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.