Type II protein secretion systems (T2SS) are molecular machines that promote specific transport of folded periplasmic proteins in Gram-negative bacteria, across a dedicated channel in the outer membrane. Secreted substrates, released to the milieu or displayed on the cell surface, contribute to bacterial adaptation to a range of habitats, from deep-sea waters to animal and plant tissues. The past decade has seen remarkable progress in structural, biochemical and functional analysis of T2SS and related systems, bringing new mechanistic insights into these dynamic complexes. This review focuses on recent advances in the field, and discusses open questions regarding the secretion mechanism. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

In Gram‐negative bacteria, type II secretion systems (T2SS) assemble inner membrane proteins of the major pseudopilin PulG (GspG) family into periplasmic filaments, which could drive protein secretion in a piston‐like manner. Three minor pseudopilins PulI, PulJ and PulK are essential for protein secretion in the Klebsiella oxytoca T2SS, but their molecular function is unknown. Here, we demonstrate that together these proteins prime pseudopilus assembly, without actively controlling its length or secretin channel opening. Using molecular dynamics, bacterial two‐hybrid assays, cysteine crosslinking and functional analysis, we show that PulI and PulJ nucleate filament assembly by forming a staggered complex in the plasma membrane. Binding of PulK to this complex results in its partial extraction from the membrane and in a 1‐nm shift between their transmembrane segments, equivalent to the major pseudopilin register in the assembled PulG filament. This promotes fully efficient pseudopilus assembly and protein secretion. Therefore, we propose that PulI, PulJ and PulK self‐assembly is thermodynamically coupled to the initiation of pseudopilus assembly, possibly setting the assembly machinery in motion.

Many Gram-negative bacteria secrete specific proteins via the type II secretion systems (T2SS). These complex machineries share with the related archaeal flagella and type IV pilus (T4P) biogenesis systems the ability to assemble thin, flexible filaments composed of small, initially inner membrane-localized proteins called "pilins." In the T2SS from Klebsiella oxytoca, periplasmic pseudopili that are essential for pullulanase (PulA) secretion extend beyond the bacterial surface and form pili when the major pilin PulG is overproduced. Here, we describe the detailed, experimentally validated structure of the PulG pilus generated from crystallographic and electron microscopy data by a molecular modeling approach. Two intermolecular salt bridges crucial for function were demonstrated using single and complementary charge inversions. Double-cysteine substitutions in the transmembrane segment of PulG led to positionspecific cross-linking of protomers in assembled pili. These biochemical data provided information on residue distances in the filament that were used to derive a refined model of the T2SS pilus at pseudoatomic resolution. PulG is organized as a right-handed helix of subunits, consistent with protomer organization in gonococcal T4P. The conserved character of residues involved in key hydrophobic and electrostatic interactions within the major pseudopilin family supports the general relevance of this model for T2SS pseudopilus structure. molecular modeling | pilus assembly | protein secretion | pseudopilus | cysteine cross-linking G ram-negative bacteria use several mechanisms to secrete toxins and hydrolases with important roles in pathogenesis and metabolism. The type II protein secretion systems (T2SS) are membrane protein complexes that transport folded proteins from the periplasm to the extracellular medium through the outer membrane channel formed by the protein secretin (reviewed in ref. 1). Between 12 and 15 proteins comprising the T2SS are essential for function, and many are highly similar to components of type IV piliation (T4P) systems (2). Beyond their similarities to T4P in composition and sequence, T2SS were found to assemble pili on the bacterial surface upon overproduction of the major pseudopilin (3), suggesting a common basic mechanism (2, 4, 5). The surface-exposed T2SS pili are regarded as artificially extended periplasmic filaments called "pseudopili" that play a crucial but poorly understood role in protein secretion. The current models propose that the inner membrane (IM) assembly platform (6) transduces the energy generated by cytoplasmic ATPase PulE to facilitate pilin extraction from the IM into the growing periplasmic filament (7). According to the piston hypothesis, pseudopilus polymerization promotes secretion by direct interaction with the substrate to push it through the secretin channel (4,8).The surface pili of the pullulanase T2SS from Klebsiella oxytoca consist essentially of the major pseudopilin subunit PulG (3). The dimensions, flexibility, and bundling propensity of ex...