Figure 1.
Simplified schematic representation of Clostridium thermocellum cellulosomal architecture.
CipA is the backbone scaffoldin protein containing 9-Type I cohesins and can accommodate up to 9-Type I dockerin bearing catalytic units. CipA also contains a Type II dockerin for cell-surface attachment via anchor proteins and a Cellulose Binding Domain for attachment to the growth substrate. C. thermocellum genome encodes five proteins with Type II cohesins, four with S-layer homology domain (SdbA, OlpB, Orf2p and Cthe0735) for cell-surface anchoring of Type II dockerin bearing CipA and one without the SLH domain (Cthe0736). Also shown is the only subunit containing a Type II dockerin (Cthe1806) – (Figure adapted with permission from Carlos Fontes, CIISA, Portugal).
Figure 2.
Clostridium thermocellum growth profile on various substrates, based upon protein levels.
Pellet protein (top panel) and supernatant protein (bottom panel) profiles of Clostridium thermocellum during growth on various biomass carbohydrates, as estimated by BCA and Bradford protein assays, respectively. Data represented is the average of two biological replicate fermentations of C. thermocellum in minimal medium containing 5 g/L total of the following carbon substrates: Cellulose with 15N labeled nitrogen source, Cellulose with 14N labeled nitrogen source, Cellulose-Xylan (3∶2 weight ratio), Cellulose-Pectin-Xylan (3∶1∶1), and Cellulose-Pectin (3∶2). Data is not shown for Z-Trim®, pretreated switchgrass and cellobiose fermentations.
Table 1.
Acetate and ethanol production during growth on pretreated switchgrass and other biomass carbohydrates.
Figure 3.
Clostridium thermocellum cellulosomal protein profile during growth on various substrates, analyzed by protein gel electrophoresis.
SDS-PAGE (4–20% Tris-HEPES-Glycine gel, coommassie stain) separation of Clostridium thermocellum cellulosomal fractions isolated using the affinity digestion method from cell-free broth of duplicate fermentations during growth on pretreated switchgrass and other biomass carbohydrates. BR1, 2 = Biological Replicate 1, 2, respectively.
Figure 4.
Weighted-Normalized Spectral Abundance Factor (Weighted-NSAF) of cellulosomal components in mass spectrometry analysis.
Weighted-NSAF data for 14N isotopologs across seven different samples is arranged in descending order of values for cellulose sample. For each cellulosomal sample, the average NSAF value of the various components was divided by the value for the scaffoldin protein CipA to obtain weighted-NSAF values. Heat plot representation shows weighted-NSAF distribution (Red, highest; Green, lowest) of 54 cellulosomal subunits. The proteins with top 10, and the next 10, highest weighted-NSAF values in each sample are highlighted in Yellow and Orange, respectively. Locus entries of newly detected cellulosome components are highlighted in Blue. Substrate legend: C = Cellulose, CX = Cellulose-Xylan, CP = Cellulose-Pectin, CPX = Cellulose-Pectin-Xylan, SWG = Pretreated Switchgrass, Cb = Cellobiose, ZT = Z-Trim®.
Figure 5.
Quantitative changes in Clostridium thermocellum cellulosome composition in response to carbon substrate – Part I
(see Figure 6 caption).
Figure 6.
Quantitative changes in Clostridium thermocellum cellulosome composition in response to carbon substrate – Part II.
Quantitative data shown is combined from two biological and two technical replicates and expressed as Log2Ratio (LowerCI, UpperCI) of cellulosomal component X during growth on substrate Y over that in 15N-labeled Cellulose cellulosome sample. Substrate key: C = Crystalline Cellulose (with 14N or 15N labeled nitrogen source), C-X = Cellulose-Xylan (5 g/L total in 3∶2 weight ratio), C-P = Cellulose-Pectin (3∶2), C-P-X = Cellulose-Pectin-Xylan (3∶1∶1), SWG = Pretreated Switchgrass (50% glucan, 8% xylan, 24% lignin), Cb = Cellobiose, Z-T = Z-Trim® (60% amorphous cellulose, 16% hemicellulose). Data was normalized to the scaffoldin CipA protein. Locus entries highlighted in Blue have not been observed experimentally prior to this study. Quantitation data highlighted in Yellow (increased expression in Substrate Y) and Orange (decreased expression in Substrate Y) satisfy the cut-off criteria. Criteria for differential expression was based on control comparison of 14N- and 15N-labeled cellulose cellulosome samples and were, (1) Log2Ratio should be >+0.4 or <−0.4 and (2) Upper/Lower Confidence Intervals should exclude Log2Ratio = 0. Structural, catalytic and/or binding module information was obtained from the following sources: http://pfam.sanger.ac.uk/; http://www.cazy.org/; http://genome.ornl.gov/microbial/cthe/. Domain key: GH = Glycoside Hydrolase, CE = Carbohydrate Esterase, PL = Polysaccharide Lyase, CBM = Carbohydrate Binding Module. Legend key: MW = Molecular Weight, Log2Ratio (LowerCI, UpperCI) - protein was quantified in both biological replicates (BR), with overlapping confidence intervals; Blank - protein not identified or quantified; d - protein quantified in only one BR, but result indicates down-regulation (UpperCI<0, Log2ratio<−0.4); D - protein quantified in both BR, but confidence intervals don't overlap. Both confidence intervals indicate down-regulation (UpperCI<0, Log2ratio<−0.4); i - identified in one BR, but no quantification result; I - identified in both BR, but no quantification result; n - protein quantified in only one BR, with result indicating no change in expression (−0.4<Log2ratio<+0.4); N - protein quantified in both BR, but confidence intervals don't overlap. Both confidence intervals indicate no change in expression (−0.4<Log2ratio<+0.4); u - protein quantified in only one BR, but result indicates up-regulation (LowerCI>0, Log2ratio>+0.4); U - protein quantified in both BR, but confidence intervals don't overlap. Both confidence intervals indicate up-regulation (LowerCI>0, Log2ratio>+0.4); A indicates that the protein was identified in both 14N and 15N forms in both BR; A single underscore indicates that the protein was identified in both 14N and 15N forms in only one BR; No underscore indicates that we did not identify both 14N and 15N forms in either BR.