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1.
Protein J ; 38(4): 389-408, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31407127

RESUMO

The translocation of proteins across membranes is a fundamental cellular function. Bacteria have evolved a striking array of pathways for delivering proteins into or across cytoplasmic membranes and, when present, outer membranes. Translocated proteins can form part of the membrane landscape, reside in the periplasmic space situated between the inner and outer membranes of Gram-negative bacteria, deposit on the cell surface, or be released to the extracellular milieu or injected directly into target cells. One protein translocation system, the general secretory pathway, is conserved in all domains of life. A second, the twin-arginine translocation pathway, is also phylogenetically distributed among most bacteria and plant chloroplasts. While all cell types have evolved additional systems dedicated to the translocation of protein cargoes, the number of such systems in bacteria is now known to exceed nine. These dedicated protein translocation systems, which include the types 1 through 9 secretion systems (T1SSs-T9SSs), the chaperone-usher pathway, and type IV pilus system, are the subject of this review. Most of these systems were originally identified and have been extensively characterized in Gram-negative or diderm (two-membrane) species. It is now known that several of these systems also have been adapted to function in Gram-positive or monoderm (single-membrane) species, and at least one pathway is found only in monoderms. This review briefly summarizes the distinctive mechanistic and structural features of each dedicated pathway, as well as the shared properties, that together account for the broad biological diversity of protein translocation in bacteria.

2.
Proc Natl Acad Sci U S A ; 116(28): 14222-14227, 2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31239340

RESUMO

Bacterial conjugation systems are members of the large type IV secretion system (T4SS) superfamily. Conjugative transfer of F plasmids residing in the Enterobacteriaceae was first reported in the 1940s, yet the architecture of F plasmid-encoded transfer channel and its physical relationship with the F pilus remain unknown. We visualized F-encoded structures in the native bacterial cell envelope by in situ cryoelectron tomography (CryoET). Remarkably, F plasmids encode four distinct structures, not just the translocation channel or channel-pilus complex predicted by prevailing models. The F1 structure is composed of distinct outer and inner membrane complexes and a connecting cylinder that together house the envelope-spanning translocation channel. The F2 structure is essentially the F1 complex with the F pilus attached at the outer membrane (OM). Remarkably, the F3 structure consists of the F pilus attached to a thin, cell envelope-spanning stalk, whereas the F4 structure consists of the pilus docked to the OM without an associated periplasmic density. The traffic ATPase TraC is configured as a hexamer of dimers at the cytoplasmic faces of the F1 and F2 structures, where it respectively regulates substrate transfer and F pilus biogenesis. Together, our findings present architectural renderings of the DNA conjugation or "mating" channel, the channel-pilus connection, and unprecedented pilus basal structures. These structural snapshots support a model for biogenesis of the F transfer system and allow for detailed comparisons with other structurally characterized T4SSs.

3.
mBio ; 10(3)2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088930

RESUMO

Helicobacter pylori colonizes about half of humans worldwide, and its presence in the gastric mucosa is associated with an increased risk of gastric adenocarcinoma, gastric lymphoma, and peptic ulcer disease. H. pylori strains carrying the cag pathogenicity island (cagPAI) are associated with increased risk of disease progression. The cagPAI encodes the Cag type IV secretion system (CagT4SS), which delivers the CagA oncoprotein and other effector molecules into human gastric epithelial cells. We visualized structures of native and mutant CagT4SS machines on the H. pylori cell envelope by cryoelectron tomography. Individual H. pylori cells contain multiple CagT4SS nanomachines, each composed of a wheel-shaped outer membrane complex (OMC) with 14-fold symmetry and an inner membrane complex (IMC) with 6-fold symmetry. CagX, CagY, and CagM are required for assembly of the OMC, whereas strains lacking Cag3 and CagT produce outer membrane complexes lacking peripheral components. The IMC, which has never been visualized in detail, is configured as six tiers in cross-section view and three concentric rings surrounding a central channel in end-on view. The IMC contains three T4SS ATPases: (i) VirB4-like CagE, arranged as a hexamer of dimers at the channel entrance; (ii) a hexamer of VirB11-like Cagα, docked at the base of the CagE hexamer; and (iii) VirD4-like Cagß and other unspecified Cag subunits, associated with the stacked CagE/Cagα complex and forming the outermost rings. The CagT4SS and recently solved Legionella pneumophila Dot/Icm system comprise new structural prototypes for the T4SS superfamily.IMPORTANCE Bacterial type IV secretion systems (T4SSs) have been phylogenetically grouped into two subfamilies. The T4ASSs, represented by the Agrobacterium tumefaciens VirB/VirD4T4SS, include "minimized" machines assembled from 12 VirB- and VirD4-like subunits and compositionally larger systems such as the Helicobacter pylori CagT4SS T4BSSs encompass systems closely related in subunit composition to the Legionella pneumophila Dot/IcmT4SS Here, we present structures of native and mutant H. pylori Cag machines determined by in situ cryoelectron tomography. We identify distinct outer and inner membrane complexes and, for the first time, visualize structural contributions of all three "signature" ATPases of T4SSs at the cytoplasmic entrance of the translocation channel. Despite their evolutionary divergence, the CagT4SS aligns structurally much more closely to the Dot/IcmT4SS than an available VirB/VirD4 subcomplex. Our findings highlight the diversity of T4SSs and suggest a structural classification scheme in which T4SSs are grouped as minimized VirB/VirD4-like or larger Cag-like and Dot/Icm-like systems.


Assuntos
Proteínas de Bactérias/genética , Helicobacter pylori/genética , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/ultraestrutura , Antígenos de Bactérias/genética , Microscopia Crioeletrônica , Ilhas Genômicas , Humanos
4.
Front Microbiol ; 10: 958, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31134011

RESUMO

The conjugative plasmid pCF10 from Enterococcus faecalis encodes a Type 4 Secretion System required for plasmid transfer. The accessory factor PcfF and relaxase PcfG initiate pCF10 transfer by forming the catalytically active relaxosome at the plasmid's origin-of-transfer (oriT) sequence. Here, we report the crystal structure of the homo-dimeric PcfF, composed of an N-terminal DNA binding Ribbon-Helix-Helix (RHH) domain and a C-terminal stalk domain. We identified key residues in the RHH domain that are responsible for binding pCF10's oriT sequence in vitro, and further showed that PcfF bends the DNA upon oriT binding. By mutational analysis and pull-down experiments, we identified residues in the stalk domain that contribute to interaction with PcfG. PcfF variant proteins defective in oriT or PcfG binding attenuated plasmid transfer in vivo, but also suggested that intrinsic or extrinsic factors might modulate relaxosome assembly. We propose that PcfF initiates relaxosome assembly by binding oriT and inducing DNA bending, which serves to recruit PcfG as well as extrinsic factors necessary for optimal plasmid processing and engagement with the pCF10 transfer machine.

5.
Microbiol Spectr ; 7(2)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30953428

RESUMO

The bacterial type IV secretion systems (T4SSs) are a functionally diverse superfamily of secretion systems found in many species of bacteria. Collectively, the T4SSs translocate DNA and monomeric and multimeric protein substrates to bacterial and eukaryotic cell types. T4SSs are composed of two large subfamilies, the conjugation machines and the effector translocators that transmit their cargoes through establishment of direct donor-target cell contacts, and a third small subfamily capable of importing or exporting substrates from or to the milieu. This review summarizes recent mechanistic and structural findings that are shedding new light on how T4SSs have evolved such functional diversity. Translocation signals are now known to be located C terminally or embedded internally in structural folds; these signals in combination with substrate-associated adaptor proteins mediate the docking of specific substrate repertoires to cognate VirD4-like receptors. For the Legionella pneumophila Dot/Icm system, recent work has elucidated the structural basis for adaptor-dependent substrate loading onto the VirD4-like DotL receptor. Advances in definition of T4SS machine structures now allow for detailed comparisons of nanomachines closely related to the Agrobacterium tumefaciens VirB/VirD4 T4SS with those more distantly related, e.g., the Dot/Icm and Helicobacter pylori Cag T4SSs. Finally, it is increasingly evident that T4SSs have evolved a variety of mechanisms dependent on elaboration of conjugative pili, membrane tubes, or surface adhesins to establish productive contacts with target cells. T4SSs thus have evolved extreme functional diversity through a plethora of adaptations impacting substrate selection, machine architecture, and target cell binding.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Sistemas de Secreção Tipo IV/química , Sistemas de Secreção Tipo IV/fisiologia , Agrobacterium tumefaciens/metabolismo , Biodiversidade , DNA Bacteriano , Helicobacter pylori/metabolismo , Legionella pneumophila/metabolismo , Transporte Proteico , Especificidade por Substrato
6.
Mol Microbiol ; 111(1): 96-117, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30264928

RESUMO

Mobile genetic elements (MGEs) encode type IV secretion systems (T4SSs) known as conjugation machines for their transmission between bacterial cells. Conjugation machines are composed of an envelope-spanning translocation channel, and those functioning in Gram-negative species additionally elaborate an extracellular pilus to initiate donor-recipient cell contacts. We report that pKM101, a self-transmissible MGE functioning in the Enterobacteriaceae, has evolved a second target cell attachment mechanism. Two pKM101-encoded proteins, the pilus-tip adhesin TraC and a protein termed Pep, are exported to the cell surface where they interact and also form higher order complexes appearing as distinct foci or patches around the cell envelope. Surface-displayed TraC and Pep are required for an efficient conjugative transfer, 'extracellular complementation' potentially involving intercellular protein transfer, and activation of a Pseudomonas aeruginosa type VI secretion system. Both proteins are also required for bacteriophage PRD1 infection. TraC and Pep are exported across the outer membrane by a mechanism potentially involving the ß-barrel assembly machinery. The pKM101 T4SS, thus, deploys alternative routing pathways for the delivery of TraC to the pilus tip or both TraC and Pep to the cell surface. We propose that T4SS-encoded, pilus-independent attachment mechanisms maximize the probability of MGE propagation and might be widespread among this translocation superfamily.


Assuntos
Adesinas Bacterianas/metabolismo , Conjugação Genética , Escherichia coli/genética , Proteínas de Fímbrias/metabolismo , Transferência Genética Horizontal , Plasmídeos , Bacteriófago PRD1/fisiologia , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Multimerização Proteica , Transporte Proteico , Sistemas de Secreção Tipo IV/metabolismo , Sistemas de Secreção Tipo VI/metabolismo , Ligação Viral
7.
Nat Commun ; 9(1): 2231, 2018 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-29884781

RESUMO

CRISPR advances genome engineering by directing endonuclease sequence specificity with a guide RNA molecule (gRNA). For precisely targeting a gene for modification, each genetic construct requires a unique gRNA. By generating a gRNA against the flippase recognition target (FRT) site, a common genetic element shared by multiple genetic collections, CRISPR-FRT circumvents this design constraint to provide a broad platform for fast, scarless, off-the-shelf genome engineering.


Assuntos
Sistemas CRISPR-Cas , DNA Nucleotidiltransferases/metabolismo , Edição de Genes/métodos , RNA Guia/metabolismo , Sítios de Ligação/genética , DNA Nucleotidiltransferases/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Técnicas de Inativação de Genes , Genoma Bacteriano/genética , Modelos Genéticos , Mutação , RNA Guia/genética
8.
Mol Microbiol ; 109(3): 291-305, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29723434

RESUMO

Gram-positive bacteria deploy type IV secretion systems (T4SSs) to facilitate horizontal gene transfer. The T4SSs of Gram-positive bacteria rely on surface adhesins as opposed to conjugative pili to facilitate mating. Enterococcus faecalis PrgB is a surface adhesin that promotes mating pair formation and robust biofilm development in an extracellular DNA (eDNA) dependent manner. Here, we report the structure of the adhesin domain of PrgB. The adhesin domain binds and compacts DNA in vitro. In vivo PrgB deleted of its adhesin domain does not support cellular aggregation, biofilm development and conjugative DNA transfer. PrgB also binds lipoteichoic acid (LTA), which competes with DNA binding. We propose that PrgB binding and compaction of eDNA facilitates cell aggregation and plays an important role in establishment of early biofilms in mono- or polyspecies settings. Within these biofilms, PrgB mediates formation and stabilization of direct cell-cell contacts through alternative binding of cell-bound LTA, which in turn promotes establishment of productive mating junctions and efficient intra- or inter-species T4SS-mediated gene transfer.


Assuntos
Junções Aderentes/fisiologia , Adesinas Bacterianas/química , Adesinas Bacterianas/metabolismo , Biofilmes/crescimento & desenvolvimento , Conjugação Genética , Enterococcus faecalis/fisiologia , Adesinas Bacterianas/genética , Linhagem Celular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Enterococcus faecalis/genética , Transferência Genética Horizontal , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Plasmídeos/química , Plasmídeos/genética , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica , Ácidos Teicoicos/química , Ácidos Teicoicos/metabolismo , Sistemas de Secreção Tipo IV
9.
Curr Top Microbiol Immunol ; 418: 233-260, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29808338

RESUMO

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.


Assuntos
Agrobacterium tumefaciens/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mutagênese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Sistemas de Secreção Tipo IV/química , Sistemas de Secreção Tipo IV/genética , Agrobacterium tumefaciens/química , Agrobacterium tumefaciens/metabolismo , Agrobacterium tumefaciens/patogenicidade , Proteínas de Bactérias/química , Proteínas Recombinantes de Fusão/metabolismo , Sistemas de Secreção Tipo IV/metabolismo
10.
Nat Microbiol ; 3(6): 678-686, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29784975

RESUMO

Type IV secretion systems (T4SSs) are complex machines used by bacteria to deliver protein and DNA complexes into target host cells1-5. Conserved ATPases are essential for T4SS function, but how they coordinate their activities to promote substrate transfer remains poorly understood. Here, we show that the DotB ATPase associates with the Dot-Icm T4SS at the Legionella cell pole through interactions with the DotO ATPase. The structure of the Dot-Icm apparatus was solved in situ by cryo-electron tomography at 3.5 nm resolution and the cytoplasmic complex was solved at 3.0 nm resolution. These structures revealed a cell envelope-spanning channel that connects to the cytoplasmic complex. Further analysis revealed a hexameric assembly of DotO dimers associated with the inner membrane complex, and a DotB hexamer associated with the base of this cytoplasmic complex. The assembly of a DotB-DotO energy complex creates a cytoplasmic channel that directs the translocation of substrates through the T4SS. These data define distinct stages in Dot-Icm machine biogenesis, advance our understanding of channel activation, and identify an envelope-spanning T4SS channel.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Legionella pneumophila/crescimento & desenvolvimento , Sistemas de Secreção Tipo IV/metabolismo , Adenosina Trifosfatases/química , Animais , Microscopia Crioeletrônica , Citoplasma/metabolismo , Regulação Bacteriana da Expressão Gênica , Legionella pneumophila/metabolismo , Camundongos , Modelos Moleculares , Multimerização Proteica , Células RAW 264.7 , Sistemas de Secreção Tipo IV/química
11.
Mol Microbiol ; 107(4): 455-471, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29235173

RESUMO

Type IV secretion systems (T4SSs) are versatile multiprotein nanomachines spanning the entire cell envelope in Gram-negative and Gram-positive bacteria. They play important roles through the contact-dependent secretion of effector molecules into eukaryotic hosts and conjugative transfer of mobile DNA elements as well as contact-independent exchange of DNA with the extracellular milieu. In the last few years, many details on the molecular mechanisms of T4SSs have been elucidated. Exciting structures of T4SS complexes from Escherichia coli plasmids R388 and pKM101, Helicobacter pylori and Legionella pneumophila have been solved. The structure of the F-pilus was also reported and surprisingly revealed a filament composed of pilin subunits in 1:1 stoichiometry with phospholipid molecules. Many new T4SSs have been identified and characterized, underscoring the structural and functional diversity of this secretion superfamily. Complex regulatory circuits also have been shown to control T4SS machine production in response to host cell physiological status or a quorum of bacterial recipient cells in the vicinity. Here, we summarize recent advances in our knowledge of 'paradigmatic' and emerging systems, and further explore how new basic insights are aiding in the design of strategies aimed at suppressing T4SS functions in bacterial infections and spread of antimicrobial resistances.


Assuntos
Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Sistemas de Secreção Tipo IV/química , Sistemas de Secreção Tipo IV/metabolismo , Animais , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Bactérias Gram-Negativas/genética , Bactérias Gram-Positivas/genética , Interações Hospedeiro-Patógeno , Humanos , Plasmídeos
14.
PLoS One ; 12(4): e0175273, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28403156

RESUMO

VirE2 is a ssDNA binding protein essential for virulence in Agrobacterium tumefaciens. A tetracysteine mutant (VirE2-TC) was prepared for in vitro and in vivo fluorescence imaging based on the ReAsH reagent. VirE2-TC was found to be biochemically active as it binds both ssDNA and the acidic secretion chaperone VirE1. It was also biologically functional in complementing virE2 null strains transforming Arabidopsis thaliana roots and Nicotiana tabacum leaves. In vitro experiments demonstrated a two-color fluorescent complex using VirE2-TC/ReAsH and Alexa Fluor 488 labeled ssDNA. In vivo, fluorescent VirE2-TC/ReAsH was detected in bacteria and in plant cells at time frames relevant to transformation.


Assuntos
Agrobacterium tumefaciens/metabolismo , Arabidopsis/microbiologia , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Tumores de Planta/microbiologia , Tabaco/microbiologia , Fatores de Virulência/metabolismo , Agrobacterium tumefaciens/citologia , Agrobacterium tumefaciens/genética , Proteínas de Bactérias/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Mutação , Imagem Óptica , Ligação Proteica , Fatores de Virulência/genética
15.
Mol Microbiol ; 105(2): 273-293, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28452085

RESUMO

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 characterized the OMCC by 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. The 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.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Conjugação Genética/genética , Agrobacterium tumefaciens/genética , Proteínas da Membrana Bacteriana Externa/fisiologia , Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Fímbrias Bacterianas/metabolismo , Ligação Proteica , Transporte Proteico/genética , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/metabolismo , Fatores de Virulência/metabolismo
16.
Mol Microbiol ; 103(3): 398-412, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27785854

RESUMO

Upon sensing of the peptide pheromone cCF10, Enterococcus faecalis cells carrying pCF10 produce three surface adhesins (PrgA, PrgB or Aggregation Substance, PrgC) and the Prg/Pcf type IV secretion system and, in turn, conjugatively transfer the plasmid at high frequencies to recipient cells. Here, we report that cCF10 induction is highly toxic to cells sustaining a deletion of prgU, a small orf located immediately downstream of prgB on pCF10. Upon pheromone exposure, these cells overproduce the Prg adhesins and display impaired envelope integrity, as evidenced by antibiotic susceptibility, misplaced division septa and cell lysis. Compensatory mutations in regulatory loci controlling expression of pCF10-encoded prg/pcf genes, or constitutive PrgU overproduction, block production of the Prg adhesins and render cells insensitive to pheromone. Cells engineered to overproduce PrgB, even independently of other pCF10-encoded proteins, have severely compromised cell envelopes and strong growth defects. PrgU has an RNA-binding fold, and prgB-prgU gene pairs are widely distributed among E. faecalis isolates and other enterococcal and staphylococcal species. Together, our findings support a model in which PrgU proteins represent a novel class of RNA-binding regulators that act to mitigate toxicity accompanying overproduction of PrgB-like adhesins in E. faecalis and other clinically-important Gram-positive species.


Assuntos
Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Oligopeptídeos/metabolismo , Feromônios/metabolismo , Sequência de Aminoácidos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Conjugação Genética/genética , DNA Bacteriano/metabolismo , Enterococcus , Regulação Bacteriana da Expressão Gênica/genética , Proteínas de Membrana/metabolismo , Oligopeptídeos/genética , Feromônios/genética , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Deleção de Sequência/genética , Atrativos Sexuais/antagonistas & inibidores , Atrativos Sexuais/genética , Atrativos Sexuais/metabolismo , Transcrição Genética/genética
17.
Curr Top Microbiol Immunol ; 413: 1-30, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29536353

RESUMO

The bacterial type IV secretion systems (T4SSs) are a highly functionally and structurally diverse superfamily of secretion systems found in many species of Gram-negative and -positive bacteria. Collectively, the T4SSs can translocate DNA and monomeric and multimeric protein substrates to a variety of bacterial and eukaryotic cell types. Detailed phylogenomics analyses have established that the T4SSs evolved from ancient conjugation machines whose original functions were to disseminate mobile DNA elements within and between bacterial species. How members of the T4SS superfamily evolved to recognize and translocate specific substrate repertoires to prokaryotic or eukaryotic target cells is a fascinating question from evolutionary, biological, and structural perspectives. In this chapter, we will summarize recent findings that have shaped our current view of the biological diversity of the T4SSs. We focus mainly on two subtypes, designated as the types IVA (T4ASS) and IVB (T4BSS) systems that respectively are represented by the paradigmatic Agrobacterium tumefaciens VirB/VirD4 and Legionella pneumophila Dot/Icm T4SSs. We present current information about the composition and architectures of these representative systems. We also describe how these and a few related T4ASS and T4BSS members evolved as specialized nanomachines through acquisition of novel domains or subunits, a process that ultimately generated extensive genetic and structural mosaicism among this secretion superfamily. Finally, we present new phylogenomics information establishing that the T4BSSs are much more broadly distributed than initially envisioned.


Assuntos
Evolução Biológica , Legionella pneumophila , Agrobacterium tumefaciens , Proteínas de Bactérias , Sistemas de Secreção Bacterianos , Biodiversidade , Sistemas de Secreção Tipo IV
18.
EcoSal Plus ; 7(1)2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27735785

RESUMO

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.


Assuntos
Agrobacterium tumefaciens/genética , Escherichia coli/genética , Sistemas de Secreção Tipo IV/química , Sistemas de Secreção Tipo IV/metabolismo , Agrobacterium tumefaciens/metabolismo , Agrobacterium tumefaciens/patogenicidade , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Conjugação Genética , DNA Bacteriano , Farmacorresistência Bacteriana/genética , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Transporte Proteico , Sistemas de Secreção Tipo IV/classificação , Sistemas de Secreção Tipo IV/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
20.
J Bacteriol ; 198(19): 2701-18, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27432829

RESUMO

UNLABELLED: Bacterial type IV secretion systems (T4SSs) are composed of two major subfamilies, conjugation machines dedicated to DNA transfer and effector translocators for protein transfer. We show here that the Escherichia coli pKM101-encoded conjugation system, coupled with chimeric substrate receptors, can be repurposed for transfer of heterologous effector proteins. The chimeric receptors were composed of the N-terminal transmembrane domain of pKM101-encoded TraJ fused to soluble domains of VirD4 homologs functioning in Agrobacterium tumefaciens, Anaplasma phagocytophilum, or Wolbachia pipientis A chimeric receptor assembled from A. tumefaciens VirD4 (VirD4At) mediated transfer of a MOBQ plasmid (pML122) and A. tumefaciens effector proteins (VirE2, VirE3, and VirF) through the pKM101 transfer channel. Equivalent chimeric receptors assembled from the rickettsial VirD4 homologs similarly supported the transfer of known or candidate effectors from rickettsial species. These findings establish a proof of principle for use of the dedicated pKM101 conjugation channel, coupled with chimeric substrate receptors, to screen for translocation competency of protein effectors from recalcitrant species. Many T4SS receptors carry sequence-variable C-terminal domains (CTDs) with unknown function. While VirD4At and the TraJ/VirD4At chimera with their CTDs deleted supported pML122 transfer at wild-type levels, ΔCTD variants supported transfer of protein substrates at strongly diminished or elevated levels. We were unable to detect binding of VirD4At's CTD to the VirE2 effector, although other VirD4At domains bound this substrate in vitro We propose that CTDs evolved to govern the dynamics of substrate presentation to the T4SS either through transient substrate contacts or by controlling substrate access to other receptor domains. IMPORTANCE: Bacterial type IV secretion systems (T4SSs) display striking versatility in their capacity to translocate DNA and protein substrates to prokaryotic and eukaryotic target cells. A hexameric ATPase, the type IV coupling protein (T4CP), functions as a substrate receptor for nearly all T4SSs. Here, we report that chimeric T4CPs mediate transfer of effector proteins through the Escherichia coli pKM101-encoded conjugation system. Studies with these repurposed conjugation systems established a role for acidic C-terminal domains of T4CPs in regulating substrate translocation. Our findings advance a mechanistic understanding of T4CP receptor activity and, further, support a model in which T4SS channels function as passive conduits for any DNA or protein substrates that successfully engage with and pass through the T4CP specificity checkpoint.


Assuntos
Conjugação Genética/fisiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Proteínas Recombinantes de Fusão , Sistemas de Secreção Tipo IV/fisiologia , DNA Bacteriano , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Domínios Proteicos
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