PMC

Development of AcP and BuP assay. (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified His-tagged butyrate kinase. Left lane, protein marker; right lane, 3.6 μg of purified butyrate kinase. KD, kilodaltons. (B) Measurement of AcP (panel I) and BuP (panel II) standards by using acetate kinase and butyrate kinase, respectively. Symbols: ▵, AcP luminescence; ▪, BuP luminescence.

Expression profiles from a buk mutant time course experiment. (A) Hierarchical cluster of differentially expressed genes. (B) Detailed view of the green cluster, next to the horizontal blue bar in panel A, which contains genes downregulated relative to the first time point (sample A). (C) Motility and chemotaxis gene clusters with BuP expression data. Red and green indicate higher and lower expression, respectively, relative to sample A. ABC, ATP binding cassette; ACP, acyl carrier protein; hook-assoc., hook-associated; Hypoth., hypothetical.

BuP, AcP, growth, and product formation kinetics for C. acetobutylicum cultures. BuP and AcP kinetics are shown for the WT (▪), the pta mutant (▵), strain M5 (red circles), and the buk mutant (⋄). BuP and AcP levels are shown for two different M5 cultures (panels A and B). Transcriptional analysis was performed with samples taken from the fermentations depicted in panel B. The dashed vertical lines indicate the onset of solventogenesis in (from left to right) the WT and the pta mutant (panel A) and in the buk mutant (panel B). The vertical yellow bars indicate peaks in BuP and AcP concentrations corresponding to the onset of acetate reutilization in the WT and the buk mutant. Symbols: ○, acetate; brown circles, acetone; ▵, butyrate; ▴, butanol; ×, A₆₀₀.

Hierarchical clusters of differentially expressed genes. (A) Genes with greater expression in the buk mutant relative to the first time point (sample A) (red cluster). (B) Genes in the buk mutant with expression patterns similar to the inverse AcP pattern. (C) Fatty acid biosynthesis operon, shown with inverse AcP data from both the buk mutant time course and the analyses of strain M5 versus the buk mutant. ABC, ATP binding protein; FAD, flavin adenine dinucleotide; HSP, heat shock protein; N term, N terminal; C ter, C terminal.