Protein Acetylation and Butyrylation Regulate the Phenotype and Metabolic Shifts of the Endospore-forming Clostridium acetobutylicum

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.

Keywords: Acetylation*; Bacteria; Enzyme Regulation*; Mass Spectrometry; Post-translational modifications*; Quantification; acylation regulation; endospore-forming Clostridium; metabolic shift; quantitative acetylome; quantitative butyrylome.

Fig. 1.

Quantitative analysis of lysine acetylome and butyrylome in three growth phases in C. acetobutylicum strain. A, Western blot analysis of the global lysing acetyllysine and butyryllysine level in protein lysates from C. acetobutylicum strain in the exponential phase (EP), transitional phase (TP), and stationary phase (SP). Coomassie blue staining was used as the loading control. B, Scatter plot of the quantifiable lysine acetylation sites identified in the EP and TP of the C. acetobutylicum strain. Log₂ of the normalized TP/EP (M/L) ratios of the quantifiable sites were used for X axis and log₁₀ of total intensities for each acetylated peptide for Y axis. Blue dots represent the sites of normalized M/L ratio less than 0.67; Red dots represent the sites of normalized M/L ratio between 0.67 and 1.5; Green dots represent the sites of normalized M/L ratio higher than 1.5. The percentage of acetylated sites in different M/L ratio ranges were presented in the pie chart. C, Scatter plot of the quantifiable lysine acetylation sites identified in the TP and SP of the C. acetobutylicum strain. Log₂ of the normalized SP/TP (H/M) ratios of the quantifiable sites were used for X axis and log₁₀ of total intensities for each acetylated peptide for Y axis. Blue dots represent the sites of normalized H/M ratio less than 0.67; Red dots represent the sites of normalized H/M ratio between 0.67 and 1.5; Green dots represent the sites of normalized H/M ratio higher than 1.5. The percentage of acetylated sites in different H/M ratio ranges were presented in the pie chart. D, Scatter plot of the quantifiable lysine butyrylation sites identified in the EP and TP of the C. acetobutylicum strain. Annotation is same in Fig. 1B. E, Scatter plot of the quantifiable lysine butyrylation sites identified in the TP and SP of the C. acetobutylicum strain. Annotation is same in Fig. 1C.

Fig. 2.

Landscape of the acylated proteins/sites in C. acetobutylicum strain. A, The number of acetylation/butyrylation sites per protein. B, The Venn diagram showing the number of overlapping acylated proteins or sites identified in our study. C, The KEGG analysis of the shared acetylated and butyrylated proteins in C. acetobutylicum strain.

Fig. 3.

The enrichment analysis of the protein with elevated acylation level in C. acetobutylicum strain. A, The KEGG analysis of acetylation sites and butyrylation sites with elevated acylation level in the transitional phase, including ribosome, biosynthesis of amino acids, purine metabolism, glycolysis/gluconeogenesis, amino acid degradation, butanoate metabolism, pentose phosphate pathway, fatty acid metabolism, pyruvate metabolism, aminoacyl-tRNA biosynthesis, oxidative phosphorylation, TCA cycle and bacterial chemotaxis. B, The quantitative acetylation and butyrylation sites in the Pyruvate-flavodoxin oxidoreductase of C. acetobutylicum strain.

Fig. 4.

The acylated states of the enzymes in the acid/solvent pathway. A, Diagram showing the metabolic enzymes involved in acidogenesis or solventogenesis. Enzymes in acidogenesis were shown in green; enzymes in solventogenesis were shown in blue; Enzymes in both the two phases were shown in yellow. B, The quantitative acylation level of pta, ack, ptb and buk in the acid formation pathway and adc, bdhA, adhe1 and adhe2 in the bioalcohol formation pathway.

Fig. 5.

Mutagenesis and enzymatic activity analysis of butyrate kinase (buk). A, The annotation of butyrate kinase in the butyrate biosynthesis pathway. B, The quantitative butyrylation sites in buk of C. acetobutylicum strain. C, Sequence alignment analysis the K239 of buk, from Exiguobacterium sibiricum (E. sibiricum), Bacillus cereus (B. cereus), Bacillus anthracis (B. anthracis), Bacillus subtilis (B. subtilis), Elusimicrobium minutum (E. minutum), Thermotoga maritima (T. maritima), Clostridium perfringens (C. perfringens) and C. acetobutylicum. D, Enzymatic activities of wild-type and K167Q, K239Q and K287Q mutant of buk. Purity of the wt and mutated proteins of buk shown by SDS-PAGE gel.

Fig. 6.

Lysine butyrylation in the bacterial chemotaxis and flagellar assembly. A, The endospore-forming process in the C. acetobutylicum strain. The phenotypes of C. acetobutylicum strain in different growth stages were shown. B, The butyrylated protein involved in the bacterial chemotaxis and flagellar assembly, including methyl-accepting chemotaxis proteins, chemotaxis signal transduction protein CheW, chemotaxis signal receiving protein CheY, chemotaxis histidine kinase CheA, chemotaxis protein CheR, flagellar motor switch protein G, flagellar switch protein FliM and chemotaxis protein MotB. Sequence alignment of CheY from E. coli and C. acetobutylicum.

Fig. 7.

Regulation of Spo0A by lysine butyrylation. A, The two butyrylation sites identified in the transcription factor Spo0A of C. acetobutylicum strain. B, Sequence alignment of Spo0A, from Bacillus anthracis (Ban), Bacillus subtilis (Bsu), Clostridium pasteurianum (Cpas) and Clostridium acetobutylicum (Cac). C, DNA-binding abilities of Spo0A and derivatives by EMSA. The amount of His-Spo0A used (10⁻⁷ × g) is shown at the top of each lane. From left to right panel: wild type Spo0A, Spo0A K45Q, Spo0A K217Q and Spo0A K217R. D, DNA-binding abilities of Bu-Spo0A and Bu-Spo0A K217R. The amount of His-Spo0A used (10⁻⁷ × g) is shown at the top of each lane. Left panel: The Bu-Spo0A K217R was incubated with probes. Right panel: Bu-Spo0A was incubated with probes. E, DNA-binding abilities of wt and Bu-wt. The amount of His-Spo0A used (10⁻⁷ × g) is shown at the top of each lane. Left panel: The wt was incubated with probes. Right panel: Bu-wt was incubated with probes.