The Synthetic Phenotype of Δ<italic toggle="yes">bamB</italic> Δ<italic toggle="yes">bamE</italic> Double Mutants Results from a Lethal Jamming of the Bam Complex by the Lipoprotein RcsF

ABSTRACT The selective permeability of the Gram-negative outer membrane (OM) is maintained by integral β-barrel outer membrane proteins (OMPs). The heteropentomeric β-barrel assembly machine (Bam) folds and inserts OMPs into the OM. Coordination of the essential proteins BamA and BamD is critical fo...

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Autores principales:	Elizabeth M. Hart, Meera Gupta, Martin Wühr, Thomas J. Silhavy
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	bacterial genetics Bam complex Escherichia coli OMP assembly Rcs stress response RcsF Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/4dd05e6662ea42ec8c5ba427e3593717
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spelling	oai:doaj.org-article:4dd05e6662ea42ec8c5ba427e35937172021-11-15T15:55:24ZThe Synthetic Phenotype of Δ<italic toggle="yes">bamB</italic> Δ<italic toggle="yes">bamE</italic> Double Mutants Results from a Lethal Jamming of the Bam Complex by the Lipoprotein RcsF10.1128/mBio.00662-192150-7511https://doaj.org/article/4dd05e6662ea42ec8c5ba427e35937172019-06-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00662-19https://doaj.org/toc/2150-7511ABSTRACT The selective permeability of the Gram-negative outer membrane (OM) is maintained by integral β-barrel outer membrane proteins (OMPs). The heteropentomeric β-barrel assembly machine (Bam) folds and inserts OMPs into the OM. Coordination of the essential proteins BamA and BamD is critical for OMP assembly and therefore the viability of the cell. The role of the nonessential lipoproteins BamBCE has yet to be characterized; however, genetic evidence suggests that they have nonoverlapping roles in OMP assembly. In this work, we quantify changes of the proteome in the conditional lethal ΔbamB ΔbamE double mutant. We show that cells lacking BamB and BamE have a global OMP defect that is a result of a lethal obstruction of an assembly-competent Bam complex by the lipoprotein RcsF. RcsF is a stress-sensing lipoprotein that is threaded through the lumen of abundant β-barrel OMPs by the Bam complex to expose the amino terminus on the cell surface. We demonstrate that simply removing this lipoprotein corrects the severe OMP assembly defect of the double mutant nearly as efficiently as a previously isolated suppressor mutation in bamA. We propose that BamB and BamE play crucial, nonoverlapping roles to coordinate the activities of BamA and BamD during OMP biogenesis. IMPORTANCE Protein assembly into lipid bilayers is an essential process that ensures the viability of diverse organisms. In Gram-negative bacteria, the heteropentomeric β-barrel assembly machine (Bam) folds and inserts proteins into the outer membrane. Due to its essentiality, outer membrane protein (OMP) assembly by the Bam complex is an attractive target for antibiotic development. Here, we show that the conditional lethal phenotype of a mutant lacking two of the three nonessential lipoproteins, BamB and BamE, is caused by lethal jamming of the stripped-down Bam complex by a normally surface-exposed lipoprotein, RcsF. The heterotrimeric Bam complex (BamA, BamD, BamC) is nearly as efficient as the wild-type complex in OMP assembly if RcsF is removed. Our study highlights the importance of BamB and BamE in regulating the interaction between BamA and BamD and expands our understanding of the role of the Bam complex in outer membrane biogenesis.Elizabeth M. HartMeera GuptaMartin WührThomas J. SilhavyAmerican Society for Microbiologyarticlebacterial geneticsBam complexEscherichia coliOMP assemblyRcs stress responseRcsFMicrobiologyQR1-502ENmBio, Vol 10, Iss 3 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	bacterial genetics Bam complex Escherichia coli OMP assembly Rcs stress response RcsF Microbiology QR1-502
spellingShingle	bacterial genetics Bam complex Escherichia coli OMP assembly Rcs stress response RcsF Microbiology QR1-502 Elizabeth M. Hart Meera Gupta Martin Wühr Thomas J. Silhavy The Synthetic Phenotype of Δ<italic toggle="yes">bamB</italic> Δ<italic toggle="yes">bamE</italic> Double Mutants Results from a Lethal Jamming of the Bam Complex by the Lipoprotein RcsF
description	ABSTRACT The selective permeability of the Gram-negative outer membrane (OM) is maintained by integral β-barrel outer membrane proteins (OMPs). The heteropentomeric β-barrel assembly machine (Bam) folds and inserts OMPs into the OM. Coordination of the essential proteins BamA and BamD is critical for OMP assembly and therefore the viability of the cell. The role of the nonessential lipoproteins BamBCE has yet to be characterized; however, genetic evidence suggests that they have nonoverlapping roles in OMP assembly. In this work, we quantify changes of the proteome in the conditional lethal ΔbamB ΔbamE double mutant. We show that cells lacking BamB and BamE have a global OMP defect that is a result of a lethal obstruction of an assembly-competent Bam complex by the lipoprotein RcsF. RcsF is a stress-sensing lipoprotein that is threaded through the lumen of abundant β-barrel OMPs by the Bam complex to expose the amino terminus on the cell surface. We demonstrate that simply removing this lipoprotein corrects the severe OMP assembly defect of the double mutant nearly as efficiently as a previously isolated suppressor mutation in bamA. We propose that BamB and BamE play crucial, nonoverlapping roles to coordinate the activities of BamA and BamD during OMP biogenesis. IMPORTANCE Protein assembly into lipid bilayers is an essential process that ensures the viability of diverse organisms. In Gram-negative bacteria, the heteropentomeric β-barrel assembly machine (Bam) folds and inserts proteins into the outer membrane. Due to its essentiality, outer membrane protein (OMP) assembly by the Bam complex is an attractive target for antibiotic development. Here, we show that the conditional lethal phenotype of a mutant lacking two of the three nonessential lipoproteins, BamB and BamE, is caused by lethal jamming of the stripped-down Bam complex by a normally surface-exposed lipoprotein, RcsF. The heterotrimeric Bam complex (BamA, BamD, BamC) is nearly as efficient as the wild-type complex in OMP assembly if RcsF is removed. Our study highlights the importance of BamB and BamE in regulating the interaction between BamA and BamD and expands our understanding of the role of the Bam complex in outer membrane biogenesis.
format	article
author	Elizabeth M. Hart Meera Gupta Martin Wühr Thomas J. Silhavy
author_facet	Elizabeth M. Hart Meera Gupta Martin Wühr Thomas J. Silhavy
author_sort	Elizabeth M. Hart
title	The Synthetic Phenotype of Δ<italic toggle="yes">bamB</italic> Δ<italic toggle="yes">bamE</italic> Double Mutants Results from a Lethal Jamming of the Bam Complex by the Lipoprotein RcsF
title_short	The Synthetic Phenotype of Δ<italic toggle="yes">bamB</italic> Δ<italic toggle="yes">bamE</italic> Double Mutants Results from a Lethal Jamming of the Bam Complex by the Lipoprotein RcsF
title_full	The Synthetic Phenotype of Δ<italic toggle="yes">bamB</italic> Δ<italic toggle="yes">bamE</italic> Double Mutants Results from a Lethal Jamming of the Bam Complex by the Lipoprotein RcsF
title_fullStr	The Synthetic Phenotype of Δ<italic toggle="yes">bamB</italic> Δ<italic toggle="yes">bamE</italic> Double Mutants Results from a Lethal Jamming of the Bam Complex by the Lipoprotein RcsF
title_full_unstemmed	The Synthetic Phenotype of Δ<italic toggle="yes">bamB</italic> Δ<italic toggle="yes">bamE</italic> Double Mutants Results from a Lethal Jamming of the Bam Complex by the Lipoprotein RcsF
title_sort	synthetic phenotype of δ<italic toggle="yes">bamb</italic> δ<italic toggle="yes">bame</italic> double mutants results from a lethal jamming of the bam complex by the lipoprotein rcsf
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/4dd05e6662ea42ec8c5ba427e3593717
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The Synthetic Phenotype of Δ<italic toggle="yes">bamB</italic> Δ<italic toggle="yes">bamE</italic> Double Mutants Results from a Lethal Jamming of the Bam Complex by the Lipoprotein RcsF

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