The mechanism of bacterial defense system DdmDE from Lactobacillus casei

Bacteria has developed a diverse array of defense mechanisms to protect against invasion by mobile genetic elements. Recent study identified a bacterial defense module DdmDE system which encodes a helicase-nuclease fusion protein DdmD and a putative prokaryotic Argonaute-like protein DdmE, imposing fitness advantages to the bacteria by eliminating invasive plasmids. However, the mechanistic basis of how DdmDE system detects and degrades plasmids is not fully understood. Here, by studying the DdmDE system from Lactobacillus casei (LcDdmDE), we found that LcDdmD is able to degrade ssDNA and nick plasmids in the presence of Mn ²⁺ , and it exhibits 5’-3’ DNA helicase activity in a ssDNA length-dependent mechanism. Meanwhile, LcDdmD serves as a sensor that utilizes DNA guide to recognize target DNA. We determined the cryo-EM structures of LcDdmD dimer bound with fork DNA, guide/target DNA-bound LcDdmE, and the complex of LcDdmDE-bubble DNA in intermediate state as well as active state. Together with functional analysis, we revealed the working mechanism of LcDdmDE system. In such a scenario, guided by ssDNA, LcDdmE recruits auto-inhibited LcDdmD dimer loading onto DNA target. Through substantial conformational changes, LcDdmD dimer dissociates into active monomer and unwind duplex for plasmid degradation. Our study provides structural insights into the mechanism of DdmDE, presenting pAgo-directed plasmid degradation by the allosterically regulated helicase-nuclease.