The putative lytic transglycosylase VirB1 from Brucella suis interacts with the type IV secretion system core components VirB8, VirB9 and VirB11

VirB1-like proteins are believed to act as lytic transglycosylases, which facilitate the assembly of type IV secretion systems via localized lysis of the peptidoglycan. This paper presents the biochemical analysis of interactions of purified Brucella suis VirB1 with core components of the type IV secretion system. Genes encoding VirB1, VirB8, VirB9, VirB10 and VirB11 were cloned into expression vectors; the affinity-tagged proteins were purified from Escherichia coli, and analyses by gel filtration chromatography showed that they form monomers or homo-multimers. Analysis of protein–protein interactions by affinity precipitation revealed that VirB1 bound to VirB9 and VirB11. The results of bicistron expression experiments followed by gel filtration further supported the VirB1–VirB9 interaction. Peptide array mapping identified regions of VirB1 that interact with VirB8, VirB9 and VirB11 and underscored the importance of the C-terminus, especially for the VirB1–VirB9 interaction. The binding sites were localized on a structure model of VirB1, suggesting that different portions of VirB1 may interact with other VirB proteins during assembly of the type IV secretion machinery.

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Protein Subassemblies of the Helicobacter pylori Cag Type IV Secretion System Revealed by Localization and Interaction Studies

Journal of Bacteriology ◽

10.1128/jb.01341-07 ◽

2008 ◽

Vol 190 (6) ◽

pp. 2161-2171 ◽

Cited By ~ 86

Author(s):

Stefan Kutter ◽

Renate Buhrdorf ◽

Jürgen Haas ◽

Wulf Schneider-Brachert ◽

Rainer Haas ◽

...

Keyword(s):

Helicobacter Pylori ◽

Protein Interactions ◽

Protein Transport ◽

Secretion System ◽

Type Iv Secretion ◽

Transport Systems ◽

Type Iv Secretion System ◽

Secretion Systems ◽

Type Iv ◽

Small Proteins

ABSTRACT Type IV secretion systems are possibly the most versatile protein transport systems in gram-negative bacteria, with substrates ranging from small proteins to large nucleoprotein complexes. In many cases, such as the cag pathogenicity island of Helicobacter pylori, genes encoding components of a type IV secretion system have been identified due to their sequence similarities to prototypical systems such as the VirB system of Agrobacterium tumefaciens. The Cag type IV secretion system contains at least 14 essential apparatus components and several substrate translocation and auxiliary factors, but the functions of most components cannot be inferred from their sequences due to the lack of similarities. In this study, we have performed a comprehensive sequence analysis of all essential or auxiliary Cag components, and we have used antisera raised against a subset of components to determine their subcellular localization. The results suggest that the Cag system contains functional analogues to all VirB components except VirB5. Moreover, we have characterized mutual stabilization effects and performed a comprehensive yeast two-hybrid screening for potential protein-protein interactions. Immunoprecipitation studies resulted in identification of a secretion apparatus subassembly at the outer membrane. Combining these data, we provide a first low-resolution model of the Cag type IV secretion apparatus.

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The Brucella suis Type IV Secretion System Assembles in the Cell Envelope of the Heterologous Host Agrobacterium tumefaciens and Increases IncQ Plasmid pLS1 Recipient Competence

Infection and Immunity ◽

10.1128/iai.74.1.108-117.2006 ◽

2006 ◽

Vol 74 (1) ◽

pp. 108-117 ◽

Cited By ~ 13

Author(s):

Anna Carle ◽

Christoph Höppner ◽

Khaled Ahmed Aly ◽

Qing Yuan ◽

Amke den Dulk-Ras ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Mammalian Cells ◽

Cell Envelope ◽

Secretion System ◽

Type Iv Secretion ◽

Type Iv Secretion System ◽

Secretion Systems ◽

Heterologous Host ◽

Type Iv ◽

Brucella Suis

ABSTRACT Pathogenic Brucella species replicate within mammalian cells, and their type IV secretion system is essential for intracellular survival and replication. The options for biochemical studies on the Brucella secretion system are limited due to the rigidity of the cells and biosafety concerns, which preclude large-scale cell culture and fractionation. To overcome these problems, we heterologously expressed the Brucella suis virB operon in the closely related α2-proteobacterium Agrobacterium tumefaciens and showed that the VirB proteins assembled into a complex. Eight of the twelve VirB proteins were detected in the membranes of the heterologous host with specific antisera. Cross-linking indicated protein-protein interactions similar to those in other type IV secretion systems, and the results of immunofluorescence analysis supported the formation of VirB protein complexes in the cell envelope. Production of a subset of the B. suis VirB proteins (VirB3-VirB12) in A. tumefaciens strongly increased its ability to receive IncQ plasmid pLS1 in conjugation experiments, and production of VirB1 further enhanced the conjugation efficiency. Plasmid recipient competence correlated with periplasmic leakage and the detergent sensitivity of A. tumefaciens, suggesting a weakening of the cell envelope. Heterologous expression thus permits biochemical characterization of B. suis type IV secretion system assembly.

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Membrane and Core Periplasmic Agrobacterium tumefaciens Virulence Type IV Secretion System Components Localize to Multiple Sites around the Bacterial Perimeter during Lateral Attachment to Plant Cells

mBio ◽

10.1128/mbio.00218-11 ◽

2011 ◽

Vol 2 (6) ◽

Cited By ~ 33

Author(s):

Julieta Aguilar ◽

Todd A. Cameron ◽

John Zupan ◽

Patricia Zambryski

Keyword(s):

Agrobacterium Tumefaciens ◽

Plant Cells ◽

Secretion System ◽

Type Iv Secretion ◽

Host Cells ◽

Type Iv Secretion System ◽

Secretion Systems ◽

Content Type ◽

Protein Substrates ◽

Type Iv

ABSTRACTType IV secretion systems (T4SS) transfer DNA and/or proteins into recipient cells. Here we performed immunofluorescence deconvolution microscopy to localize the assembled T4SS by detection of its native components VirB1, VirB2, VirB4, VirB5, VirB7, VirB8, VirB9, VirB10, and VirB11 in the C58 nopaline strain ofAgrobacterium tumefaciens, following induction of virulence (vir) gene expression. These different proteins represent T4SS components spanning the inner membrane, periplasm, or outer membrane. Native VirB2, VirB5, VirB7, and VirB8 were also localized in theA. tumefaciensoctopine strain A348. Quantitative analyses of the localization of all the above Vir proteins in nopaline and octopine strains revealed multiple foci in single optical sections in over 80% and 70% of the bacterial cells, respectively. Green fluorescent protein (GFP)-VirB8 expression followingvirinduction was used to monitor bacterial binding to live host plant cells; bacteria bind predominantly along their lengths, with few bacteria binding via their poles or subpoles.vir-induced attachment-defective bacteria or bacteria without the Ti plasmid do not bind to plant cells. These data support a model where multiplevir-T4SS around the perimeter of the bacterium maximize effective contact with the host to facilitate efficient transfer of DNA and protein substrates.IMPORTANCETransfer of DNA and/or proteins to host cells through multiprotein type IV secretion system (T4SS) complexes that span the bacterial cell envelope is critical to bacterial pathogenesis. Early reports suggested that T4SS components localized at the cell poles. Now, higher-resolution deconvolution fluorescence microscopy reveals that all structural components of theAgrobacterium tumefaciens vir-T4SS, as well as its transported protein substrates, localize to multiple foci around the cell perimeter. These results lead to a new model ofA. tumefaciensattachment to a plant cell, whereA. tumefacienstakes advantage of the multiplevir-T4SS along its length to make intimate lateral contact with plant cells and thereby effectively transfer DNA and/or proteins through thevir-T4SS. The T4SS ofA. tumefaciensis among the best-studied T4SS, and the majority of its components are highly conserved in different pathogenic bacterial species. Thus, the results presented can be applied to a broad range of pathogens that utilize T4SS.

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Protein-Protein Interactions among Helicobacter pylori Cag Proteins

Journal of Bacteriology ◽

10.1128/jb.00066-06 ◽

2006 ◽

Vol 188 (13) ◽

pp. 4787-4800 ◽

Cited By ~ 53

Author(s):

Valerie J. Busler ◽

Victor J. Torres ◽

Mark S. McClain ◽

Oscar Tirado ◽

David B. Friedman ◽

...

Keyword(s):

Helicobacter Pylori ◽

Protein Interactions ◽

Secretion System ◽

Type Iv Secretion ◽

Type Iv Secretion System ◽

Protein Protein Interactions ◽

Chromosomal Dna ◽

2D Dige ◽

Type Iv ◽

H Pylori

ABSTRACT Many Helicobacter pylori isolates contain a 40-kb region of chromosomal DNA known as the cag pathogenicity island (PAI). The risk for development of gastric cancer or peptic ulcer disease is higher among humans infected with cag PAI-positive H. pylori strains than among those infected with cag PAI-negative strains. The cag PAI encodes a type IV secretion system that translocates CagA into gastric epithelial cells. To identify Cag proteins that are expressed by H. pylori during growth in vitro, we compared the proteomes of a wild-type H. pylori strain and an isogenic cag PAI deletion mutant using two-dimensional difference gel electrophoresis (2D-DIGE) in multiple pH ranges. Seven Cag proteins were identified by this approach. We then used a yeast two-hybrid system to detect potential protein-protein interactions among 14 Cag proteins. One heterotypic interaction (CagY/7 with CagX/8) and two homotypic interactions (involving H. pylori VirB11/ATPase and Cag5) were similar to interactions previously reported to occur among homologous components of the Agrobacterium tumefaciens type IV secretion system. Other interactions involved Cag proteins that do not have known homologues in other bacterial species. Biochemical analysis confirmed selected interactions involving five of the proteins that were identified by 2D-DIGE. Protein-protein interactions among Cag proteins are likely to have an important role in the assembly of the H. pylori type IV secretion apparatus.

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Involvement of a Plasmid-Encoded Type IV Secretion System in the Plant Tissue Watersoaking Phenotype of Burkholderia cenocepacia

Journal of Bacteriology ◽

10.1128/jb.186.18.6015-6024.2004 ◽

2004 ◽

Vol 186 (18) ◽

pp. 6015-6024 ◽

Cited By ~ 41

Author(s):

Amanda S. Engledow ◽

Enrique G. Medrano ◽

Eshwar Mahenthiralingam ◽

John J. LiPuma ◽

Carlos F. Gonzalez

Keyword(s):

Plant Tissue ◽

Protein S ◽

Secretion System ◽

Type Iv Secretion ◽

Site Directed Mutagenesis ◽

Burkholderia Cenocepacia ◽

Type Iv Secretion System ◽

Directed Mutagenesis ◽

Secretion Systems ◽

Type Iv

ABSTRACT Burkholderia cenocepacia strain K56-2, a representative of the Burkholderia cepacia complex, is part of the epidemic and clinically problematic ET12 lineage. The strain produced plant tissue watersoaking (ptw) on onion tissue, which is a plant disease-associated trait. Using plasposon mutagenesis, mutants in the ptw phenotype were generated. The translated sequence of a disrupted gene (ptwD4) from a ptw-negative mutant showed homology to VirD4-like proteins. Analysis of the region proximal to the transfer gene homolog identified a gene cluster located on the 92-kb resident plasmid that showed homology to type IV secretion systems. The role of ptwD4, ptwC, ptwB4, and ptwB10 in the expression of ptw activity was determined by conducting site-directed mutagenesis. The ptw phenotype was not expressed by K56-2 derivatives with a disruption in ptwD4, ptwB4, or ptwB10 but was observed in a derivative with a disruption in ptwC. Complementation of ptw-negative K56-2 derivatives in trans resulted in complete restoration of the ptw phenotype. In addition, analysis of culture supernatants revealed that the putative ptw effector(s) was a secreted, heat-stable protein(s) that caused plasmolysis of plant protoplasts. A second chromosomally encoded type IV secretion system with complete homology to the VirB-VirD system was identified in K56-2. Site-directed mutagenesis of key secretory genes in the VirB-VirD system did not affect expression of the ptw phenotype. Our findings indicate that in strain K56-2, the plasmid-encoded Ptw type IV secretion system is responsible for the secretion of a plant cytotoxic protein(s).

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AtlA Functions as a Peptidoglycan Lytic Transglycosylase in the Neisseria gonorrhoeae Type IV Secretion System

Journal of Bacteriology ◽

10.1128/jb.00531-07 ◽

2007 ◽

Vol 189 (15) ◽

pp. 5421-5428 ◽

Cited By ~ 39

Author(s):

Petra L. Kohler ◽

Holly L. Hamilton ◽

Karen Cloud-Hansen ◽

Joseph P. Dillard

Keyword(s):

Neisseria Gonorrhoeae ◽

Secretion System ◽

Type Iv Secretion ◽

Type Iv Secretion System ◽

Secretion Systems ◽

Lytic Transglycosylases ◽

Type Iv ◽

Lytic Transglycosylase ◽

Enzymatic Function ◽

Type Iv Secretion Systems

ABSTRACT Type IV secretion systems require peptidoglycan lytic transglycosylases for efficient secretion, but the function of these enzymes is not clear. The type IV secretion system gene cluster of Neisseria gonorrhoeae encodes two peptidoglycan transglycosylase homologues. One, LtgX, is similar to peptidoglycan transglycosylases from other type IV secretion systems. The other, AtlA, is similar to endolysins from bacteriophages and is not similar to any described type IV secretion component. We characterized the enzymatic function of AtlA in order to examine its role in the type IV secretion system. Purified AtlA was found to degrade macromolecular peptidoglycan and to produce 1,6-anhydro peptidoglycan monomers, characteristic of lytic transglycosylase activity. We found that AtlA can functionally replace the lambda endolysin to lyse Escherichia coli. In contrast, a sensitive measure of lysis demonstrated that AtlA does not lyse gonococci expressing it or gonococci cocultured with an AtlA-expressing strain. The gonococcal type IV secretion system secretes DNA during growth. A deletion of ltgX or a substitution in the putative active site of AtlA severely decreased DNA secretion. These results indicate that AtlA and LtgX are actively involved in type IV secretion and that AtlA is not involved in lysis of gonococci to release DNA. This is the first demonstration that a type IV secretion peptidoglycanase has lytic transglycosylase activity. These data show that AtlA plays a role in type IV secretion of DNA that requires peptidoglycan breakdown without cell lysis.

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VirB8: a conserved type IV secretion system assembly factor and drug targetThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Membrane Proteins in Health and Disease.

Biochemistry and Cell Biology ◽

10.1139/o06-148 ◽

2006 ◽

Vol 84 (6) ◽

pp. 890-899 ◽

Cited By ~ 44

Author(s):

Christian Baron

Keyword(s):

Function Analysis ◽

Model Organism ◽

Secretion System ◽

Type Iv Secretion ◽

Type Iv Secretion System ◽

Secretion Systems ◽

Type Iv ◽

Assembly Factor ◽

Type Iv Secretion Systems ◽

Multiple Interactions

Type IV secretion systems are used by many Gram-negative bacteria for the translocation of macromolecules (proteins, DNA, or DNA–protein complexes) across the cell envelope. Among them are many pathogens for which type IV secretion systems are essential virulence factors. Type IV secretion systems comprise 8–12 conserved proteins, which assemble into a complex spanning the inner and the outer membrane, and many assemble extracellular appendages, such as pili, which initiate contact with host and recipient cells followed by substrate translocation. VirB8 is an essential assembly factor for all type IV secretion systems. Biochemical, cell biological, genetic, and yeast two-hybrid analyses showed that VirB8 undergoes multiple interactions with other type IV secretion system components and that it directs polar assembly of the membrane-spanning complex in the model organism Agrobacterium tumefaciens. The availability of the VirB8 X-ray structure has enabled a detailed structure–function analysis, which identified sites for the binding of VirB4 and VirB10 and for self-interaction. Due to its multiple interactions, VirB8 is an excellent model for the analysis of assembly factors of multiprotein complexes. In addition, VirB8 is a possible target for drugs that target its protein–protein interactions, which would disarm bacteria by depriving them of their essential virulence functions.

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Novel Type IV Secretion System Involved in Propagation of Genomic Islands

Journal of Bacteriology ◽

10.1128/jb.01327-06 ◽

2006 ◽

Vol 189 (3) ◽

pp. 761-771 ◽

Cited By ~ 87

Author(s):

Mario Juhas ◽

Derrick W. Crook ◽

Ioanna D. Dimopoulou ◽

Gerton Lunter ◽

Rosalind M. Harding ◽

...

Keyword(s):

Gene Cluster ◽

Genomic Island ◽

Secretion System ◽

Type Iv Secretion ◽

Genomic Islands ◽

Phenotypic Traits ◽

Type Iv Secretion System ◽

Genome Plasticity ◽

Secretion Systems ◽

Type Iv

ABSTRACT Type IV secretion systems (T4SSs) mediate horizontal gene transfer, thus contributing to genome plasticity, evolution of infectious pathogens, and dissemination of antibiotic resistance and other virulence traits. A gene cluster of the Haemophilus influenzae genomic island ICEHin1056 has been identified as a T4SS involved in the propagation of genomic islands. This T4SS is novel and evolutionarily distant from the previously described systems. Mutation analysis showed that inactivation of key genes of this system resulted in a loss of phenotypic traits provided by a T4SS. Seven of 10 mutants with a mutation in this T4SS did not express the type IV secretion pilus. Correspondingly, disruption of the genes resulted in up to 100,000-fold reductions in conjugation frequencies compared to those of the parent strain. Moreover, the expression of this T4SS was found to be positively regulated by one of its components, the tfc24 gene. We concluded that this gene cluster represents a novel family of T4SSs involved in propagation of genomic islands.

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VirB1* Promotes T-Pilus Formation in the vir-Type IV Secretion System of Agrobacterium tumefaciens

Journal of Bacteriology ◽

10.1128/jb.00480-07 ◽

2007 ◽

Vol 189 (18) ◽

pp. 6551-6563 ◽

Cited By ~ 40

Author(s):

John Zupan ◽

Cheryl A. Hackworth ◽

Julieta Aguilar ◽

Doyle Ward ◽

Patricia Zambryski

Keyword(s):

Protein Interactions ◽

Secretion System ◽

Type Iv Secretion ◽

Type Iv Secretion System ◽

Protein Protein Interactions ◽

Additional Function ◽

Type Iv ◽

Lytic Transglycosylase ◽

Peptidoglycan Cell Wall ◽

Two Hybrid

ABSTRACT The vir-type IV secretion system of Agrobacterium is assembled from 12 proteins encoded by the virB operon and virD4. VirB1 is one of the least-studied proteins encoded by the virB operon. Its N terminus is a lytic transglycosylase. The C-terminal third of the protein, VirB1*, is cleaved from VirB1 and secreted to the outside of the bacterial cell, suggesting an additional function. We show that both nopaline and octopine strains produce abundant amounts of VirB1* and perform detailed studies on nopaline VirB1*. Both domains are required for wild-type virulence. We show here that the nopaline type VirB1* is essential for the formation of the T pilus, a subassembly of the vir-T4SS composed of processed and cyclized VirB2 (major subunit) and VirB5 (minor subunit). A nopaline virB1 deletion strain does not produce T pili. Complementation with full-length VirB1 or C-terminal VirB1*, but not the N-terminal lytic transglycosylase domain, restores T pili containing VirB2 and VirB5. T-pilus preparations also contain extracellular VirB1*. Protein-protein interactions between VirB1* and VirB2 and VirB5 were detected in the yeast two-hybrid assay. We propose that VirB1 is a bifunctional protein required for virT4SS assembly. The N-terminal lytic transglycosylase domain provides localized lysis of the peptidoglycan cell wall to allow insertion of the T4SS. The C-terminal VirB1* promotes T-pilus assembly through protein-protein interactions with T-pilus subunits.

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