The surface of Gram-negative bacteria is composed of the inner membrane, cell wall, and outer membrane. While the cell wall is crucial for growth under typical conditions and is a target for antibiotics like penicillin, bacteria can also grow in a cell wall-less state known as the L-form under specific conditions. However, the outer membrane remains essential regardless of the L-form. One of the key components of the outer membrane is outer membrane proteins (OMPs), which have β-barrel transmembrane domains and are assembled by the Beta-barrel Assembly Machinery (BAM) complex. Recent studies suggest that membrane tension, provided by the cell wall, drives this assembly. This raises the question of how Gram-negative bacteria achieve OMP assembly in L-form, where the cell wall is absent, resulting in no tension condition. We aimed to identify factors crucial for L-form growth through real-time observation and mutant analysis.
We identified that BamC, a previously poorly understood subunit of the BAM complex, is essential for L-form growth. BamC was required for OMP assembly under conditions that a plasmolysis condition reduces outer membrane tension, such as cultivation in high-osmolarity media. Topology analysis revealed that during the logarithmic growth phase, BamC exposes about 70% of its C-terminal region extracellular. This topology was similar to Sam35, the subunit of the Sorting Assembly Machinery (SAM) complex which is the homologue of the BAM complex in mitochondria. Because mitochondria always do not have a cell wall, they assemble outer membrane protein without membrane tension. Thus, BamC likely performs a similar function in the L-form state as mitochondrial sorting machinery.