Regulation of Lactobacillus acidophilus gene expression by dietary compounds via N4-methylcytosine modifications: a novel approach to probiotic engineering

Abstract

Lactobacillus acidophilus is a probiotic integral to the human gut microbiome and associated with numerous health benefits. While it is well-established that dietary compounds can affect gene functionality within the gut microbiome, the precise mechanisms remain unclear. In this study, we demonstrate that specific dietary compounds, such as genistein and resveratrol, significantly regulate gene expression in Lactobacillus acidophilus (LA). Our findings reveal that each dietary compound uniquely influences gene transcription and metabolic activity, with the key insight that N4-methylcytosine (m4C), a relatively understudied methyl modification, is the primary mechanism driving transcriptional regulation. For instance, in LA cells treated with genistein, 76 genes were upregulated and 130 genes were downregulated. Notably, a mucus-binding protein gene, crucial for the bacteria's adherence to epithelial cells and important for probiotic functionality, showed a remarkable 38-fold upregulation due to demethylation of m4C in the promoter region. The melibiose operon regulatory protein gene was upregulated 78-fold, correlating with increased melibiose (a prebiotic) concentration in metabolomic analyses. The cro/C1-type HTH domain gene, which represses temperate bacteriophages, was significantly downregulated 10-fold with genistein but upregulated 9-fold with sodium butyrate (SB). Interestingly, different dietary compounds varied in their effects on this gene. Genome editing in bacteria typically involves complex laborious, multi-step methods such as suicide plasmids. Although CRISPR-Cas technologies has revolutionised eukaryotic genome editing, they are less widely favoured for bacterial genome editing. This study showed that each dietary compound uniquely influences the gene expression profile of LA and present potential for the metabolic engineering of probiotic/bacterial strain modification without the need for genetic modification tools and offer a biotechnological approach to modulating the human gut microbiome.