scholarly journals Novel Bacterial Surface Display Systems Based on Outer Membrane Anchoring Elements from the Marine Bacterium Vibrio anguillarum

2008 ◽  
Vol 74 (14) ◽  
pp. 4359-4365 ◽  
Author(s):  
Zhao Yang ◽  
Qin Liu ◽  
Qiyao Wang ◽  
Yuanxing Zhang
Keyword(s):  
Outer Membrane ◽  
Signal Sequence ◽  
Surface Display ◽  
Vibrio Anguillarum ◽  
Display System ◽  
Outer Membrane Lipoprotein ◽  
Surface Localization ◽  
Terminal Amino ◽  
Membrane Lipoprotein ◽  
Display Systems

ABSTRACT Surface display of heterologous peptides and proteins such as receptors, antigens, and enzymes on live bacterial cells is of considerable value for various biotechnological and industrial applications. In this study, a series of novel cell surface display systems were examined by using Vibrio anguillarum outer membrane protein and outer membrane lipoprotein as anchoring motifs. These display systems consist of (i) the signal sequence and first 11 N-terminal amino acids of V. anguillarum outer membrane lipoprotein Wza, or the signal sequence and first 9 N-terminal amino acids of the mature major Escherichia coli lipoprotein Lpp, and (ii) transmembrane domains of V. anguillarum outer membrane proteins Omporf1, OmpU, or Omp26La. In order to assay the translocation efficiency of constructed display systems in bacteria, green fluorescent protein (GFP) was inserted to the systems and the results of GFP surface localization confirmed that four of the six surface display systems could successfully display GFP on the E. coli surface. For assaying its potential application in live bacteria carrier vaccines, an excellent display system Wza-Omporf1 was fused with the major capsid protein (MCP) of large yellow croaker iridovirus and introduced into attenuated V. anguillarum strain MVAV6203, and subsequent analysis of MCP surface localization proved that the novel display system Wza-Omporf1 could function as a strong tool in V. anguillarum carrier vaccine development.

scholarly journals Genes Required for Plasmid R64 Thin-Pilus Biogenesis: Identification and Localization of Products of the pilK, pilM, pilO, pilP, pilR, and pilT Genes

2002 ◽  
Vol 184 (2) ◽  
pp. 444-451 ◽  
Author(s):  
Daisuke Sakai ◽  
Teruya Komano
Keyword(s):  
Outer Membrane ◽  
Subcellular Fractionation ◽  
His Tag ◽  
Outer Membrane Lipoprotein ◽  
Pilus Biogenesis ◽  
Periplasmic Proteins ◽  
Terminal Amino ◽  
Membrane Lipoprotein ◽  
Prepilin Peptidase ◽  
Plasmid R64

ABSTRACT We have previously shown that the pilL, pilN, pilQ, pilS, pilU, and pilV genes of plasmid R64 encode outer membrane lipoprotein, secretin, cytoplasmic ATPase, major pilin, prepilin peptidase, and minor pilin, respectively, which are required for thin-pilus formation. In this work, we characterized the products of the remaining essential genes, pilK, pilM, pilO, pilP, pilR, and pilT, with regard to their localization and processing. Overexpression systems containing pilM, pilO, and pilP genes fused with N-terminal glutathione S-transferase (GST) or a His tag were constructed. Overproduced proteins were purified and used to raise specific antibodies. Localization of PilM, PilO, and PilP proteins was performed by Western blot analysis with anti-GST-PilM, anti-PilO, and anti-PilP antibodies, respectively. The pilK, pilR, and pilT products were produced with a C-terminal His tag and then detected by anti-His tag antibody. Subcellular fractionation experiments with Escherichia coli cells producing R64 thin pili revealed that PilK, PilM, and PilR are inner membrane proteins, and PilP and PilT are periplasmic proteins. PilO protein was localized to the outer membrane in the presence of other Pil proteins, whereas it was localized to the cytoplasm in the absence of these proteins. Furthermore, the cleavage site of PilP protein was determined by N-terminal amino acid sequencing of purified mature PilP protein. We predict that PilK, PilM, PilO, PilP, and PilT proteins function as the components of the pilin transport apparatus and thin-pilus basal body.

scholarly journals Outer membrane lipoprotein biogenesis: Lol is not the end

2015 ◽  
Vol 370 (1679) ◽  
pp. 20150030 ◽  
Author(s):  
Anna Konovalova ◽  
Thomas J. Silhavy
Keyword(s):  
Outer Membrane ◽  
Signal Sequence ◽  
Cysteine Residue ◽  
Lipid Modification ◽  
Mature Protein ◽  
Bam Complex ◽  
Beta Barrel ◽  
Outer Membrane Lipoprotein ◽  
Lipid Moiety ◽  
Membrane Lipoprotein

Bacterial lipoproteins are lipid-anchored proteins that contain acyl groups covalently attached to the N-terminal cysteine residue of the mature protein. Lipoproteins are synthesized in precursor form with an N-terminal signal sequence (SS) that targets translocation across the cytoplasmic or inner membrane (IM). Lipid modification and SS processing take place at the periplasmic face of the IM. Outer membrane (OM) lipoproteins take the localization of lipoproteins (Lol) export pathway, which ends with the insertion of the N-terminal lipid moiety into the inner leaflet of the OM. For many lipoproteins, the biogenesis pathway ends here. We provide examples of lipoproteins that adopt complex topologies in the OM that include transmembrane and surface-exposed domains. Biogenesis of such lipoproteins requires additional steps beyond the Lol pathway. In at least one case, lipoprotein sequences reach the cell surface by being threaded through the lumen of a beta-barrel protein in an assembly reaction that requires the heteropentomeric Bam complex. The inability to predict surface exposure reinforces the importance of experimental verification of lipoprotein topology and we will discuss some of the methods used to study OM protein topology.

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