Understanding the cogs in a bacterial machine

Written by
Department of Molecular Biology, Princeton University, Molecular Biology Staff
May 24, 2019

The Bam machinery (in purple) helps thread RcsF (in green) through several outer membrane proteins (in blue). But in the absence of BamB and BamE, RcsF becomes irreversibly attached to BamA, jamming up the machinery and preventing other outer membrane proteins from being folded and inserted into the outer membrane. (Credit: Betsy Hart/Silhavy Lab)

In order to grow and survive, bacteria such as E. coli rely on a large number of barrel-shaped proteins that are inserted into their outer membrane and allow nutrients and other molecules to enter and exit the cell. These outer membrane proteins (OMPs) are assembled and inserted into the outer membrane by a group of proteins called the b-barrel assembly machine (Bam). Because of its importance to the bacterial cell, this machinery is an attractive target for developing new antibiotics, but how the different components work together to insert OMPs into the outer membrane is unclear. A new paper from the Silhavy and Wühr labs reveals how two components, known as BamB and BamE, coordinate the machine’s activity to prevent it from becoming jammed.

Two components of the Bam complex, BamA and BamD, are absolutely essential; bacteria lacking either one of these proteins are unable to survive. In contrast, cells lacking one of the other three components, BamB, BamC, or BamE, are mostly fine; it seems that the role of these three accessory proteins is to improve the efficiency of the Bam machinery as it folds and inserts OMPs into the outer membrane.

Nevertheless, bacteria lacking BamB and BamE have severe growth defects. One possibility is that these two proteins perform the same function and that cells can cope when one, but not both, of them are missing. “Alternatively, these proteins may have evolved to perform specialized functions, and when both of these functions are compromised, the assembly of certain OMPs is more strongly affected than others,” says Betsy Hart, a graduate student in Thomas Silhavy’s lab.

To investigate the roles of BamB and BamE, Hart and Silhavy collaborated with Martin Wühr and his graduate student Meera Gupta, who used a quantitative mass spectrometry approach called TMTc+ to compare the levels of bacterial cell proteins in the presence and absence of the two Bam components.

Most OMPs were significantly reduced in cells lacking BamB and BamE, suggesting that the loss of these two accessory proteins causes a global block in OMP assembly. However, the researchers found that this global block was caused by a specific defect in the assembly of a particular subset of OMPs that enclose a protein called RcsF.

RcsF helps bacteria detect and repair defects in their outer membrane. To do this, RcsF is threaded through the center of certain OMPs by the Bam machinery so that its lipid tail is exposed at the cell surface. This process is less efficient in bacteria lacking BamE, but it appears to become irretrievably broken when cells also lack BamB.

Hart and colleagues determined that in the absence of BamB and BamE, RcsF becomes irreversibly attached to BamA, jamming up the Bam machinery so that it can no longer fold and insert any type of OMP into the outer membrane. The researchers found that deleting the gene encoding RcsF unblocked the Bam machinery and allowed BamB/BamE-deficient bacteria to efficiently assemble OMPs and grow as fast as normal cells.

Based on their analyses, Hart and colleagues think that BamB and BamE have distinct roles in the assembly of RcsF/OMP complexes. BamE appears to control the interaction between BamA and RcsF and may be required to translocate the lipid tail of RcsF to the outer leaflet of the outer membrane. BamB, on the other hand, appears to monitor the assembly process and prevent the machinery from becoming jammed if BamE is absent. “We now want to investigate how these two nonessential accessory proteins perform these specialized functions,” Hart says.

E.M. Hart, M. Gupta, M. Wühr, and T.J. Silhavy. The Synthetic Phenotype of ΔbamB ΔbamE Double Mutants Results from a Lethal Jamming of the Bam Complex by the Lipoprotein RcsF. mBio 10:e00662-19. (2019). DOI: 10.1128/mBio.00662-19