Abstract
Clostridium acetobutylicum is a strict anaerobic, endospore-forming bacterium, which is used for the production of the high energy biofuel butanol in metabolic engineering. The life cycle of C. acetobutylicum can be divided into two phases, with acetic and butyric acids being produced in the exponential phase (acidogenesis) and butanol formed in the stationary phase (solventogenesis). During the transitional phase from acidogenesis to solventogenesis and latter stationary phase, concentration peaks of the metabolic intermediates butyryl phosphate and acetyl phosphate are observed. As an acyl group donor, acyl-phosphate chemically acylates protein substrates. However, the regulatory mechanism of lysine acetylation and butyrylation involved in the phenotype and solventogenesis of C. acetobutylicum remains unknown. In our study, we conducted quantitative analysis of protein acetylome and butyrylome to explore the dynamic change of lysine acetylation and butyrylation in the exponential phase, transitional phase, and stationary phase of C. acetobutylicum Total 458 lysine acetylation sites and 1078 lysine butyrylation sites were identified in 254 and 373 substrates, respectively. Bioinformatics analysis uncovered the similarities and differences between the two acylation modifications in C. acetobutylicum Mutation analysis of butyrate kinase and the central transcriptional factor Spo0A was performed to characterize the unique role of lysine butyrylation in the metabolic pathway and sporulation process of C. acetobutylicum Moreover, quantitative proteomic assays were performed to reveal the relationship between protein features (e.g. gene expression level and lysine acylation level) and metabolites in the three growth stages. This study expanded our knowledge of lysine acetylation and butyrylation in Clostridia and constituted a resource for functional studies on lysine acylation in bacteria.
Highlights
N-lysine acetylation is a reversible, dynamic and conserved post-translational modification (PTM), which plays pivotal roles in cellular physiological and pathological functions in both eukaryotes and prokaryotes [1, 2]
The first Butyrylome in Clostridium phosphate (BuP) concentration peak was identified during the transitional phase, which coincides with the onset of solvent production, whereas the second peak appears when butyric acid is reused during the solventogenesis phase
Peptides isolated from the exponential phase were labeled using “L” dimethyl labeling reagents (i.e. CH2O and NaBH3CN), peptides isolated from the transitional phase were labeled using “M” dimethyl labeling reagents (i.e. CD2O and NaBH3CN), and peptides isolated from the stationary phase were labeled using “H” labeling reagents (i.e. 13CD2O and NaBD3CN)
Summary
N-lysine acetylation is a reversible, dynamic and conserved post-translational modification (PTM), which plays pivotal roles in cellular physiological and pathological functions in both eukaryotes and prokaryotes [1, 2]. We conducted quantitative protein acetylome and butyrylome analysis to explore the dynamic changes of lysine acetylation and butyrylation during the exponential phase, transitional phase and stationary phase of the C. acetobutylicum life cycle.
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