The inner membrane protein YhdP modulates the rate of anterograde phospholipid flow in Escherichia coli

The outer membrane (OM) of Gram-negative bacteria is a selective permeability barrier that allows uptake of nutrients while simultaneously protecting the cell from harmful compounds. The basic pathways and molecular machinery responsible for transporting lipopolysaccharides (LPS), lipoproteins, and β-barrel proteins to the OM have been identified, but very little is known about phospholipid (PL) transport. To identify genes capable of affecting PL transport, we screened for genetic interactions with mlaA *, a mutant in which anterograde PL transport causes the inner membrane (IM) to shrink and eventually rupture; characterization of mlaA *-mediated lysis suggested that PL transport can occur via a high-flux, diffusive flow mechanism. We found that YhdP, an IM protein involved in maintaining the OM permeability barrier, modulates the rate of PL transport during mlaA *-mediated lysis. Deletion of yhdP from mlaA * reduced the rate of IM transport to the OM by 50%, slowing shrinkage of the IM and delaying lysis. As a result, the weakened OM of Δ ydhP cells was further compromised and ruptured before the IM during mlaA *-mediated death. These findings demonstrate the existence of a high-flux, diffusive pathway for PL flow in Escherichia coli that is modulated by YhdP. Significance Statement The outer membrane (OM) of Gram-negative bacteria serves as a barrier that protects cells from harmful chemical compounds, including many antibiotics. Understanding how bacteria build this barrier is an important step in engineering strategies to circumvent it. A long-standing mystery in the field is how phospholipids (PLs) are transported from the inner membrane (IM) to the OM. We previously discovered that a mutation in the gene mlaA causes rapid flow of PLs to the OM, eventually resulting in IM rupture. Here, we found that deletion of the gene yhdP delayed cell death in the mlaA mutant by slowing flow of PLs to the OM. These findings reveal a high-flux, diffusive pathway for PL transport in Gram-negative bacteria modulated by YhdP. ### Competing Interest Statement The authors have declared no competing interest.