Mutational and Topological Analysis of the Escherichia coli BamA Protein

Douglas F Browning,Mark Jeeves,Timothy J Wells,Amanda E Rossiter,Ian R Henderson,Vassiliy N Bavro,Timothy J Knowles,Jessica L Mason,Catherine A Wardius,Sophie A Matthews,Yanina R Sevastsyanovich,Adam F Cunningham,Michael Overduin

doi:10.1371/journal.pone.0084512

Douglas F Browning, Mark Jeeves + Show 11 more

Open Access

https://doi.org/10.1371/journal.pone.0084512

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Journal: PLoS ONE	Publication Date: Dec 23, 2013
Citations: 66	License type: CC BY 4.0

Affiliation: University of Birmingham

Abstract

The multi-protein β-barrel assembly machine (BAM) of Escherichia coli is responsible for the folding and insertion of β-barrel containing integral outer membrane proteins (OMPs) into the bacterial outer membrane. An essential component of this complex is the BamA protein, which binds unfolded β-barrel precursors via the five polypeptide transport-associated (POTRA) domains in its N-terminus. The C-terminus of BamA contains a β-barrel domain, which tethers BamA to the outer membrane and is also thought to be involved in OMP insertion. Here we mutagenize BamA using linker scanning mutagenesis and demonstrate that all five POTRA domains are essential for BamA protein function in our experimental system. Furthermore, we generate a homology based model of the BamA β-barrel and test our model using insertion mutagenesis, deletion analysis and immunofluorescence to identify β-strands, periplasmic turns and extracellular loops. We show that the surface-exposed loops of the BamA β-barrel are essential.

Highlights

The outer membranes of Gram-negative bacteria serve as a barrier to protect cells from toxic compounds such as antibiotics and detergents
Deletion analysis of BamA polypeptide transport– associated (POTRA) domains suggested that POTRA3, POTRA4 and POTRA5 are essential for function but that POTRA1 and POTRA2 are dispensable, cells expressing these deletion proteins grew extremely poorly [6,7]
Linker scanning mutagenesis of BamA BamA is an essential protein in E. coli and homologues are found in all Gram-negative bacteria [3,5]

Summary

Introduction

The outer membranes of Gram-negative bacteria serve as a barrier to protect cells from toxic compounds such as antibiotics and detergents. OMP insertion is achieved by the multi-protein b-barrel assembly machine (BAM) complex, which consists of the essential OMP BamA and four accessory lipoproteins (BamB, BamC, BamD and BamE) [1,2]. This is an evolutionary conserved molecular machine, components of which are found in all Gram-negative bacteria as well as eukaryotic mitochondria and chloroplasts [3,4]. BamA is an essential protein in E. coli and belongs to the Omp family of proteins [3,5] It consists of an N-terminal periplasmic domain composed of five polypeptide transport– associated (POTRA) motifs (POTRA1 to POTRA5) and a Cterminal b–barrel domain, which anchors the protein in the outer membrane [1,2]. In addition to their role of scaffolding the BAM lipoproteins, POTRA domains bind unfolded OMPs and are thought to be responsible for delivering them to the outer membrane for insertion [6,9,10,12]

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Conclusion