Journal: Applied and Environmental Microbiology
Article Title: Clostridium scindens ATCC 35704: Integration of Nutritional Requirements, the Complete Genome Sequence, and Global Transcriptional Responses to Bile Acids
Figure Lengend Snippet: Growth and bile acid metabolism by Clostridium scindens ATCC 35704. (A) Growth profiles after a minimum of three sequential transfers under each culture condition: ♦, brain heart infusion (BHI) broth; ○, undefined medium (UM; DM with 0.1% yeast extract); Δ, defined medium (DM); ▲, PO 4 -buffered DM; ■, minimal medium (MM); and □, PO 4 -buffered MM. (B) Bile acid metabolism in DM and MM (left to right): lane 1, cholate (CA) standard; lane 2, deoxycholate (DCA) standard; lane 3, DM and C. scindens 35704; lane 4, DM with CA, and C. scindens 35704; lane 5, DM modified to contain tryptophan as the sole amino acid plus CA and C. scindens 35704; lane 6, DM modified to contain riboflavin, pantothenic acid, and pyridoxal HCl as sole vitamins plus CA and C. scindens 35704; and lane 7, MM with CA and C. scindens 35704. The initial concentration of CA and DCA in standards and CA in cultures was 100 µM. (C) Growth profiles in PO 4 -buffered DM in the absence (control, ▲) and presence of cholic acid (CA, □) or deoxycholic acid (DCA, ●). Arrows indicate addition of 50 μM bile acid, and the star indicates addition of RNAprotect for RNA-Seq analysis as well as removal of a 1-ml aliquot for the [24- 14 C]CA conversion assay. (D) Determination of the rate of conversion of [24- 14 C]CA to [24- 14 C]DCA. The inset is the autoradiograph of TLC-separated [24- 14 C]CA metabolites. CA and 7-oxo-DCA were scraped and counted, and DCA and 3-oxo-DCA were scraped and counted.
Article Snippet: Cells were quenched with RNAprotect (Qiagen) and stored at −80°C until further processing.
Techniques: Modification, Concentration Assay, RNA Sequencing Assay, Autoradiography, Thin Layer Chromatography