A number of studies have demonstrated the

ability of C. thermocellum to control scaffoldin and cellulase mRNA [25–28] and protein [29–32] levels in response to substrate type and growth rate, whereby cellulosome gene expression is positively regulated through binding of cellulose and xylan to anti-σ factors, preventing their binding to alternative σ factors required for cellulosome expression [33, 34], and find more negatively regulated by cellobiose via a carbon catabolite repression mechanism [28, 31]. A few studies have looked CBL0137 cost at expression levels of genes encoding proteins involved in central metabolism and end-product formation. Stevenson and Weimer have looked at expression levels of 17 genes involved in cellulose degradation, intracellular phosphorylation, catabolite repression, and fermentation end-product formation in response to substrate and growth rate [35]. More recently, microarray studies have looked at overall gene expression levels and global changes in mRNA levels in response to substrate and dilution rate [36] and growth phase in cellulose-grown batch cultures [37]. To date, there have been no reports of global protein expression levels of C. thermocellum. We have now completed

TH-302 the first proteomic study of cellobiose-grown batch culture C. thermocellum cell-free extracts to determine relative abundances of metabolic proteins and responses in their expression levels during different growth phases. Shotgun two-dimensional high performance liquid chromatography-tandem mass spectrometry (2D-HPLC-MS/MS) was used to determine protein relative abundance indexes (RAI), calculated as the number of spectral counts (SpC) divided by molecular mass (Mr) of protein, in exponential phase cell-free extracts. Differences in protein expression levels between exponential

and stationary phase cell-free extracts labeled with isobaric only tags for relative and absolute quantitation (iTRAQ) were determined using 4-plex 2D-HPLC-MS/MS. Materials and methods Organism, media, and growth The type strain of Clostridium thermocellum, DSM 1237 (equivalent to ATCC 27405), obtained from the German Type Culture collection, was employed for all growth experiments. Fresh cultures were maintained by routinely transferring 10% (v/v) mid-exponential phase inoculum into complex 1191 medium as previously described [4] containing 2.2 g L-1 (11.8 mM) cellobiose. Cultures were grown at 60°C and stored anaerobically at 4°C. All chemicals were reagent grade and were obtained from Sigma Chemical Co (St. Loius, MO) unless otherwise specified. All gases were purchased from Welder’s Supply (Winnipeg, MB, Canada). Cells for end-product and proteomic analysis were grown in triplicate in anaerobic Balch tubes (26 mL; Bellco Glass Inc., Vineland, NJ) in 10 mL of 1191 medium (pH 7.2) on 2.2 g L-1 cellobiose.