Bacterial Phage Receptors, Versatile Tools for Display of Polypeptides on the Cell Surface

ABSTRACT Four outer membrane proteins of Escherichia coli were examined for their capabilities and limitations in displaying heterologous peptide inserts on the bacterial cell surface. The T7 tag or multiple copies of the myc epitope were inserted into loops 4 and 5 of the ferrichrome and phage T5 receptor FhuA. Fluorescence-activated cell sorting analysis showed that peptides of up to 250 amino acids were efficiently displayed on the surface of E. coli as inserts within FhuA. Strains expressing FhuA fusion proteins behaved similarly to those expressing wild-type FhuA, as judged by phage infection and colicin sensitivity. The vitamin B12 and phage BF23 receptor BtuB could display peptide inserts of at least 86 amino acids containing the T7 tag. In contrast, the receptors of the phages K3 and λ, OmpA and LamB, accepted only insertions in their respective loop 4 of up to 40 amino acids containing the T7 tag. The insertion of larger fragments resulted in inefficient transport and/or assembly of OmpA and LamB fusion proteins into the outer membrane. Cells displaying a foreign peptide fused to any one of these outer membrane proteins were almost completely recovered by magnetic cell sorting from a large pool of cells expressing the relevant wild-type platform protein only. Thus, this approach offers a fast and simple screening procedure for cells displaying heterologous polypeptides. The combination of FhuA, along with with BtuB and LamB, should provide a comprehensive tool for displaying complex peptide libraries of various insert sizes on the surface of E. coli for diverse applications.

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LamB, OmpA and OmpC are key altered outer membrane proteins in E. coli response to isopropanol

Toxicology Letters ◽

10.1016/j.toxlet.2011.05.742 ◽

2011 ◽

Vol 205 ◽

pp. S216

Author(s):

S. Wang ◽

D. Zhang ◽

X. Lin ◽

H. Li ◽

X. Peng

Keyword(s):

Membrane Proteins ◽

Outer Membrane ◽

Outer Membrane Proteins ◽

E Coli

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Why do the outer membrane proteins OmpF from E. coli and OprP from P. aeruginosa prefer trimers? Simulation studies

Journal of Molecular Graphics and Modelling ◽

10.1016/j.jmgm.2016.02.002 ◽

2016 ◽

Vol 65 ◽

pp. 1-7 ◽

Cited By ~ 9

Author(s):

Jitti Niramitranon ◽

Mark SP Sansom ◽

Prapasiri Pongprayoon

Keyword(s):

Membrane Proteins ◽

Outer Membrane ◽

Outer Membrane Proteins ◽

Simulation Studies ◽

E Coli

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BaeR participates in cephalosporins susceptibility by regulating the expression level of outer membrane proteins in Escherichia coli

The Journal of Biochemistry ◽

10.1093/jb/mvaa100 ◽

2020 ◽

Author(s):

Shuaiyang Wang ◽

Chunbo You ◽

Fareed Qumar Memon ◽

Geyin Zhang ◽

Yawei Sun ◽

...

Keyword(s):

Escherichia Coli ◽

Membrane Proteins ◽

Outer Membrane ◽

Efflux Pump ◽

Outer Membrane Proteins ◽

Expression Level ◽

Antibiotics Resistance ◽

Dilution Method ◽

Expression Levels ◽

E Coli

Abstract The two-component system BaeSR participates in antibiotics resistance of Escherichia coli. To know whether the outer membrane proteins involve in the antibiotics resistance mediated by BaeSR, deletion of acrB was constructed and the recombined plasmid p-baeR was introduced into E. coli K12 and K12△acrB. Minimum inhibitory concentrations (MICs) of antibacterial agents were determined by 2-fold broth micro-dilution method. Gene expressions related with major outer membrane proteins and multidrug efflux pump-related genes were determined by real-time quantitative reverse transcription polymerase chain reaction. The results revealed that the MICs of K12ΔacrB to the tested drugs except for gentamycin and amikacin decreased 2- to 16.75-folds compared with those of K12. When BaeR was overexpressed, the MICs of K12ΔacrB/p-baeR to ceftiofur and cefotaxime increased 2.5- and 2-fold, respectively, compared with their corresponding that of K12△acrB. At the same time, the expression levels of ompC, ompF, ompW, ompA and ompX showed significant reduction in K12ΔacrB/p-baeR as compared with K12△acrB. Moreover, the expression levels of ompR, marA, rob and tolC also significantly ‘decreased’ in K12ΔacrB/p-baeR. These findings indicated that BaeR overproduction can decrease cephalosporins susceptibility in acrB-free E. coli by decreasing the expression level of outer membrane proteins.

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Extrusion of actin-positive strands from HEp-2 and Int 407 cells caused by outer membrane preparations of enteropathogenicEscherichia coliand specific attachment of wild type bacteria to the strands

Canadian Journal of Microbiology ◽

10.1139/w01-058 ◽

2001 ◽

Vol 47 (8) ◽

pp. 727-734 ◽

Cited By ~ 4

Author(s):

Sukumaran Sunil Kumar ◽

Vasantha Malladi ◽

Krishnan Sankaran ◽

Richard Haigh ◽

Peter Williams ◽

...

Keyword(s):

Escherichia Coli ◽

Membrane Proteins ◽

Outer Membrane ◽

Outer Membrane Proteins ◽

Three Dimensional ◽

Secretory Proteins ◽

Enteropathogenic Escherichia Coli ◽

Wild Type ◽

Cytopathic Effects ◽

Cytoskeletal Elements

Enteropathogenic Escherichia coli (EPEC) causes persistent infantile diarrhoea. This nontoxigenic E. coli exhibits a complicated pathogenic mechanism in which its outer membrane proteins and type III secretory proteins damage intestinal epithelium and cause diarrhoea. In accordance with this, our previous study using HEp-2 cells demonstrated cytopathic effects caused by cell-free outer membrane preparations of EPEC. In this study, we report the extrusion of actin-positive strands from HEp-2 and Int 407 cells when treated with outer membrane preparations. An interesting observation of this work, perhaps relevant to the characteristic localized three-dimensional colony formation of EPEC, is the attachment of a wild type EPEC strain to these actin-positive strands.Key words: enteropathogenic Escherichia coli, actin, outer membrane proteins, cytoskeletal elements.

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Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane

Journal of Bacteriology ◽

10.1128/jb.00135-08 ◽

2008 ◽

Vol 190 (11) ◽

pp. 4001-4016 ◽

Cited By ~ 26

Author(s):

Wallace A. Kaserer ◽

Xiaoxu Jiang ◽

Qiaobin Xiao ◽

Daniel C. Scott ◽

Matthew Bauler ◽

...

Keyword(s):

Amino Acids ◽

Outer Membrane ◽

Cell Envelope ◽

Outer Membrane Proteins ◽

Binding Motif ◽

Inner Membrane ◽

E Coli ◽

Hybrid Proteins ◽

C Terminus ◽

N Terminus

ABSTRACT We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB + bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepAΔ3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.

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Localized adherence by enteropathogenic Escherichia coli is an inducible phenotype associated with the expression of new outer membrane proteins.

Journal of Experimental Medicine ◽

10.1084/jem.174.5.1167 ◽

1991 ◽

Vol 174 (5) ◽

pp. 1167-1177 ◽

Cited By ~ 64

Author(s):

J Vuopio-Varkila ◽

G K Schoolnik

Keyword(s):

Escherichia Coli ◽

Membrane Proteins ◽

Outer Membrane ◽

De Novo ◽

Outer Membrane Proteins ◽

Temporal Correlation ◽

Enteropathogenic Escherichia Coli ◽

E Coli

Enteropathogenic Escherichia coli grow as discrete colonies on the mucous membranes of the small intestine. A similar pattern can be demonstrated in vitro; termed localized adherence (LA), it is characterized by the presence of circumscribed clusters of bacteria attached to the surfaces of cultured epithelial cells. The LA phenotype was studied using B171, an O111:NM enteropathogenic E. coli (EPEC) strain, and HEp-2 cell monolayers. LA could be detected 30-60 min after exposure of HEp-2 cells to B171. However, bacteria transferred from infected HEp-2 cells to fresh monolayers exhibited LA within 15 min, indicating that LA is an inducible phenotype. Induction of the LA phenotype was found to be associated with de novo protein synthesis and changes in the outer membrane proteins, including the production of a new 18.5-kD polypeptide. A partial NH2-terminal amino acid sequence of this polypeptide was obtained and showed it to be identical through residue 12 to the recently described bundle-forming pilus subunit of EPEC. Expression of the 18.5-kD polypeptide required the 57-megadalton enteropathogenic E. coli adherence plasmid previously shown to be required for the LA phenotype in vitro and full virulence in vivo. This observation, the correspondence of the 18.5-kD polypeptide to an EPEC-specific pilus protein, and the temporal correlation of its expression with the development of the LA phenotype suggest that it may contribute to the EPEC colonial mode of growth.

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Export and assembly of outer membrane proteins in E. coli

Protein Export and Membrane Biogenesis - Advances in Cellular and Molecular Biology of Membranes and Organelles ◽

10.1016/s1874-5172(06)80011-8 ◽

1995 ◽

pp. 145-173 ◽

Cited By ~ 1

Author(s):

Jan Tommassen ◽

Hans de Cock

Keyword(s):

Membrane Proteins ◽

Outer Membrane ◽

Outer Membrane Proteins ◽

E Coli

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Functions of the BamBCDE Lipoproteins Revealed by Bypass Mutations in BamA

Journal of Bacteriology ◽

10.1128/jb.00401-20 ◽

2020 ◽

Vol 202 (21) ◽

Author(s):

Elizabeth M. Hart ◽

Thomas J. Silhavy

Keyword(s):

Membrane Proteins ◽

Outer Membrane ◽

Outer Membrane Proteins ◽

Gram Negative Bacteria ◽

Wild Type ◽

Gram Negative ◽

Bam Complex ◽

Essential Function ◽

The Individual

ABSTRACT The heteropentomeric β-barrel assembly machine (BAM complex) is responsible for folding and inserting a diverse array of β-barrel outer membrane proteins (OMPs) into the outer membrane (OM) of Gram-negative bacteria. The BAM complex contains two essential proteins, the β-barrel OMP BamA and a lipoprotein BamD, whereas the auxiliary lipoproteins BamBCE are individually nonessential. Here, we identify and characterize three bamA mutations, the E-to-K change at position 470 (bamAE470K), the A-to-P change at position 496 (bamAA496P), and the A-to-S change at position 499 (bamAA499S), that suppress the otherwise lethal ΔbamD, ΔbamB ΔbamC ΔbamE, and ΔbamC ΔbamD ΔbamE mutations. The viability of cells lacking different combinations of BAM complex lipoproteins provides the opportunity to examine the role of the individual proteins in OMP assembly. Results show that, in wild-type cells, BamBCE share a redundant function; at least one of these lipoproteins must be present to allow BamD to coordinate productively with BamA. Besides BamA regulation, BamD shares an additional essential function that is redundant with a second function of BamB. Remarkably, bamAE470K suppresses both, allowing the construction of a BAM complex composed solely of BamAE470K that is able to assemble OMPs in the absence of BamBCDE. This work demonstrates that the BAM complex lipoproteins do not participate in the catalytic folding of OMP substrates but rather function to increase the efficiency of the assembly process by coordinating and regulating the assembly of diverse OMP substrates. IMPORTANCE The folding and insertion of β-barrel outer membrane proteins (OMPs) are conserved processes in mitochondria, chloroplasts, and Gram-negative bacteria. In Gram-negative bacteria, OMPs are assembled into the outer membrane (OM) by the heteropentomeric β-barrel assembly machine (BAM complex). In this study, we probe the function of the individual BAM proteins and how they coordinate assembly of a diverse family of OMPs. Furthermore, we identify a gain-of-function bamA mutant capable of assembling OMPs independently of all four other BAM proteins. This work advances our understanding of OMP assembly and sheds light on how this process is distinct in Gram-negative bacteria.

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