scholarly journals Bam complex-mediated assembly of bacterial outer membrane proteins synthesized in an in vitro translation system

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sunyia Hussain ◽  
Janine H. Peterson ◽  
Harris D. Bernstein
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
In Vitro Translation ◽  
Translation System ◽  
Bam Complex ◽  
Bacterial Outer Membrane

scholarly journals Formation and function of bacterial outer membrane proteins- incorporated liposomes using an in vitro translation system

2021 ◽  
Author(s):  
Koki Kamiya
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Lipid Membranes ◽  
Outer Membrane Proteins ◽  
Sucrose Density Gradient ◽  
In Vitro Translation ◽  
Translation System ◽  
Patch Clamp Technique ◽  
E Coli

Abstract Outer membrane proteins (OMPs), located on the outer membrane of gram-negative bacteria, have a β-strand structure and form nanopores, which allow passage of ions, sugars, and small molecules. Recently, OMPs have been used as sensing elements to detect biological molecules. OMPs are normally expressed and purified from E. coli.. Although the cell-free synthesis of OMPs, such as OmpA and OmpG, is achieved in the presence of liposomes and periplasmic chaperones, the amount of OmpA and OmpG incorporated into the nano-sized liposomes is not clear. In this study, after in vitro translation, the incorporation of OmpG into purified nano-sized liposomes, with various lipid compositions, was investigated. Liposomes containing the synthesized OmpG were purified using a stepwise sucrose density gradient. We report that liposomes prepared with the E. coli lipid extract (PE/PG) had the highest amount of OmpG incorporated compared to liposomes with other lipid compositions, as detected by recording the current across the OmpG containing liposomes using the patch clamp technique. This study reveals some of the requirements for the insertion and refolding of OMPs synthesized by the in vitro translation system into lipid membranes, which plays a role in the biological sensing of various molecules.

scholarly journals Versatile in vitro system to study translocation and functional integration of bacterial outer membrane proteins

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Derrick Norell ◽  
Alexander Heuck ◽  
Thuy-Anh Tran-Thi ◽  
Hansjörg Götzke ◽  
Françoise Jacob-Dubuisson ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Functional Integration ◽  
In Vitro System ◽  
Bacterial Outer Membrane

scholarly journals The role of BamA in the biogenesis of beta-barrel membrane proteins

2014 ◽  
Vol 70 (a1) ◽  
pp. C578-C578
Author(s):  
Nicholas Noinaj ◽  
Adam Kuszak ◽  
Curtis Balusek ◽  
JC Gumbart ◽  
Petra Lukacik ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Crystal Structures ◽  
Outer Membrane Proteins ◽  
Hydrophobic Surface ◽  
Md Simulations ◽  
Structural Features ◽  
Bam Complex ◽  
Beta Barrel

Beta-barrel membrane proteins are essential for nutrient import, signaling, motility, and survival. In Gram-negative bacteria, the beta-barrel assembly machinery (BAM) complex is responsible for the biogenesis of beta-barrel outer membrane proteins (OMPs), with homologous complexes found in mitochondria and chloroplasts. Despite their essential roles, exactly how these OMPs are formed remains unknown. The BAM complex consists of a central and essential component called BamA (an OMP itself) and four lipoproteins called BamB-E. While the structure of the lipoproteins have been reported, the structure of full length BamA has been elusive. Recently though, we described the structure of BamA from two species of bacteria: Neisseria gonorrhoeae and Haemophilus ducreyi. BamA consists of a large periplasmic domain attached to a 16-strand transmembrane beta-barrel domain. Together, our crystal structures and molecule dynamics (MD) simulations revealed several structural features which gave clues to the mechanism by which BamA catalyzes beta-barrel assembly. The first is that the interior cavity is accessible in one BamA structure and conformationally closed in the other. Second, an exterior rim of the beta-barrel has a distinctly narrowed hydrophobic surface, locally destabilizing the outer membrane. Third, the beta-barrel can undergo lateral opening, suggesting a route from the interior cavity in BamA into the outer membrane. And fourth, a surface exposed exit pore positioned above the lateral opening site which may play a role in the biogenesis of extracellular loops. In this presentation, the crystal structures and MD simulations of BamA will be presented along with our work looking at the role of these four structural features in the role of BamA within the BAM complex.

scholarly journals Proteins Released by Helicobacter pylori In Vitro

2002 ◽  
Vol 184 (22) ◽  
pp. 6155-6162 ◽  
Author(s):  
Nayoung Kim ◽  
David L. Weeks ◽  
Jai Moo Shin ◽  
David R. Scott ◽  
Mary K. Young ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Membrane Proteins ◽  
Dna Binding ◽  
Outer Membrane ◽  
Nalidixic Acid ◽  
Outer Membrane Proteins ◽  
Gastric Epithelium ◽  
Synthesis Inhibitor ◽  
Vitro Analysis

ABSTRACT Secretion of proteins by Helicobacter pylori may contribute to gastric inflammation and epithelial damage. An in vitro analysis was designed to identify proteins released by mechanisms other than nonspecific lysis. The radioactivity of proteins in the supernatant was compared with that of the intact organism by two-dimensional gel phosphorimaging following a 4-h pulse-chase. The ratio of the amount of UreB, a known cytoplasmic protein, in the supernatant to that in the pellet was found to be 0.25, and this was taken as an index of lysis during the experiments (n = 6). Ratios greater than that of UreB were used to distinguish proteins that were selectively released into the medium. Thus, proteins enriched more than 10-fold in the supernatant compared to UreB were identified by mass spectrometry. Sixteen such proteins were present in the supernatant: VacA; a conserved secreted protein (HP1286); putative peptidyl cis-trans isomerase (HP0175); six proteins encoded by HP0305, HP0231, HP0973, HP0721, HP0129, and HP0902; thioredoxin (HP1458); single-stranded-DNA-binding 12RNP2 precursor (HP0827); histone-like DNA-binding protein HU (HP0835); ribosomal protein L11 (HP1202); a putative outer membrane protein (HP1564); and outer membrane proteins Omp21 (HP0913) and Omp20 (HP0912). All except HP0902, thioredoxin, HP0827, HP0835, and HP1202 had a signal peptide. When nalidixic acid, a DNA synthesis inhibitor, was added to inhibit cell division but not protein synthesis, to decrease possible contamination due to outer membrane shedding, two outer membrane proteins (Omp21 and Omp20) disappeared from the supernatant, and the amount of VacA also decreased. Thus, 13 proteins were still enriched greater than 10-fold in the medium after nalidixic acid treatment, suggesting these were released specifically, possibly by secretion. These proteins may be implicated in H. pylori-induced effects on the gastric epithelium.

Bacterial ß-Barrel Outer Membrane Proteins

2009 ◽  
pp. 182-207
Author(s):  
Pantelis G. Bagos ◽  
Stavros J. Hamodrakas
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Bacterial Cell ◽  
Outer Membrane Proteins ◽  
Structural Features ◽  
Structure And Function ◽  
Structural Motif ◽  
Functional Roles ◽  
Bacterial Outer Membrane ◽  
And Function

ß-barrel outer membrane proteins constitute the second and less well-studied class of transmembrane proteins. They are present exclusively in the outer membrane of Gram-negative bacteria and presumably in the outer membrane of mitochondria and chloroplasts. During the last few years, remarkable advances have been made towards an understanding of their functional and structural features. It is now wellknown that ß-barrels are performing a large variety of biologically important functions for the bacterial cell. Such functions include acting as specific or non-specific channels, receptors for various compounds, enzymes, translocation channels, structural proteins, and adhesion proteins. All these functional roles are of great importance for the survival of the bacterial cell under various environmental conditions or for the pathogenic properties expressed by these organisms. This chapter reviews the currently available literature regarding the structure and function of bacterial outer membrane proteins. We emphasize the functional diversity expressed by a common structural motif such as the ß-barrel, and we provide evidence from the current literature for dozens of newly discovered families of transmembrane ß-barrels.

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

1991 ◽  
Vol 174 (5) ◽  
pp. 1167-1177 ◽  
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.

scholarly journals Catecholamine-Modulated Novel Surface-Exposed Adhesin LIC20035 ofLeptospiraspp. Binds Host Extracellular Matrix Components and Is Recognized by the Host during Infection

2017 ◽  
Vol 84 (6) ◽  
Author(s):  
Karukriti Kaushik Ghosh ◽  
Aman Prakash ◽  
Vinayagamurthy Balamurugan ◽  
Manish Kumar
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Membrane Surface ◽  
Outer Membrane Proteins ◽  
Extracellular Matrices ◽  
Content Type ◽  
Extracellular Matrix Components ◽  
Gene Transcripts ◽  
Host Tissues

ABSTRACTIn this study, the effect of the host stress hormone catecholamine onLeptospiragene transcripts encoding outer membrane proteins was investigated. There was no impact of catecholamine supplementation on thein vitrogrowth pattern ofLeptospira interrogans; however, 7 genes out of 41 were differentially transcribed, and the effect was reversed to the basal level in the presence of the antagonist propranolol. Comprehensive analysis of one of the differentially regulated proteins, LIC20035 (in serovar Copenhageni)/LB047 (in serovar Lai) (due to catecholamine supplementation), revealed immunogenicity and ability to adhere to host extracellular matrices. Protease accessibility assay and phase partition of integral membrane proteins ofLeptospirashowed LIC20035/LB047 to be an outer membrane surface-exposed protein. The recombinant LIC20035 protein can be serologically detected using human/bovine sera positive for leptospirosis. Moreover, the recombinant LIC20035 can bind to diverse host extracellular matrices, with a higher affinity toward collagen and chondroitin sulfate.IMPORTANCELeptospirosis is a neglected tropical disease of global importance. This study aimed to identify outer membrane proteins of pathogenicLeptospiraresponding to host chemical signals like catecholamines, with the potential to serve as virulence factors, new serodiagnostic antigens, and vaccine candidates. This study mimicked the plausible means by whichLeptospiraduring infection and hormonal stress intercepts host catecholamines to disseminate in host tissues.

scholarly journals Functions of the BamBCDE Lipoproteins Revealed by Bypass Mutations in BamA

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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