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

2008 ◽  
Vol 191 (1) ◽  
pp. 161-168 ◽  
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.

scholarly journals Green Fluorescent Chimeras Indicate Nonpolar Localization of Pullulanase Secreton Components PulL and PulM

2006 ◽  
Vol 188 (8) ◽  
pp. 2928-2935 ◽  
Author(s):  
Nienke Buddelmeijer ◽  
Olivera Francetic ◽  
Anthony P. Pugsley
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescent Protein ◽  
Cell Envelope ◽  
Klebsiella Oxytoca ◽  
Subcellular Location ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Native Proteins ◽  
Type Ii Secretion System ◽  
Green Fluorescent

ABSTRACT The Klebsiella oxytoca pullulanase secreton (type II secretion system) components PulM and PulL were tagged at their N termini with green fluorescent protein (GFP), and their subcellular location was examined by fluorescence microscopy and fractionation. When produced at moderate levels without other secreton components in Escherichia coli, both chimeras were envelope associated, as are the native proteins. Fluorescent GFP-PulM was evenly distributed over the cell envelope, with occasional brighter foci. Under the same conditions, GFP-PulL was barely detectable in the envelope by fluorescence microscopy. When produced together with all other secreton components, GFP-PulL exhibited circumferential fluorescence, with numerous brighter patches. The envelope-associated fluorescence of GFP-PulL was almost completely abolished when native PulL was also produced, suggesting that the chimera cannot compete with PulL for association with other secreton components. The patches of GFP-PulL might represent functional secretons, since GFP-PulM also appeared in similar patches. GFP-PulM and GFP-PulL both appeared in spherical polar foci when made at high levels. In K. oxytoca, GFP-PulM was evenly distributed over the cell envelope, with few patches, whereas GFP-PulL showed only weak envelope-associated fluorescence. These data suggest that, in contrast to their Vibrio cholerae Eps secreton counterparts (M. Scott, Z. Dossani, and M. Sandkvist, Proc. Natl. Acad. Sci. USA 98:13978-13983, 2001), PulM and PulL do not localize specifically to the cell poles and that the Pul secreton is distributed over the cell surface.

scholarly journals The c-Type Cytochrome OmcA Localizes to the Outer Membrane upon Heterologous Expression in Escherichia coli

2008 ◽  
Vol 190 (14) ◽  
pp. 5127-5131 ◽  
Author(s):  
James W. Donald ◽  
Matthew G. Hicks ◽  
David J. Richardson ◽  
Tracy Palmer
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Shewanella Oneidensis ◽  
Type Ii Secretion ◽  
Type Ii ◽  
E Coli ◽  
Expression In Escherichia Coli ◽  
Type Ii Secretion System ◽  
Surface Localization ◽  
K 12

ABSTRACT We have functionally produced the outer membrane cytochrome OmcA from Shewanella oneidensis in Escherichia coli. Substrate accessibility experiments indicate that OmcA is surface exposed in an E. coli B strain but not in a K-12 strain. We show that a functional type II secretion system is required for surface localization.

scholarly journals Porcine Enterotoxigenic Escherichia coli Strains Differ in Their Capacity To Secrete Enterotoxins through Varying YghG Levels

2020 ◽  
Vol 86 (24) ◽  
Author(s):  
Haixiu Wang ◽  
Raquel Sanz Garcia ◽  
Eric Cox ◽  
Bert Devriendt
Keyword(s):  
Escherichia Coli ◽  
Secretion System ◽  
Farm Animals ◽  
Health Concern ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Content Type ◽  
E Coli ◽  
Type Ii Secretion System ◽  
Heat Labile

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) strains are important pathogens for humans and farm animals such as pigs. Porcine ETEC strains induce diarrhea through the production of heat-labile enterotoxin (LT) and/or heat-stable enterotoxins (pSTa/STb). Although LT secretion levels differ between porcine ETEC strains, and this has been linked to virulence, it is unclear whether ST secretion levels also differ between porcine ETEC strains. In addition, the molecular mechanism underlying different LT secretion levels has not been elucidated. In this work, multiple porcine ETEC strains were assessed for their capacity to produce and secrete the enterotoxins LT, pSTa, and STb. The strains differed greatly in their capacity to secrete LT, pSTa, and STb. Remarkably, in some strains, periplasmic production did not correlate with their ability to secrete LT, resulting in high periplasmic production and low LT secretion levels. Furthermore, the results indicated that the type II secretion system (T2SS) protein YghG plays a regulatory role in controlling LT secretion levels. These findings highlight YghG as an important mediator of the secretion of the heat-labile enterotoxin LT by porcine ETEC strains and provide better insights into ETEC enterotoxin secretion. IMPORTANCE Enterotoxigenic E. coli strains are a major health concern. Enterotoxins secreted by enterotoxigenic E. coli are crucial for diarrhea induction. Enterotoxin secretion levels differ between strains; however, it is currently unclear what drives these differences. The discrepancy in the production and secretion capacities of enterotoxins in ETEC is important to clarify their function involved in diarrhea induction. Our results further deepen our understanding of how type II secretion system (T2SS) components of ETEC control enterotoxin secretion levels and may lay the foundation for a better understanding of ETEC molecular pathogenesis.

scholarly journals YghG (GspSβ) Is a Novel Pilot Protein Required for Localization of the GspSβType II Secretion System Secretin of Enterotoxigenic Escherichia coli

2012 ◽  
Vol 80 (8) ◽  
pp. 2608-2622 ◽  
Author(s):  
Timothy G. Strozen ◽  
Gang Li ◽  
S. Peter Howard
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Secretion System ◽  
Enterotoxigenic Escherichia Coli ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Secretion Systems ◽  
Content Type ◽  
Insertional Inactivation ◽  
Prepilin Peptidase

ABSTRACTThe enterotoxigenicEscherichia coli(ETEC) pathotype, characterized by the prototypical strain H10407, is a leading cause of morbidity and mortality in the developing world. A major virulence factor of ETEC is the type II secretion system (T2SS) responsible for secretion of the diarrheagenic heat-labile enterotoxin (LT). In this study, we have characterized the two type II secretion systems, designated alpha (T2SSα) and beta (T2SSβ), encoded in the H10407 genome and describe the prevalence of both systems in otherE. colipathotypes. Under laboratory conditions, the T2SSβis assembled and functional in the secretion of LT into culture supernatant, whereas the T2SSαis not. Insertional inactivation of the three genes located upstream ofgspCβ(yghJ,pppA, andyghG) in the atypical T2SSβoperon revealed that YghJ is not required for assembly of the GspDβsecretin or secretion of LT, that PppA is likely the prepilin peptidase required for the function of T2SSβ, and that YghG is required for assembly of the GspDβsecretin and thus function of the T2SSβ. Mutational and physiological analysis further demonstrated that YghG (redesignated GspSβ) is a novel outer membrane pilotin protein that is integral for assembly of the T2SSβby localizing GspDβto the outer membrane, whereupon GspDβforms the macromolecular secretin multimer through which T2SSβsubstrates are translocated.

scholarly journals Multiple approaches towards understanding the type II secretion system

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.

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

Microbiology ◽  
2006 ◽  
Vol 152 (8) ◽  
pp. 2257-2264 ◽  
Author(s):  
Teena Mehta ◽  
Susan E. Childers ◽  
Richard Glaven ◽  
Derek R. Lovley ◽  
Tünde Mester
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Secretion System ◽  
Geobacter Sulfurreducens ◽  
Extracellular Electron Transfer ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Oxide Reduction ◽  
Type Ii Secretion System

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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