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Combining genetic methods, mass spectrometry, and biochemical approaches, we show that the multiple peptide resistance factor (MprF) homolog LysX from\n                    <jats:italic toggle=\"yes\">Corynebacterium pseudotuberculosis<\/jats:italic>\n                    and two mycobacterial species is responsible for Lys-DAG synthesis. LysX is conserved in most Actinobacteria and was previously implicated in the synthesis of another modified lipid, lysyl-phosphatidylglycerol (Lys-PG), in\n                    <jats:italic toggle=\"yes\">Mycobacterium tuberculosis<\/jats:italic>\n                    . Although we detected low levels of Lys-PG in the membrane of\n                    <jats:italic toggle=\"yes\">C. pseudotuberculosis<\/jats:italic>\n                    , our data suggest that Lys-PG is not directly synthesized by LysX and may require an additional downstream pathway, which is as yet undefined. Our results show that LysX in\n                    <jats:italic toggle=\"yes\">C. pseudotuberculosis<\/jats:italic>\n                    is a major factor of resistance against a variety of positively charged antibacterial agents, including cationic antimicrobial peptides (e.g., human peptide LL-37 and polymyxin B) and aminoglycosides (e.g., gentamycin and apramycin). Deletion of\n                    <jats:italic toggle=\"yes\">lysX<\/jats:italic>\n                    caused an increase in cellular membrane permeability without dissipation of the membrane potential, suggesting that loss of the protein does not result in mechanical damage to the cell membrane. Furthermore,\n                    <jats:italic toggle=\"yes\">lysX<\/jats:italic>\n                    -deficient cells exhibited an attenuated virulence phenotype in a\n                    <jats:italic toggle=\"yes\">Galleria mellonella<\/jats:italic>\n                    infection model, supporting a role for LysX during infection. Altogether, Lys-DAG represents a novel molecular determinant for antimicrobial resistance and virulence that may be widespread in Actinobacteria and points to a richer landscape than previously realized of lipid components contributing to overall membrane physiology in this important bacterial phylum.\n                  <\/jats:p>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      In the past two decades, tRNA-dependent modification of membrane phosphatidylglycerol has been implicated in altering the biochemical properties of the cell surface, thereby enhancing the antimicrobial resistance and virulence of various bacterial pathogens. Here, we show that in several Actinobacteria, the multifunctional protein LysX attaches lysine to diacylglycerol instead of phosphatidylglycerol. We found that lysyl-diacylglycerol (Lys-DAG) confers high levels of resistance against various cationic antimicrobial peptides and aminoglycosides and also enhances virulence. Our data show that Lys-DAG is a lipid commonly found in important actinobacterial pathogens, including\n                      <jats:italic toggle=\"yes\">Mycobacterium<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">Corynebacterium<\/jats:italic>\n                      species.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.01429-23","type":"journal-article","created":{"date-parts":[[2023,9,28]],"date-time":"2023-09-28T09:03:17Z","timestamp":1695891797000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":5,"title":["The MprF homolog LysX synthesizes lysyl-diacylglycerol contributing to antibiotic resistance and virulence"],"prefix":"10.1128","volume":"11","author":[{"given":"Cameron P.","family":"Gill","sequence":"first","affiliation":[{"name":"Burnett School of Biomedical Sciences, College of Medicine, University of Central 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