Summary
Type IV secretion systems (T4SSs) are versatile multiprotein nanomachines spanning the entire cell envelope in Gram‐negative and Gram‐positive bacteria. They play important roles through the ...contact‐dependent secretion of effector molecules into eukaryotic hosts and conjugative transfer of mobile DNA elements as well as contact‐independent exchange of DNA with the extracellular milieu. In the last few years, many details on the molecular mechanisms of T4SSs have been elucidated. Exciting structures of T4SS complexes from Escherichia coli plasmids R388 and pKM101, Helicobacter pylori and Legionella pneumophila have been solved. The structure of the F‐pilus was also reported and surprisingly revealed a filament composed of pilin subunits in 1:1 stoichiometry with phospholipid molecules. Many new T4SSs have been identified and characterized, underscoring the structural and functional diversity of this secretion superfamily. Complex regulatory circuits also have been shown to control T4SS machine production in response to host cell physiological status or a quorum of bacterial recipient cells in the vicinity. Here, we summarize recent advances in our knowledge of ‘paradigmatic’ and emerging systems, and further explore how new basic insights are aiding in the design of strategies aimed at suppressing T4SS functions in bacterial infections and spread of antimicrobial resistances.
Type IV secretion systems (T4SSs) are highly sophisticated nanomachines in the cell envelope of many bacteria. They exhibit crucial roles during infection of humans by the secretion of effector proteins, conjugative transfer of DNA and exchange of DNA with the extracellular environment. In this MicroReview, we summarize recent progress on T4SS composition, assembly and structure and highlight how basic understanding of their functions is aiding in the design of novel strategies for antimicrobial therapies.
Bacterial conjugation systems are members of the large type IV secretion system (T4SS) superfamily. Conjugative transfer of F plasmids residing in the Enterobacteriaceae was first reported in the ...1940s, yet the architecture of F plasmid-encoded transfer channel and its physical relationship with the F pilus remain unknown. We visualized F-encoded structures in the native bacterial cell envelope by in situ cryoelectron tomography (CryoET). Remarkably, F plasmids encode four distinct structures, not just the translocation channel or channel-pilus complex predicted by prevailing models. The F1 structure is composed of distinct outer and inner membrane complexes and a connecting cylinder that together house the envelope-spanning translocation channel. The F2 structure is essentially the F1 complex with the F pilus attached at the outer membrane (OM). Remarkably, the F3 structure consists of the F pilus attached to a thin, cell envelope-spanning stalk, whereas the F4 structure consists of the pilus docked to the OM without an associated periplasmic density. The traffic ATPase TraC is configured as a hexamer of dimers at the cytoplasmic faces of the F1 and F2 structures, where it respectively regulates substrate transfer and F pilus biogenesis. Together, our findings present architectural renderings of the DNA conjugation or “mating” channel, the channel–pilus connection, and unprecedented pilus basal structures. These structural snapshots support a model for biogenesis of the F transfer system and allow for detailed comparisons with other structurally characterized T4SSs.
The translocation of proteins across membranes is a fundamental cellular function. Bacteria have evolved a striking array of pathways for delivering proteins into or across cytoplasmic membranes and, ...when present, outer membranes. Translocated proteins can form part of the membrane landscape, reside in the periplasmic space situated between the inner and outer membranes of Gram-negative bacteria, deposit on the cell surface, or be released to the extracellular milieu or injected directly into target cells. One protein translocation system, the general secretory pathway, is conserved in all domains of life. A second, the twin-arginine translocation pathway, is also phylogenetically distributed among most bacteria and plant chloroplasts. While all cell types have evolved additional systems dedicated to the translocation of protein cargoes, the number of such systems in bacteria is now known to exceed nine. These dedicated protein translocation systems, which include the types 1 through 9 secretion systems (T1SSs–T9SSs), the chaperone–usher pathway, and type IV pilus system, are the subject of this review. Most of these systems were originally identified and have been extensively characterized in Gram-negative or diderm (two-membrane) species. It is now known that several of these systems also have been adapted to function in Gram-positive or monoderm (single-membrane) species, and at least one pathway is found only in monoderms. This review briefly summarizes the distinctive mechanistic and structural features of each dedicated pathway, as well as the shared properties, that together account for the broad biological diversity of protein translocation in bacteria.
The bacterial type IV secretion systems (T4SSs) translocate DNA and protein substrates to bacterial or eukaryotic target cells generally by a mechanism dependent on direct cell-to-cell contact. The ...T4SSs encompass two large subfamilies, the conjugation systems and the effector translocators. The conjugation systems mediate interbacterial DNA transfer and are responsible for the rapid dissemination of antibiotic resistance genes and virulence determinants in clinical settings. The effector translocators are used by many Gram-negative bacterial pathogens for delivery of potentially hundreds of virulence proteins to eukaryotic cells for modulation of different physiological processes during infection. Recently, there has been considerable progress in defining the structures of T4SS machine subunits and large machine subassemblies. Additionally, the nature of substrate translocation sequences and the contributions of accessory proteins to substrate docking with the translocation channel have been elucidated. A DNA translocation route through the Agrobacterium tumefaciens VirB/VirD4 system was defined, and both intracellular (DNA ligand, ATP energy) and extracellular (phage binding) signals were shown to activate type IV-dependent translocation. Finally, phylogenetic studies have shed light on the evolution and distribution of T4SSs, and complementary structure-function studies of diverse systems have identified adaptations tailored for novel functions in pathogenic settings. This review summarizes the recent progress in our understanding of the architecture and mechanism of action of these fascinating machines, with emphasis on the ‘archetypal’ A. tumefaciens VirB/VirD4 T4SS and related conjugation systems. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.
•We review structural information about bacterial type IV secretion systems (T4SSs).•We discuss adaptations to type IV machines that evolved for specialized functions.•We summarize functions of subunits and subassemblies of archetypal T4SSs.•We review mechanisms for substrate presentation to the bacterial T4SSs.•We present models depicting the substrate translocation route through T4SSs.
Escherichia coli and other Gram-negative and -positive bacteria employ type IV secretion systems (T4SSs) to translocate DNA and protein substrates, generally by contact-dependent mechanisms, to other ...cells. The T4SSs functionally encompass two major subfamilies, the conjugation systems and the effector translocators. The conjugation systems are responsible for interbacterial transfer of antibiotic resistance genes, virulence determinants, and genes encoding other traits of potential benefit to the bacterial host. The effector translocators are used by many Gram-negative pathogens for delivery of potentially hundreds of virulence proteins termed effectors to eukaryotic cells during infection. In E. coli and other species of Enterobacteriaceae, T4SSs identified to date function exclusively in conjugative DNA transfer. In these species, the plasmid-encoded systems can be classified as the P, F, and I types. The P-type systems are the simplest in terms of subunit composition and architecture, and members of this subfamily share features in common with the paradigmatic Agrobacterium tumefaciens VirB/VirD4 T4SS. This review will summarize our current knowledge of the E. coli systems and the A. tumefaciens P-type system, with emphasis on the structural diversity of the T4SSs. Ancestral P-, F-, and I-type systems were adapted throughout evolution to yield the extant effector translocators, and information about well-characterized effector translocators also is included to further illustrate the adaptive and mosaic nature of these highly versatile machines.
A large subfamily of the type IV secretion systems (T4SSs), termed the conjugation systems, transmit mobile genetic elements (MGEs) among many bacterial species. In the initiating steps of ...conjugative transfer, DNA transfer and replication (Dtr) proteins assemble at the origin‐of‐transfer (oriT) sequence as the relaxosome, which nicks the DNA strand destined for transfer and couples the nicked substrate with the VirD4‐like substrate receptor. Here, we defined contributions of the Dtr protein TraK, a predicted member of the Ribbon‐Helix‐Helix (RHH) family of DNA‐binding proteins, to transfer of DNA and protein substrates through the pKM101‐encoded T4SS. Using a combination of cross‐linking/affinity pull‐downs and two‐hybrid assays, we determined that TraK self‐associates as a probable tetramer and also forms heteromeric contacts with pKM101‐encoded TraI relaxase, VirD4‐like TraJ receptor, and VirB11‐like and VirB4‐like ATPases, TraG and TraB, respectively. TraK also promotes stable TraJ–TraB complex formation and stimulates binding of TraI with TraB. Finally, TraK is required for or strongly stimulates the transfer of cognate (pKM101, TraI relaxase) and noncognate (RSF1010, MobA relaxase) substrates. We propose that TraK functions not only to nucleate pKM101 relaxosome assembly, but also to activate the TrapKM101 T4SS via interactions with the ATPase energy center positioned at the channel entrance.
DNA transfer and replication (Dtr) proteins initiate DNA transfer through T4SSs by binding oriT sequences and nicking the strand destined for transfer. We discovered that the Dtr factor TraK, a putative RHH DNA‐binding protein, activates the pKM101‐encoded T4SS through binding the three ATPases (TraJ, TraG, TraB) situated at the channel entrance. TraK‐dependent channel activation is required for or strongly stimulates the intercellular transfer of all tested DNA and protein substrates.
Type IV secretion systems (T4SSs) are versatile secretion systems that are found in both Gram-negative and Gram-positive bacteria and secrete a wide range of substrates, from single proteins to ...protein-protein and protein-DNA complexes. They usually consist of 12 components that are organized into ATP-powered, double-membrane-spanning complexes. The structures of single soluble components or domains have been solved, but an understanding of how these structures come together has only recently begun to emerge. This Review focuses on the structural advances that have been made over the past 10 years and how the corresponding structural insights have helped to elucidate many of the details of the mechanism of type IV secretion.
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