Cross-species chimeras reveal BamA POTRA and β-barrel domains must be fine-tuned for efficient OMP insertion.

Douglas F Browning,Vassiliy N Bavro,Jessica L Mason,Ian R Henderson,Yanina R Sevastsyanovich,Amanda E Rossiter,Timothy J Knowles,Timothy J Wells,James W Donald,Tracy Palmer,Adam F Cunningham,Mark Jeeves,Michael Overduin

doi:10.1111/mmi.13052

Abstract

Summary BAM is a conserved molecular machine, the central component of which is BamA. Orthologues of BamA are found in all Gram‐negative bacteria, chloroplasts and mitochondria where it is required for the folding and insertion of β‐barrel containing integral outer membrane proteins (OMPs) into the outer membrane. BamA binds unfolded β‐barrel precursors via the five polypeptide transport‐associated (POTRA) domains at its N‐terminus. The C‐terminus of BamA folds into a β‐barrel domain, which tethers BamA to the outer membrane and is involved in OMP insertion. BamA orthologues are found in all Gram‐negative bacteria and appear to function in a species‐specific manner. Here we investigate the nature of this species‐specificity by examining whether chimeric E scherichia coli BamA fusion proteins, carrying either the β‐barrel or POTRA domains from various BamA orthologues, can functionally replace E . coli BamA. We demonstrate that the β‐barrel domains of many BamA orthologues are functionally interchangeable. We show that defects in the orthologous POTRA domains can be rescued by compensatory mutations within the β‐barrel. These data reveal that the POTRA and barrel domains must be precisely aligned to ensure efficient OMP insertion.

Highlights

The insertion of β-barrel containing integral outer membrane proteins (OMPs) into the outer membrane of Escherichia coli is achieved by the multi-protein β-barrel assembly machine, the BAM complex (Knowles et al, 2009; Hagan et al, 2011)
Orthologues of BamA are found in all Gram-negative bacteria, chloroplasts and mitochondria where it is required for the folding and insertion of β-barrel containing integral outer membrane proteins (OMPs) into the outer membrane
We investigate the nature of this speciesspecificity by examining whether chimeric Escherichia coli BamA fusion proteins, carrying either the β-barrel or polypeptide transport-associated (POTRA) domains from various BamA orthologues, can functionally replace E. coli BamA

Summary

Introduction

The insertion of β-barrel containing integral outer membrane proteins (OMPs) into the outer membrane of Escherichia coli is achieved by the multi-protein β-barrel assembly machine, the BAM complex (Knowles et al, 2009; Hagan et al, 2011). The N-terminal periplasmic domain of BamAEc is composed of five polypeptide transport-associated (POTRA) motifs (POTRA1 to POTRA5) and a C-terminal β-barrel domain, which anchors the protein in the outer membrane (Knowles et al, 2009; Hagan et al, 2011). BamC and BamE do not bind BamAEc directly, but associate with the BAM complex through BamD (Kim et al, 2007). In E. coli, many of the loops are essential for BamAEc function, in particular L6, which is partially located within the barrel lumen because of an interaction between the barrel wall and the conserved VRGF amino acid motif at its tip (Delattre et al, 2010; Leonard-Rivera and Misra, 2012; Browning et al, 2013; Noinaj et al, 2013; Rigel et al, 2013; Albrecht et al, 2014; Ni et al, 2014)

Results

Discussion

Conclusion