Characterization

of Cbp subunits revealed that CbpA (Cthe_0393) binds only to cellotriose, CbpB (Cthe_1020) binds to cellodextrins of different lengths (G2-G5), while CbpC selleck compound and CbpD (Cthe_2128 and Cthe_2446, respectively) preferentially bind to G3-G5 cellodextrins [34]. Given the absence of cellodextrins longer than cellobiose (G2) in our growth medium, the absence of the latter transporters Cthe_2125-2128 and Cthe_2446-2449 is not surprising. While high expression levels of cellotriose ABC transporter were a bitsurprising given the cells were grown on cellobiose, studies have shown that C. thermocellum and other cellulolytic bacteria (ie. Fibrobacter succinogenes) are capable of producing cellotriose during growth on cellobiose via reversible cellodextrin phosphorylases [69, 70]. While the 2.8-fold increase in Cthe_1020 expression and Cell Cycle inhibitor 2.6-fold decrease in Cthe_0391 expression in stationary phase was statistically significant (V diff  > 1), the other subunits of these transporters did not follow suit. Conversion of cellobiose to end-products Glycolysis In C. thermocellum, conversion of glucose to phosphoenolpyruvate (PEP)

occurs via the Embden-Meyerhoff-Parnas pathway (Figure  2a, Additional file 4). All glycolytic proteins were detected in the top 20% (RAI > 0.83) of total proteins detected by 2D-HPLC-MS/MS, with a few exceptions. Glucose-6-P isomerase (Cthe_0217) had a RAI = 0.28, and one of the two encoded glucose

kinases (Cthe_0390) was not detected. While Idoxuridine glyceraldehyde-3-P dehydrogenase was the most highly expressed protein (RAI = 21.1) of all proteins detected, expression of subsequent proteins encoded in the predicted operon (Cthe_0137-0140) decreased respectively with increasing gene distance from glyceraldehyde-3-P dehydrogenase, suggesting transcriptional and/or post-transcriptional regulation of the operon. Protein expression profiles show that interconversion of fructose-1-P to fructose-1,6-bisphosphate can occur via pyrophosphate (PPi)-dependent 6-P-fructokinase (RAI = 5.64), which was detected at higher levels than ATP-dependent 6-P-fructokinases Cthe_1261 and Cthe_0389 (RAI = 1.47 and 1.06, respectively). Of the two encoded fructose-1,6-P aldolases (Cthe_0349 and Cthe_2938), only Cthe_0349 was detected. While seven copies of putative phosphoglycerate mutase are encoded, Cthe_0140, which is encoded in a predicted operon containing glyceraldehydes-3-P dehydrogenase, phosphoglycerate kinase, and triosephosphate isomerase (Cthe_0137-0139) shows maximal expression throughout fermentation, consistent with mRNA expression profiles on cellulose [37]. Expression of phosphoglycerate mutase Cthe_0946, Cthe_1292, and Cthe_0707 were also detected, albeit at lower levels than Cthe_0140, while Cthe_1435, Cthe_2449, and Cthe_3153 were not detected.