A ubiquitin-like protein controls assembly of a bacterial Type VIIb secretion system

Type VII secretion systems (T7SS) are crucial bacterial nanomachines that mediate interbacterial competition and host-pathogen interactions in Gram-positive bacteria. Despite their importance, the structural basis for assembly and substrate transport in T7SSb, a widely distributed T7SS variant, remains poorly understood. Here, we present the cryo-EM structure of the T7SSb core complex from Bacillus subtilis, revealing how a ubiquitin-like protein, YukD, coordinates assembly of the secretion machinery. YukD forms extensive interactions with the central channel component YukB and promotes its association with the pseudokinase YukC, creating a stable building block for channel assembly. Using microscopy and competition assays, we demonstrate that YukD is essential for proper T7SSb complex formation and contact-dependent bacterial killing. Structural modeling suggests this YukD-dependent assembly mechanism is conserved across diverse Gram-positive bacteria. Our findings reveal how bacteria have adapted a ubiquitin-like protein as a structural regulator for assembling a large secretion complex.