Abstract
The type IV filament superfamily is a widespread group of molecular machines involved in natural transformation, motility, adhesion, nutrient uptake, and secretion of a wide spectrum of protein substrates. The gram-negative bacterium Caulobacter vibrioides expresses the tight adherence (Tad) pilus, a type IV machine involved in surface colonization. Here we investigated the proteins involved in Tad pilus production by ΦCbK resistance screening and the order of machine assembly and its polar remnant by fluorescence tagging. Using electron cryo-tomography and subtomogram averaging of wild-type and mutant strains, we resolved the Tad pilus machine architecture and built an integrative model of its structure. The resulting model suggests the individual roles of multiple Tad proteins. Together the data also reveals the Tad pilus machine's assembly order.