Abstract
Bacterial type IV secretion systems (T4SSs) are widespread nanomachines specialized in the transport across the cell envelope of various types of molecules including mobile genetic elements during conjugation. Despite their prevalence in Gram-positive bacteria, including relevant pathogens, their assembly and functioning remain unknown. This study addresses these gaps by investigating VirB8 proteins, known to be central components of conjugative T4SSs in Gram-positive bacteria. However, the functional packing and precise role of VirB8 in T4SSs biology remain undefined. Our findings elucidate the nature of VirB8 proteins as cell wall components, where they multimerize and exhibit a conserved assembly pattern, distinct from VirB8 in Gram-negative bacteria. We also demonstrate that VirB8 proteins interact with other T4SS subunits and DNA, indicating their pivotal role in the building of the DNA translocation channel across the cell wall. We lastly propose a distinct architecture for conjugative T4SSs in Gram-positive bacteria compared to their Gram-negative counterparts, possibly attributed to the differences in the cell wall structure.