An In Vitro Assay for Outer Membrane Protein Assembly by the BAM Complex

The outer-membrane protein OmpW of Vibrio cholerae was studied with respect to its structure, functional properties and regulation of expression. On SDS-PAGE, the membrane-associated form of OmpW protein (solubilized by either 0·1 % or 2 % SDS at 25 °C) migrated as a monomer of 19 kDa that changed to 21 kDa on boiling. The protein was hyperexpressed in Escherichia coli in the histidine-tagged form and the purified His6-OmpW (heated or unheated) migrated as a 23 kDa protein on SDS-PAGE. Circular dichroism and Fourier-transform infrared spectroscopic analyses of the recombinant protein showed the presence of β-structures (∼40 %) with minor amounts (8–15 %) of α-helix. These results were consistent with those obtained by computational analysis of the sequence data of the protein using the secondary structure prediction program Jnet. The recombinant protein did not exhibit any porin-like property in a liposome-swelling assay. An antiserum to the purified protein induced a moderate level (66·6 % and 33·3 % at 1 : 50 and 1 : 100 dilutions, respectively) of passive protection against live vibrio challenge in a suckling mouse model. OmpW-deficient mutants of V. cholerae strains were generated by insertion mutagenesis. In a competitive assay in mice, the intestinal colonization activities of these mutants were found to be either only marginally diminished (for O1 strains) or 10-fold less (for an O139 strain) as compared to those of the corresponding wild-type strains. The OmpW protein was expressed in vivo as well as in vitro in liquid culture medium devoid of glucose. Interestingly, the glucose-dependent regulation of OmpW expression was less prominent in a ToxR− mutant of V. cholerae. Further, the expression of OmpW protein was found to be dependent on in vitro cultural conditions such as temperature, salinity, and availability of nutrients or oxygen. These results suggest that the modulation of OmpW expression by environmental factors may be linked to the adaptive response of the organism under stress conditions.

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Purification of the Major Outer Membrane Protein of Azospirillum brasilense, Its Affinity to Plant Roots, and Its Involvement in Cell Aggregation

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.4.555 ◽

2001 ◽

Vol 14 (4) ◽

pp. 555-561 ◽

Cited By ~ 44

Author(s):

Saul Burdman ◽

Gabriella Dulguerova ◽

Yaacov Okon ◽

Edouard Jurkevitch

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Azospirillum Brasilense ◽

Outer Membrane Proteins ◽

Cell Aggregation ◽

Major Outer Membrane Protein ◽

Adhesion Assay ◽

Fab Fragments

The major outer membrane protein (MOMP) of the nitrogen-fixing rhizobacterium Azospirillum brasilense strain Cd was purified and isolated by gel filtration, and antiserum against this protein was obtained. A screening of the binding of outer membrane proteins (OMPs) of A. brasilense to membrane-immobilized root extracts of various plant species revealed different affinities for the MOMP, with a stronger adhesion to extracts of cereals in comparison with legumes and tomatoes. Moreover, this protein was shown to bind to roots of different cereal seedlings in an in vitro adhesion assay. Incubation of A. brasilense cells with MOMP-antiserum led to fast agglutination, indicating that the MOMP is a surface-exposed protein. Cells incubated with Fab fragments obtained from purified MOMP-antiserum immunoglobulin G exhibited significant inhibition of bacterial aggregation as compared with controls. Bacteria preincubated with Fab fragments showed weaker adhesion to corn roots in comparison to controls without Fab fragments. These findings suggest that the A. brasilense MOMP acts as an adhesin involved in root adsorption and cell aggregation of this bacterium.

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A novel O -linked glycan modulates Campylobacter jejuni major outer membrane protein-mediated adhesion to human histo-blood group antigens and chicken colonization

Open Biology ◽

10.1098/rsob.130202 ◽

2014 ◽

Vol 4 (1) ◽

pp. 130202 ◽

Cited By ~ 33

Author(s):

Jafar Mahdavi ◽

Necmettin Pirinccioglu ◽

Neil J. Oldfield ◽

Elisabet Carlsohn ◽

Jeroen Stoof ◽

...

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Blood Group ◽

Campylobacter Jejuni ◽

Outer Membrane Protein ◽

Ex Vivo ◽

Binding Capacity ◽

Major Outer Membrane Protein ◽

Blood Group Antigens

Campylobacter jejuni is an important cause of human foodborne gastroenteritis; strategies to prevent infection are hampered by a poor understanding of the complex interactions between host and pathogen. Previous work showed that C. jejuni could bind human histo-blood group antigens (BgAgs) in vitro and that BgAgs could inhibit the binding of C. jejuni to human intestinal mucosa ex vivo. Here, the major flagella subunit protein (FlaA) and the major outer membrane protein (MOMP) were identified as BgAg-binding adhesins in C. jejuni NCTC11168 . Significantly, the MOMP was shown to be O- glycosylated at Thr 268 ; previously only flagellin proteins were known to be O- glycosylated in C. jejuni . Substitution of MOMP Thr 268 led to significantly reduced binding to BgAgs. The O- glycan moiety was characterized as Gal(β1–3)-GalNAc(β1–4)-GalNAc(β1–4)-GalNAcα1-Thr 268 ; modelling suggested that O- glycosylation has a notable effect on the conformation of MOMP and this modulates BgAg-binding capacity. Glycosylation of MOMP at Thr 268 promoted cell-to-cell binding, biofilm formation and adhesion to Caco-2 cells, and was required for the optimal colonization of chickens by C. jejuni , confirming the significance of this O- glycosylation in pathogenesis.

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Involvement and necessity of the Cpx regulon in the event of aberrant β-barrel outer membrane protein assembly

Molecular Microbiology ◽

10.1111/j.1365-2958.2009.07042.x ◽

2010 ◽

Vol 75 (4) ◽

pp. 1033-1046 ◽

Cited By ~ 27

Author(s):

Henri Gerken ◽

Owen P. Leiser ◽

Drew Bennion ◽

Rajeev Misra

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Protein Assembly

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Structure of a Fragment of the Bacterial Outer Membrane Protein Assembly Machinery

The FASEB Journal ◽

10.1096/fasebj.21.5.a41-a ◽

2007 ◽

Vol 21 (5) ◽

Author(s):

Daniel E. Kahne

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Protein Assembly ◽

Bacterial Outer Membrane

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Immunogenicity of in vitro folded outer membrane protein PorA ofNeisseria meningitidis

FEMS Immunology & Medical Microbiology ◽

10.1111/j.1574-695x.2000.tb01434.x ◽

2000 ◽

Vol 27 (3) ◽

pp. 227-233 ◽

Cited By ~ 17

Author(s):

Carmen Jansen ◽

Betsy Kuipers ◽

Jenny Biezen ◽

Hans Cock ◽

Peter Ley ◽

...

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein

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In vitro trimerization of outer membrane protein PhoE

Biochimie ◽

10.1016/0300-9084(90)90143-5 ◽

1990 ◽

Vol 72 (2-3) ◽

pp. 177-182 ◽

Cited By ~ 21

Author(s):

H. de Cock ◽

D. Hekstra ◽

J. Tommassen

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein

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A growing toolbox of techniques for studying β-barrel outer membrane protein folding and biogenesis

Biochemical Society Transactions ◽

10.1042/bst20160020 ◽

2016 ◽

Vol 44 (3) ◽

pp. 802-809 ◽

Cited By ~ 9

Author(s):

Jim E. Horne ◽

Sheena E. Radford

Keyword(s):

Protein Folding ◽

Membrane Protein ◽

Outer Membrane ◽

Single Molecule ◽

Outer Membrane Protein ◽

Lipid Membrane ◽

Water Soluble ◽

Membrane Protein Folding ◽

Over 40 Years

Great strides into understanding protein folding have been made since the seminal work of Anfinsen over 40 years ago, but progress in the study of membrane protein folding has lagged behind that of their water soluble counterparts. Researchers in these fields continue to turn to more advanced techniques such as NMR, mass spectrometry, molecular dynamics (MD) and single molecule methods to interrogate how proteins fold. Our understanding of β-barrel outer membrane protein (OMP) folding has benefited from these advances in the last decade. This class of proteins must traverse the periplasm and then insert into an asymmetric lipid membrane in the absence of a chemical energy source. In this review we discuss old, new and emerging techniques used to examine the process of OMP folding and biogenesis in vitro and describe some of the insights and new questions these techniques have revealed.

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