Bacteria have evolved a remarkable array of sophisticated nanomachines to export various virulence factors across the bacterial cell envelope. In recent years, considerable progress has been made ...towards elucidating the structural and molecular mechanisms of the six secretion systems (types I-VI) of Gram-negative bacteria, the unique mycobacterial type VII secretion system, the chaperone-usher pathway and the curli secretion machinery. These advances have greatly enhanced our understanding of the complex mechanisms that these macromolecular structures use to deliver proteins and DNA into the extracellular environment or into target cells. In this Review, we explore the structural and mechanistic relationships between these single- and double-membrane-embedded systems, and we briefly discuss how this knowledge can be exploited for the development of new antimicrobial strategies.
Pili are crucial virulence factors for many Gram-negative pathogens. These surface structures provide bacteria with a link to their external environments by enabling them to interact with, and attach ...to, host cells, other surfaces or each other, or by providing a conduit for secretion. Recent high-resolution structures of pilus filaments and the machineries that produce them, namely chaperone-usher pili, type IV pili, conjugative type IV secretion pili and type V pili, are beginning to explain some of the intriguing biological properties that pili exhibit, such as the ability of chaperone-usher pili and type IV pili to stretch in response to external forces. By contrast, conjugative pili provide a conduit for the exchange of genetic information, and recent high-resolution structures have revealed an integral association between the pilin subunit and a phospholipid molecule, which may facilitate DNA transport. In addition, progress in the area of cryo-electron tomography has provided a glimpse of the overall architecture of the type IV pilus machinery. In this Review, we examine recent advances in our structural understanding of various Gram-negative pilus systems and discuss their functional implications.
Bacterial type IV secretion systems (T4SSs) are a functionally diverse translocation superfamily. They consist mainly of two large subfamilies: (i) conjugation systems that mediate interbacterial DNA ...transfer and (ii) effector translocators that deliver effector macromolecules into prokaryotic or eukaryotic cells. A few other T4SSs export DNA or proteins to the milieu, or import exogenous DNA. The T4SSs are defined by 6 or 12 conserved “core” subunits that respectively elaborate “minimized” systems in Gram‐positive or ‐negative bacteria. However, many “expanded” T4SSs are built from “core” subunits plus numerous others that are system‐specific, which presumptively broadens functional capabilities. Recently, there has been exciting progress in defining T4SS assembly pathways and architectures using a combination of fluorescence and cryoelectron microscopy. This review will highlight advances in our knowledge of structure–function relationships for model Gram‐negative bacterial T4SSs, including “minimized” systems resembling the Agrobacterium tumefaciens VirB/VirD4 T4SS and “expanded” systems represented by the Helicobacter pylori Cag, Legionella pneumophila Dot/Icm, and F plasmid‐encoded Tra T4SSs. Detailed studies of these model systems are generating new insights, some at atomic resolution, to long‐standing questions concerning mechanisms of substrate recruitment, T4SS channel architecture, conjugative pilus assembly, and machine adaptations contributing to T4SS functional versatility.
Type IV secretion systems (T4SSs) are a functionally diverse superfamily of translocation nanomachines deployed by most species of bacteria. Collectively, T4SSs mediate the transfer of mobile genetic elements or protein toxins to other bacteria, or effector proteins or other macromolecules to eukaryotic cells to aid in infection. Recent structural and fluorescence approaches are generating exciting new insights into how T4SSs assemble in the cell envelope and recruit and translocate substrates to other cells.
Considerable progress has been made in recent years in the structural and molecular biology of type IV secretion systems in Gram-negative bacteria. The latest advances have substantially improved our ...understanding of the mechanisms underlying the recruitment and delivery of DNA and protein substrates to the extracellular environment or target cells. In this Review, we aim to summarize these exciting structural and molecular biology findings and to discuss their functional implications for substrate recognition, recruitment and translocation, as well as the biogenesis of extracellular pili. We also describe adaptations necessary for deploying a breadth of processes, such as bacterial survival, host-pathogen interactions and biotic and abiotic adhesion. We highlight the functional and structural diversity that allows this extremely versatile secretion superfamily to function under different environmental conditions and in different bacterial species. Additionally, we emphasize the importance of further understanding the mechanism of type IV secretion, which will support us in combating antimicrobial resistance and treating type IV secretion system-related infections.
Conjugation is one of the most important processes that bacteria utilize to spread antibiotic resistance genes among bacterial populations. Interbacterial DNA transfer requires a large double ...membrane-spanning nanomachine called the type 4 secretion system (T4SS) made up of the inner-membrane complex (IMC), the outer-membrane core complex (OMCC) and the conjugative pilus. The iconic F plasmid-encoded T4SS has been central in understanding conjugation for several decades, however atomic details of its structure are not known. Here, we report the structure of a complete conjugative OMCC encoded by the pED208 plasmid from E. coli, solved by cryo-electron microscopy at 3.3 Å resolution. This 2.1 MDa complex has a unique arrangement with two radial concentric rings, each having a different symmetry eventually contributing to remarkable differences in protein stoichiometry and flexibility in comparison to other OMCCs. Our structure suggests that F-OMCC is a highly dynamic complex, with implications for pilus extension and retraction during conjugation.
Conjugative pili are widespread bacterial appendages that play important roles in horizontal gene transfer, in spread of antibiotic resistance genes, and as sites of phage attachment. Among ...conjugative pili, the F “sex” pilus encoded by the F plasmid is the best functionally characterized, and it is also historically the most important, as the discovery of F-plasmid-mediated conjugation ushered in the era of molecular biology and genetics. Yet, its structure is unknown. Here, we present atomic models of two F family pili, the F and pED208 pili, generated from cryoelectron microscopy reconstructions at 5.0 and 3.6 Å resolution, respectively. These structures reveal that conjugative pili are assemblies of stoichiometric protein-phospholipid units. We further demonstrate that each pilus type binds preferentially to particular phospholipids. These structures provide the molecular basis for F pilus assembly and also shed light on the remarkable properties of conjugative pili in bacterial secretion and phage infection.
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•The atomic structures of the F pilus and an F-like pilus are solved by cryo-EM•The structure reveals an assembly of a protein-phospholipid complex unit•Mutations at subunit-lipid interfaces affect phage infection and conjugation•The structure elucidates the molecular basis for DNA transfer and phage attachment
Conjugative pili, the appendages that enable DNA transfer between bacteria, are not made only of protein but, as near-atomic resolution cryoEM reveals, are assemblies of stoichiometric protein-phospholipid units, with the phospholipid component suited to electrochemically facilitating DNA transport.
The genome of Pseudomonas aeruginosa encodes three type VI secretion systems, each comprising a dozen distinct proteins, which deliver toxins upon T6SS sheath contraction. The least conserved T6SS ...component, TssA, has variations in size which influence domain organisation and structure. Here we show that the TssA Nt1 domain interacts directly with the sheath in a specific manner, while the C-terminus is essential for oligomerisation. We built chimeric TssA proteins by swapping C-termini and showed that these can be functional even when made of domains from different TssA sub-groups. Functional specificity requires the Nt1 domain, while the origin of the C-terminal domain is more permissive for T6SS function. We identify two regions in short TssA proteins, loop and hairpin, that contribute to sheath binding. We propose a docking mechanism of TssA proteins with the sheath, and a model for how sheath assembly is coordinated by TssA proteins from this position.
Conjugation is used by bacteria to propagate antimicrobial resistance (AMR) in the environment. Central to this process are widespread conjugative F-pili that establish the connection between donor ...and recipient cells, thereby facilitating the spread of IncF plasmids among enteropathogenic bacteria. Here, we show that the F-pilus is highly flexible but robust at the same time, properties that increase its resistance to thermochemical and mechanical stresses. By a combination of biophysical and molecular dynamics methods, we establish that the presence of phosphatidylglycerol molecules in the F-pilus contributes to the structural stability of the polymer. Moreover, this structural stability is important for successful delivery of DNA during conjugation and facilitates rapid formation of biofilms in harsh environmental conditions. Thus, our work highlights the importance of F-pilus structural adaptations for the efficient spread of AMR genes in a bacterial population and for the formation of biofilms that protect against the action of antibiotics.
We employ hydrodynamical simulations using the moving-mesh code arepo to investigate the role of energy and momentum input from active galactic nuclei (AGN) in driving large-scale galactic outflows. ...We start by reproducing analytic solutions for both energy- and momentum-driven outflowing shells in simulations of a spherical isolated dark matter potential with gas in hydrostatic equilibrium and with no radiative cooling. We confirm that for this simplified setup, galactic outflows driven by a momentum input rate of order L
Edd/c can establish an M
BH–σ relation with slope and normalization similar to that observed. We show that momentum input at a rate of L
Edd/c is however insufficient to drive efficient outflows once cooling and gas inflows as predicted by cosmological simulations at resolved scales are taken into account. We argue that observed large-scale AGN-driven outflows are instead likely to be energy-driven and show that such outflows can reach momentum fluxes exceeding 10L
Edd/c within the innermost 10 kpc of the galaxy. The outflows are highly anisotropic, with outflow rates and a velocity structure found to be inadequately described by spherical outflow models. We verify that the hot energy-driven outflowing gas is expected to be strongly affected by metal-line cooling, leading to significant amounts ( ≳ 109 M⊙) of entrained cold gas.
Abstract
Genome replication is a fundamental biological activity shared by all organisms. Chromosomal replication proceeds bidirectionally from origins, requiring the loading of two helicases, one ...for each replisome. However, the molecular mechanisms underpinning helicase loading at bacterial chromosome origins (oriC) are unclear. Here we investigated the essential DNA replication initiation protein DnaD in the model organism Bacillus subtilis. A set of DnaD residues required for ssDNA binding was identified, and photo-crosslinking revealed that this ssDNA binding region interacts preferentially with one strand of oriC. Biochemical and genetic data support the model that DnaD recognizes a new single-stranded DNA (ssDNA) motif located in oriC, the DnaD Recognition Element (DRE). Considered with single particle cryo-electron microscopy (cryo-EM) imaging of DnaD, we propose that the location of the DRE within oriC orchestrates strand-specific recruitment of helicase during DNA replication initiation. These findings significantly advance our mechanistic understanding of bidirectional replication from a bacterial chromosome origin.
Graphical Abstract
Graphical Abstract
In Bacillus subtilis, the basal unwinding sequence at the origin of replication oriC was known to comprise two DnaA boxes (DnaAboxes6/7, grey triangles) and a single-strand DNA binding region on the bottom strand (DnaA-trios, green triangles). The master initiator protein DnaA (green circles) binds both of these regions and can provide a scaffold to load a first helicase on bottom strand. Here we discovered a new element of the unwinding region located opposite the DnaA-trios and recognised by the replication protein DnaD, which we named the DnaD Recognition Element (DRE). We propose that binding of DnaD to the DRE promotes a second helicase loading event, providing a pathway to achieve bidirectional DNA replication initiation at a bacterial chromosome origin.
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