The outer membrane of Gram-negative bacteria consists of two leaflets that differ in their composition: the inner leaflet contains mostly phospholipids (PL) whereas the outer leaflet is mostly composed of lipopolysaccharides (LPS). LPS provide a physical barrier against hydrophobic molecules like antibiotics and bile salts, therefore, the balance of the two major components is essential for viability. To maintain the optimal ratio of LPS and PL, their biosynthesis pathways are connected at multiple sites, resulting in a complex protein network (Möller et al., 2023). Since an overproduction of LPS leads to cell death, the first committed enzyme in the LPS pathway, LpxC, is posttranslationally degraded by the protease FtsH, which is facilitated by the adaptor protein LapB. The adaptor function of LapB is inhibited by the inner membrane protein YejM (LapC) upon lack of LPS, resulting in stabilisation of LpxC. Interestingly, the yejM gene is located downstream of yejL, which encodes for a so far uncharacterised protein. Unlike other proteins involved in the regulatory interplay, YejL is not essential. However, YejL is conserved among gammaproteobacteria, contains 75 amino acids and forms a dimer with a tight conformation.

To investigate the function of YejL in the complex regulatory network, we performed protein-protein interaction studies using bacterial two-hybrid, pull down, microscale thermophoresis (MST) and far western dot blot analysis. We identified various proteins involved in outer membrane biosynthesis that might interact with YejL. The bacterial two-hybrid assay and MST showed that the first protein of the PL pathway, FabZ, as well as the first protein of the LPS pathway, LpxA, bind to YejL. In addition, MST and far western dot blot revealed an interaction between YejL and MreB which coordinates cell shape.

Overall, our studies indicate that YejL is likely involved in the regulatory interplay of outer membrane biogenesis.