A proteomic and transcriptional view of acidogenic and solventogenic steady-state cells of Clostridium acetobutylicum in a chemostat culture

Abstract

The complex changes in the life cycle of Clostridium acetobutylicum, a promising biofuel producer, are not well understood. During exponential growth, sugars are fermented to acetate and butyrate, and in the transition phase, the metabolism switches to the production of the solvents acetone and butanol accompanied by the initiation of endospore formation. Using phosphate-limited chemostat cultures at pH 5.7, C. acetobutylicum was kept at a steady state of acidogenic metabolism, whereas at pH 4.5, the cells showed stable solvent production without sporulation. Novel proteome reference maps of cytosolic proteins from both acidogenesis and solventogenesis with a high degree of reproducibility were generated. Yielding a 21% coverage, 15 protein spots were specifically assigned to the acidogenic phase, and 29 protein spots exhibited a significantly higher abundance in the solventogenic phase. Besides well-known metabolic proteins, unexpected proteins were also identified. Among these, the two proteins CAP0036 and CAP0037 of unknown function were found as major striking indicator proteins in acidogenic cells. Proteome data were confirmed by genome-wide DNA microarray analyses of the identical cultures. Thus, a first systematic study of acidogenic and solventogenic chemostat cultures is presented, and similarities as well as differences to previous studies of batch cultures are discussed.

Fig. 1

“Master” fermentation of C. acetobutylicum. Monitored 240 h after starting the medium supply at t₀. a shows pH (filled upright triangles) and optical density (empty squares); b illustrates the pH (filled upright triangles) and the fermentation products butyrate (filled squares), acetate (filled inverse triangles), butanol (empty upright triangles), acetone (empty circles), and ethanol (filled circles). Roman numbers highlight the four different phases, I: starting of continuous culture; II: establishing of steady-state growth at pH 5.7; III: switch of pH from 5.7 to 4.5; IV: establishing of steady-state growth at pH 4.5 of the fermentation. Arrows indicated sampling points for proteome and transcriptome analyses at the end of phase II and phase IV

Fig. 2

Representative colloidal Coomassie-stained 2-D gel of cytosolic proteins from cells grown at pH 5.7. The numbers highlight protein spots with higher abundance in acidogenic cells; respective proteins are listed in Table 1

Fig. 3

Representative colloidal Coomassie-stained 2-D gel of cytosolic proteins from cells grown at pH 4.5. The numbers highlight protein spots with higher abundance in solventogenic cells; respective proteins are listed in Table 2

Fig. 4

Overview of the transcript levels during acidogenesis at pH 5.7 and solventogenesis at pH 4.5. Log expression ratios of acidogenesis to solventogenesis are shown. All genes with log values (as logarithms to the basis of 2) higher than 1.6 are significantly induced at pH 5.7, and genes with a negative log of less than −1.6 were significantly induced at pH 4.5. According to this definition, all genes between the dashed lines were expected to be not significantly influenced

Fig. 5

Northern blot analyses of cap0037 and cap0036. Total RNA samples (15 μg per lane in a and b; 1 μg in c) of C. acetobutylicum cells grown at pH 5.7 and pH 4.5 (as indicated) were hybridized with DIG-labeled probes against cap0037 (a), cap0036 (b), or 16S rRNA transcript (c) as control of RNA integrity