Search

Additional file 12: Fig. S7. Characterisation of the recombinant bacterial CAZymes encoded by the endosymbionts. DNS reducing sugars assays showing activities on a number of substrates for LpsGH5_8, LpsGH11, LpsGH134a and LpsGH134b. CMC = carboxymethyl cellulose, LBG = locust bean gum. The nanomoles of sugars released by the different proteins cannot be compared quantitatively as different amounts were used for the assay. File format .DOCX.

Additional file 8: Table S2. CAZy families. List of the CAZy families mentioned in the results, with the description of the activities that have been recorded for enzymes listed in each family. The information has been gathered from the CAZy database ( http://www.cazy.org ). File format .DOCX.

Additional file 1: Figure S1. Experiment location. Figure S2. Coverage estimates for each 16S rRNA amplicon library for all biological replicates across each time point. Figure S3. CAZyme families identified within the metatranscriptomic databases presented as transcripts per million. Figure S4. CAZyme producing genera and their respective CAZyme contributions. Figure S5. Productivity index for CAZyme producing taxa at family level resolution. Figure S6. Phylogenetic distribution of CAZyme classes at class resolution for week one. Figure S7. Phylogenetic distribution of CAZyme classes at class resolution for week three. Figure S8. Phylogenetic distribution of CAZyme classes at class resolution for week five. Figure S9. Phylogenetic distribution of CAZyme classes at class resolution for week ten. Figure S10. Functional classification of proteins within the metasecretome. Figure S11. Fungal profiles and OTU richness elucidated from internal transcribed spacer region 2 amplicon sequencing across the 16-week time course. Figure S12. Nutrient acquisition strategy of fungi profile across the 16-week time course. Figure S13. Bacteria profiles elucidated from 16S rRNA sequence homology. Figure S14. Bacteria profiles elucidated from 16S rRNA sequence homology. Table S1. Transect position of the five biological replicates in Welwick salt marsh. Table S2. Sequence reads throughout RNA sequence processing and assembly. Table S3. Commands for the replication of the 16S rRNA amplicon database processing pipeline.

Additional file 14: Table S6. Primers used for the cloning of the bacterial proteins LpsGH5_8, LpsGH11, LpsGH134a and LpsGH134b. LpsAA10A could not be amplified from the cDNA and therefore a synthetic version of the gene was codon-optimised for E-coli expression. File format .DOCX.

Additional file 2: Fig. S2. Searching for the opening of the duct of Deshayes in the shipworm digestive system. Left, transverse sections through the shell valve, from the anterior to posterior adductor muscle (A-E). Right, 3D rendered model of the whole shipworm, with the green highlighted region showing the position of the transverse cross section displayed on the left. File format .DOCX.

Additional file 10: Table S4. Details of the proteins for which heterologous recombinant expression from symbionts cDNA was successful. The annotation was performed against the NCBI non-redundant database, the signal peptides were identified with the Signal P4.1 server and the CAZy domains were searched using dbCAN. Dis. bonds = disulfide bonds. File format .DOCX.

Additional file 1: Fig. S1. Scanning electron microscopic images of the gills and food groove of L. pedicellatus. A) Close up of the gills lamellae to show the numerous cilia that capture food and draw it to the food groove. B) Close up of the food groove to show the numerous cilia (most of the mucus is lost during critical point drying). File format .DOCX.

Additional file 6: Fig. S6. Immunogold labelling of LpsGH5_8 in L. pedicellatus using pre-immune serum (negative control). A) SEM image illustrating the anatomical position of the gills, food groove, caecum and mouth in the shipworm body. B-D) Immunogold labelling of the lumen of the food groove (B), lumen of the caecum (C), and the gills (D) performed with pre-immune serum. No gold particles are observed in any of the samples. B = bacteria. File format .DOCX.

Additional file 11: Table S5. The amino acid sequences of the bacterial proteins LpsGH5_8, LpsGH11, LpsGH134a, LpsGH134b and LpsAA10A. Text in red represents the signal peptide. File format .DOCX.

Additional file 7: Table S1. Details of the proteins identified through proteomic analysis of the crystalline style. The annotation was performed against the NCBI non-redundant database, the signal peptides were identified with the Signal P4.1 server and the CAZy domains were searched using dbCAN. Many of the contigs were out of frame or not full length (particularly the bacterial ones) and therefore the identification of the signal peptide was not possible. File format .DOCX.

Additional file 1: Table S1. The functional annotations of proteins identified in the proteome of PfMig188. Table S2. The distribution of upregulated proteins across the different secretomes containing sugarcane bagasse.

Additional file 4: Fig. S4. Bacterial symbionts in L. pedicellatus. A) TEM image of a gill bacteriocyte. Arrows indicate the numerous rod-shaped bacteria in the cell. B) Close-up view of gill bacteria (TEM). C) Detailed TEM showing a cross section of a gill bacterium and some of its features (periplasmic space and membranes). File format .DOCX.

Additional file 5: Fig. S5. SDS-PAGE and nitrocellulose western blot obtained by loading 0.6 ��g of the recombinant purified bacterial LpsGH5_8 (without the appended CBM). A) Coomassie Brilliant Blue stained SDS-PAGE gel of the purified protein. B) Western blot detection performed with the purified pre-immune serum. C) Western blot detection performed with the purified anti-protein serum. File format .DOCX.

Additional file 3: Fig. S3. Scanning electron microscopy of L. pedicellatus. A dissected specimen with the mantle removed, showing the food groove connecting the gills to the mouth. File format .DOCX.

Additional file 13: Fig. S8. MALDI-TOF MS analysis of in vitro activity assays with purified LpsAA10A under the same experimental conditions as in Fig. 5 C. Panels from a to d show spectra of products obtained after incubation of the enzyme with 4 mg mL-1 Avicel (a), 4 mg mL-1 Avicel plus 4 mM gallic acid (b), 4 mg mL-1 Avicel plus 2 ��M LPMO (c) and 4 mg mL-1 Avicel plus 2 ��M LPMO and 4 mM gallic acid (d). In panels a to d, 100% relative intensity represents 1.3 �� 104 arbitrary units (a.u.). The panels from e to g show spectra of products obtained after incubation of 4 mg mL-1 PASC (e), 4 mg mL-1 PASC plus 4 mM gallic acid (f) and 4 mg mL-1 PASC plus 2 ��M LPMO (g). In panels e to g, 100% relative intensity represents 1.0 �� 104 arbitrary units. File format .DOCX.

Additional file 9: Table S3. CAZy families of the caecum. Table showing the relative abundance (calculated from the emPAI score) of eukaryotic and prokaryotic CAZy families identified in the proteomic analysis of the caecum content of L. pedicellatus. File format .DOCX.

Additional file 1. Containing Q-Q plots of GWAS

Additional file 7. Full derivation of the fitness function used for in silico evolution of organic acid production.

Additional file 4. Detailed calculation for estimated gains of using a marker in breeding

Additional file 5: Table S15. Index numbers of mutations and corresponding reactions and mutation effects.

Additional file 1. The file provides the Additional Tables S1–S14.

Additional file 7. Field trial GPS coordinates

Additional file 9. Containing a report of Genotyping by Sequencing (GBS) – Reference Pipeline

Additional file 7. Field trial GPS coordinates

Additional file 7. Full derivation of the fitness function used for in silico evolution of organic acid production.

Additional file 6. The file provides the additional Figures S1–S15.

Additional file 9. Containing a report of Genotyping by Sequencing (GBS) – Reference Pipeline

Additional file 4. Detailed calculation for estimated gains of using a marker in breeding

Additional file 5: Table S15. Index numbers of mutations and corresponding reactions and mutation effects.

Additional file 1. The file provides the Additional Tables S1–S14.

Additional file 7: Figure S6. The Na+ Site on the TtAA10A Surface. The modelled sodium ion is shown as a grey sphere with the groups that coordinate it shown as sticks coloured by atom type. The 2Fobsâ Fcalc map is shown as a blue wire mesh contoured at 1Ď . The sodium ion is octahedrally coordinated with three main chain carbonyl groups and three water molecules. Sodium was assigned at this site based on the coordinating bond lengths and this was the ion that gave a B-factor following refinement closest to the surrounding protein atoms.

Additional file 4: Table S1. TtAA10A Data collection and refinement statistics.

Additional file 6: Figure S5. SEC-MALLS analysis of TtAA10A, where the solid red line indicates the refractive index, the dashed line is the light scattering (these are essentially identical so indistinguishable), dotted line the UV response at 280 nm. The central line inside the peak is representative of the molar mass (2.44 × 104 Da), indicating that TtAA10A forms a monomer in solution.

Additional file 3: Figure S3. EPR Cu titration reveals the presence of two distinct Cu sites. EPR copper titration experiment in which the protein was pre-treated with EDTA to remove the copper (as shown by the lack of EPR signal). Addition of 0.2 equivalents of Cu (to the concentration of protein) produced a single Cu species indicative of the histidine brace. Addition of a further 0.2 equivalents caused a change in the spectra with appearance of a second set of hyperfine peaks in the parallel region. The clearest change is seen after 0.6 equivalents of Cu have been added, where multiple peaks in the spectra indicate two Cu species. Loss of resolution in the last two spectra may be due to signal dilution.

Additional file 1. Additional tables, figures, and additional text.

Additional file 1: Table S1. Number of sequences, OTUs and alpha indices from Ion Torrent sequencing of 16S and 18S amplicons of samples. Table S2. Number of raw, rRNA and quality filtered sequences from the RNA-seq metatranscriptomics. Table S3. Number of unassigned and assigned spectra through analysis of biotin-labelled (BF) and supernatant (SNT) fractions of the wheat straw cultures.

Additional file 2: Figure S1. Morphological changes of the wheat straw biomass collected from weekly time points. Figure S2. Rarefaction analysis of prokaryotic (a) and eukaryotic (b) community from the wheat straw cultures based on rRNA amplicon sequencing. Figure S3. Overview of the wheat straw degrading community metatranscriptome. Figure S4. Overview of the metasecretome of wheat straw degrading community. Figure S5. Comparison of Clusters of Orthologous Groups (COGs) in the metasecretome (MP) and metatranscriptome (MT).

Synteny map depicting the alignment and localization of M. sinensis mapped markers to the Sorghum bicolor (L.) Moench genome. Linkage groups of the female map are designated as ‘a’ or ‘b; linkage groups of the male map are designated as ‘c’ or ‘d’. The position of mapped QTLs is also shown. For each QTL, colored boxes indicate 1-LOD support intervals while extension bars delimit 2-LOD support intervals. (PDF 5499 kb)

Additional file 1. Additional figures and tables.

Additional file 5: Figure S4. Electrostatic Surface Potential for TtAA10A. The electrostatic charge distribution for TtAA10A has been mapped onto the protein surface using the APBS plug-in for PyMol at Âąâ 10 KBT/e. The histidine brace, secondary copper binding site and sodium binding site are outlined with black lines showing the negatively charged areas to which the ions bind.

Additional file 1: Table S1. Relative abundance of microbes in the enriched consortium compared to the original consortium. Table S2 Carbohydrate-Binding Modules (CBM) family proteins observed in the rice YSB gut consortium. Table S3 Carbohydrate Esterases (CE) family proteins observed in the rice YSB gut consortium. Table S4 Relative ranking of top 18 CAZy family proteins of different classes as observed in the rice YSB gut consortium based on emPAI score. Figure S1 (a) Reduction in rice straw weight after 7 days of incubation with different gut consortium, with uninoculated medium as a control. (b) Glucose release after incubation of supernatant of different consortium with rice straw for 7 days. Figure S2 Change in community structure as a result of enrichment. Figure S3 Alkali-treated (a) and untreated (b) rice straw hydrolysis and product analysis.

Additional file 1: Figure S1. MALDI-TOF MS analysis of in vitro negative control activity assays with purified TtAA10A, under the same experimental conditions as in Fig. 2a. The panels show spectra of products obtained after incubation of 4 mg/mL Avicel (a), Avicel with 4 mM gallic acid (b) and Avicel with 2 µM TtAA10A (c). The spectra show no detectable amounts of native or oxidised cello-oligosaccharides. Relative intensity represents 1.23 × 103.

Additional file 1: Table S1. Relative abundance of microbes in the enriched consortium compared to the original consortium. Table S2 Carbohydrate-Binding Modules (CBM) family proteins observed in the rice YSB gut consortium. Table S3 Carbohydrate Esterases (CE) family proteins observed in the rice YSB gut consortium. Table S4 Relative ranking of top 18 CAZy family proteins of different classes as observed in the rice YSB gut consortium based on emPAI score. Figure S1 (a) Reduction in rice straw weight after 7 days of incubation with different gut consortium, with uninoculated medium as a control. (b) Glucose release after incubation of supernatant of different consortium with rice straw for 7 days. Figure S2 Change in community structure as a result of enrichment. Figure S3 Alkali-treated (a) and untreated (b) rice straw hydrolysis and product analysis.

Additional file 1: Figure S1. MALDI-TOF MS analysis of in vitro negative control activity assays with purified TtAA10A, under the same experimental conditions as in Fig. 2a. The panels show spectra of products obtained after incubation of 4 mg/mL Avicel (a), Avicel with 4 mM gallic acid (b) and Avicel with 2 µM TtAA10A (c). The spectra show no detectable amounts of native or oxidised cello-oligosaccharides. Relative intensity represents 1.23 × 103.

Additional file 5: Figure S4. Electrostatic Surface Potential for TtAA10A. The electrostatic charge distribution for TtAA10A has been mapped onto the protein surface using the APBS plug-in for PyMol at Âąâ 10 KBT/e. The histidine brace, secondary copper binding site and sodium binding site are outlined with black lines showing the negatively charged areas to which the ions bind.