thermocellum. Overall, the gene expression patterns revealed a
coordinated response by C. thermocellum Microbiology inhibitor to conditions of altering substrate availability during cellulose batch fermentations. C. thermocellum modulates the composition of cellulosomes released into the environment in stationary phase and enhances signal transduction, chemotaxis mechanisms probably for sensing of substrate gradients resulting from the action of cell-free cellulosomes. C. thermocellum also increases expression of genes involved in cellular motility function, potentially to orient the movement of cells towards available nutrient sources in the environment. Such a coordinated cellular strategy should increase its chances of survival under conditions akin to feast and famine that are frequently encountered in natural ecosystems. To our knowledge, this is the first study looking at the transcriptional response of C. thermocellum at a global level and provides the foundation for future research using natural biomass as growth substrates. Methods Fermentation
C. thermocellum ATCC 27405 wild-type strain was a gift from Prof. Herb Strobel at the University of Kentucky, Lexington, KY. Batch fermentations were conducted in 3 L BioStat B jacketed glass fermentors (Sartorius Stedim Biotech, Bohemia, NY) using a 2 L working buy AZD4547 volume of MTC medium (mineral salt medium containing 1 g/L yeast extract; ) at 58°C and 300 rpm, with pH controlled at 7.0 using 3N NaOH. Fermentors with medium containing only the carbon substrate, 5 g/L RepSox datasheet crystalline cellulose (Avicel® PH105, FMC Biopolymer, Philadelphia, PA), were sparged with ultra-high purity nitrogen and vigorously agitated overnight, followed by addition of the remaining medium components and sparged for an MycoClean Mycoplasma Removal Kit additional 2-3 hrs with nitrogen gas. A 10% v/v inoculum of overnight (16-20 hrs) 5 g/L Avicel® bottle cultures was used to inoculate the fermentors and the gas inlet/exhaust lines were clamped post inoculation. Protein and metabolite analysis Well-mixed 2 mL aliquots of cultures were harvested
at regular intervals and centrifuged quickly to separate into pellet and supernatant samples for protein analysis of pellet fractions and HPLC analysis of extracellular metabolites, respectively. Cell growth was monitored based on increase in protein content within the total solids present in the pellet fraction, including the Avicel® substrate . Briefly, the solid pellet was washed with de-ionized water and the cells were lysed using 0.2N NaOH/1% w/v SDS solution, cell debris were pelleted and removed, and protein concentration in the clear supernatant was estimated using the bicinchoninic acid protein assay (Pierce Chemical, Rockford, IL). Metabolite analysis was performed using a LaChrom Elite system (Hitachi High Technologies America, Inc., Pleasanton, CA) equipped with a refractive index detector (Model L-2490).