A CELL WALL HYDROLASE ENABLES BACTERIAL INTERSPECIES NANOTUBE FORMATION

Bacteria display an array of contact dependent interaction systems to facilitate direct cell-to-cell communication. Using Bacillus subtilis as a model organism, we have previously shown a novel type of bacterial communication mediated by intercellular bridges, which we named nanotubes. We found that via these membranous tubes bacteria can exchange cytoplasmic content, including small molecules, proteins, and non-conjugative plasmids. To explore the molecular components comprising nanotubes, we purified nanotube associated proteins and identified them by Mass spectrometry. Using this approach, we identified the phosphodiesterase YmdB, as a key player in nanotube formation. Here we report that a cell wall remodeling enzyme- LytB, found in the nanotube fraction, is required for intercellular molecular exchange and nanotube formation. We found that LytB forms foci on the cell wall at the site of nanotube emergence, and this localization is entirely dependent on YmdB. Strikingly, HR-SEM analysis revealed that LytB migrates along nanotubes to reach nearby neighboring cells. Furthermore, this delivery occurs in an interspecies manner, as LytB was found to be localized to sites of nanotube invasion on the surface of recipient Bacillus megaterium and Staphylococcus aureus cells. An array of biochemical and cell biological analyses revealed that LytB has a cell wall lytic activity that can damage the cell wall of various Gram positive bacteria. Further, the lack of LytB led to perturbation in interspecies molecular exchange. We propose that LytB localizes to site of nanotube emergence, tearing the cell wall to enable nanotube protrusion. LytB then travels along the growing nanotube to reach the surface of a nearby recipient cell, where it pinches the cell wall and facilitate nanotube access and attachment.