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  1. Article ; Online: The Escherichia coli Outer Membrane β-Barrel Assembly Machinery (BAM) Crosstalks with the Divisome

    Elisa Consoli / Joen Luirink / Tanneke den Blaauwen

    International Journal of Molecular Sciences, Vol 22, Iss 12101, p

    2021  Volume 12101

    Abstract: The BAM is a macromolecular machine responsible for the folding and the insertion of integral proteins into the outer membrane of diderm Gram-negative bacteria. In Escherichia coli , it consists of a transmembrane β-barrel subunit, BamA, and four outer ... ...

    Abstract The BAM is a macromolecular machine responsible for the folding and the insertion of integral proteins into the outer membrane of diderm Gram-negative bacteria. In Escherichia coli , it consists of a transmembrane β-barrel subunit, BamA, and four outer membrane lipoproteins (BamB-E). Using BAM-specific antibodies, in E. coli cells, the complex is shown to localize in the lateral wall in foci. The machinery was shown to be enriched at midcell with specific cell cycle timing. The inhibition of septation by aztreonam did not alter the BAM midcell localization substantially. Furthermore, the absence of late cell division proteins at midcell did not impact BAM timing or localization. These results imply that the BAM enrichment at the site of constriction does not require an active cell division machinery. Expression of the Tre1 toxin, which impairs the FtsZ filamentation and therefore midcell localization, resulted in the complete loss of BAM midcell enrichment. A similar effect was observed for YidC, which is involved in the membrane insertion of cell division proteins in the inner membrane. The presence of the Z-ring is needed for preseptal peptidoglycan (PG) synthesis. As BAM was shown to be embedded in the PG layer, it is possible that BAM is inserted preferentially simultaneously with de novo PG synthesis to facilitate the insertion of OMPs in the newly synthesized outer membrane.
    Keywords Escherichia coli ; β-barrel assembly machinery ; BAM complex ; divisome ; Sec machinery ; immunolabelling ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
    Subject code 570
    Language English
    Publishing date 2021-11-01T00:00:00Z
    Publisher MDPI AG
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: Overexpression of the Bam Complex Improves the Production of Chlamydia trachomatis MOMP in the E. coli Outer Membrane

    Dung T. Huynh / Wouter S. P. Jong / Gregory M. Koningstein / Peter van Ulsen / Joen Luirink

    International Journal of Molecular Sciences, Vol 23, Iss 7393, p

    2022  Volume 7393

    Abstract: A licensed Chlamydia trachomatis (Ct) vaccine is not yet available. Recombinant Chlamydia trachomatis major outer membrane protein ( Ct -MOMP), the most abundant constituent of the chlamydial outer membrane complex, is considered the most attractive ... ...

    Abstract A licensed Chlamydia trachomatis (Ct) vaccine is not yet available. Recombinant Chlamydia trachomatis major outer membrane protein ( Ct -MOMP), the most abundant constituent of the chlamydial outer membrane complex, is considered the most attractive candidate for subunit-based vaccine formulations. Unfortunately, Ct -MOMP is difficult to express in its native structure in the E. coli outer membrane (OM). Here, by co-expression of the Bam complex, we improved the expression and localization of recombinant Ct -MOMP in the E. coli OM. Under these conditions, recombinant Ct -MOMP appeared to assemble into a β-barrel conformation and express domains at the cell surface indicative of correct folding. The data indicate that limited availability of the Bam complex can be a bottleneck for the production of heterologous OM vaccine antigens, information that is also relevant for strategies aimed at producing recombinant OMV-based vaccines.
    Keywords Chlamydia trachomatis major outer membrane protein ; β-barrel assembly machinery ; outer membrane protein ; E. coli ; OMV-based vaccine ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
    Subject code 612
    Language English
    Publishing date 2022-07-01T00:00:00Z
    Publisher MDPI AG
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: Interrogating the Essential Bacterial Cell Division Protein FtsQ with Fragments Using Target Immobilized NMR Screening (TINS)

    Marjolein Glas / Eiso AB / Johan Hollander / Gregg Siegal / Joen Luirink / Iwan de Esch

    International Journal of Molecular Sciences, Vol 20, Iss 15, p

    2019  Volume 3684

    Abstract: The divisome is a large protein complex that regulates bacterial cell division and therefore represents an attractive target for novel antibacterial drugs. In this study, we report on the ligandability of FtsQ, which is considered a key component of the ... ...

    Abstract The divisome is a large protein complex that regulates bacterial cell division and therefore represents an attractive target for novel antibacterial drugs. In this study, we report on the ligandability of FtsQ, which is considered a key component of the divisome. For this, the soluble periplasmic domain of Escherichia coli FtsQ was immobilized and used to screen a library of 1501 low molecular weight (< 300 Da), synthetic compounds for those that interact with the protein. A primary screen was performed using target immobilized NMR screening (TINS) and yielded 72 hits. Subsequently, these hits were validated in an orthogonal assay. At first, we aimed to do this using surface plasmon resonance (SPR), but the lack of positive control hampered optimization of the experiment. Alternatively, a two-dimensional heteronuclear single quantum coherence (HSQC) NMR spectrum of FtsQ was obtained and used to validate these hits by chemical shift perturbation (CSP) experiments. This resulted in the identification of three fragments with weak affinity for the periplasmic domain of FtsQ, arguing that the ligandability of FtsQ is low. While this indicates that developing high affinity ligands for FtsQ is far from straightforward, the identified hit fragments can help to further interrogate FtsQ interactions.
    Keywords bacterial cell division ; antibacterials ; Escherichia coli ; fragment screening ; divisome ; FtsQ ; NMR ; TINS ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
    Subject code 540 ; 500
    Language English
    Publishing date 2019-07-01T00:00:00Z
    Publisher MDPI AG
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article ; Online: Comparing autotransporter β-domain configurations for their capacity to secrete heterologous proteins to the cell surface.

    Wouter S P Jong / Maaike Schillemans / Corinne M Ten Hagen-Jongman / Joen Luirink / Peter van Ulsen

    PLoS ONE, Vol 13, Iss 2, p e

    2018  Volume 0191622

    Abstract: Monomeric autotransporters have been extensively used for export of recombinant proteins to the cell surface of Gram-negative bacteria. A bottleneck in the biosynthesis of such constructs is the passage of the outer membrane, which is facilitated by the ... ...

    Abstract Monomeric autotransporters have been extensively used for export of recombinant proteins to the cell surface of Gram-negative bacteria. A bottleneck in the biosynthesis of such constructs is the passage of the outer membrane, which is facilitated by the β-domain at the C terminus of an autotransporter in conjunction with the Bam complex in the outer membrane. We have evaluated eight β-domain constructs for their capacity to secrete fused proteins to the cell surface. These constructs derive from the monomeric autotransporters Hbp, IgA protease, Ag43 and EstA and the trimeric autotransporter Hia, which all were selected because they have been previously used for secretion of recombinant proteins. We fused three different protein domains to the eight β-domain constructs, being a Myc-tag, the Hbp passenger and a nanobody or VHH domain, and assessed expression, membrane insertion and surface exposure. Our results show that expression levels differed considerably between the constructs tested. The constructs that included the β-domains of Hbp and IgA protease appeared the most efficient and resulted in expression levels that were detectable on Coomassie-stained SDS-PAGE gels. The VHH domain appeared the most difficult fusion partner to export, probably due to its complex immunoglobulin-like structure with a tertiary structure stabilized by an intramolecular disulfide bond. Overall, the Hbp β-domain compared favorably in exporting the fused recombinant proteins, because it showed in every instance tested a good level of expression, stable membrane insertion and clear surface exposure.
    Keywords Medicine ; R ; Science ; Q
    Subject code 500
    Language English
    Publishing date 2018-01-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  5. Article ; Online: Posttranslational insertion of small membrane proteins by the bacterial signal recognition particle.

    Ruth Steinberg / Andrea Origi / Ana Natriashvili / Pinku Sarmah / Mariya Licheva / Princess M Walker / Claudine Kraft / Stephen High / Joen Luirink / Wei Q Shi / Martin Helmstädter / Maximilian H Ulbrich / Hans-Georg Koch

    PLoS Biology, Vol 18, Iss 9, p e

    2020  Volume 3000874

    Abstract: Small membrane proteins represent a largely unexplored yet abundant class of proteins in pro- and eukaryotes. They essentially consist of a single transmembrane domain and are associated with stress response mechanisms in bacteria. How these proteins are ...

    Abstract Small membrane proteins represent a largely unexplored yet abundant class of proteins in pro- and eukaryotes. They essentially consist of a single transmembrane domain and are associated with stress response mechanisms in bacteria. How these proteins are inserted into the bacterial membrane is unknown. Our study revealed that in Escherichia coli, the 27-amino-acid-long model protein YohP is recognized by the signal recognition particle (SRP), as indicated by in vivo and in vitro site-directed cross-linking. Cross-links to SRP were also observed for a second small membrane protein, the 33-amino-acid-long YkgR. However, in contrast to the canonical cotranslational recognition by SRP, SRP was found to bind to YohP posttranslationally. In vitro protein transport assays in the presence of a SecY inhibitor and proteoliposome studies demonstrated that SRP and its receptor FtsY are essential for the posttranslational membrane insertion of YohP by either the SecYEG translocon or by the YidC insertase. Furthermore, our data showed that the yohP mRNA localized preferentially and translation-independently to the bacterial membrane in vivo. In summary, our data revealed that YohP engages an unique SRP-dependent posttranslational insertion pathway that is likely preceded by an mRNA targeting step. This further highlights the enormous plasticity of bacterial protein transport machineries.
    Keywords Biology (General) ; QH301-705.5
    Subject code 570
    Language English
    Publishing date 2020-09-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  6. Article: Salmonella outer membrane vesicles displaying high densities of pneumococcal antigen at the surface offer protection against colonization

    Kuipers, Kirsten / Corinne M. ten Hagen-Jongman / Elles Simonetti / Fred van Opzeeland / Joen Luirink / Maria H. Daleke-Schermerhorn / Marien I. de Jonge / Wouter S.P. Jong

    Vaccine. 2015 Apr. 21, v. 33, no. 17

    2015  

    Abstract: Bacterial outer membrane vesicles (OMVs) are attractive vaccine formulations because they have intrinsic immunostimulatory properties. In principle, heterologous antigens incorporated into OMVs will elicit specific immune responses, especially if ... ...

    Abstract Bacterial outer membrane vesicles (OMVs) are attractive vaccine formulations because they have intrinsic immunostimulatory properties. In principle, heterologous antigens incorporated into OMVs will elicit specific immune responses, especially if presented at the vesicle surface and thus optimally exposed to the immune system. In this study, we explored the feasibility of our recently developed autotransporter Hbp platform, designed to efficiently and simultaneously display multiple antigens at the surface of bacterial OMVs, for vaccine development. Using two Streptococcus pneumoniae proteins as model antigens, we showed that intranasally administered Salmonella OMVs displaying high levels of antigens at the surface induced strong protection in a murine model of pneumococcal colonization, without the need for a mucosal adjuvant. Importantly, reduction in bacterial recovery from the nasal cavity was correlated with local production of antigen-specific IL-17A. Furthermore, the protective efficacy and the production of antigen-specific IL-17A, and local and systemic IgGs, were all improved at increased concentrations of the displayed antigen. This discovery highlights the importance of an adequate antigen expression system for development of recombinant OMV vaccines. In conclusion, our findings demonstrate the suitability of the Hbp platform for development of a new generation of OMV vaccines, and illustrate the potential of using this approach to develop a broadly protective mucosal pneumococcal vaccine.
    Keywords adjuvants ; animal models ; immune response ; immune system ; immunoglobulin G ; interleukin-17 ; nasal cavity ; Salmonella ; Streptococcus pneumoniae ; surface antigens ; vaccine development ; vaccines
    Language English
    Dates of publication 2015-0421
    Size p. 2022-2029.
    Publishing place Elsevier Ltd
    Document type Article
    ZDB-ID 605674-x
    ISSN 1873-2518 ; 0264-410X
    ISSN (online) 1873-2518
    ISSN 0264-410X
    DOI 10.1016/j.vaccine.2015.03.010
    Database NAL-Catalogue (AGRICOLA)

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  7. Article ; Online: Characterization of ftsZ mutations that render Bacillus subtilis resistant to MinC.

    Inês Filipa Fernandes de Oliveira / Anabela de Sousa Borges / Viola Kooij / Jeremy Bartosiak-Jentys / Joen Luirink / Dirk-Jan Scheffers

    PLoS ONE, Vol 5, Iss 8, p e

    2010  Volume 12048

    Abstract: Cell division in Bacillus subtilis occurs precisely at midcell. Positional control of cell division is exerted by two mechanisms: nucleoid occlusion, through Noc, which prevents division through nucleoids, and the Min system, where the combined action of ...

    Abstract Cell division in Bacillus subtilis occurs precisely at midcell. Positional control of cell division is exerted by two mechanisms: nucleoid occlusion, through Noc, which prevents division through nucleoids, and the Min system, where the combined action of the MinC, D and J proteins prevents formation of the FtsZ ring at cell poles or recently completed division sites.We used a genetic screen to identify mutations in ftsZ that confer resistance to the lethal overexpression of the MinC/MinD division inhibitor. The FtsZ mutants were purified and found to polymerize to a similar or lesser extent as wild type FtsZ, and all mutants displayed reduced GTP hydrolysis activity indicative of a reduced polymerization turnover. We found that even though the mutations conferred in vivo resistance to MinC/D, the purified FtsZ mutants did not display strong resistance to MinC in vitro.Our results show that in B. subtilis, overproduction of MinC can be countered by mutations that alter FtsZ polymerization dynamics. Even though it would be very likely that the FtsZ mutants found depend on other Z-ring stabilizing proteins such as ZapA, FtsA or SepF, we found this not to be the case. This indicates that the cell division process in B. subtilis is extremely robust.
    Keywords Medicine ; R ; Science ; Q
    Subject code 571
    Language English
    Publishing date 2010-08-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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