Overproduction of β-barrel outer membrane proteins in Escherichia coli BL21(DE3) induces hypervesiculation

Abstract

Aim: Gram-negative bacteria release outer membrane vesicles (OMVs) that fulfill many functions including survival during stress conditions, delivery of virulence factors, and nutrient acquisition. Additionally, they are increasingly used as an alternative for live bacteria in vaccine development and as a platform for bioengineering. Recently, OMVs have also been applied to express recombinant outer membrane proteins (OMPs) in their natural context as an alternative to the cumbersome reconstitution in liposomes. Here, we use an Escherichia coli strain that lacks four major OMPs for selective expression of the β-barrel assembly machinery (BAM) complex and PhoE in OMVs.

Methods: OMV production of Escherichia coli BL21(DE3) and its omp8 derivative upon overexpression of BAM and PhoE is compared and characterized.

Results: We find that overexpression of the BAM complex and PhoE causes a strong hypervesiculation phenotype, and the OMVs produced are intact and appear to recruit the BamA subunit of BAM and PhoE in their correctly folded and assembled conformations.

Conclusion: While the exact mechanism of hypervesiculation remains to be elucidated, it contributes to the suitability of the BL21(DE3)omp8 host strain to produce recombinant OMP-enriched OMVs that can be used for various purposes, including structural analysis.

Keywords

Outer membrane vesicles / β-barrel proteins / BL21(DE3) / BAM complex

References

[1] Guest RL.Cracking outer membrane biogenesis.Biochim Biophys Acta Mol Cell Res2023;1870:119405 PMCID:PMC9878550

[2] Lundstedt E,Ruiz N.Assembly and maintenance of lipids at the bacterial outer membrane.Chem Rev2021;121:5098-123 PMCID:PMC7981291

[3] Deghelt M,Govers SK.The outer membrane and peptidoglycan layer form a single mechanical device balancing turgor.BioRxiv2023;

[4] Schwechheimer C.Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions.Nat Rev Microbiol2015;13:605-19 PMCID:PMC5308417

[5] Sartorio MG,Feldman MF.Bacterial outer membrane vesicles: from discovery to applications.Annu Rev Microbiol2021;75:609-30 PMCID:PMC8500939

[6] Liu H,Zhang T.Engineered bacterial extracellular vesicles for osteoporosis therapy.Chem Eng J2022;450:138309

[7] Krishnan N,Holay M,Fang RH.Bacterial membrane vesicles for vaccine applications.Adv Drug Deliv Rev2022;185:114294

[8] Suri K,Huang D,Amiji M.Bacterial extracellular vesicle applications in cancer immunotherapy.Bioact Mater2023;22:551-66 PMCID:PMC9637733

[9] van Ulsen P, Rahman Su, Jong WS, Daleke-Schermerhorn MH, Luirink J. Type V secretion: from biogenesis to biotechnology.Biochim Biophys Acta2014;1843:1592-611

[10] Gerritzen MJH,Wijffels RH,Stork M.Bioengineering bacterial outer membrane vesicles as vaccine platform.Biotechnol Adv2017;35:565-74

[11] Daleke-Schermerhorn MH,Soprova Z.Decoration of outer membrane vesicles with multiple antigens by using an autotransporter approach.Appl Environ Microbiol2014;80:5854-65 PMCID:PMC4178611

[12] van den Berg van Saparoea HB, Houben D, de Jonge MI, Jong WSP, Luirink J. Display of recombinant proteins on bacterial outer membrane vesicles by using protein ligation.Appl Environ Microbiol2018;84:e02567-17 PMCID:PMC5881043

[13] Bernstein HD.Type V secretion: the autotransporter and two-partner secretion pathways.EcoSal Plus2010;4:20 PMCID:PMC4229083

[14] Phan TH,Huynh DT,Luirink J.Overproducing the BAM complex improves secretion of difficult-to-secrete recombinant autotransporter chimeras.Microb Cell Fact2021;20:176 PMCID:PMC8419823

[15] Huynh DT,Koningstein GM,Luirink J.Overexpression of the bam complex improves the production of Chlamydia trachomatis MOMP in the E. coli outer membrane.Int J Mol Sci2022;23 PMCID:PMC9266984

[16] Prilipov A,Van Gelder P,Koebnik R.Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins from E. coli.FEMS Microbiol Lett1998;163:65-72

[17] Roman-Hernandez G,Bernstein HD.Reconstitution of bacterial autotransporter assembly using purified components.Elife2014;3:e04234 PMCID:PMC4174580

[18] Jong WS,Ruijter E,Genevaux P.YidC is involved in the biogenesis of the secreted autotransporter hemoglobin protease.J Biol Chem2010;285:39682-90 PMCID:PMC3000949

[19] Consoli E,den Blaauwen T.The Escherichia coli outer membrane β-barrel assembly machinery (BAM) anchors the peptidoglycan layer by spanning it with all subunits.Int J Mol Sci2021;22:1853 PMCID:PMC7918090

[20] Steenhuis M,de Munnik SM.Inhibition of autotransporter biogenesis by small molecules.Mol Microbiol2019;112:81-98 PMCID:PMC6850105

[21] Bakelar J,Noinaj N.The structure of the β-barrel assembly machinery complex.Science2016;351:180-6 PMCID:PMC4883095

[22] Thoma J,Kalbermatter D,Fotiadis D.Protein-enriched outer membrane vesicles as a native platform for outer membrane protein studies.Commun Biol2018;1:23 PMCID:PMC6123736

[23] Tommassen J.PHO-regulon of Escherichia coli K12: a minireview.Ann Microbiol1982;133:243-9

[24] Noinaj N,Buchanan SK.Heat modifiability of outer membrane proteins from gram-negative bacteria.Methods Mol Biol2015;1329:51-6 PMCID:PMC4880013

[25] Burgess NK,Stanley AM.Beta-barrel proteins that reside in the Escherichia coli outer membrane in vivo demonstrate varied folding behavior in vitro.J Biol Chem2008;283:26748-58 PMCID:PMC3258919

[26] Hussain S.The Bam complex catalyzes efficient insertion of bacterial outer membrane proteins into membrane vesicles of variable lipid composition.J Biol Chem2018;293:2959-73 PMCID:PMC5827433

[27] Cowan SW,Rummel G.Crystal structures explain functional properties of two E. coli porins.Nature1992;358:727-33

[28] MacDonald IA.Offense and defense: microbial membrane vesicles play both ways.Res Microbiol2012;163:607-18 PMCID:PMC3518640

[29] Rigel NW,Ricci DP.BamE modulates the Escherichia coli beta-barrel assembly machine component BamA.J Bacteriol2012;194:1002-8 PMCID:PMC3294770

[30] Hall SCL,Sridhar P.Surface-tethered planar membranes containing the β-barrel assembly machinery: a platform for investigating bacterial outer membrane protein folding.Biophys J2021;120:5295-308 PMCID:PMC8715194

[31] Rigel NW,Silhavy TJ.Conformation-specific labeling of BamA and suppressor analysis suggest a cyclic mechanism for β-barrel assembly in Escherichia coli.Proc Natl Acad Sci U S A2013;110:5151-6 PMCID:PMC3612609

[32] Noinaj N,Gumbart JC.Structural insight into the biogenesis of β-barrel membrane proteins.Nature2013;501:385-90 PMCID:PMC3779476

[33] Wu T,Ruiz N,Silhavy TJ.Identification of a multicomponent complex required for outer membrane biogenesis in Escherichia coli.Cell2005;121:235-45

[34] Balhuizen MD,Haagsman HP.Outer membrane vesicle induction and isolation for vaccine development.Front Microbiol2021;12:629090 PMCID:PMC7889600

[35] Zhao X,Bu Y,Dong Z.Review on bacterial outer membrane vesicles: structure, vesicle formation, separation and biotechnological applications.Microb Cell Fact2025;24:27 PMCID:PMC11749425

[36] Mertes V,Mascherin E.Recombinant expression of Yersinia ruckeri outer membrane proteins in Escherichia coli extracellular vesicles.Protein Expr Purif2024;215:106409

[37] Rath P,Schaefer R.High-throughput screening of BAM inhibitors in native membrane environment.Nat Commun2023;14:5648 PMCID:PMC10499997

[38] Meuskens I,Chauhan N,Leo JC.A new strain collection for improved expression of outer membrane proteins.Front Cell Infect Microbiol2017;7:464 PMCID:PMC5681912

[39] Benn G,Prakaash D.OmpA controls order in the outer membrane and shares the mechanical load.Proc Natl Acad Sci U S A2024;121:e2416426121 PMCID:PMC11648852

[40] Tan WB.How bacteria establish and maintain outer membrane lipid asymmetry.Annu Rev Microbiol2024;78:553-73

[41] Roier S,Cakar F.A novel mechanism for the biogenesis of outer membrane vesicles in Gram-negative bacteria.Nat Commun2016;7:10515 PMCID:PMC4737802

[42] Benn G,Inns PG.Phase separation in the outer membrane of Escherichia coli.Proc Natl Acad Sci U S A2021;118:e2112237118 PMCID:PMC8612244

[43] Rassam P,Birkholz O.Supramolecular assemblies underpin turnover of outer membrane proteins in bacteria.Nature2015;523:333-6 PMCID:PMC4905513

[44] Gunasinghe SD,Stubenrauch CJ.The WD40 protein BamB mediates coupling of BAM complexes into assembly precincts in the bacterial outer membrane.Cell Rep2018;23:2782-94