scholarly journals Roles of Internal Cysteines in the Function, Localization, and Reactivity of the TraV Outer Membrane Lipoprotein Encoded by the F Plasmid

2002 ◽  
Vol 184 (11) ◽  
pp. 3126-3129 ◽  
Author(s):  
Robin L. Harris ◽  
Philip M. Silverman
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Osmotic Shock ◽  
Wild Type ◽  
F Plasmid ◽  
Outer Membrane Lipoprotein ◽  
Numerous Cell ◽  
Mixed Disulfides ◽  
Membrane Lipoprotein

ABSTRACT We have examined the functional role of two internal cysteine residues of the F-plasmid TraV outer membrane lipoprotein. Each was mutated to a serine separately and together to yield three mutant traV genes: traV C10S, traV C18S, and traV C10S/C18S. All three cysteine mutations complemented a traV mutant for DNA donor activity and for sensitivity to donor-specific bacteriophage; however, when measured by a transduction assay, the donor-specific DNA bacteriophage sensitivities of the traV C18S and, especially, traV C10S/C18S mutant strains were significantly less than those of the traV + and traV C10S strains. Thus, unlike the Agrobacterium tumefaciens T-plasmid-encoded VirB7 outer membrane lipoprotein, TraV does not require either internal cysteine to retain significant biological activity. By Western blot analysis, all three mutant TraV proteins were shown to accumulate in the outer membrane. However, by nonreducing gel electrophoresis, wild-type TraV and especially the TraVC18S mutant were shown to form mixed disulfides with numerous cell envelope proteins. This was not observed with the TraVC10S or TraVC10S/C18S proteins. Thus, it appears that TraV C10 is unusually reactive and that this reactivity is reduced by C18, perhaps by intramolecular oxidation. Finally, whereas the TraVC10S and TraVC18S proteins fractionated primarily with the outer membrane, as did the wild-type protein, the TraVC10S/C18S protein was found in osmotic shock fluid and inner membrane fractions as well as outer membrane fractions. Hence, at least one cysteine is required for the efficient localization of TraV to the outer membrane.

scholarly journals Role of the Pilot Protein YscW in the Biogenesis of the YscC Secretin in Yersinia enterocolitica

2004 ◽  
Vol 186 (16) ◽  
pp. 5366-5375 ◽  
Author(s):  
Peter Burghout ◽  
Frank Beckers ◽  
Emmie de Wit ◽  
Ria van Boxtel ◽  
Guy R. Cornelis ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Yersinia Enterocolitica ◽  
Amino Acid Residues ◽  
Wild Type ◽  
C Terminus ◽  
Outer Membrane Lipoprotein ◽  
Protein Secretion System ◽  
Membrane Lipoprotein ◽  
Type Iii Protein Secretion

ABSTRACT The YscC secretin is a major component of the type III protein secretion system of Yersinia enterocolitica and forms an oligomeric structure in the outer membrane. In a mutant lacking the outer membrane lipoprotein YscW, secretion is strongly reduced, and it has been proposed that YscW plays a role in the biogenesis of the secretin. To study the interaction between the secretin and this putative pilot protein, YscC and YscW were produced in trans in a Y. enterocolitica strain lacking all other components of the secretion machinery. YscW expression increased the yield of oligomeric YscC and was required for its outer membrane localization, confirming the function of YscW as a pilot protein. Whereas the pilot-binding site of other members of the secretin family has been identified in the C terminus, a truncated YscC derivative lacking the C-terminal 96 amino acid residues was functional and stabilized by YscW. Pulse-chase experiments revealed that ∼30 min were required before YscC oligomerization was completed. In the absence of YscW, oligomerization was delayed and the yield of YscC oligomers was strongly reduced. An unlipidated form of the YscW protein was not functional, although it still interacted with the secretin and caused mislocalization of YscC even in the presence of wild-type YscW. Hence, YscW interacts with the unassembled YscC protein and facilitates efficient oligomerization, likely at the outer membrane.

Structure of the conservedFrancisellavirulence protein FvfA

2017 ◽  
Vol 73 (10) ◽  
pp. 814-821
Author(s):  
Subramania Kolappan ◽  
Karen Y. Lo ◽  
Chiao Ling Jennifer Shen ◽  
Julian A. Guttman ◽  
Lisa Craig
Keyword(s):  
Outer Membrane ◽  
Osmotic Shock ◽  
Single Amino Acid ◽  
Human Pathogen ◽  
Crystal Forms ◽  
Membrane Perturbation ◽  
C Terminus ◽  
Outer Membrane Lipoprotein ◽  
Membrane Lipoprotein ◽  
Similarities And Differences

Francisella tularensisis a potent human pathogen that invades and survives in macrophage and epithelial cells. Two identical proteins, FTT_0924 fromF. tularensissubsp.tularensisand FTL_1286 fromF. tularensissubsp.holarcticaLVS, have previously been identified as playing a role in protection of the bacteria from osmotic shock and its survival in macrophages. FTT_0924 has been shown to localize to the inner membrane, with its C-terminus exposed to the periplasm. Here, crystal structures of theF. novicidahomologue FTN_0802, which we call FvfA, in two crystal forms are reported at 1.8 Å resolution. FvfA differs from FTT_0924 and FTL_1286 by a single amino acid. FvfA has a DUF1471 fold that closely resembles theEscherichia coliouter membrane lipoprotein RscF, a component of a phosphorelay pathway involved in protecting bacteria from outer membrane perturbation. The structural and functional similarities and differences between these proteins and their implications forF. tularensispathogenesis are discussed.

scholarly journals SciN Is an Outer Membrane Lipoprotein Required for Type VI Secretion in Enteroaggregative Escherichia coli

2008 ◽  
Vol 190 (22) ◽  
pp. 7523-7531 ◽  
Author(s):  
Marie-Stéphanie Aschtgen ◽  
Christophe S. Bernard ◽  
Sophie De Bentzmann ◽  
Roland Lloubès ◽  
Eric Cascales
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Biofilm Formation ◽  
Cell Envelope ◽  
Gene Clusters ◽  
Type Vi Secretion ◽  
Outer Membrane Lipoprotein ◽  
In Vivo Labeling ◽  
Membrane Lipoprotein

ABSTRACT Enteroaggregative Escherichia coli (EAEC) is a pathogen implicated in several infant diarrhea or diarrheal outbreaks in areas of endemicity. Although multiple genes involved in EAEC pathogenesis have been identified, the overall mechanism of virulence is not well understood. Recently, a novel secretion system, called type VI secretion (T6S) system (T6SS), has been identified in EAEC and most animal or plant gram-negative pathogens. T6SSs are multicomponent cell envelope machines responsible for the secretion of at least two putative substrates, Hcp and VgrG. In EAEC, two copies of T6S gene clusters, called sci-1 and sci-2, are present on the pheU pathogenicity island. In this study, we focused our work on the sci-1 gene cluster. The Sci-1 apparatus is probably composed of all, or a subset of, the 21 gene products encoded on the cluster. Among these subunits, some are shared by all T6SSs identified to date, including a ClpV-type AAA+ ATPase (SciG) and an IcmF (SciS) and an IcmH (SciP) homologue, as well as a putative lipoprotein (SciN). In this study, we demonstrate that sciN is a critical gene necessary for T6S-dependent secretion of the Hcp-like SciD protein and for biofilm formation. We further show that SciN is a lipoprotein, as shown by the inhibition of its processing by globomycin and in vivo labeling with [3H]palmitic acid. SciN is tethered to the outer membrane and exposed in the periplasm. Sequestration of SciN at the inner membrane by targeting the +2 residue responsible for lipoprotein localization (Gly2Asp) fails to complement an sciN mutant for SciD secretion and biofilm formation. Together, these results support a model in which SciN is an outer membrane lipoprotein exposed in the periplasm and essential for the Sci-1 apparatus function.

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