No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Cryo-EM structure of the Agrobacterium tumefaciens T4SS-associated T-pilus reveals stoichiometric protein-phospholipid assembly
Abstract
Agrobacterium tumefaciens causes crown gall disease in plants by the horizontal transfer of oncogenic DNA. The conjugation is mediated by the VirB/D4 type 4 secretion system (T4SS) that assembles an extracellular filament, the T-pilus, and is involved in mating pair formation between A. tumefaciens and the recipient plant cell. Here, we present a 3 Å cryoelectron microscopy (cryo-EM) structure of the T-pilus solved by helical reconstruction. Our structure reveals that the T-pilus is a stoichiometric assembly of the VirB2 major pilin and phosphatidylglycerol (PG) phospholipid with 5-start helical symmetry. We show that PG head groups and the positively charged Arg 91 residues of VirB2 protomers form extensive electrostatic interactions in the lumen of the T-pilus. Mutagenesis of Arg 91 abolished pilus formation. While our T-pilus structure is architecturally similar to previously published conjugative pili structures, the T-pilus lumen is narrower and positively charged, raising questions of whether the T-pilus is a conduit for ssDNA transfer.
... Bacterial conjugation is mediated by the type IV secretion system (T4SS)-a membrane-embedded nanomachine capable of producing a hollow extracellular appendage, known as the conjugative pilus, which projects from the outer membrane of the donor cell and is responsible for the interaction with the recipient cell (1). In Gram-negative bacteria, many high-resolution structures have been determined using cryo-electron microscopy (cryo-EM) for components of the secretion system, including the parts of the T4SS associated with both inner and outer membranes and the pilus, for model organisms such as Escherichia coli, Agrobacterium tumefaciens, Klebsiella pneumoniae, and Legionella pneumophila (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). The conjugative pilus has a lumen diameter of approximately 15 Å and is composed of many copies of the TraA subunit that folds into an all α-helical structure containing three α-helices. ...
... This was viewed as consistent with the role of the pilus in transferring DNA. However, the structure of the A. tumefaciens T-pilus revealed a positively charged lumen (2,3,15), seemingly in conflict with this notion of DNA transfer. An explanation was suggested that the properties of the T-pilus lumen may have evolved as a compromise between transferring negatively charged DNA and positively charged effector proteins (3,16). ...
... Extension allows the pilus to survey the surroundings and attach to a recipient cell and then retract, pulling the recipient cell into juxtaposition, followed by the formation of stable mating junctions defined by the contact of the mating pairs' cell envelopes (Movies S1 to S3; Fig. S5C) (1). All solved structures of conjugation pili, both bacterial and archaeal, show a tight association of lipids with TraA subunits, with a 1:1 stoichio metric ratio for bacterial pili (2)(3)(4)(5)15). It was suggested that one of the functions of this pilin:lipid association would be to facilitate reinsertion of the pilus subunits within the membrane during retraction events (5). ...
Bacterial conjugation, a process of horizontal gene transfer, plays a key role in promoting the spread of antimicrobial resistance among human pathogens. The mechanism of conjugation involves the development of a conjugative pilus that forms a physical bridge between two bacterial cells and the subsequent unidirectional transfer of single-stranded DNA (ssDNA) complexed with a protein from the donor to the recipient cell. Atomic structures exist for many of the components of the type IV secretion system (T4SS), responsible for the nucleoprotein secretion, but little is known about the events preceding gene transfer, specifically what is the extent of the participation of the conjugative pilus in ssDNA transfer? There has been a longstanding debate about whether its main role is to bring a donor and a recipient cell into physical juxtaposition and form a mating junction that allows for ssDNA transfer via the T4SS machinery complex or whether ssDNA is actually transferred through the lumen of the pilus. Here, through a combination of maleimide labeling of the conjugative pilus and SeqA-YFP labeling of the transferred ssDNA, we visualize the process of bacterial conjugation in real time. We discover that the conjugative pilus is capable of transferring the ssDNA at a distance, between physically separated cells, and thus conclude that a physical mating junction is not essential for conjugative gene transfer.
IMPORTANCE
Bacteria are constantly exchanging DNA, which constitutes horizontal gene transfer. While some of these occurs by a non-specific process called natural transformation, some occurs by a specific mating between a donor and a recipient cell. In specific conjugation, the mating pilus is extended from the donor cell to make contact with the recipient cell, but whether DNA is actually transferred through this pilus or by another mechanism involving the type IV secretion system complex without the pilus has been an open question. Using Escherichia coli , we show that DNA can be transferred through this pilus between a donor and a recipient cell that has not established a tight mating junction, providing a new picture for the role of this pilus.
... At this time, high-resolution structures have been presented for several T4SS subunits, including the VirD4 T4CP and the VirB4 and VirB11 ATPases that energize substrate recruitment and trafficking [5,8,9]. Additionally, structures have been solved of OMCCs from several model T4SSs [10][11][12][13][14][15] and a number of conjugative pili [16][17][18][19]. Most recently, a nearly intact T4SS encoded by conjugative plasmid R388 (T4SS R388 ) was solved at near-atomic resolution [5]. ...
Bacterial type IV secretion systems (T4SSs) are a versatile family of macromolecular translocators, collectively able to recruit diverse DNA and protein substrates and deliver them to a wide range of cell types. Presently, there is little understanding of how T4SSs recognize substrate repertoires and form productive contacts with specific target cells. Although T4SSs are composed of a number of conserved subunits and adopt certain conserved structural features, they also display considerable compositional and structural diversity. Here, we explored the structural bases underlying the functional versatility of T4SSs through systematic deletion and subunit swapping between two conjugation systems encoded by the distantly-related IncF plasmids, pED208 and F. We identified several regions of intrinsic flexibility among the encoded T4SSs, as evidenced by partial or complete functionality of chimeric machines. Swapping of VirD4-like TraD type IV coupling proteins (T4CPs) yielded functional chimeras, indicative of relaxed specificity at the substrate—TraD and TraD—T4SS interfaces. Through mutational analyses, we further delineated domains of the TraD T4CPs contributing to recruitment of cognate vs heterologous DNA substrates. Remarkably, swaps of components comprising the outer membrane core complexes, a few F-specific subunits, or the TraA pilins supported DNA transfer in the absence of detectable pilus production. Among sequenced enterobacterial species in the NCBI database, we identified many strains that harbor two or more F-like plasmids and many F plasmids lacking one or more T4SS components required for self-transfer. We confirmed that host cells carrying co-resident, non-selftransmissible variants of pED208 and F elaborate chimeric T4SSs, as evidenced by transmission of both plasmids. We propose that T4SS plasticity enables the facile assembly of functional chimeras, and this intrinsic flexibility at the structural level can account for functional diversification of this superfamily over evolutionary time and, on a more immediate time-scale, to proliferation of transfer-defective MGEs in nature.
... AlphaFold2 54 modeling of the mature CagC monomer lacking the leader peptide 54-56 revealed a predicted helical structure that closely resembles the experimentally resolved structures of conjugative pilins VirB2 57,58 and TraA 59 ( Fig. 2a and Supplementary Fig. 1b,c). In contrast to a previous report suggesting that conjugative pilins are cyclic in nature 60 , superimposition of predicted CagC structures with monomeric VirB2 and TraA revealed a slightly U-shaped protomer corresponding to structural conformations in which the free pilin termini were oriented towards the exterior of assembled canonical T-pilus or F-pilus filaments [57][58][59] (Fig. 2a). ...
Structural asymmetry within secretion system architecture is fundamentally important for apparatus diversification and biological function. However, the mechanism by which symmetry mismatch contributes to nanomachine assembly and interkingdom effector translocation are undefined. Here, we show that architectural asymmetry orchestrates dynamic substrate selection and enables trans-kingdom DNA conjugation through the Helicobacter pylori cag type IV secretion system ( cag T4SS). Structural analyses of asymmetric units within the cag T4SS periplasmic ring complex (PRC) revealed intermolecular π-π stacking interactions that coordinate DNA binding and license trans-kingdom conjugation without disrupting the translocation of protein and peptidoglycan effector molecules. Additionally, we identified a novel proximal translocation channel gating mechanism that regulates cargo loading and governs substrate transport across the outer membrane. We thus propose a model whereby the organization and geometry of architectural symmetry mismatch exposes π−π interfaces within the PRC to facilitate DNA transit through the cag T4SS translocation channel.
... Evidence suggests that conjugative pili can serve as translocation conduits through which ssDNA and unfolded relaxases can be delivered into recipient cells at long range (82). In support of this hypothesis, the E. coli F pilus (81) and A. tumefaciens T pilus (83,84) lumens are lined by inner membrane-derived phospholipids which impart a weak negative charge that may facilitate DNA passage (81). Conflicting observations support an indirect role of pili in substrate delivery to target cells. ...
The versatile type IV secretion system (T4SS) nanomachine plays a pivotal role in bacterial pathogenesis and the propagation of antibiotic resistance determinants throughout microbial populations. In addition to paradigmatic DNA conjugation machineries, diverse T4SSs enable the delivery of multifarious effector proteins to target prokaryotic and eukaryotic cells, mediate DNA export and uptake from the extracellular milieu, and in rare examples, facilitate transkingdom DNA translocation. Recent advances have identified new mechanisms underlying unilateral nucleic acid transport through the T4SS apparatus, highlighting both functional plasticity and evolutionary adaptations that enable novel capabilities. In this review, we describe the molecular mechanisms underscoring DNA translocation through diverse T4SS machineries, emphasizing the architectural features that implement DNA exchange across the bacterial membrane and license transverse DNA release across kingdom boundaries. We further detail how recent studies have addressed outstanding questions surrounding the mechanisms by which nanomachine architectures and substrate recruitment strategies contribute to T4SS functional diversity.
The increase of antimicrobial resistance constitutes a significant threat to human health. One of the mechanisms responsible for the spread of resistance to antimicrobials is the transfer of plasmids between bacteria by conjugation. This process is mediated by type IV secretion systems (T4SS) and previous studies have provided in vivo evidence for interactions between DNA and components of the T4SS. Here, we purified TraD and TraE, two inner membrane proteins from the Escherichia coli pKM101 T4SS. Using electrophoretic mobility shift assays and fluorescence polarization we showed that the purified proteins both bind single-stranded and double-stranded DNA in the nanomolar affinity range. The previously identified conjugation inhibitor BAR-072 inhibits TraE DNA binding in vitro, providing evidence for its mechanism of action. Site-directed mutagenesis identified conserved amino acids that are required for conjugation that may be targets for the development of more potent conjugation inhibitors.
Conjugation, the major driver of the spread of antimicrobial resistance genes, relies on a conjugation pilus for DNA transfer. Conjugative pili, such as the F–pilus, are dynamic tubular structures, composed of a polymerized pilin, that mediate the initial donor–recipient interactions, a process known as mating pair formation (MPF). IncH are low copy number plasmids, traditionally considered broad host range, which are found in bacteria infecting both humans and animals. The reference IncHI1 plasmid R27, isolated from Salmonella enterica serovar Typhi, encodes the conjugative H–pilus subunit TrhA containing 74 residues after cleavage of the signal sequence. Here, we show that the H–pilus forms long filamentous structures that mediate MPF, and describe its cryo electron–microscopic (cryo–EM) structure at 2.2 Å resolution. Like the F pilus, the H-pilin subunits form helical assemblies with phospholipid molecules at a stochiometric ratio of 1:1. While there were previous reports that the T–pilus from Agrobacterium tumefaciens was composed of cyclic subunits, three recent cryo-EM structures of the T–pilus found no such cyclization. Here, we report that the H–pilin is cyclic, with a covalent bond connecting the peptide backbone between the N– and C–termini. Both the cryo–EM map and mass spectrometry revealed cleavage of the last five residues of the pilin, followed by cyclization via condensation of the amine and carboxylate residues. The cyclic nature of the pilin could stabilize the pilus and may explain the high incidence of IncH plasmid dissemination.
Bacterial type IV secretion systems (T4SSs) are a versatile family of macromolecular translocators, collectively able to recruit diverse DNA and protein substrates and deliver them to a wide range of cell types. Presently, there is little understanding of how T4SSs recognize substrate repertoires and form productive contacts with specific target cells. Although T4SSs are composed of a number of conserved subunits and adopt certain conserved structural features, they also display considerable compositional and structural diversity. Here, we explored the structural bases underlying the functional versatility of T4SSs through systematic deletion and subunit swapping between two conjugation systems encoded by the distantly-related IncF plasmids, pED208 and F. We identified several regions of intrinsic flexibility among the encoded T4SSs, as evidenced by partial or complete functionality of chimeric machines. Swapping of VirD4-like TraD type IV coupling proteins (T4CPs) yielded functional chimeras, indicative of relaxed specificity at the substrate - TraD and TraD - T4SS interfaces. Through mutational analyses, we further delineated domains of the TraD T4CPs contributing to recruitment of cognate vs heterologous DNA substrates. Remarkably, swaps of components comprising the outer membrane core complexes, a few F-specific subunits, or the TraA pilins supported DNA transfer in the absence of detectable pilus production. Among sequenced enterobacterial species in the NCBI database, we identified many strains that harbor two or more F-like plasmids and many F plasmids lacking one or more T4SS components required for self-transfer. We confirmed that host cells carrying co-resident, non-selftransmissible variants of pED208 and F elaborate chimeric T4SSs, as evidenced by transmission of both plasmids. We propose that T4SS plasticity enables the facile assembly of functional chimeras, and this intrinsic flexibility at the structural level can account for functional diversification of this superfamily over evolutionary time and, on a more immediate time-scale, to proliferation of transfer-defective MGEs in nature.
AUTHOR SUMMARY
Mobile genetic elements (MGEs) comprise a diverse group of extrachromosomal plasmids or integrated DNA fragments that are widely distributed among many bacterial species. MGEs typically encode conjugation systems dedicated to their transmission to other bacteria, and also code for resistance to antibiotics or virulence or other fitness traits. The conjugation systems, along with an equally medically important group of translocators devoted to the interkingdom delivery of protein effectors by pathogenic species, comprise the superfamily of type IV secretion systems (T4SSs). Recent studies have defined many mechanistic and structural features of the T4SSs, yet there remains little understanding of how T4SSs recruit specific DNA or protein substrates, elaborate functional channels, and in some cases build attachment organelles termed conjugative pili. We explored the mechanics of T4SS machine function by systematically exchanging individual components between two distinct conjugation systems functioning in enterobacterial species. Through construction of chimeric machines, and further mutational analyses, we identified subunits or protein domains of conjugation machines specifying recruitment of distinct DNA substrates or selectively contributing to assembly of translocation channels or conjugative pili. Such features of T4SSs are prime targets for development of inhibitory strategies aimed at blocking T4SS functions for therapeutic intervention.
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.
Deep learning excels at cryo-tomographic image restoration and segmentation tasks but is hindered by a lack of training data. Here we introduce cryo-TomoSim (CTS), a MATLAB-based software package that builds coarse-grained models of macromolecular complexes embedded in vitreous ice and then simulates transmitted electron tilt series for tomographic reconstruction. We then demonstrate the effectiveness of these simulated datasets in training different deep learning models for use on real cryotomographic reconstructions. Computer-generated ground truth datasets provide the means for training models with voxel-level precision, allowing for unprecedented denoising and precise molecular segmentation of datasets. By modeling phenomena such as a three-dimensional contrast transfer function, probabilistic detection events, and radiation-induced damage, the simulated cryo-electron tomograms can cover a large range of imaging content and conditions to optimize training sets. When paired with small amounts of training data from real tomograms, networks become incredibly accurate at segmenting in situ macromolecular assemblies across a wide range of biological contexts.
Summary
By pairing rapidly synthesized Cryo-ET data with computed ground truths, deep learning models can be trained to accurately restore and segment real tomograms of biological structures both in vitro and in situ .
Conjugation is a major mechanism of horizontal gene transfer promoting the spread of antibiotic resistance among human pathogens. It involves establishing a junction between a donor and a recipient cell via an extracellular appendage known as the mating pilus. In bacteria, the conjugation machinery is encoded by plasmids or transposons and typically mediates the transfer of cognate mobile genetic elements. Much less is known about conjugation in archaea. Here, we determine atomic structures by cryo-electron microscopy of three conjugative pili, two from hyperthermophilic archaea (Aeropyrum pernix and Pyrobaculum calidifontis) and one encoded by the Ti plasmid of the bacterium Agrobacterium tumefaciens, and show that the archaeal pili are homologous to bacterial mating pili. However, the archaeal conjugation machinery, known as Ced, has been ‘domesticated’, that is, the genes for the conjugation machinery are encoded on the chromosome rather than on mobile genetic elements, and mediates the transfer of cellular DNA.
Bacterial conjugation is the fundamental process of unidirectional transfer of DNAs, often plasmid DNAs, from a donor cell to a recipient cell1. It is the primary means by which antibiotic resistance genes spread among bacterial populations2,3. In Gram-negative bacteria, conjugation is mediated by a large transport apparatus—the conjugative type IV secretion system (T4SS)—produced by the donor cell and embedded in both its outer and inner membranes. The T4SS also elaborates a long extracellular filament—the conjugative pilus—that is essential for DNA transfer4,5. Here we present a high-resolution cryo-electron microscopy (cryo-EM) structure of a 2.8 megadalton T4SS complex composed of 92 polypeptides representing 8 of the 10 essential T4SS components involved in pilus biogenesis. We added the two remaining components to the structural model using co-evolution analysis of protein interfaces, to enable the reconstitution of the entire system including the pilus. This structure describes the exceptionally large protein–protein interaction network required to assemble the many components that constitute a T4SS and provides insights on the unique mechanism by which they elaborate pili. Cryo-electron microscopy structures of a 2.8 megadalton bacterial type IV secretion system encoded by the plasmid R388 and comprising 92 polypeptides provide insights into the stepwise mechanism of pilus assembly.
Immune cells, such as macrophages, reprogram their lipid metabolism in response to the activation of pattern recognition receptors (e.g., TLRs, NLRs) and cytokine receptors (e.g., interferons, interleukins). Profiling these changes can be achieved with shotgun mass spectrometry. This protocol provides step-by-step instructions on the generation and stimulation of bone marrow-derived macrophages (BMDMs), sample collection, and lipid extraction for profiling the macrophage lipidome.
For complete details on the use and execution of this protocol, please refer to Hsieh et al. (2020).
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 six or twelve 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.
Bacterial conjugation, also referred to as bacterial sex, is a major horizontal gene transfer mechanism through which DNA is transferred from a donor to a recipient bacterium by direct contact. Conjugation is universally conserved among bacteria and occurs in a wide range of environments (soil, plant surfaces, water, sewage, biofilms, and host-associated bacterial communities). Within these habitats, conjugation drives the rapid evolution and adaptation of bacterial strains by mediating the propagation of various metabolic properties, including symbiotic lifestyle, virulence, biofilm formation, resistance to heavy metals, and, most importantly, resistance to antibiotics. These properties make conjugation a fundamentally important process, and it is thus the focus of extensive study. Here, we review the key steps of plasmid transfer by conjugation in Gram-negative bacteria, by following the life cycle of the F factor during its transfer from the donor to the recipient cell. We also discuss our current knowledge of the extent and impact of conjugation within an environmentally and clinically relevant bacterial habitat, bacterial biofilms.
Single-stranded RNA bacteriophages (ssRNA phages) infect Gram-negative bacteria via a single maturation protein (Mat), which attaches to a retractile pilus of the host. Here we present structures of the ssRNA phage MS2 in complex with the Escherichia coli F-pilus, showing a network of hydrophobic and electrostatic interactions at the Mat-pilus interface. Moreover, binding of the pilus induces slight orientational variations of the Mat relative to the rest of the phage capsid, priming the Mat-connected genomic RNA (gRNA) for its release from the virions. The exposed tip of the attached Mat points opposite to the direction of the pilus retraction, which may facilitate the translocation of the gRNA from the capsid into the host cytosol. In addition, our structures determine the orientation of the assembled F-pilin subunits relative to the cell envelope, providing insights into the F-like type IV secretion systems.
Agrobacterium tumefaciens transfers oncogenic T‐DNA via the type IV secretion system (T4SS) into plants causing tumor formation. The acvB gene encodes a virulence factor of unknown function required for plant transformation. Here we specify AcvB as a periplasmic lysyl‐phosphatidylglycerol (L‐PG) hydrolase, which modulates L‐PG homeostasis. Via functional characterization of recombinant AcvB variants, we showed that the C‐terminal domain of AcvB (residues 232–456) is sufficient for full enzymatic activity and defined key residues for catalysis. Absence of the hydrolase resulted in ~10‐fold increase of L‐PG in Agrobacterium membranes and abolished T‐DNA transfer and tumor formation. Overproduction of the L‐PG synthase gene (lpiA) in wild type A. tumefaciens resulted in a similar increase of the L‐PG content (~7‐fold) and a virulence defect even in the presence of intact AcvB. These results suggest that elevated L‐PG amounts (either by overproduction of the synthase or absence of the hydrolase) are responsible for the virulence phenotype. Gradually increasing the L‐PG content by complementation with different acvB variants revealed that cellular L‐PG levels above 3% of total phospholipids interfere with T‐DNA transfer. Cumulatively, this study identified AcvB as a novel virulence factor required for membrane‐lipid homeostasis and T‐DNA transfer.
This article is protected by copyright. All rights reserved.
This paper introduces ISOLDE, a new software package designed to provide an intuitive environment for high-fidelity interactive remodelling/refinement of macromolecular models into electron-density maps. ISOLDE combines interactive molecular-dynamics flexible fitting with modern molecular-graphics visualization and established structural biology libraries to provide an immersive interface wherein the model constantly acts to maintain physically realistic conformations as the user interacts with it by directly tugging atoms with a mouse or haptic interface or applying/removing restraints. In addition, common validation tasks are accelerated and visualized in real time. Using the recently described 3.8 Å resolution cryo-EM structure of the eukaryotic minichromosome maintenance (MCM) helicase complex as a case study, it is demonstrated how ISOLDE can be used alongside other modern refinement tools to avoid common pitfalls of low-resolution modelling and improve the quality of the final model. A detailed analysis of changes between the initial and final model provides a somewhat sobering insight into the dangers of relying on a small number of validation metrics to judge the quality of a low-resolution model.
Natural and Artificial Strategies to Control the Conjugative Transmission of Plasmids, Page 1 of 2
Abstract
Antibiotics have saved the lives of countless people suffering from bacterial infections since Alexander Fleming discovered penicillin in 1928 ( 1 ). Nevertheless, this success was accompanied by the emergence of antibiotic resistance (AbR). It is thought that AbR arose originally as a self-protection mechanism of producer organisms ( 2 ). AbR genes rapidly disseminated through the biosphere as a result of the selection pressure established by human application of antibiotics ( 3 ). Resistance mechanisms capable of rendering newly discovered drugs ineffective emerged with astonishing speed, rapidly reaching human pathogens and increasingly invalidating newer antimicrobial therapies ( 4 ). Altogether, >20,000 potential resistance genes of nearly 400 types have been predicted from bacterial genome sequences ( 5 ). The danger created by the ever-increasing number of pathogens resistant to conventional antibiotics is further increased by a significant drop in the development of new antimicrobial compounds ( 6 ). This situation demands solutions to prevent the hundreds of thousands of people dying each year as a result of AbR from becoming millions ( 7 ). Proposed strategies include more-accurate prescription policies and a controlled use and release of antibiotics in animal husbandry and agriculture, restrictions difficult to implement on a global scale ( 3 ).
The implementation of Agrobacterium tumefaciens as a transformation tool revolutionized approaches to discover and understand gene functions in a large number of fungal species. A. tumefaciens mediated transformation (AtMT) is one of the most transformative technologies for research on fungi developed in the last 20 years, a development arguably only surpassed by the impact of genomics. AtMT has been widely applied in forward genetics, whereby generation of strain libraries using random T-DNA insertional mutagenesis, combined with phenotypic screening, has enabled the genetic basis of many processes to be elucidated. Alternatively, AtMT has been fundamental for reverse genetics, where mutant isolates are generated with targeted gene deletions or disruptions, enabling gene functional roles to be determined. When combined with concomitant advances in genomics, both forward and reverse approaches using AtMT have enabled complex fungal phenotypes to be dissected at the molecular and genetic level. Additionally, in several cases AtMT has paved the way for the development of new species to act as models for specific areas of fungal biology, particularly in plant pathogenic ascomycetes and in a number of basidiomycete species. Despite its impact, the implementation of AtMT has been uneven in the fungi. This review provides insight into the dynamics of expansion of new research tools into a large research community and across multiple organisms. As such, AtMT in the fungi, beyond the demonstrated and continuing power for gene discovery and as a facile transformation tool, provides a model to understand how other technologies that are just being pioneered, e.g. CRISPR/Cas, may play roles in fungi and other eukaryotic species.
Recent studies have shown that conjugation systems of Gram-negative bacteria are composed of distinct inner and outer membrane core complexes (IMCs and OMCCs, respectively). Here, we functionally characterized the OMCC, focusing first on a cap domain that forms a channel across the outer membrane. Strikingly, the OMCC caps of the Escherichia coli pKM101 Tra and Agrobacterium tumefaciens VirB/VirD4 systems are completely dispensable for substrate transfer, but required for formation of conjugative pili. The pKM101 OMCC cap and extended pilus also are dispensable for activation of a Pseudomonas aeruginosa type VI secretion system (T6SS). Chimeric conjugation systems composed of the IMCpKM101 joined to OMCCs from the A. tumefaciens VirB/VirD4, E. coli R388 Trw, and Bordetella pertussis Ptl systems support conjugative DNA transfer in E. coli and trigger P. aeruginosa T6SS killing, but not pilus production. The A. tumefaciens VirB/VirD4 OMCC, solved by transmission electron microscopy, adopts a cage structure similar to the pKM101 OMCC. Our findings establish that OMCCs are highly structurally and functionally conserved - but also intrinsically conformationally flexible - scaffolds for translocation channels. Furthermore, the OMCC cap and a pilus tip protein coregulate pilus extension but are not required for channel assembly or function. This article is protected by copyright. All rights reserved.
We describe a new implementation for the reconstruction of helical assemblies in the empirical Bayesian framework of RELION. Our approach calculates optimal linear filters for the 3D reconstruction by embedding helical symmetry operators in Fourier-space, and deals with deviations from perfect helical symmetry through Gaussian-shaped priors on the orientations of individual segments. By incorporating our approach into the standard pipeline for single-particle analysis in RELION, our implementation aims to be easily accessible for non-experienced users. Although our implementation does not solve the problem that grossly incorrect structures can be obtained when the wrong helical symmetry is imposed, we show for four different test cases that it is capable of reconstructing structures to near-atomic resolution.
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.
The first reports on opines date back to the midfifties, when Morel (1956) and Lioret (1956) independently presented their results on, respectively, the metabolism of arginine and the identification of unusual amino acids in Agrobacterium-induced crown gall tumors, at a meeting of the French Society for Plant Physiology. These tumor compounds were later purified and identified (Figure 1) as lysopine (Biemann et al., 1960), octopine (Ménagé and Morel, 1964), octopinic acid (Ménagé and Morel, 1965), nopaline (Goldmann et al., 1969) and collectively termed opines (reviews: Tempé and Schell, 1977; Tempé and Goldmann, 1982). The significance of the metabolic perturbation undergone by the crown gall cells remained unclear for several years mostly because the specificity of opines as markers of these tissues was long debated (reviewed by Tempé and Goldmann, 1982). Three observations, however, were milestones in the understanding of the role of opines in the interaction: 1) Agrobacterium can degrade opines (Lejeune and Jubier, 1968); 2) the nature of opines synthesized in a tumor depends on the inciting Agrobacterium tumefaciens strains, not on the plant as it had been initially proposed (Goldmann et al., 1968; Petit et al., 1970); and 3) the correlation between opine degradation by agrobacteria and opine synthesis in plant cells is strict: that is, a given A. tumefaciens strain can only degrade the opines synthesized by the tumors it induced (Petit et al., 1970; reviewed by Tempé and Petit, 1983). Interestingly, these features were immediately interpreted as an indication of a possible gene transfer by Petit and Tourneur (1972) — a hypothesis that was first suggested by Braun (1947) and Klein (1954) (reviewed in Braun, 1982 and in Tempé and Petit, 1983) — whereas it was understood only years later that the production of opines by the crown gall cells provided the pathogen with a selective growth advantage (Schell et al., 1979; Tempé et al., 1979). This understanding resulted also from the discovery of the Ti plasmids (Zaenen et al., 1974; Van Larebeke et al., 1974; Watson et al., 1975), the elucidation of the tumorigenesis mechanism (Chilton et al., 1977; De Beuckeleer et al., 1978; Thomashow et al., 1980; Willmitzer et al., 1980, see also other chapters in this book), the localization of the genes involved in opine synthesis and degradation on the Ti plasmids (Bomhoff et al., 1976; Montoya et al., 1977) and the demonstration of the opine-induced, conjugal activity of these plasmids (Kerr et al., 1977; Petit et al., 1978a). All these data were combined and the fundamental role of opines in the Agrobacterium-plant interaction was rationalized by the originators of “the opine concept” (Tempé et al., 1979) and “the genetic colonization theory” (Schell et al., 1979) which define opines as follows. Opines are small-size molecules, the presence of which in the crown gall tumor is triggered by the pathogen to support its multiplication and to promote the dissemination of its virulence determinants (previous reviews on this topics: Dessaux et al., 1992, 1993; Gelvin, 1992).
We have recently completed a full rearchitecturing of the Rosetta molecular modeling program, generalizing and expanding its existing functionality. The new architecture enables the rapid prototyping of novel protocols by providing easy-to-use interfaces to powerful tools for molecular modeling. The source code of this rearchitecturing has been released as Rosetta3 and is freely available for academic use. At the time of its release, it contained 470,000 lines of code. Counting currently unpublished protocols at the time of this writing, the source includes 1,285,000 lines. Its rapid growth is a testament to its ease of use. This chapter describes the requirements for our new architecture, justifies the design decisions, sketches out central classes, and highlights a few of the common tasks that the new software can perform.
Agrobacterium tumefaciens is a phytopathogenic bacterium that causes crown gall disease by transferring transferred DNA (T-DNA) into the plant genome. The translocation process is mediated by the type IV secretion system (T4SS) consisting of the VirD4 coupling protein and 11 VirB proteins (VirB1 to VirB11). All VirB proteins are required for the production of T-pilus, which consists of processed VirB2 (T-pilin) and VirB5 as major and minor subunits, respectively. VirB2 is an essential component of T4SS, but the roles of VirB2 and the assembled T-pilus in Agrobacterium virulence and the T-DNA transfer process remain unknown. Here, we generated 34 VirB2 amino acid substitution variants to study the functions of VirB2 involved in VirB2 stability, extracellular VirB2/T-pilus production and virulence of A. tumefaciens. From the capacity for extracellular VirB2 production (ExB2+ or ExB2-) and tumorigenesis on tomato stems (Vir+ or Vir-), the mutants could be classified into three groups: ExB2-/Vir-, ExB2-/Vir+, and ExB2+/Vir+. We also confirmed by electron microscopy that five ExB2-/Vir+ mutants exhibited a wild-type level of virulence with their deficiency in T-pilus formation. Interestingly, although the five T-pilus-/Vir+ uncoupling mutants retained a wild-type level of tumorigenesis efficiency on tomato stems and/or potato tuber discs, their transient transformation efficiency in Arabidopsis seedlings was highly attenuated. In conclusion, we have provided evidence for a role of T-pilus in Agrobacterium transformation process and have identified the domains and amino acid residues critical for VirB2 stability, T-pilus biogenesis, tumorigenesis, and transient transformation efficiency.
Many cellular processes critically depend on the membrane composition. In this review, we focus on the biosynthesis and physiological roles of membrane lipids in the plant pathogen Agrobacterium tumefaciens. The major components of A. tumefaciens membranes are the phospholipids (PLs), phosphatidylethanolamine (PE), phosphatidylglycerol, phosphatidylcholine (PC) and cardiolipin, and ornithine lipids (OLs). Under phosphate-limited conditions, the membrane composition shifts to phosphate-free lipids like glycolipids, OLs and a betaine lipid. Remarkably, PC and OLs have opposing effects on virulence of A. tumefaciens. OL-lacking A. tumefaciens mutants form tumors on the host plant earlier than the wild type suggesting a reduced host defense response in the absence of OLs. In contrast, A. tumefaciens is compromised in tumor formation in the absence of PC. In general, PC is a rare component of bacterial membranes but amount to ~22% of all PLs in A. tumefaciens. PC biosynthesis occurs via two pathways. The phospholipid N-methyltransferase PmtA methylates PE via the intermediates monomethyl-PE and dimethyl-PE to PC. In the second pathway, the membrane-integral enzyme PC synthase (Pcs) condenses choline with CDP-diacylglycerol to PC. Apart from the virulence defect, PC-deficient A. tumefaciens pmtA and pcs double mutants show reduced motility, enhanced biofilm formation and increased sensitivity towards detergent and thermal stress. In summary, there is cumulative evidence that the membrane lipid composition of A. tumefaciens is critical for agrobacterial physiology and tumor formation.
RELION, for REgularized LIkelihood OptimizatioN, is an open-source computer program for the refinement of macromolecular structures by single-particle analysis of electron cryo-microscopy (cryo-EM) data. Whereas alternative approaches often rely on user expertise for the tuning of parameters, RELION uses a Bayesian approach to infer parameters of a statistical model from the data. This paper describes developments that reduce the computational costs of the underlying maximum a posteriori (MAP) algorithm, as well as statistical considerations that yield new insights into the accuracy with which the relative orientations of individual particles may be determined. A so-called gold-standard Fourier shell correlation (FSC) procedure to prevent overfitting is also described. The resulting implementation yields high-quality reconstructions and reliable resolution estimates with minimal user intervention and at acceptable computational costs.
TrbC propilin is the precursor of the pilin subunit TrbC of IncP conjugative pili in Escherichia coli.Likewise, its homologue, VirB2 propilin, is processed into T pilin of the Ti plasmid T pilus in Agrobacterium tumefaciens. TrbC and VirB2 propilin are truncated post-translationally at the N terminus by the removal of a 36/47-residue leader peptide,
respectively. TrbC propilin undergoes a second processing step by the removal of 27 residues at the C terminus by host-encoded
functions followed by the excision of four additional C-terminal residues by a plasmid-borne serine protease. The final product
TrbC of 78 residues is cyclized via an intramolecular covalent head-to-tail peptide bond. The T pilin does not undergo additional
truncation but is likewise cyclized. The circular structures of these pilins, as verified by mass spectrometry, represent
novel primary configurations that conform and assemble into the conjugative apparatus.
X-ray crystallography is a critical tool in the study of biological systems. It is able to provide information that has been a prerequisite to understanding the fundamentals of life. It is also a method that is central to the development of new therapeutics for human disease. Significant time and effort are required to determine and optimize many macromolecular structures because of the need for manual interpretation of complex numerical data, often using many different software packages, and the repeated use of interactive three-dimensional graphics. The Phenix software package has been developed to provide a comprehensive system for macromolecular crystallographic structure solution with an emphasis on automation. This has required the development of new algorithms that minimize or eliminate subjective input in favor of built-in expert-systems knowledge, the automation of procedures that are traditionally performed by hand, and the development of a computational framework that allows a tight integration between the algorithms. The application of automated methods is particularly appropriate in the field of structural proteomics, where high throughput is desired. Features in Phenix for the automation of experimental phasing with subsequent model building, molecular replacement, structure refinement and validation are described and examples given of running Phenix from both the command line and graphical user interface.
We have recently completed a full re-architecturing of the ROSETTA molecular modeling program, generalizing and expanding its existing functionality. The new architecture enables the rapid prototyping of novel protocols by providing easy-to-use interfaces to powerful tools for molecular modeling. The source code of this rearchitecturing has been released as ROSETTA3 and is freely available for academic use. At the time of its release, it contained 470,000 lines of code. Counting currently unpublished protocols at the time of this writing, the source includes 1,285,000 lines. Its rapid growth is a testament to its ease of use. This chapter describes the requirements for our new architecture, justifies the design decisions, sketches out central classes, and highlights a few of the common tasks that the new software can perform.
Agrobacterium VirB2 pilin is required for assembly of the VirB/VirD4 type IV secretion system (T4SS). The propilin is processed by signal sequence cleavage and covalent linkage of the N and C termini, and the cyclized pilin integrates into the inner membrane (IM) as a pool for assembly of the secretion channel and T pilus. Here, by use of the substituted cysteine accessibility method (SCAM), we defined the VirB2 IM topology and then identified distinct contributions of the T4SS ATPase subunits to the pilin structural organization. Labeling patterns of Cys-substituted pilins exposed to the membrane-impermeative, thiol-reactive reagent 3-(N-maleimidopropionyl)biocytin (MPB) supported a topology model in which two hydrophobic stretches comprise transmembrane domains, an intervening hydrophilic loop (residues 90 to 94) is cytoplasmic, and the hydrophilic N and C termini joined at residues 48 and 121 form a periplasmic loop. Interestingly, the VirB4 ATPase, but not a Walker A nucleoside triphosphate (NTP) binding motif mutant, induced (i) MPB labeling of Cys94, a residue that in the absence of the ATPase is located in the cytoplasmic loop, and (ii) release of pilin from the IM upon osmotic shock. These findings, coupled with evidence for VirB2-VirB4 complex formation by coimmunoprecipitation, support a model in which VirB4 functions as a dislocation motor to extract pilins from the IM during T4SS biogenesis. The VirB11 ATPase functioned together with VirB4 to induce a structural change in the pilin that was detectable by MPB labeling, suggestive of a role for VirB11 as a modulator of VirB4 dislocase activity.
Phosphatidylcholine (PC) is the most abundant phospholipid in eukaryotic membranes, whereas only a limited number of bacteria are able to synthesize PC. Intriguingly, many of the bacteria with PC-containing membranes interact with eukaryotic hosts. PC is one of the major membrane lipids in the phytopathogenic bacterium Agrobacterium tumefaciens. The presence of PC is critical for diverse cellular processes like motility, biofilm formation, stress resistance, and virulence. The exact role of PC in these processes is unknown. Here, we examined the global consequences of the complete loss of PC at the proteomic and transcriptomic levels. Both strategies validated the impaired virulence gene induction responsible for the virulence defect of the PC-deficient mutant. In addition, the proteomic approach revealed a limited subset of proteins with altered abundance including the reduced flagellar proteins FlaA and FlaB, which explains the motility defect of the PC mutant. At the whole-genome level, the loss of PC was correlated with altered expression of up to 13% of all genes, most encoding membrane or membrane-associated proteins and proteins with functions in the extracytoplasmic stress response. Our integrated analysis revealed that A. tumefaciens dynamically remodels its membrane protein composition in order to sustain normal growth in the absence of PC.
Coot is a molecular-graphics application for model building and validation of biological macromolecules. The program displays electron-density maps and atomic models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are ‘discoverable’ through familiar user-interface elements (menus and toolbars) or by intuitive behaviour (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallographic community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.
Agrobacterium tumefaciens VirB proteins assemble a type IV secretion apparatus and a T-pilus for secretion of DNA and proteins into plant cells. The pilin-like protein VirB3, a membrane protein of unknown topology, is required for the assembly of the T-pilus and for T-DNA secretion. Using PhoA and green fluorescent protein (GFP) as periplasmic and cytoplasmic reporters, respectively, we demonstrate that VirB3 contains two membrane-spanning domains and that both the N and C termini of the protein reside in the cytoplasm. Fusion proteins with GFP at the N or C terminus of VirB3 were fluorescent and, like VirB3, localized to a cell pole. Biochemical fractionation studies demonstrated that VirB3 proteins encoded by three Ti plasmids, the octopine Ti plasmid pTiA6NC, the supervirulent plasmid pTiBo542, and the nopaline Ti plasmid pTiC58, are inner membrane proteins and that VirB4 has no effect on membrane localization of pTiA6NC-encoded VirB3 (pTiA6NC VirB3). The pTiA6NC and pTiBo542 VirB2 pilins, like VirB3, localized to the inner membrane. The pTiC58 VirB4 protein was earlier found to be essential for stabilization of VirB3. Stabilization of pTiA6NC VirB3 requires not only VirB4 but also two additional VirB proteins, VirB7 and VirB8. A binary interaction between VirB3 and VirB4/VirB7/VirB8 is not sufficient for VirB3 stabilization. We hypothesize that bacteria use selective proteolysis as a mechanism to prevent assembly of unproductive precursor complexes under conditions that do not favor assembly of large macromolecular structures.
Comparative whole-genome analyses have demonstrated that horizontal gene transfer (HGT) provides a significant contribution to prokaryotic genome innovation. The evolution of specific prokaryotes is therefore tightly linked to the environment in which they live and the communal pool of genes available within that environment. Here we use the term supergenome to describe the set of all genes that a prokaryotic 'individual' can draw on within a particular environmental setting. Conjugative plasmids can be considered particularly successful entities within the communal pool, which have enabled HGT over large taxonomic distances. These plasmids are collections of discrete regions of genes that function as 'backbone modules' to undertake different aspects of overall plasmid maintenance and propagation. Conjugative plasmids often carry suites of 'accessory elements' that contribute adaptive traits to the hosts and, potentially, other resident prokaryotes within specific environmental niches. Insight into the evolution of plasmid modules therefore contributes to our knowledge of gene dissemination and evolution within prokaryotic communities. This communal pool provides the prokaryotes with an important mechanistic framework for obtaining adaptability and functional diversity that alleviates the need for large genomes of specialized 'private genes'.
EDP208 conjugative pili contain a single polypeptide subunit of 11,500 daltons with a blocked N-terminus. This N-terminal blocking moiety was identified as an N-acetyl group by 1H nuclear magnetic resonance analysis of an N-terminal tripeptide isolated from pronase digests of EDP208 pilin. Limited acid hydrolysis of the tripeptide allowed its sequence to be determined as acetyl-NH-Thr-Asp-Leu. Trypsin digestion of EDP208 pilin resulted in the quantitative release of a fragment containing 12 residues from the N-terminus of the protein. The sequence of this dodecapeptide was determined to be acetyl-NH-Thr-Asp-Leu-Leu-Ala-Gly-Gly-Lys-Asp-Val-Asp-Lys.
Agrobacterium tumefaciens is a natural genetic engineer that transfers DNA into plants, which is the most applied process for generation of genetically modified plants. DNA transfer is mediated by a type IV secretion system in the cell envelope and extracellular T-pili. We here report the cryo-electron microscopic structures of the T-pilus at 3.2-Å resolution and of the plasmid pKM101-determined N-pilus at 3-Å resolution. Both pili contain a main pilus protein (VirB2 in A. tumefaciens, TraM in pKM101) and phospholipids arranged in a five-start helical assembly. They contain positively charged amino acids in the lumen, and the lipids are positively charged in the T-pilus (phosphatidylcholine) conferring overall positive charge. Mutagenesis of the lumen-exposed Arg91 in VirB2 results in protein destabilization and loss of pilus formation. Our results reveal that different phospholipids can be incorporated into type IV secretion pili and that the charge of the lumen may be of functional importance.
Differential mobility spectrometry (DMS) is highly useful for shotgun lipidomic analysis because it overcomes difficulties in measuring isobaric species within a complex lipid sample and allows for acyl tail characterization of phospholipid species. Despite these advantages, the resulting workflow presents technical challenges, including the need to tune the DMS before every batch to update compensative voltages settings within the method. The Sciex Lipidyzer platform uses a Sciex 5500 QTRAP with a DMS (SelexION), an LC system configured for direction infusion experiments, an extensive set of standards designed for quantitative lipidomics, and a software package (Lipidyzer Workflow Manager) that facilitates the workflow and rapidly analyzes the data. Although the Lipidyzer platform remains very useful for DMS-based shotgun lipidomics, the software is no longer updated for current versions of Analyst and Windows. Furthermore, the software is fixed to a single workflow and cannot take advantage of new lipidomics standards or analyze additional lipid species. To address this multitude of issues, we developed Shotgun Lipidomics Assistant (SLA), a Python-based application that facilitates DMS-based lipidomics workflows. SLA provides the user with flexibility in adding and subtracting lipid and standard MRMs. It can report quantitative lipidomics results from raw data in minutes, comparable to the Lipidyzer software. We show that SLA facilitates an expanded lipidomics analysis that measures over 1450 lipid species across 17 (sub)classes. Lastly, we demonstrate that the SLA performs isotope correction, a feature that was absent from the original software.
Compositional data are nonnegative data carrying relative, rather than absolute, information—these are often data with a constant-sum constraint on the sample values, for example, proportions or percentages summing to 1% or 100%, respectively. Ratios between components of a composition are important since they are unaffected by the particular set of components chosen. Logarithms of ratios (logratios) are the fundamental transformation in the ratio approach to compositional data analysis—all data thus need to be strictly positive, so that zero values present a major problem. Components that group together based on domain knowledge can be amalgamated (i.e., summed) to create new components, and this can alleviate the problem of data zeros. Once compositional data are transformed to logratios, regular univariate and multivariate statistical analysis can be performed, such as dimension reduction and clustering, as well as modeling. Alternative methodologies that come close to the ideals of the logratio approach are also considered, especially those that avoid the problem of data zeros, which is particularly acute in large bioinformatic data sets.
Conjugative pili are important in mediating bacterial conjugation and horizontal gene transfer. Since plasmid transfer can include antibiotic-resistance genes, conjugation is an important mechanism in the spread of antibiotic resistance. Filamentous bacteriophages have been shown to exist in two different structural classes: those with a 5-fold rotational symmetry and those with a one-start helix with approximately 5 subunits per turn. Structures for the F and the F-like pED208 conjugation pilus have shown that they have 5-fold rotational symmetry. Here, we report the cryoelectron-microscopic structure of conjugative pili from carbapenem-resistant Klebsiella pneumoniae, encoded on the IncFIIK pKpQIL plasmid, at 3.9 Å resolution and show that it has a one-start helix. These results establish that conjugation pili can exist in at least two structural classes, consistent with other results showing that relatively small perturbations are needed to change the helical symmetry of polymers.
NAMD is a molecular dynamics program designed for high-performance simulations of very large biological objects on CPU- and GPU-based architectures. NAMD offers scalable performance on petascale parallel supercomputers consisting of hundreds of thousands of cores, as well as on inexpensive commodity clusters commonly found in academic environments. It is written in C++ and leans on Charm++ parallel objects for optimal performance on low-latency architectures. NAMD is a versatile, multipurpose code that gathers state-of-the-art algorithms to carry out simulations in apt thermodynamic ensembles, using the widely popular CHARMM, AMBER, OPLS, and GROMOS biomolecular force fields. Here, we review the main features of NAMD that allow both equilibrium and enhanced-sampling molecular dynamics simulations with numerical efficiency. We describe the underlying concepts utilized by NAMD and their implementation, most notably for handling long-range electrostatics; controlling the temperature, pressure, and pH; applying external potentials on tailored grids; leveraging massively parallel resources in multiple-copy simulations; and hybrid quantum-mechanical/molecular-mechanical descriptions. We detail the variety of options offered by NAMD for enhanced-sampling simulations aimed at determining free-energy differences of either alchemical or geometrical transformations and outline their applicability to specific problems. Last, we discuss the roadmap for the development of NAMD and our current efforts toward achieving optimal performance on GPU-based architectures, for pushing back the limitations that have prevented biologically realistic billion-atom objects to be fruitfully simulated, and for making large-scale simulations less expensive and easier to set up, run, and analyze. NAMD is distributed free of charge with its source code at www.ks.uiuc.edu.
The Agrobacterium tumefaciens VirB/VirD4 translocation machine is a member of a superfamily of translocators designated as type IV secretion systems (T4SSs) that function in many species of gram-negative and gram-positive bacteria. T4SSs evolved from ancestral conjugation systems for specialized purposes relating to bacterial colonization or infection. A. tumefaciens employs the VirB/VirD4 T4SS to deliver oncogenic DNA (T-DNA) and effector proteins to plant cells, causing the tumorous disease called crown gall. This T4SS elaborates both a cell-envelope-spanning channel and an extracellular pilus for establishing target cell contacts. Recent mechanistic and structural studies of the VirB/VirD4 T4SS and related conjugation systems in Escherichia coli have defined T4SS architectures, bases for substrate recruitment, the translocation route for DNA substrates, and steps in the pilus biogenesis pathway. In this review, we provide a brief history of A. tumefaciens VirB/VirD4 T4SS from its discovery in the 1980s to its current status as a paradigm for the T4SS superfamily. We discuss key advancements in defining VirB/VirD4 T4SS function and structure, and we highlight the power of in vivo mutational analyses and chimeric systems for identifying mechanistic themes and specialized adaptations of this fascinating nanomachine.
Plant genetic transformation heavily relies on the bacterial pathogen Agrobacterium tumefaciens as a powerful tool to deliver genes of interest into a host plant. Inside the plant nucleus, the transferred DNA is capable of integrating into the plant genome for inheritance to the next generation (i.e. stable transformation). Alternatively, the foreign DNA can transiently remain in the nucleus without integrating into the genome but still be transcribed to produce desirable gene products (i.e. transient transformation). From the discovery of A. tumefaciens to its wide application in plant biotechnology, numerous aspects of the interaction between A. tumefaciens and plants have been elucidated. This article aims to provide a comprehensive review of the biology and the applications of Agrobacterium-mediated plant transformation, which may be useful for both microbiologists and plant biologists who desire a better understanding of plant transformation, protein expression in plants, and plant-microbe interaction.
UCSF ChimeraX is next-generation software for the visualization and analysis of molecular structures, density maps, 3D microscopy, and associated data. It addresses challenges in the size, scope, and disparate types of data attendant with cutting-edge experimental methods, while providing advanced options for high-quality rendering (interactive ambient occlusion, reliable molecular surface calculations, etc.) and professional approaches to software design and distribution. This paper highlights some specific advances in the areas of visualization and usability, performance, and extensibility. ChimeraX is free for noncommercial use and is available from http://www.rbvi.ucsf.edu/chimerax/ for Windows, Mac, and Linux. This article is protected by copyright. All rights reserved.
The Mosaic Type IV Secretion Systems, Page 1 of 2
Abstract
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.
Agrobacterium tumefaciens T pili are long semi-rigid, flexuous filaments of 10 nm diameter that are primarily composed of T pilin cyclized protein subunits. The cyclic character of T pilin apparently confers a high level of structural stability on the T pilus. Purified T pili subjected to extreme environmental conditions such as acid and alkali, including glycerol remained relatively unaffected morphologically. T pili lost their semi-rigidity when subjected to high temperatures and high pH, and dissociated into donut shaped subunits when exposed to Triton X-100. Sodium dodecyl sulfate increased the uptake of uranyl acetate exposing a 2 nm wide lumen running the length of the T pilus filament.
CTFFIND is a widely-used program for the estimation of objective lens defocus parameters from transmission electron micrographs. Defocus parameters are estimated by fitting a model of the microscope's contrast transfer function (CTF) to an image's amplitude spectrum. Here we describe modifications to the algorithm which make it significantly faster and more suitable for use with images collected using modern technologies such as dose fractionation and phase plates. We show that this new version preserves the accuracy of the original algorithm while allowing for higher throughput. We also describe a measure of the quality of the fit as a function of spatial frequency and suggest this can be used to define the highest resolution at which CTF oscillations were successfully modeled.
Copyright © 2015. Published by Elsevier Inc.
zCompositions is an R package for the imputation of left-censored data under a compositional approach. It is pertinent when the analyst assumes that the relevant information is contained on the relative variation structure of the data. For instance, in cases where the experimental data are simultaneously measured amounts related to a same total weight or volume. The approach is used in fields like geochemistry of waters or sedimentary rocks, environmental studies related to air pollution, physicochemical analysis of glass fragments in forensic science, among many others. In these fields, rounded zeros and nondetects are usually regarded as left-censored data that hamper any subsequent data analysis. The implemented methods consider aspects of relevance for a compositional approach such as scale invariance, subcompositional coherence or preserving the multivariate relative structure of the data. Based on solid statistical frameworks, it comprises the ability to deal with single and varying censoring thresholds, consistent treatment of closed and non-closed data, exploratory tools, multiple imputation, MCMC, robust and non-parametric alternatives, and recent proposals for count data. Key methodological aspects, new contributions, computational implementation and the practical application of the approach are discussed.
T-pilus biogenesis uses a conserved transmembrane nucleoprotein- and protein-transport apparatus for the transport of cyclic T-pilin subunits to the Agrobacterium cell surface. T-pilin subunits are processed from full-length VirB2 pro-pilin into a cyclized peptide, a rapid reaction that is Agrobacterium specific and can occur in the absence of Ti-plasmid genes.
Previous studies have implicated the obligatory requirement for the vir regulon (or "virulon") of the Ti plasmid for the transfer of oncogenes from Agrobacterium tumefaciens to plant cells. The machinery used in this horizontal gene transfer has been long thought to be a transformation or conjugative delivery system. Based on recent protein sequence comparisons, the proteins encoded by the virB operon are strikingly similar to proteins involved in the synthesis and assembly of conjugative pili such as the conjugative pilus of F plasmid in Escherichia coli. The F pilus is composed of TraA pilin subunits derived from TraA propilin. In the present study, evidence is provided showing that the counterpart of TraA is VirB2, which like TraA propilin is processed into a 7.2-kDa product that comprises the pilus subunit as demonstrated by biochemical and electron microscopic analyses. The processed VirB2 protein is present exocellularly on medium on which induced A. tumefaciens had grown and appears as thin filaments of 10 nm that react specifically to VirB2 antibody. Exocellular VirB2 is produced abundantly at 19 degreesC as compared with 28 degreesC, an observation that parallels the effect of low temperature on the production of vir gene-specific pili observed previously (K. J. Fullner, L. C. Lara, and E. W. Nester, Science 273:1107-1109, 1996). Export of the processed VirB2 requires other virB genes since mutations in these genes cause the loss of VirB2 pilus formation and result in processed VirB2 accumulation in the cell. The presence of exocellular processed VirB2 is directly correlated with the formation of pili, and it appears as the major protein in the purified pilus preparation. The evidence provides a compelling argument for VirB2 as the propilin whose 7.2-kDa processed product is the pilin subunit of the promiscuous conjugative pilus, hereafter called the "T pilus" of A. tumefaciens.
SWISS-MODEL pioneered the field of automated modeling as the first protein modeling service on the Internet. In combination with the visualization tool Swiss-PdbViewer, the Internet-based Workspace and the SWISS-MODEL Repository, it provides a fully integrated sequence to structure analysis and modeling platform. This computational environment is made freely available to the scientific community with the aim to hide the computational complexity of structural bioinformatics and encourage bench scientists to make use of the ever-increasing structural information available. Indeed, over the last decade, the availability of structural information has significantly increased for many organisms as a direct consequence of the complementary nature of comparative protein modeling and experimental structure determination. This has a very positive and enabling impact on many different applications in biomedical research as described in this paper.
Agrobacterium tumefaciens VirB10 couples inner membrane (IM) ATP energy consumption to substrate transfer through the VirB/D4 type IV secretion (T4S) channel and also mediates biogenesis of the virB-encoded T pilus. Here, we determined the functional importance of VirB10 domains denoted as the: (i) N-terminal cytoplasmic region, (ii) transmembrane (TM) alpha-helix, (iii) proline-rich region (PRR) and (iv) C-terminal beta-barrel domain. Mutations conferring a transfer- and pilus-minus (Tra(-), Pil(-)) phenotype included PRR deletion and beta-barrel substitution mutations that prevented VirB10 interaction with the outer membrane (OM) VirB7-VirB9 channel complex. Mutations permissive for substrate transfer but blocking pilus production (Tra(+), Pil(-)) included a cytoplasmic domain deletion and TM domain insertion mutations. Another class of Tra(+) mutations also selectively disrupted pilus biogenesis but caused release of pilin monomers to the milieu; these mutations included deletions of alpha-helical projections extending from the beta-barrel domain. Our findings, together with results of Cys accessibility studies, indicate that VirB10 stably integrates into the IM, extends via its PRR across the periplasm, and interacts via its beta-barrel domain with the VirB7-VirB9 channel complex. The data further support a model that distinct domains of VirB10 regulate formation of the secretion channel or the T pilus.
Transcription of the virG gene of Agrobacterium tumefaciens was previously shown to be expressed from two tandem promoters and to be responsive to three stimuli: plant-released phenolic compounds, phosphate starvation, and acidic media. In this report, I describe a set of deletions and other alterations of the 5' end of virG that show that the upstream promoter (P1) is necessary for induction by phenolic compounds and by phosphate starvation, whereas the downstream promoter (P2) is induced by acidic media. Upstream of promoter P1 there are three copies of a family of sequences (vir boxes) found near all VirA, VirG-inducible promoters. Site-directed mutagenesis of these sequences showed that vir box I and vir box III but not vir box II are needed for induction of P1 by acetosyringone. Induction of P1 by phosphate starvation requires vir box III (or an overlapping site), whereas vir box I and vir box II are not needed. The relative importance of promoters P1 and P2 in vir gene induction was tested by measuring the expression of a virB::lacZ fusion in strains containing mutations at either promoter P1 or P2. Mutations in either promoter significantly attenuated the expression of virB, indicating that both promoters play important roles in vir gene induction.
The virulence regulon of the Agrobacterium tumefaciens TiC58 plasmid is composed of six operons, virA, virB, virG, virC, virD and virE, which direct the transfer of T-DNA into plant cells. The 9.5 kbp virB operon is the largest of these operons and its entire nucleotide sequence was determined and found to contain eleven open reading frames (ORFs). Gene fusions of each VirB ORF to T7 phi 10 were made and overexpressed in Escherichia coli to confirm that they encode proteins of predicted size. Hydrophobic analysis of these peptide sequences revealed nine proteins that contain hydrophobic spanning regions including signal-peptide-like sequences. These data suggest that the majority of VirB proteins may associate with bacterial cell membranes, while the two additional proteins possess a potential ATP-binding site. Strong homologies in amino acid sequences were observed between nopaline- and octopine-type plasmids. Specific differences in amino acid sequence encoded by VirB ORFs of nopaline and octopine Ti plasmid and a functional role of the gene products are discussed.
Agrobacterium tumefaciens genetically transforms plant cells by transferring a copy of its T-DNA to the plant where it is integrated and stably maintained. In the presence of wounded plant cells this process is activated and mediated by the products of the vir genes which are grouped into six distinct loci. The largest is the virB locus spanning 9.5 kb. Transposon mutagenesis studies have shown that virB gene products are required for virulence but their functions remain largely unknown. To provide information relevant to understanding the function of VirB polypeptides, the nucleotide sequence of the virB operon from a nopaline plasmid, pTiC58, is presented here. Eleven open reading frames (ORFs) are predicted from this sequence. The predicted sizes of 10 of the 11 VirB polypeptides are verified by specific expression in Escherichia coli. Only the product of the smallest ORF potentially encoding a 5.8 kDa polypeptide has not been detected. The initiation of translation of five virB ORFs occurs at codons that overlap the termination codons of the ORF immediately upstream; thus, translational coupling may be an important mechanism for efficient translation of the large virB polycistronic mRNA. Based on hydropathy plot analysis nine of the virB ORFs encode proteins that may interact with membranes; these data support the earlier hypothesis that virB gene products may form a membrane pore or channel to mediate exit of the T-DNA copy (T-strands) from Agrobacterium into the plant cell. A comparison of the two published octopine virB sequences with the nopaline sequence presented here is made.
The complete sequence of a 1.4-kilobase PstI fragment containing the F transfer genes traA, -L, and -E is presented. The traA reading frame has been located both genetically and by comparing the primary structure of F pilin (the traA product) predicted by the DNA sequence to the amino acid composition and sequence of N- and C-terminal peptides isolated from purified F pilin. Taken together, these data show that there is a leader peptide of 51 amino acids and that F pilin contains 70 amino acids, giving molecular weights of 13,200 for F propilin and 7,200 for mature F pilin. Secondary structure predictions for F pilin revealed a reverse turn that precedes the sequence Ala-Met-Ala51, a classic signal peptidase cleavage site. The N-terminal alanine residue is blocked by an acetyl group as determined by 1H-nuclear magnetic resonance spectroscopy. The traL and traE genes encode proteins of molecular weights 10,350 and 21,200, respectively. According to DNA sequence predictions, these proteins do not contain signal peptide leader sequences. Secondary structure predictions for these proteins are in accord with traLp and traEp being membrane proteins in which hydrophobic regions capable of spanning the membrane are linked by sequences that form turns and carry positively charged residues capable of interacting with the membrane surface.