Integrative Dynamic Proteomics and Acyl-Proteomics Analyses in Clostridium butyricum

Clostridium butyricum (C. butyricum) is a notable butyrate-producing intestinal probiotic that has been utilized for the enhancement and treatment of various intestinal and extraintestinal diseases. Butyrate and acetate, as significant metabolites of this bacterium, are short-chain fatty acids (SCFAs) that play crucial roles in maintaining intestinal stability and safeguarding host health. However, the regulatory functions of lysine butyrylation (Kbu) and acetylation (Kac), mediated by these metabolites, remain inadequately understood. In this study, we performed a comprehensive dynamic multiomic analysis encompassing protein expression, Kbu, and Kac in C. butyricum. Our multiomic analysis identified a total of 2622 proteins, 1887 Kbu sites, and 2188 Kac sites. Subsequent bioinformatic analyses revealed that biological functions such as gene expression and energy metabolism exhibited dynamic changes throughout the growth cycle at both the proteome and post-translational modification levels. Enzymatic experiments demonstrated that Kbu modified key active sites of important substrates, including alanine-tRNA ligase at K794, GMP synthase at K384, and probable butyrate kinase at K269. This study enhances the current understanding of lysine acylations in bacteria and focuses on elucidating the finely tuned regulatory mechanisms of Kbu during the growth and development of C. butyricum.