Ilnam Kang (@ilnamkang.bsky.social)

Type IV Secretion System Drives Lipid Mixing Type IV secretion systems (T4SSs) are versatile molecular machines used by bacteria to secrete protein effectors into host cells, promoting pathogenesis, and to transfer DNA between bacteria through conjugation, driving horizontal gene transfer. Most, like Dot/Icm of the pathogen Legionella pneumophila ( L. pneumophila) or Escherichia coli ( E. coli) RK2, are primed for substrate delivery only upon contact with a target membrane, but mechanisms are unknown. A pilus could bind a receptor to initiate priming, but many T4SSs, especially those that deliver effectors, lack a pilus. Here, we present evidence that T4SSs are primed by direct contact with target membrane lipids. Combining fluorescence assays with genetics and biochemistry, we found that Dot/Icm drives lipid exchange between bacterial cells and between bacteria and synthetic membranes containing only lipids. Lipid exchange requires membrane contact but does not require ATP hydrolysis or even full complex assembly. Minimally, the outer membrane core complex protein DotG needs to be present in at least one of the apposed membranes. We similarly observed lipid mixing with the simpler E. coli RK2 T4SS, where we could follow lipid mixing and plasmid transfer simultaneously. We found that lipid mixing always preceded or accompanied plasmid transfer, suggesting it may be part of the contact-dependent priming mechanism. Lipid mixing was inhibited or promoted by lipids that inhibit or promote membrane fusion, respectively. Lipids inhibiting lipid mixing also inhibited substrate transfer. Together, our results suggest that initial contact between DotG outer segments and target membrane lipids promotes lipid mixing as part of the mechanism that primes T4SS for substrate translocation. Highlights 1. The Legionella pneumophila Dot/Icm secretion system requires contact with a target membrane for effector translocation, but how membrane contact primes the machinery for this is unknown. 2. We found that Dot/Icm drives contact-dependent lipid mixing between bacteria or between bacteria and liposomes, showing that Dot/Icm priming does not require a protein receptor. 3. Lipid mixing also occurs with the simpler Type IVA system ( Escherichia coli RK2) during conjugation, suggesting lipid mixing is a general feature of Type IV Secretion System function. 4. The core complex protein DotG is sufficient to drive lipid mixing, suggesting DotG-target membrane interactions may destabilize the target membrane. ### Competing Interest Statement The authors have declared no competing interest. National Institutes of Health, R01NS122388, R37AI041699 https://www.biorxiv.org/content/10.64898/2026.01.14.699567v1