Recognition and Assembly of Mutant Outer Membrane Protein A by the β-Barrel Assembly Machine in Escherichia coli

Abstract

The outer membrane (OM) of Gram-negative bacteria is a lipid bilayer containing β-barrel proteins. Assembly of outer membrane proteins (OMPs) is essential in Gramnegative bacteria such as Escherichia coli and is catalyzed by the β-barrel assembly machine (Bam). How Bam recognizes and folds nascent OMP substrates is poorly understood. The essential Bam components BamA and BamD are known to interact with substrates independently. A conserved signature sequence, termed the β-signal, has been identified in the C-termini of many OMPs and is hypothesized to target OMPs to Bam. This sequence is necessary for binding of some OMPs to BamA and BamD in vitro, but its in vivo role in recognition of OMPs by Bam remains unclear. Four mutations to the proposed β-signal of OmpA, a model OMP, were shown to reduce levels of mature OmpA. One of these mutations, ompAG186R, caused significant impairment to OM biogenesis, resulting in dominant drug permeability defects. Synthetic lethality with ΔbamB indicates ompAG186R disrupts normal Bam interactions. Induction of the σE response and the essentiality of the protease DegP in the mutant strain suggest misfolding and impaired assembly of mutant protein. However, this is contradicted by phage sensitivity assays, which demonstrate assembly of OmpAG186R, and growth and heat modifiability assays, which indicate proper Bam function. Formation of large pore OmpA in the mutant strain best explains these data. OmpA can adopt two different β- barrel conformations, a small and large pore, but the large pore form has yet to be isolated or characterized in vivo. This study may therefore be useful in shedding light on not only the stepwise process of OMP biogenesis but also the in vivo properties of large pore OmpA.