The BAM complex drives assembly of β-barrel proteins into the outer membrane of Gram-negative bacteria. It is composed of five subunits: BamA, BamB, BamC, BamD and BamE. BamC is apparently a non-essential component. Its N-terminus carries a highly conserved region, crucial for stable interaction with BamD. Immunoprecipitation, immunofluorescence and protease-sensitivity assays show that the C-terminal domain of BamC, comprised of two helix-grip motifs, is exposed on the surface of E. coli (Webb et al., 2012).
Pathogenic bacteria assemble proteins into their outer membranes to colonize host environments and to avoid recognition by the immune system. As a surface component of the outer membrane, and one that is immunogenic, BamC deserves special attention. The function of BamC is not known, nor is the mechanism by which it is assembled into the outer membrane.
We sought to answer this question using a genetic screening approach. The screen exploits a synthetic lethal approach where bamC mutation has been introduced into a library of 3818 E. coli mutants, each one of which lacks a non-essential gene. The library has been arrayed into a format suitable for high-throughput screening with a Singer RoToR robotics platform. The double mutant collection library will be surveyed to select for mutants that cannot assemble PET (plasmid encoded toxin)- amylase hybrid. The amylase protein portion of the hybrid is exported to the extracellular milieu facilitating the use of starch as the sole carbon source. Double mutants that fail to correctly assemble the PET β-barrel cannot grow in media containing starch as the sole carbon source. We hope from this study to map out the genes involved in assembly of β-barrels in the outer membrane with regards to BamC.