Overexpression of diglucosyldiacylglycerol synthase leads to daptomycin resistance in <i>Bacillus subtilis</i>

The lipopeptide antibiotic daptomycin exhibits bactericidal activity against Gram-positive bacteria by forming a complex with phosphatidylglycerol (PG) and lipid II in the cell membrane, causing membrane perforation. With the emergence of daptomycin-resistant bacteria, understanding the mechanisms of bacterial resistance to daptomycin has gained great importance. In this study, we aimed to identify the genetic factors contributing to daptomycin resistance in <i>Bacillus subtilis</i>, a model Gram-positive bacterium. Our findings demonstrated that overexpression of <i>ugtP</i>, which encodes diglucosyldiacylglycerol synthase, induces daptomycin resistance in <i>B. subtilis</i>. Specifically, overexpression of <i>ugtP</i> resulted in increased levels of diglucosyldiacylglycerol (Glc₂DAG) and decreased levels of acidic phospholipids cardiolipin and PG, as well as the basic phospholipid lysylphosphatidylglycerol. However, <i>ugtP</i> overexpression did not alter the cell surface charge and the susceptibility to the cationic antimicrobial peptide nisin or the cationic surfactant hexadecyltrimethylammonium bromide. Furthermore, by serial passaging in the presence of daptomycin, we obtained daptomycin-resistant mutants carrying <i>ugtP</i> mutations. These mutants showed increased levels of Glc₂DAG and a >4-fold increase in the minimum inhibitory concentration of daptomycin. These results suggest that increased Glc₂DAG levels, driven by <i>ugtP</i> overexpression, modify the phospholipid composition and confer daptomycin resistance in <i>B. subtilis</i> without altering the cell surface charge of the bacteria.IMPORTANCEDaptomycin is one of the last-resort drugs for the treatment of methicillin-resistant <i>Staphylococcus aureus</i> infections, and the emergence of daptomycin-resistant bacteria has become a major concern. Understanding the mechanism of daptomycin resistance is important for establishing clinical countermeasures against daptomycin-resistant bacteria. In the present study, we found that overexpression of <i>ugtP</i>, which encodes diglucosyldiacylglycerol synthase, induces daptomycin resistance in <i>B. subtilis</i>, a model Gram-positive bacteria. The overexpression of <i>UgtP</i> increased diglucosyldiacylglycerol levels, resulting in altered phospholipid composition and daptomycin resistance. These findings are important for establishing clinical strategies against daptomycin-resistant bacteria, including their detection and management.