compost in China [84], and C. saccharolyticus, isolated from a hot spring in New Zealand [68] shared a higher percent identity values with C. changbaiensis (94.2–96.2%, Fig. 2 and Table S2). In addition to ANIb supporting the close evolutionary relationship between C. saccharolyticus, Caldicellulosiruptor sp. F32 and C. changbaiensis, all three species also clustered on the basis of their core-genome nucleotide sequence (Fig. 3a), and orthologous amino acid sequences (Fig. 3b). This is consistent with a prior phylogenetic analysis which included contigs from environmental metagenomes, where C. saccharolyticus and Caldicellulosiruptor sp. F32 also clustered together in a maximum-likelihood phylogenetic tree [46]. Across all three measures of similarity and digital DNA–DNA hybridization (dDDH) [51], C. saccharolyticus is more closely related to Caldicellulosiruptor sp. F32 (ANIb > 96% and dDDH > 69%) than to C. changbaiensis (ANIb > 94% and dDDH > 59%). The level of similarity between this cluster, however, is not as high as that shared among the Caldicellulosiruptor species isolated from Iceland which share over 97% similarity based on ANIb (Table S2) and 78% similarity based on dDDH, meeting the genome-based taxonomic criteria to be classified as single species. Fig. 2 Open in new tabDownload slide Heatmap representation of the average nucleotide identity for 14 genome sequenced species from the genus Caldicellulosiruptor. Average nucleotide identity (ANIb) was calculated on the basis of legacy BLASTn sequence identity over 1020 nt sequence fragments. ANIb values of all 14 genomes are represented by a heat plot ranging from blue (75% < ANIb < 90%), white (90% < ANIb < 95%) to red (ANIb > 95%). Pyani (https://github.com/widdowquinn/pyani) was used to calculate ANIb values and generate the clustered heatmap. Hierarchal cluster dendrograms were generated on the basis of similar ANIb values across each species. ANIb values are reported in Table S2. Calace, C. acetigenus; Cbes, C. bescii; Calcha, C. changbaiensis; Caldan, C. danielii; Calhy, C. hydrothermalis; Calkr, C. kristjanssonii; Calkro, C. kronotskyensis; Calla, C. lactoaceticus; Calmo, C. morganii; Calna, C. naganoensis; COB47, C. obsidiansis; Calow, C. owensensis; Csac, C. saccharolyticus; F32, C. sp. F32 Fig. 3 Open in new tabDownload slide Phylogenomic trees constructed from core genome nucleotide or protein alignment for all 14 Caldicellulosiruptor genomes. a Maximum-likelihood phylogenomic tree constructed from the core genome nucleotide alignment. All nodes were supported with a bootstrap value of 100. b Maximum-likelihood phylogenomic tree constructed from the alignment of 329 orthologous protein clusters using PhyloPhlAn [67]. All nodes were supported with bootstrap values ≥ 95.9, except the node denoted by a green star (C. kristjanssonii and C. lactoaceticus) which had a significantly lower bootstrap value of 37.5. Both trees shared little similarity with regards to topology; however, most genomes continued to cluster based on biogeography. In both cases, C. changbaiensis clustered with Caldicellulosiruptor sp. F32 and C. saccharolyticus C. changbaiensis encodes for carbohydrate and nitrogen metabolism-related genes not present in the genome of C. saccharolyticus Despite synteny between the genomes of C. changbaiensis, C. saccharolyticus and Caldicellulosiruptor sp. F32 (Fig. S1), there are regions of the C. changbaiensis genome that are absent in C. saccharolyticus or Caldicellulosiruptor sp. F32. Given the permanent draft status of Caldicellulosiruptor sp. F32 (2.38 Mb, 127 contigs) [53], we compared the gene inventories of C. changbaiensis and C. saccharolyticus, as both genomes were assembled into single, complete contigs. Overall, the C. changbaiensis genome encodes for 352 genes not present in the C. saccharolyticus genome. Among these genes are included chemotaxis-related genes including three methyl-accepting chemotaxis proteins not found in the C. saccharolyticus genome (Table S3), highlighting the diverse set of regulatory networks in the Caldicellulosiruptor pangenome. One unique diguanylate cyclase enzyme is also encoded for by C. changbaiensis, which is located in a locus that encodes for purine catabolism enzymes, and may play a role in nitrogen scavenging. Interestingly, C. changbaiensis also encodes for enzymes involved in proline reduction, one of the reductive pathways in the Stickland reaction [33]. This is the first observation of a Caldicellulosiruptor encoding for enzymes involved in amino acid fermentation, and warrants further investigation to determine if C. changbaiensis is capable of catalyzing a full Stickland reaction, in addition to fermentation of carbohydrates. Nineteen out of the 352 genes are annotated as CAZymes (Table S3), and includes those encoded by a rhamnogalacturonan degradation locus that also encodes for an ATP-binding cassette (ABC) transporter previously observed to be upregulated by C. bescii during growth on switchgrass [87], supporting its potential role in transporting sugars released by enzymes in this locus. In addition to this locus, C. changbaiensis also encodes for an annotated rhamnogalacturonan acetylesterase (Genbank accession AZT91282) that is absent from the C. saccharolyticus genome. As expected, based on their phenotypes [4, 7], both C. saccharolyticus and C. changbaiensis encode for a glucan degradation locus (GDL) and a xylan degradation locus (XDL) [6] that encode for modular enzymes involved in cellulose (GDL) or xylan (XDL) hydrolysis. However, when comparing the GDL organization between C. changbaiensis and C. saccharolyticus, differences were also noted (Fig. 4). Most striking is the inclusion of CelC (GH10–CBM3–CBM3–CBM3–GH48) in the C. changbaiensis GDL which is absent from the C. saccharolyticus GDL. CelC is a multifunctional, modular enzyme critical for the hydrolysis of plant biomass [28] and a key member of a superior ternary mixture of cellulases (‘ACE’ cellulases) for in vitro hydrolysis of microcrystalline cellulose [27]. Fig. 4 Open in new tabDownload slide Modular multifunctional enzymes encoded for by the glucan degradation locus. Glucan degradation loci were selected on the basis of the presence of “ACE” cellulases (Cbes, Calcha, Cdan, Calkro, Calna). or significant similarity between genomes (Csac). ACE cellulases: CelA, CelC and CelE. Circles represent the glycoside hydrolase (GH) domains; rectangles represent the carbohydrate binding module (CBM) domains. GH5, green circles; GH9, red circles; GH10, violet circles; GH 44, blue circles; GH48, gray circles; GH74, orange circles. CBM3, gray rectangles; CBM22, pink rectangles. Cbes, C. bescii; Calcha, C. changbaiensis; Cdan, C. danielii; Calkro, C. kronotskyensis; Calna, C. naganoensis; Csac, C. saccharolyticus C. changbaiensis exhibits different abilities to grow on polysaccharides versus C. bescii To benchmark the physiological ability of C. changbaiensis to grow on plant-related polysaccharides, we compared its doubling time during exponential growth on representative plant polysaccharides to C. bescii (Table 2). Multiple studies have demonstrated that C. bescii is more cellulolytic than other Caldicellulosiruptor species, including C. saccharolyticus [46, 80, 87], and enzymes encoded by its GDL have also been extensively characterized [9–11, 14, 22, 28, 40, 70, 79, 81–83]. As such, C. bescii has emerged as the benchmark species for comparison to newly sequenced species [6, 46], or newly discovered enzymes [26, 46]. Overall, C. changbaiensis grows slower on microcrystalline cellulose than C. bescii, with a 38% longer doubling time during growth on crystalline cellulose; however, both cultures grew at similar rates on xylan. On both glucomannan and pectin, C. changbaiensis grew faster with 35% lower doubling times (Table 2). Doubling time of C. changbaiensis or C. bescii grown on carbohydrates Polysaccharide . g Cbes (h) . g Calcha (h) . Microcrystalline cellulose 3.93 ± 0.157 5.43 ± 0.304 Beechwood xylan 2.55 ± 0.211 2.54 ± 0.428 Glucomannan 3.22 ± 0.62 2.08 ± 0.025 Pectin 3.48 ± 0.224 2.26 ± 0.167 Rhamnose 2.80 ± 0.007 2.57 ± 0.027 Arabinogalactan 2.73 ± 0.034 2.32 ± 0.023 Polygalacturonic acid 2.08 ± 0.000 2.27 ± 0.012 Fucose 4.70 ± 0.037 5.30 ± 0.041 Polysaccharide . g Cbes (h) . g Calcha (h) . Microcrystalline cellulose 3.93 ± 0.157 5.43 ± 0.304 Beechwood xylan 2.55 ± 0.211 2.54 ± 0.428 Glucomannan 3.22 ± 0.62 2.08 ± 0.025 Pectin 3.48 ± 0.224 2.26 ± 0.167 Rhamnose 2.80 ± 0.007 2.57 ± 0.027 Arabinogalactan 2.73 ± 0.034 2.32 ± 0.023 Polygalacturonic acid 2.08 ± 0.000 2.27 ± 0.012 Fucose 4.70 ± 0.037 5.30 ± 0.041 Open in new tab Doubling time of C. changbaiensis or C. bescii grown on carbohydrates Polysaccharide . g Cbes (h) . g Calcha (h) . Microcrystalline cellulose 3.93 ± 0.157 5.43 ± 0.304 Beechwood xylan 2.55 ± 0.211 2.54 ± 0.428 Glucomannan 3.22 ± 0.62 2.08 ± 0.025 Pectin 3.48 ± 0.224 2.26 ± 0.167 Rhamnose 2.80 ± 0.007 2.57 ± 0.027 Arabinogalactan 2.73 ± 0.034 2.32 ± 0.023 Polygalacturonic acid 2.08 ± 0.000 2.27 ± 0.012 Fucose 4.70 ± 0.037 5.30 ± 0.041 Polysaccharide . g Cbes (h) . g Calcha (h) . Microcrystalline cellulose 3.93 ± 0.157 5.43 ± 0.304 Beechwood xylan 2.55 ± 0.211 2.54 ± 0.428 Glucomannan 3.22 ± 0.62 2.08 ± 0.025 Pectin 3.48 ± 0.224 2.26 ± 0.167 Rhamnose 2.80 ± 0.007 2.57 ± 0.027 Arabinogalactan 2.73 ± 0.034 2.32 ± 0.023 Polygalacturonic acid 2.08 ± 0.000 2.27 ± 0.012 Fucose 4.70 ± 0.037 5.30 ± 0.041 Open in new tab The differential ability of C. changbaiensis and C. bescii to grow on pectin and glucomannan is not unexpected, as other species have been demonstrated to grow at various rated on plant biomass, which is comprised of polysaccharides such as xylan, pectin and glucomannan. For example, C. saccharolyticus was observed to grow slower on plant biomass in comparison to C. bescii [80] and C. kronotskyensis [87] and another observation where C. danielii grew approximately 50% faster than C. bescii, C. morganii and C. naganoensis on plant biomass [46]. When comparing the genomes of C. changbaiensis and C. bescii, C. changbaiensis encodes for 411 genes not encoded by C. bescii, including the 120 genes not previously observed in the Caldicellulosiruptor pangenome. We expect that the differences in growth rates on carbohydrates are in part related to differences in gene inventory. In fact, the C. changbaiensis gene inventory encoding for carbohydrate active enzymes includes 13 genes not found in the C. bescii genome, including an annotated β-mannanase (glycoside hydrolase [GH] family 26) and two mannooligosaccharide phosphorylases (GH130). This additional β-mannanase and phosphorylases likely contribute to the enhanced growth of C. changbaiensis on glucomannan (Table 2). The lower doubling time on pectin is surprising, however, given that C. changbaiensis does not encode for the pectinase cluster that is located in the C. bescii genome immediately downstream of the GDL. C. bescii gene deletion strains lacking the pectinase cluster were impaired in their growth on both pectin-rich plant biomass and pectin [21], indicating that C. changbaiensis has evolved alternate mechanisms to deconstruct or metabolize pectin. Screening the C. changbaiensis genome for pectin-related enzymes did not identify any genes encoding for polysaccharide lyases (PL) that were unique in comparison to C. bescii, for example, the rhamnogalacturonan degradation locus is present in both species; however, genes encoding for representatives from GH families 43, 51 (α-l-arabinofuranosidases) and 95 (α-fucosidase) were present. One possible scenario is that these enzymes participate in the hydrolysis of carbohydrate side chains from pectin [57]. Another plausible explanation is that C. changbaiensis has evolved to import and efficiently ferment a broader range of carbohydrates released during growth on plant biomass, including uronic acids, and/or the deoxy sugars fucose and rhamnose. To test these possibilities, we compared the doubling rates of C. bescii and C. changbaiensis during grown on deoxy sugars (rhamnose or fucose), arabinogalactan and polygalacturonate (Table 2). Interestingly, C. bescii grew slightly faster on fucose and polygalacturonic acid in comparison to C. changbaiensis, while the latter grew slightly faster on rhamnose and arbabinogalactan. Given that C. bescii can grow rigorously on polygalacturonic acid, we propose that C. changbaiensis is better able to handle the methylated or acetylated state of pectin and, in turn, can grow faster on pectin. Organization of the C. changbaiensis glucan degradation locus Caldicellulosiruptor changbaiensis was originally described as strongly cellulolytic [4] and accordingly, its genome encodes for a GDL that shares a similar organization with other strongly cellulolytic members of the genus. Since C. bescii was able to grow at a faster rate on microcrystalline cellulose than C. changbaiensis (Table 2), we opted to focus on the comparison of GDL between these two species. The GDL from both species is remarkedly similar, with only CelD possessing a different arrangement of catalytic and non-catalytic domains (GH10–CBM3–GH5) from C. changbaiensis, and truncated versions of CelE (GH9–CBM3–GH5) and CelF (GH74–CBM3) present (Fig. 4). Prior in vitro biochemical analyses on the synergy of cellulase mixtures from C. bescii had observed that a ternary mixture of CelA, CelC and CelE worked synergistically to hydrolyze cellulose as well as a mixture of all six C. bescii cellulases [27]. One could hypothesize, then, that members of the genus Caldicellulosiruptor that possess all three of these enzymes would be among the most cellulolytic. Three additional species, C. kronotskyensis, C. danielii, and C. naganoensis, also share a similar organization of their GDL [46], including the presence of CelA, CelC and CelE. The contributions of CelD and CelF to cellulose hydrolysis or solubilization are low [27, 28] and likely not to impact the ability of C. changbaiensis to efficiently hydrolyze cellulose. Indeed, C. changbaiensis can solubilize microcrystalline cellulose (Fig. 5); however, the amount of cellulose solubilized was 22.4% lower than the amount solubilized by C. bescii, which is similar to the performance of C. saccharolyticus when compared to C. bescii [24.8% lower, 87]. This result begs the question if the mere presence of the ACE cellulases is sufficient to meet the C. bescii benchmark for hydrolysis of cellulose. One explanation could be that the C. changbaiensis CelE ortholog may not be as efficient in cellulose hydrolysis since it is lacking two CBM3 modules. However, the nearly equal reduction of cellulose solubilization by both C. bescii gene deletion strains incapable of producing CelA–CelC versus CelA–CelE does not support this possibility [28]. Furthermore, CelE truncations that possessed the GH9 catalytic domain and three or two CBM3 domains were equally capable of microcrystalline cellulose hydrolysis [70], making it unlikely that the loss of a CBM3 domain from the C. changbaiensis CelA ortholog hampered its activity. Fig. 5 Open in new tabDownload slide Solubilization of microcrystalline cellulose by C. bescii and C. changbaiensis. Cellulose solubilization is reported as percent cellulose mass loss after 5 days of batch growth with C. bescii, C. changbaiensis or an abiotic control. Uninoculated control, abiotic cellulose solubilization in LOD medium. Error bars represent standard error (n = 3). The asterisk over columns denotes a p value < 0.05 as determined by a t test Alternately, sequence divergence of ACE cellulase orthologs may play a larger role in the catalytic capacity of cellulolytic members from the genus Caldicellulosiruptor. Of the ACE cellulases, CelA is the key enzyme for cellulose hydrolysis, supported by its unique hydrolysis mechanism [9], the severe reduction in cellulose hydrolysis by C. bescii celA gene deletion mutants [28, 86], and biochemical analysis of GDL enzyme synergy [27]. Prior comparison of CelA orthologs from C. bescii and C. danielii found CbCelA to be a superior enzyme [46], indicating that GDL sequences have diverged during speciation, making it likely that the ACE cellulases from C. changbaiensis may not demonstrate the same catalytic efficiency as C. bescii. Attachment of C. bescii and C. changbaiensis to plant polysaccharides Aside from comparisons of catalytic ability, we also compared the ability of C. changbaiensis versus C. bescii planktonic cells to bind to insoluble substrates (xylan and cellulose). A decrease in the planktonic cell density (PCD) after exposure to the substrate compared to the PCD of the negative controls without substrate is indicative of cells binding to the substrate. Surprisingly, we saw no such decrease in PCD for C. changbaiensis cultured on xylan after incubation with cellulose or xylan (Fig. 6a, b). This inability of C. changbaiensis to attach to xylan or cellulose after growth on xylan is surprising, given that xylan is a major polysaccharide constituent of lignocellulose, and would likely serve as a chemical signal. Since no xylan or cellulose attachment proteins are produced in response to growth on xylan, C. changbaiensis appears to act as a specialist, responding only to cellulose. Regardless, when C. changbaiensis is grown on cellulose, it maintains an ability to attach to cellulose (29% cells attached), which is slightly lower than the relative amount of C. bescii cells attached to cellulose (33% attached, Fig. 6c). Surprisingly, when C. bescii cells cultured on xylan were tested for attachment to either xylan or cellulose, there was a significant decrease in (PCD) of indicating that C. bescii cells grown on xylan are producing proteins capable of attaching to xylan (33% attachment, Fig. 6a) or cellulose (68% attachment, Fig. 6b). While we expected to see cells from cultures grown on xylan attaching to xylan, interestingly, C. bescii cell attachment was most pronounced when cells were grown on xylan and incubated with cellulose (Fig. 6b). The ability of C. bescii to attach to cellulose (Fig. 6b, c) is in large part due to the presence of tāpirins since a C. bescii tāpirin deletion mutant was impaired in cellular attachment to cellulose [47]. Fig. 6 Open in new tabDownload slide Comparison of the ability of C. bescii or C. changbaiensis planktonic cells to attach to polysaccharides. Titles above bar charts indicate the carbon source for growth/binding substrate. a, b When cells are grown on xylan, only planktonic C. bescii cells were able to attach to xylan or cellulose. c Cells grown on cellulose as the carbon source and exposed to cellulose as the binding substrate. Planktonic cell densities (PCD), enumerated by epifluorescence microscopy are plotted on the y-axis. Green columns indicate PCD without binding substrate and purple columns indicate PCD with the binding substrate. *A p value < 0.01 as determined by a t test. All assays had n = 6 biological replicates The C. changbaiensis genome encodes for atypical tāpirin genes Another notable difference observed between C. changbaiensis and C. bescii during growth on cellulose is the lack of floc formation by C. changbaiensis (Fig. 7). Based on this discrepancy between C. changbaiensis and C. bescii, we examined the genomic context of the type IV pilus locus encoded by the C. changbaiensis genome in comparison to strongly to weakly cellulolytic Caldicellulosiruptor species (Fig. 8). The T4P locus is found in the genome in all members of the Caldicellulosiruptor, and is also located upstream of the GDL in the genomes of strongly cellulolytic species [5, 6]. Most notably, while a full T4P locus is present in the C. changbaiensis genome, standard tāpirin genes which encode for proteins that bind with high affinity to cellulose are absent [5, 47]. Instead, two genes with little to no homology to the standard tāpirins are located directly downstream of the T4P locus which we will refer to as atypical tāpirins. The proteins encoded for by these genes are not unique to C. changbaiensis, as both C. acetigenus and C. ownesensis also encode for homologous atypical tāpirins; however, this is the first report of atypical tāpirin genes encoded for by a strongly cellulolytic Caldicellulosiruptor. All three species encode for a hypothetical protein and a von Willebrand Factor A protein (yellow arrows, Fig. 8). Caldicellulosiruptor changbaiensis shares a similar genomic context with C. acetigenus and C. owensensis at the 3′ end of the T4P locus, inclusive of the atypical C. changbaiensis tāpirins. Both classes of atypical tāpirins are orthologs, and they share 74.33–74.72% and 68.01–70.02% amino sequence similarity with the first and second atypical tāpirins encoded by C. owensensis and C. acetigenus. Prior proteomics data collected from cellulose-bound, supernatant and whole cell lysate protein fractions determined that both atypical tāpirins are produced by C. owensensis in response to cellulose [6] and, in fact, both atypical tāpirins from C. owensensis were enriched in the supernatant and cellulose-bound fractions, supporting their potential role in cell attachment to cellulose. Fig. 7 Open in new tabDownload slide Flocculation of C. bescii cells cultured on chemically defined medium and microcrystalline cellulose. a Formation of a floc of C. bescii cells around microcrystalline cellulose (diameter, 20 µm) while planktonic C. changbaiensis cells (cloudiness) are visible. b Same serum bottles as in a; however, the bottles were vigorously mixed. The C. bescii floc remains intact, while both microcrystalline cellulose and cells are mixed in the C. changbaiensis culture Fig. 8 Open in new tabDownload slide Genomic context for the location of the tāpirins from strongly to weakly cellulolytic Caldicellulosiruptor species. All tāpirin genes are encoded downstream of a type IV pilus locus (in peach). Different colors represent standard (blue and green) versus atypical (yellow) tāpirins. Blue arrows represent standard tāpirin classes 1 and 2 encoded by strongly cellulolytic C. bescii, C. danielii, C. kronotskyensis, C. morganii (only class 2), C. naganoensis (only class 1), C. obsidiansis, C. saccharolyticus and Caldicellulosiruptor sp. F32. The green arrow represents the tāpirin encoded by C. hydrothermalis which shares structural similarity with tāpirins from strongly cellulolytic species. Yellow arrows represent atypical tāpirins encoded by C. changbaiensis (Genbank accessions: WP_127352232.1 and WP_127352233.1), C. acetigenus and C. owensensis. Grey rectangles indicate the presence of the GDL downstream of the type IV pilus locus and tāpirins. Atypical tāpirin 1 is annotated as a hypothetical protein and atypical tāpirin 2 is annotated as a von Willebrand factor A protein for C. changbaiensis, C. acetigenus and C. owensensis. Cbes, C. bescii; Calhy, C. hydrothermalis; Calcha, C. changbaiensis Calow, C. owensensis and Cace, C. acetigenus. Approximate amino acid length are noted in the arrows This observed sequence divergence between the atypical tāpirins from strongly and weakly cellulolytic species is similar to the tāpirin encoded for by C. hydrothermalis which shares little amino acid sequence homology with classical tāpirins, but shares a similar tertiary structure, and is capable of occupying more sites on crystalline cellulose in comparison to classical tāpirins [47]. Production of tāpirins with an affinity to cellulose likely plays a role in the ability of weakly cellulolytic members of the genus to adhere to cellulose and benefit from the cellooligosaccharides released by the action of cellulases [77]. The atypical tāpirins, originally only observed in the genomes of weakly cellulolytic species, may also serve as cellulose adhesins; however, further in-depth biochemical characterization of both atypical tāpirin proteins is required to confirm their function. Conclusions Overall, the Caldicellulosiruptor pangenome remains open, and is expected to gain approximately 63 new genes with each additional species sequenced (Fig. 1a). The addition of a second species isolated from China indicates that the diversity of Caldicellulosiruptor species from this region is higher than those isolated from Iceland; however, the level of observed diversity is not as high as those species isolated from Kamchatka, Russia or New Zealand on the basis of ANIb (Figs. 2, 3). Caldicellulosiruptor changbaiensis encodes for a GDL (Fig. 4) similar in organization as C. bescii; however, this species is not as cellulolytic as C. bescii on the basis of doubling time (Table 2) and cellulose solubilization (Fig. 5). However, C. changbaiensis does grow faster on the plant polysaccharides, glucomannan and pectin. Caldicellulosiruptor changbaiensis also fails to form a floc during growth on microcrystalline cellulose (Fig. 7), a phenotype previously described for C. bescii [85]; however, both species are capable of attaching to cellulose (Fig. 6). Interestingly, C. bescii retains an ability to attach to cellulose when previously grown on xylan, while C. changbaiensis does not (Fig. 6b) indicating that the two species respond differently to soluble carbohydrates present in their environment. Tāpirins were previously demonstrated to be key cellulose adhesins for strongly [5] to weakly cellulolytic [47] members of the genus Caldicellulosiruptor. Surprisingly, C. changbaiensis does not encode for the classical tāpirins, and instead encodes for atypical tāpirins, one of which possesses a von Willebrand type A protein domain (Fig. 8). These atypical tāpirins are homologous to those encoded for by weakly cellulolytic C. owensensis and C. acetigenus; however, this may not indicate that the atypical tāpirins are not involved in the attachment to cellulose, as the divergent classical tāpirin encoded for by C. hydrothermalis binds at a high density to cellulose [47]. The combined lack of standard tāpirins, along with the ability to attach to cellulose indicates that C. changbaiensis evolved a different strategy to attach to cellulose that we have not yet observed in other strongly cellulolytic Caldicellulosiruptor. Further study on the biophysical properties of these atypical tāpirins is warranted to assess their ability to interact with plant polysaccharides, including cellulose. Acknowledgements The authors wish to acknowledge the support of an Oakland University Provost Award, awarded to C. Mendoza. References 1. 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Zverlov V , Mahr S, Riedel K, Bronnenmeier K Properties and gene structure of a bifunctional cellulolytic enzyme (CelA) from the extreme thermophile ‘Anaerocellum thermophilum’ with separate glycosyl hydrolase family 9 and 48 catalytic domains Microbiology 1998 144 457 465 10.1099/00221287-144-2-457 Google Scholar Crossref Search ADS PubMed WorldCat Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. © Society for Industrial Microbiology 2019 This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) © Society for Industrial Microbiology 2019
TI - Genomic and physiological analyses reveal that extremely thermophilic Caldicellulosiruptor changbaiensis deploys uncommon cellulose attachment mechanisms
JO - Journal of Industrial Microbiology & Biotechnology
DO - 10.1007/s10295-019-02222-1
DA - 2019-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/genomic-and-physiological-analyses-reveal-that-extremely-thermophilic-phdLxATR0d
SP - 1251
EP - 1263
VL - 46
IS - 9-10
DP - DeepDyve
ER -