Analysis of Dot/Icm Type IVB Secretion System Subassemblies by Cryoelectron Tomography Reveals Conformational Changes Induced by DotB Binding

ABSTRACT Type IV secretion systems (T4SSs) are sophisticated nanomachines used by many bacterial pathogens to translocate protein and DNA substrates across a host cell membrane. Although T4SSs have important roles in promoting bacterial infections, little is known about the biogenesis of the apparatus and the mechanism of substrate transfer. Here, high-throughput cryoelectron tomography (cryo-ET) was used to visualize Legionella pneumophila T4SSs (also known as Dot/Icm secretion machines) in both the whole-cell context and at the cell pole. These data revealed the distribution patterns of individual Dot/Icm machines in the bacterial cell and identified five distinct subassembled intermediates. High-resolution in situ structures of the Dot/Icm machine derived from subtomogram averaging revealed that docking of the cytoplasmic DotB (VirB11-related) ATPase complex onto the DotO (VirB4-related) ATPase complex promotes a conformational change in the secretion system that results in the opening of a channel in the bacterial inner membrane. A model is presented for how the Dot/Icm apparatus is assembled and for how this machine may initiate the transport of cytoplasmic substrates across the inner membrane. IMPORTANCE Many bacteria use type IV secretion systems (T4SSs) to translocate proteins and nucleic acids into target cells, which promotes DNA transfer and host infection. The Dot/Icm T4SS in Legionella pneumophila is a multiprotein nanomachine that is known to translocate over 300 different protein effectors into eukaryotic host cells. Here, advanced cryoelectron tomography and subtomogram analysis were used to visualize the Dot/Icm machine assembly and distribution in a single L. pneumophila cell. Extensive classification and averaging revealed five distinct intermediates of the Dot/Icm machine at high resolution. Comparative analysis of the Dot/Icm machine and subassemblies derived from wild-type cells and several mutants provided a structural basis for understanding mechanisms that underlie the assembly and activation of the Dot/Icm machine.

Type IV Secretion Systems Diversity in the Acidithiobacillus Genus

Dispersal between genomes of certain mobile genetic elements and their gene cargo depends on conjugative type IV secretion systems. In this work, variants of these nanomachines, tra and trb, have been profiled in publicly available genomes of the genus Acidithiobacillus and in a set of relevant strains. Our analyses show that the trb system is of broad distribution, being present in most of the strains analyzed. In turn, the tra type is present in fewer strains of A. ferrooxidans, A. ferrivorans, A. ferriphilus and A. thiooxidans, and generally correlates with the presence of larger ICE in the respective genomes. Herein, sequence conservation, genomic context, integration site and synteny analyses are performed to infer functionality of the T4SS systems of the acidithiobacilli.