Morphological and gene expression changes C. acetobutylicum undergoes during exponential, transitional, and stationary phases. (a) Growth and acid and solvent production curves as they relate to morphological and transcriptional changes during sporulation. The gray bar indicates the beginning of the transitional phase as determined by solvent production. A600 with microarray sample (filled squares); A600 (open squares); butyrate (filled circles); butanol (filled triangles). Roman numerals correspond with those in (b), and bars and numbers along the top correspond to the clusters in (c). (b) Morphological changes during sporulation. When stained with Syto-9 (green) and PI (red), vegetative cells take on a predominantly red color (I and II). At peak butanol production, swollen, cigar-shaped clostridial-form cells appear (arrow in III), which stain almost equally with both dyes, and persist until late stationary phase. Towards the end of solvent production (IV), endospore (arrow 1) forms are visible, and clostridial (arrow 2) forms are still present. As the culture enters late stationary phase (V and VI), cells stain almost exclusively green, regardless of morphology. All cell types are still present, including free spores (arrows in V and VI), and vegetative cells identified by their motility. (c) Average expression profiles for each K-means cluster generated using a moving average trendline with period 3. (d) Expression of the 814 genes (rows) at 25 timepoints (columns, hours 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 44, 48, 54, 58, and 66). Genes with higher expression than the reference RNA are shown in red and those with lower expression as green. Saturated expression levels: ten-fold difference.