Differentiation of gram-negative intermembrane phospholipid transporter function by intrinsic substrate preference

The outer membrane of diderm Gram-negative bacteria acts as a barrier from chemical and physical stress. Anterograde phospholipid transport to the outer membrane has long been an area of intense investigation and, in E. coli K-12, it has recently been shown to be mediated by three related proteins: YhdP, TamB, and YdbH, which appear to provide hydrophobic channels for phospholipid diffusion, with YhdP and TamB playing the major roles. However, YhdP and TamB have different phenotypes suggesting distinct phospholipid transport functions. We investigated these functions using the synthetic cold sensitivity of a strain with ΔyhdP (but not ΔtamB or ΔydbH) and ΔfadR, a transcriptional regulator allowing switching between fatty acid degradation and synthesis and regulating unsaturated fatty acid production. Deletion of tamB, forcing phospholipid transport to YdbH, suppresses the ΔyhdP ΔfadR cold sensitivity suggesting this phenotype is due to TamB dysfunction. Increased levels of cardiolipin and fatty acid saturation are necessary for cold sensitivity and lowering levels of either suppresses this sensitivity. Our data support a model where YhdP primarily transports more saturated phospholipids, TamB primarily transports phospholipids with more than one carbon unsaturation, and cardiolipin obstructs TamB by selectively clogging its channel. Thus, the multiple phospholipid transporters may allow the saturation state of the outer membrane to be regulated independently of the inner membrane by altering the YhdP-TamB activity ratio. Maintaining membrane physical integrity and function under changing conditions may require envelope remodeling including altered phospholipid composition and intermembrane trafficking. Our data provide a potential mechanism for this regulation. Importance Gram-negative bacteria possess an impermeable outer membrane protecting against environmental stress and antibiotics. Outer membrane phospholipid transport remained mysterious until recently when YhdP, TamB, and YdbH were implicated in phospholipid transport between the inner and outer membranes of E. coli. TamB also modulates phospholipid transport in a distantly related diderm Fermicute. Here, we investigate functional differentiation between YhdP and TamB. Our data strongly suggest YhdP’s and TamB’s functions are distinguished by the saturation state of phospholipids with YhdP preferentially transporting more saturated phospholipids and TamB transporting more unsaturated phospholipids. Cardiolipin headgroup specificity may also contribute to TamB inhibition, perhaps due to the bulky nature of cardiolipin inhibiting the passage of other phospholipids. Diversification of function between YhdP and TamB provides a mechanism for regulation of phospholipid composition, and possibly the mechanical strength and permeability of the outer membrane, and so the cell’s intrinsic antibiotic resistance, in changing environmental conditions.