YdbL directly modulates YdbH-YnbE bridge formation to maintain <i>Escherichia coli</i> outer membrane homeostasis

ABSTRACT Gram-negative bacteria pose a threat to global healthcare mainly because their outer membrane (OM) provides an intrinsic barrier to many antimicrobials. Key to this barrier function is the asymmetric structure of the OM, with phospholipids constituting the inner leaflet and lipopolysaccharides, the outer leaflet. Although the mechanism of phospholipid transport between the inner membrane (IM) and OM remains poorly understood, recent studies implicate TamB, YhdP, and YdbH as functionally redundant proteins mediating this process in Escherichia coli . Accordingly, the collective loss of these three paralogs is lethal, and any one of them is sufficient for growth. YdbH is anchored to the IM, and its periplasmic repeating β-sheet groove domain interacts with the OM lipoprotein YnbE via β-strand augmentation to form an intermembrane bridge. Additionally, YnbE multimerizes, and the periplasmic protein YdbL is proposed to modulate YnbE multimerization to facilitate its stacking on the C-terminus of YdbH. Here, we demonstrate that excess YdbL specifically inhibits the function of the YdbH-YnbE complex since overexpression of ydbL causes lethality in the Δ yhdP Δ tamB double mutant, but the presence of both ydbH and ynbE in trans abrogates this lethality. We resolve high-resolution structural data for YdbL and ascertain its interaction site with the YnbE C-terminal α-helix, with residues mediating this interface highly conserved and critical for YdbL function. Finally, we show that YdbL is protected from degradation by the protease DegP when complexed with YnbE. Overall, our data support a model in which YdbL ensures proper YdbH-YnbE intermembrane bridge formation by directly interacting with YnbE. IMPORTANCE The mechanism underlying phospholipid transport between the inner and outer membranes of gram-negative bacteria remains enigmatic. Bacterial bridge-like protein systems such as the YdbH-YnbE complex resemble proteins found at membrane contact sites between eukaryotic organelles. These proteins are proposed to mediate intermembrane phospholipid transport, which is essential for growth of the outer membrane (OM). Here, we define the role of YdbL, a periplasmic protein that specifically modulates the YdbH-YnbE system. YdbL directly interacts with YnbE and controls the formation of the YdbH-YnbE complex. Additionally, we reveal that YdbL is selectively degraded by the periplasmic protease DegP. We propose a regulatory model that connects the YdbH-YnbE complex assembly and controls the levels of YdbL, providing new insight into OM homeostasis in gram-negative bacteria.