Membrane-binding of the bacterial dynamin-like protein DynA is essential for effective phage defence

Abstract

Bacterial dynamin-like proteins are large GTPases that play crucial roles in membrane dynamics. DynA in B. subtilis, a two-headed bacterial dynamin-like protein, possesses membrane-binding and tethering functions in trans. The formation of large DynA clusters on membranes in response to pore-forming antibiotics and phages demonstrates its potential role in maintaining membrane integrity under various environmental stresses. In this study, we identified the membrane-binding site of B. subtilis DynA within the D1 domain of the protein that includes positively charged lysine residues K360 and K367, as well as hydrophobic phenylalanine residues F363, F364, and F365. For experimental validation, recombinant proteins with mutations in the lysine or phenylalanine residues were produced and used in liposome binding assays. Non-conservative mutations lead to a complete loss of DynAs membrane-binding capability. In vivo data showed that the membrane-binding mutants exhibit significantly increased susceptibility to phage infection compared to wild-type strains, further emphasizing the importance of DynAs membrane interaction in conferring phage resistance. Our findings bridge the gap between the structural characteristics of DynA and its functional implications in maintaining membrane integrity and mediating phage resistance.