Binding of <i>Bacillus subtilis</i> dynamin‐like protein <scp>DynA</scp> to the bacterial membrane is essential for effective phage defense

Bacterial dynamin-like proteins are large GTPases that play crucial roles in membrane dynamics. Bacillus subtilis dynamin-like protein A (DynA), a two-headed bacterial dynamin-like protein, possesses membrane-binding and membrane-tethering functions in trans. The formation of large DynA clusters on bacterial membranes in response to pore-forming antibiotics and phages demonstrates its potential role in maintaining bacterial membrane integrity under various environmental stresses. In this study, we identified the membrane-binding site of B. subtilis DynA within the D1 subunit 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 amino acid substitutions in the lysine and phenylalanine residues were produced and used in liposome binding assays. Nonconservative substitutions led to a complete loss of DynA's membrane-binding capability. In vivo data showed strains with DynA variants lacking membrane-binding capability exhibit significantly increased susceptibility to phage infection compared with wild-type cells, further emphasizing the importance of DynA's membrane interaction in conferring phage resistance. Our findings bridge the gap between the structural characteristics of DynA and its functional implications in maintaining bacterial membrane integrity and mediating phage resistance.