Abstract
Type IV secretion (T4S) systems are versatile bacterial secretion systems mediating transport of protein and/or DNA. T4S systems are generally composed of 11 VirB proteins and 1 VirD protein (VirD4). The VirB1‐11 proteins assemble to form a secretion machinery and a pilus while the VirD4 protein is responsible for substrate recruitment. The structure of VirD4 in isolation is known; however, its structure bound to the VirB1‐11 apparatus has not been determined. Here, we purify a T4S system with VirD4 bound, define the biochemical requirements for complex formation and describe the protein–protein interaction network in which VirD4 is involved. We also solve the structure of this complex by negative stain electron microscopy, demonstrating that two copies of VirD4 dimers locate on both sides of the apparatus, in between the VirB4 ATPases. Given the central role of VirD4 in type IV secretion, our study provides mechanistic insights on a process that mediates the dangerous spread of antibiotic resistance genes among bacterial populations.
Highlights
Type IV secretion (T4S) systems are membrane embedded multiprotein complexes, which are present, both in Gram-negative and Gram-positive bacteria, as well as archaea (Wallden et al, 2010; Trokter et al, 2014; Costa et al, 2015)
The second group of T4S systems are responsible for DNA release or uptake to or from the extracellular milieu, respectively: these T4S systems operate in bacterial species such as Neisseria gonorrhoeae and Helicobacter pylori (Karnholz et al, 2006; Ramsey et al, 2011)
For the purpose of this study, three requirements for cloning were considered key: (i) sufficient material for negative stain electron microscopy (NS-EM) analysis should be obtained, (ii) tags should be engineered within the vectors employed, and (iii) all combinations of constructs should drive the production of functional T4S systems
Summary
Type IV secretion (T4S) systems are membrane embedded multiprotein complexes, which are present, both in Gram-negative and Gram-positive bacteria, as well as archaea (Wallden et al, 2010; Trokter et al, 2014; Costa et al, 2015). They are used to transport a variety of biomolecules across the bacterial envelope. The first group of T4S systems are called conjugative T4S systems and members of this group transfer DNA from a donor to a recipient cell (de la Cruz et al, 2010) This process contributes to the spread of antibiotic resistance genes among different bacterial species and is instrumental in bacterial adaptation to environmental changes (Thomas & Nielsen, 2005). T4S systems can translocate effector molecules into eukaryotic cells, which can result in many diseases such as Legionnaires’ disease caused by Legionella pneumophila or brucellosis caused by Brucella spp (Llosa et al, 2009; Nagai & Kubori, 2011; Terradot & Waksman, 2011)
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