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bioRxiv · 10.64898/2025.12.18.695078

Pili are essential for conjugation also in many Gram-positive bacteria

ter Beek, J.·Svedberg, D.·Deane-Alder, K.·Mateus, A.·Berntsson, R. P.-A.
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Abstract

Type IV secretion systems (T4SS) enable the spread of antibiotic resistance and other virulence factors. In Gram-positive bacteria, T4SSs have long been thought to lack VirB2-like proteins that form conjugative pili and instead rely on adhesins for cell-cell contacts. Yet, it has remained unclear how subsequent DNA transfer (conjugation) is physically mediated. Here we identify a VirB2-like protein, PrgFB2, from the clinically isolated conjugative plasmid pCF10 in Enteroccocus faecalis and show that it is essential for conjugation. Structural modeling confidently predicts a pilus-like assembly for PrgFB2. We validate this prediction through mutagenesis, conjugation assays, and targeted chemical labeling. By combining various machine learning bioinformatic techniques, we analysed >1000 Gram-positive conjugative plasmids from diverse species, including major pathogens, and identified pili forming VirB2-like proteins in almost all of them. Our findings overturn the prevailing view that Gram-positive T4SSs lack pili. This discovery provides a new framework for understanding horizontal gene transfer and highlights critical targets for combating antimicrobial resistance and virulence in Gram-positive bacteria.

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ter Beek, J., Svedberg, D., Deane-Alder, K., Mateus, A., Berntsson, R. P.-A.. 2025-12-18. Pili are essential for conjugation also in many Gram-positive bacteria. https://doi.org/10.64898/2025.12.18.695078

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