A putative multicopper protein secreted by an atypical type II secretion system involved in the reduction of insoluble electron acceptors in Geobacter sulfurreducens

Extracellular electron transfer onto Fe(III) oxides in Geobacter sulfurreducens is considered to require proteins that must be exported to the outer surface of the cell. In order to investigate this, the putative gene for OxpG, the pseudopilin involved in a type II general secretion pathway of Gram-negative bacteria, was deleted. The mutant was unable to grow with insoluble Fe(III) oxide as the electron acceptor. Growth on soluble Fe(III) was not affected. An analysis of proteins that accumulated in the periplasm of the oxpG mutant, but not in the wild-type, led to the identification of a secreted protein, OmpB. OmpB is predicted to be a multicopper protein, with highest homology to the manganese oxidase, MofA, from Leptothrix discophora. OmpB contains a potential Fe(III)-binding site and a fibronectin type III domain, suggesting a possible role for this protein in accessing Fe(III) oxides. OmpB was localized to the membrane fraction of G. sulfurreducens and in the supernatant of growing cultures, consistent with the type II secretion system exporting OmpB. A mutant in which ompB was deleted had the same phenotype as the oxpG mutant, suggesting that the failure to export OmpB was responsible for the inability of the oxpG-deficient mutant to reduce Fe(III) oxide. This is the first report that proposes a role for a multicopper oxidase-like protein in an anaerobic organism. These results further emphasize the importance of outer-membrane proteins in Fe(III) oxide reduction and suggest that outer-membrane proteins other than c-type cytochromes are required for Fe(III) oxide reduction in Geobacter species.

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Type II Secretion System Secretin PulD Localizes in Clusters in the Escherichia coli Outer Membrane

Journal of Bacteriology ◽

10.1128/jb.01138-08 ◽

2008 ◽

Vol 191 (1) ◽

pp. 161-168 ◽

Cited By ~ 36

Author(s):

Nienke Buddelmeijer ◽

Martin Krehenbrink ◽

Frédéric Pecorari ◽

Anthony P. Pugsley

Keyword(s):

Escherichia Coli ◽

Fluorescence Microscopy ◽

Outer Membrane ◽

Cellular Localization ◽

Fluorescent Protein ◽

Sodium Dodecyl ◽

Secretion System ◽

Type Ii Secretion ◽

Type Ii ◽

Type Ii Secretion System

ABSTRACT The cellular localization of a chimera formed by fusing a monomeric red fluorescent protein to the C terminus of the Klebsiella oxytoca type II secretion system outer membrane secretin PulD (PulD-mCherry) in Escherichia coli was determined in vivo by fluorescence microscopy. Like PulD, PulD-mCherry formed sodium dodecyl sulfate- and heat-resistant multimers and was functional in pullulanase secretion. Chromosome-encoded PulD-mCherry formed fluorescent foci on the periphery of the cell in the presence of high (plasmid-encoded) levels of its cognate chaperone, the pilotin PulS. Subcellular fractionation demonstrated that the chimera was located exclusively in the outer membrane under these circumstances. A similar localization pattern was observed by fluorescence microscopy of fixed cells treated with green fluorescent protein-tagged affitin, which binds with high affinity to an epitope in the N-terminal region of PulD. At lower levels of (chromosome-encoded) PulS, PulD-mCherry was less stable, was located mainly in the inner membrane, from which it could not be solubilized with urea, and did not induce the phage shock response, unlike PulD in the absence of PulS. The fluorescence pattern of PulD-mCherry under these conditions was similar to that observed when PulS levels were high. The complete absence of PulS caused the appearance of bright and almost exclusively polar fluorescent foci.

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Multiple approaches towards understanding the type II secretion system

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314094224 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C577-C577

Author(s):

Connie Lu ◽

Young-un Park ◽

Konstantin Korotkov ◽

Wei Mi ◽

Stewart Turley ◽

...

Keyword(s):

Outer Membrane ◽

Secretion System ◽

Crystal Formation ◽

Type Ii Secretion ◽

Type Ii ◽

Inner Membrane ◽

Membrane Pore ◽

Membrane Complex ◽

Type Ii Secretion System ◽

Folded Proteins

Transport of folded proteins across membranes is a feat accomplished by few biomacromolecular machines. One of the machineries able to do so is the sophisticated type II secretion system (T2SS). It can translocate key virulence factors from the bacterial periplasm into the lumen of the gut of the human host. A prime example is the secretion of cholera toxin by Vibrio cholerae. The T2SS consists of ~12 different proteins, most of these present in multiple copies, organized into three subassemblies: (i) the Inner Membrane Platform; (ii) the Pseudopilus in the periplasm, which acts most likely as a piston pushing exoproteins through the outer membrane pore; (iii) the Outer Membrane Complex, allowing passage of ~100 kDa folded proteins. We have determined crystal structures from more than a dozen T2SS domains, yet, a full understanding of the architecture and mechanism of action of the T2SS remains a formidable challenge. Our approaches include the use of "assistant-multimers" to promote recalcitrant multimer formation and of nanobodies to overcome reluctant crystal formation. The Inner Membrane Platform is interacting with the secretion ATPase GspE which most likely needs to be hexameric for full activity. Full-length GspE co-crystallized with its major partner, the cytoplasmic domain of GspL, revealed a tremendous flexibility of this ATPase, and, most unexpectedly, also the organization of the same linear arrangement of cyto-GspL domains throughout three entirely different crystal forms. Two very different hexamers of GspE were elucidated by linking the GspE subunit to the subunit of Hcp1, which successfully acted as an "assistant hexamer", inducing hexamer formation by GspE. The dodecameric nature of the ~ 850 kDa GspD, the major component of the Outer Membrane Complex, evident in earlier electron microscopy studies, was observed in the dodecameric ring-like helix in crystals of its N-terminal domain. The contacts between GspD and the inner-membrane protein GspC will be discussed as well as the remarkably frequent occurrence of dimers of Inner Membrane Platform domains. How dimers are co-assembled with an ATPase hexamer with C6 symmetry and the Outer Membrane Complex dodecamer with C12 symmetry remains one of the many fascinating outstanding questions of the T2SS.

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Functional Characterization of EpsC, a Component of the Type II Secretion System, in the Pathogenicity of Vibrio vulnificus

Infection and Immunity ◽

10.1128/iai.05351-11 ◽

2011 ◽

Vol 79 (10) ◽

pp. 4068-4080 ◽

Cited By ~ 12

Author(s):

Won Hwang ◽

Na Yeon Lee ◽

Juri Kim ◽

Mi-Ae Lee ◽

Kun-Soo Kim ◽

...

Keyword(s):

Vibrio Vulnificus ◽

Outer Membrane Proteins ◽

Functional Characterization ◽

Secretion System ◽

Host Cells ◽

Type Ii Secretion ◽

Type Ii ◽

Content Type ◽

Type Ii Secretion System ◽

Secreted Factors

ABSTRACTEpsC, one of the components comprising the type II secretion system (T2SS), was isolated from a human-pathogenic bacterium,Vibrio vulnificus, to evaluate its role in eliciting virulence. AnespC-deleted mutant ofV. vulnificusdisplayed a reduced cytotoxicity to the human cell line HEp-2 and an attenuated virulence in a mouse model. This mutant exhibited dramatic defects in the secretion of diverse extracellular proteins, such as outer membrane proteins, transporters, and the known secreted factors, notably, a hemolysin (VvhA) and an elastase (VvpE). A defect in its secretion of proteins was restored by intranscomplementation of the intactepsCgene. Analyses of cellular fractions revealed that VvhA and VvpE of theΔepsCmutant were not excreted outside the cell but were present mainly in the periplasmic space. Examination of aV. vulnificusmutant deficient in TolC, a component of the T1SS, showed that it is not involved in the secretion of VvhA and VvpE but that it is necessary for the secretion of another major toxin ofV. vulnificus, RtxA. Therefore, the T2SS is required forV. vulnificuspathogenicity, which is mediated by at least two secreted factors, VvhA and VvpE, via facilitating the secretion and exposure of these factors to host cells.

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Faculty Opinions recommendation of A putative multicopper protein secreted by an atypical type II secretion system involved in the reduction of insoluble electron acceptors in Geobacter sulfurreducens.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1033757.388949 ◽

2006 ◽

Author(s):

Tracy Palmer

Keyword(s):

Secretion System ◽

Electron Acceptors ◽

Geobacter Sulfurreducens ◽

Type Ii Secretion ◽

Type Ii ◽

Type Ii Secretion System ◽

Insoluble Electron Acceptors

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Alterations in Peptidoglycan Cross-Linking Suppress the Secretin Assembly Defect Caused by Mutation of GspA in the Type II Secretion System

Journal of Bacteriology ◽

10.1128/jb.00617-16 ◽

2017 ◽

Vol 199 (8) ◽

Cited By ~ 3

Author(s):

Elizabeth M. Vanderlinde ◽

Timothy G. Strozen ◽

Sara B. Hernández ◽

Felipe Cava ◽

S. Peter Howard

Keyword(s):

Outer Membrane ◽

Secretion System ◽

Cross Linking ◽

Type Ii Secretion ◽

Type Ii ◽

Assembly Model ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Type Ii Secretion System

ABSTRACT In Gram-negative bacteria, the peptidoglycan (PG) cell wall is a significant structural barrier for outer membrane protein assembly. In Aeromonas hydrophila, outer membrane multimerization of the type II secretion system (T2SS) secretin ExeD requires the function of the inner membrane assembly factor complex ExeAB. The putative mechanism of the complex involves the reorganization of PG and localization of ExeD, whereby ExeA functions by interacting with PG to form a site for secretin assembly and ExeB forms an interaction with ExeD. This mechanism led us to hypothesize that increasing the pore size of PG would circumvent the requirement for ExeA in the assembly of the ExeD secretin. Growth of A. hydrophila in 270 mM Gly reduced PG cross-links by approximately 30% and led to the suppression of secretin assembly defects in exeA strains and in those expressing ExeA mutants by enabling localization of the secretin in the outer membrane. We also established a heterologous ExeD assembly system in Escherichia coli and showed that ExeAB and ExeC are the only A. hydrophila proteins required for the assembly of the ExeD secretin in E. coli and that ExeAB-independent assembly of ExeD can occur upon overexpression of the d,d-carboxypeptidase PBP 5. These results support an assembly model in which, upon binding to PG, ExeA induces multimerization and pore formation in the sacculus, which enables ExeD monomers to interact with ExeB and assemble into a secretin that both is inserted in the outer membrane and crosses the PG layer to interact with the inner membrane platform of the T2SS. IMPORTANCE The PG layer imposes a strict structural impediment for the assembly of macromolecular structures that span the cell envelope and serve as virulence factors in Gram-negative species. This work revealed that by decreasing PG cross-linking by growth in Gly, the absolute requirement for the PG-binding activity of ExeA in the assembly of the ExeD secretin was alleviated in A. hydrophila. In a heterologous assembly model in E. coli, expression of the carboxypeptidase PBP 5 could relieve the requirement for ExeAB in the assembly of the ExeD secretin. These results provide some mechanistic details of the ExeAB assembly complex function, in which the PG-binding and oligomerization functions of ExeAB are used to create a pore in the PG that is required for secretin assembly.

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