Figure 1.
The R. flavipes worker-caste digestive tract and common protist gut symbionts.
(A) Photograph showing host and symbiont fractions used in the current research. (B) Drawing of the R. flavipes worker digestive tract showing the esophagus (E), salivary glands (SG), foregut (FG), midgut (MG), Malpighian tubules (MT), and hindgut (HG) with the paunch that houses microbial symbionts. Gut regions that served as host and symbiont fractions in the current study are indicated. (C) Common protozoan hindgut symbionts of R. flavipes: Dinenympha (Din.), Pyrsonympha (Pyrs.), Trichonympha (Tricho.), Spirotrichonympha (Spiro.), and Holomastigotes (Holo.). Termite gut photo by J.A. Smith.
Figure 2.
Monosaccharides released from pine lignocellulose by R. flavipes host (SG/FG/MG), symbiont (hindgut), and whole gut fractions.
(A) Glucose and pentose released in 10-hr assays. Bars with different letters (a,b,c or x,y,z) are significantly different by Tukey's HSD test (p<0.05). P-values above the bars indicate significant differences in glucose and pentose released within the different gut fractions. (B) Comparison of expected and observed glucose and pentose released. Expected values were determined by adding host and symbiont fraction results from (A) above, while observed values are the whole gut results from (A) above. P-values above bars indicate significant differences for expected and observed monosaccharide release. See Tables S1, S2, S3, and S4 for ANOVA summaries. Bars in both panels indicate average ± std. error determined from three colonies with triplicate determinations each.
Figure 3.
Glucose released from pine lignocellulose and beechwood xylan by three recombinant enzymes that represent genes sampled from the R. flavipes host gut transcriptome.
(A) Pine wood contains a combination of cellulose, hemicellulose and lignin; whereas (B) beechwood xylan contains only hemicellulose and lignin. The three recombinant enzymes tested were (1) the Cell-1 endoglucanase, (2) the β-glu beta-glucosidase, and (3) the LacA laccase (see text for details). Each enzyme was tested alone and in two- and three-way combinations. As shown in (A), >300-fold synergy in glucose release occurred when combining Cell-1 and β-glu, but output was reduced in the presence of LacA, likely via end-product inhibition. As shown in (B), Cell-1 and β-glu attained >70-fold synergy when combined, and the addition of LacA resulted in greater glucose output, presumably via lignin-hemicellulose dissociation. Bars within graphs with the same letters are not significantly different by Tukey's HSD test (p<0.05). Whole-model ANOVA results indicating significance are shown.
Figure 4.
Evidence of end-product inhibition that limits host digestive capabilities.
(A) Glucose inhibition of beta-glucosidase activity by the recombinant β-glu enzyme. Results show decreasing turnover of the model substrate p-nitrophenyl-β-D-glucopyranoside (pNPG) in the presence of increasing concentrations of free glucose. (B) Lineweaver-Burk double-reciprocal plot showing impacts of 10 mM glucose on pNPG activity by the recombinant β-glu enzyme across a range of pNPG concentrations. Results show a shift in both km and Vmax, suggesting that glucose un-competitively inhibits β-glu via interaction with the enzyme-substrate complex. (C) Results of time-course assays comparing the two enzyme combination of Cell-1+β-glu to the three enzyme combination of Cell-1+ β-glu+LacA. Results show continual glucose output through the 24-hr assays, as well as reduced activity for the three-enzyme combination. Reduced activity for the three enzyme combination presumably occurs through end-product inhibition of β-glu as shown in (A) and (B). Combined results from (A), (B) and (C) suggest a host catalytic deficiency that is offset by symbiont cellulose and hemicellulose digestive capabilities.