scholarly journals Export of the Pseudopilin XcpT of the Pseudomonas aeruginosa Type II Secretion System via the Signal Recognition Particle-Sec Pathway

2006 ◽  
Vol 189 (5) ◽  
pp. 2069-2076 ◽  
Author(s):  
Jorik Arts ◽  
Ria van Boxtel ◽  
Alain Filloux ◽  
Jan Tommassen ◽  
Margot Koster
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Protein Complexes ◽  
Signal Recognition Particle ◽  
Leader Peptide ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Signal Recognition ◽  
Amino Acid Residues ◽  
Reporter Protein ◽  
Type Iv

ABSTRACT Type IV pilins and pseudopilins are found in various prokaryotic envelope protein complexes, including type IV pili and type II secretion machineries of gram-negative bacteria, competence systems of gram-positive bacteria, and flagella and sugar-binding structures in members of the archaeal kingdom. The precursors of these proteins have highly conserved N termini, consisting of a short, positively charged leader peptide, which is cleaved off by a dedicated peptidase during maturation, and a hydrophobic stretch of approximately 20 amino acid residues. Which pathway is involved in the inner membrane translocation of these proteins is unknown. We used XcpT, the major pseudopilin from the type II secretion machinery of Pseudomonas aeruginosa, as a model to study this process. Transport of an XcpT-PhoA hybrid was shown to occur in the absence of other Xcp components in P. aeruginosa and in Escherichia coli. Experiments with conditional sec mutants and reporter-protein fusions showed that this transport process involves the cotranslational signal recognition particle targeting route and is dependent on a functional Sec translocon.

scholarly journals Type II Protein Secretion in Pseudomonas aeruginosa: the Pseudopilus Is a Multifibrillar and Adhesive Structure

2003 ◽  
Vol 185 (9) ◽  
pp. 2749-2758 ◽  
Author(s):  
Éric Durand ◽  
Alain Bernadac ◽  
Geneviève Ball ◽  
Andrée Lazdunski ◽  
James N. Sturgis ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Infections ◽  
Hydrolytic Enzymes ◽  
Macromolecular Complex ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Secretion Systems ◽  
Type Iv Pilus ◽  
Type Iv ◽  
Type Iv Pilin

ABSTRACT The type II secretion pathway of Pseudomonas aeruginosa is involved in the extracellular release of various toxins and hydrolytic enzymes such as exotoxin A and elastase. This pathway requires the function of a macromolecular complex called the Xcp secreton. The Xcp secreton shares many features with the machinery involved in type IV pilus assembly. More specifically, it involves the function of five pilin-like proteins, the XcpT-X pseudopilins. We show that, upon overexpression, the XcpT pseudopilin can be assembled in a pilus, which we call a type II pseudopilus. Image analysis and filtering of electron micrographs indicated that these appendages are composed of individual fibrils assembled together in a bundle structure. Our observations thus revealed that XcpT has properties similar to those of type IV pilin subunits. Interestingly, the assembly of the type II pseudopilus is not exclusively dependent on the Xcp machinery but can be supported by other similar machineries, such as the Pil (type IV pilus) and Hxc (type II secretion) systems of P. aeruginosa. In addition, heterologous pseudopilins can be assembled by P. aeruginosa into a type II pseudopilus. Finally, we showed that assembly of the type II pseudopilus confers increased bacterial adhesive capabilities. These observations confirmed the ability of pseudopilins to form a pilus structure and raise questions with respect to their function in terms of secretion and adhesion, two crucial biological processes in the course of bacterial infections.

scholarly journals Signal Recognition Particle-Dependent Inner Membrane Targeting of the PulG Pseudopilin Component of a Type II Secretion System

2006 ◽  
Vol 189 (5) ◽  
pp. 1783-1793 ◽  
Author(s):  
Olivera Francetic ◽  
Nienke Buddelmeijer ◽  
Shawn Lewenza ◽  
Carol A. Kumamoto ◽  
Anthony P. Pugsley
Keyword(s):  
Signal Sequence ◽  
Klebsiella Oxytoca ◽  
Signal Recognition Particle ◽  
Secretion System ◽  
Membrane Targeting ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Signal Recognition ◽  
Inner Membrane ◽  
Type Ii Secretion System

ABSTRACT The pseudopilin PulG is an essential component of the pullulanase-specific type II secretion system from Klebsiella oxytoca. PulG is the major subunit of a short, thin-filament pseudopilus, which presumably elongates and retracts in the periplasm, acting as a dynamic piston to promote pullulanase secretion. It has a signal sequence-like N-terminal segment that, according to studies with green and red fluorescent protein chimeras, anchors unassembled PulG in the inner membrane. We analyzed the early steps of PulG inner membrane targeting and insertion in Escherichia coli derivatives defective in different protein targeting and export factors. The β-galactosidase activity in strains producing a PulG-LacZ hybrid protein increased substantially when the dsbA, dsbB, or all sec genes tested except secB were compromised by mutations. To facilitate analysis of native PulG membrane insertion, a leader peptidase cleavage site was engineered downstream from the N-terminal transmembrane segment (PrePulG*). Unprocessed PrePulG* was detected in strains carrying mutations in secA, secY, secE, and secD genes, including some novel alleles of secY and secD. Furthermore, depletion of the Ffh component of the signal recognition particle (SRP) completely abolished PrePulG* processing, without affecting the Sec-dependent export of periplasmic MalE and RbsB proteins. Thus, PulG is cotranslationally targeted to the inner membrane Sec translocase by SRP.

scholarly journals Purification and Three-Dimensional Electron Microscopy Structure of the Neisseria meningitidis Type IV Pilus Biogenesis Protein PilG

2007 ◽  
Vol 189 (17) ◽  
pp. 6389-6396 ◽  
Author(s):  
Richard F. Collins ◽  
Muhammad Saleem ◽  
Jeremy P. Derrick
Keyword(s):  
Electron Microscopy ◽  
Neisseria Meningitidis ◽  
Metal Ion ◽  
Polyacrylamide Gel Electrophoresis ◽  
Three Dimensional ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Type Iv Pilus ◽  
Type Iv ◽  
Pilus Biogenesis

ABSTRACT Type IV pili are surface-exposed retractable fibers which play a key role in the pathogenesis of Neisseria meningitidis and other gram-negative pathogens. PilG is an integral inner membrane protein and a component of the type IV pilus biogenesis system. It is related by sequence to the extensive GspF family of secretory proteins, which are involved in type II secretion processes. PilG was overexpressed and purified from Escherichia coli membranes by detergent extraction and metal ion affinity chromatography. Analysis of the purified protein by perfluoro-octanoic acid polyacrylamide gel electrophoresis showed that PilG formed dimers and tetramers. A three-dimensional (3-D) electron microscopy structure of the PilG multimer was determined using single-particle averaging applied to samples visualized by negative staining. Symmetry analysis of the unsymmetrized 3-D volume provided further evidence that the PilG multimer is a tetramer. The reconstruction also revealed an asymmetric bilobed structure approximately 125 Å in length and 80 Å in width. The larger lobe within the structure was identified as the N terminus by location of Ni-nitrilotriacetic acid nanogold particles to the N-terminal polyhistidine tag. We propose that the smaller lobe corresponds to the periplasmic domain of the protein, with the narrower “waist” region being the transmembrane section. This constitutes the first report of a 3-D structure of a member of the GspF family and suggests a physical basis for the role of the protein in linking cytoplasmic and periplasmic protein components of the type II secretion and type IV pilus biogenesis systems.

scholarly journals Cyclic Di-GMP and VpsR Induce the Expression of Type II Secretion in Vibrio cholerae

2017 ◽  
Vol 199 (19) ◽  
Author(s):  
Rudolph E. Sloup ◽  
Ashley E. Konal ◽  
Geoffrey B. Severin ◽  
Michelle L. Korir ◽  
Mira M. Bagdasarian ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Protein Complexes ◽  
Second Messenger ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Secretion Systems ◽  
Content Type ◽  
Severe Diarrhea ◽  
Wide Range ◽  
Genes Encoding

ABSTRACT Vibrio cholerae is a human pathogen that alternates between growth in environmental reservoirs and infection of human hosts, causing severe diarrhea. The second messenger cyclic di-GMP (c-di-GMP) mediates this transition by controlling a wide range of functions, such as biofilms, virulence, and motility. Here, we report that c-di-GMP induces expression of the extracellular protein secretion (eps) gene cluster, which encodes the type II secretion system (T2SS) in V. cholerae. Analysis of the eps genes confirmed the presence of two promoters located upstream of epsC, the first gene in the operon, one of which is induced by c-di-GMP. This induction is directly mediated by the c-di-GMP-binding transcriptional activator VpsR. Increased expression of the eps operon did not impact secretion of extracellular toxin or biofilm formation but did increase expression of the pseudopilin protein EpsG on the cell surface. IMPORTANCE Type II secretion systems (T2SSs) are the primary molecular machines by which Gram-negative bacteria secrete proteins and protein complexes that are folded and assembled in the periplasm. The substrates of T2SSs include extracellular factors, such as proteases and toxins. Here, we show that the widely conserved second messenger cyclic di-GMP (c-di-GMP) upregulates expression of the eps genes encoding the T2SS in the pathogen V. cholerae via the c-di-GMP-dependent transcription factor VpsR.

scholarly journals Role of fimV in type II secretion system-dependent protein secretion of Pseudomonas aeruginosa on solid medium

Microbiology ◽  
2011 ◽  
Vol 157 (7) ◽  
pp. 1945-1954 ◽  
Author(s):  
Gérard P. F. Michel ◽  
Anthony Aguzzi ◽  
Geneviève Ball ◽  
Chantal Soscia ◽  
Sophie Bleves ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Solid Medium ◽  
Early Stage ◽  
Growth Conditions ◽  
Classical Type ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Secretion Systems ◽  
Type Ii Secretion System ◽  
Dependent Protein

Although classical type II secretion systems (T2SSs) are widely present in Gram-negative bacteria, atypical T2SSs can be found in some species. In Pseudomonas aeruginosa, in addition to the classical T2SS Xcp, it was reported that two genes, xphA and xqhA, located outside the xcp locus were organized in an operon (PaQa) which encodes the orphan PaQa subunit. This subunit is able to associate with other components of the classical Xcp machinery to form a functional hybrid T2SS. In the present study, using a transcriptional lacZ fusion, we found that the PaQa operon was more efficiently expressed (i) on solid LB agar than in liquid LB medium, (ii) at 25 °C than at 37 °C and (iii) at an early stage of growth. These results suggested an adaptation of the hybrid system to particular environmental conditions. Transposon mutagenesis led to the finding that vfr and fimV genes are required for optimal expression of the orphan PaQa operon in the defined growth conditions used. Using an original culturing device designed to monitor secretion on solid medium, the ring-plate system, we found that T2SS-dependent secretion of exoproteins, namely the elastase LasB, was affected in a fimV deletion mutant. Our findings led to the discovery of an interplay between FimV and the global regulator Vfr triggering the modulation of the level of Vfr and consequently the modulation of T2SS-dependent secretion on solid medium.

Type II Secretion and Type IV Pili of Francisella

2007 ◽  
Vol 1105 (1) ◽  
pp. 187-201 ◽  
Author(s):  
A. FORSBERG ◽  
T. GUINA
Keyword(s):  
Type Iv Pili ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Type Iv

scholarly journals Type IV Fimbrial Biogenesis Is Required for Protease Secretion and Natural Transformation in Dichelobacter nodosus

2007 ◽  
Vol 189 (14) ◽  
pp. 5022-5033 ◽  
Author(s):  
Xiaoyan Han ◽  
Ruth M. Kennan ◽  
Dane Parker ◽  
John K. Davies ◽  
Julian I. Rood
Keyword(s):  
Natural Transformation ◽  
Secretion System ◽  
Extracellular Protease ◽  
Classical Type ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Dichelobacter Nodosus ◽  
Type Iv ◽  
Type Ii Secretion System ◽  
Protease Secretion

ABSTRACT The objective of this study was to develop an understanding of the molecular mechanisms by which type IV fimbrial biogenesis, natural transformation, and protease secretion are linked in the ovine foot rot pathogen, Dichelobacter nodosus. We have shown that like the D. nodosus fimbrial subunit FimA, the pilin-like protein PilE and the FimN, FimO, and FimP proteins, which are homologs of PilB, PilC, and PilD from Pseudomonas aeruginosa, are essential for fimbrial biogenesis and natural transformation, indicating that transformation requires an intact type IV fimbrial apparatus. The results also showed that extracellular protease secretion in the fimN, fimO, fimP, and pilE mutants was significantly reduced, which represents the first time that PilB, PilC, and PilE homologs have been shown to be required for the secretion of unrelated extracellular proteins in a type IV fimbriate bacterium. Quantitative real-time PCR analysis of the three extracellular protease genes aprV2, aprV5, and bprV showed that the effects on protease secretion were not mediated at the transcriptional level. Bioinformatic analysis did not identify a classical type II secretion system, and the putative fimbrial biogenesis gene pilQ was the only outer membrane secretin gene identified. Based on these results, it is postulated that in D. nodosus, protease secretion occurs by a type II secretion-related process that directly involves components of the type IV fimbrial biogenesis machinery, which represents the only type II secretion system encoded by the small genome of this highly evolved pathogen.

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