Functional diversification of gram-negative intermembrane phospholipid transporters by intrinsic substrate preference

The outer membrane of Gram-negative bacteria is a barrier to chemical and physical stress. Phospholipid transport between the inner and outer membranes has been an area of intense investigation and, in E. coli K-12, it has recently been shown to be mediated by YhdP, TamB, and YdbH, which are suggested to provide hydrophobic channels for phospholipid diffusion, with YhdP and TamB playing the major roles. However, YhdP and TamB have different phenotypes suggesting distinct functions. We investigated these functions using synthetic cold sensitivity (at 30 °C) of a strain with deletion of yhdP , but not tamB or ydbH , and fadR , a transcriptional regulator controlling fatty acid degradation and unsaturated fatty acid production. Deletion of tamB , redirecting phospholipid transport to YdbH, suppresses the Δ yhdP Δ fadR cold sensitivity suggesting this phenotype is directly related to phospholipid transport. The Δ yhdP Δ fadR strain shows a greater increase in cardiolipin upon transfer to the non-permissive temperature and genetically lowering cardiolipin levels can suppress cold sensitivity. These data also reveal a qualitative difference between cardiolipin synthases in E. coli , as deletion of clsA and clsC suppresses cold sensitivity but deletion of clsB does not despite lower cardiolipin levels. In addition to increased cardiolipin, increased fatty acid saturation is necessary for cold sensitivity and lowering this level genetically or through supplementation of oleic acid suppresses the cold sensitivity of the Δ yhdP Δ fadR strain. A parsimonious explanation for our data is that YhdP and TamB have differential substrate transport preferences, most likely with YhdP preferentially transporting more saturated phospholipids and TamB preferentially transporting more unsaturated phospholipids. We envision cardiolipin contributing to this transport preference by sterically clogging TamB-mediated transport of saturated phospholipids. Thus, our data provide a potential mechanism for independent control of the phospholipid composition of the inner and outer membranes in response to changing conditions. Author Summary Gram-negative bacteria possess an impermeable outer membrane, which protects 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 . Similar roles for YhdP and/or TamB have been suggested in both closely and distantly related gram-negative bacteria. Here, given the transporters’ apparent partial redundancy, we investigated functional differentiation between YhdP and TamB. Our data demonstrate that YhdP and TamB have differential involvement with fatty acid and phospholipid metabolism. In fact, transport of higher than normal levels of cardiolipin and saturated phospholipids by TamB at a non-permissive temperature is lethal. From these data, we suggest a model where the functions of YhdP and TamB are distinguished by their preference for phospholipid transport with YhdP preferentially transporting more saturated phospholipids and TamB transporting more unsaturated phospholipids. Cardiolipin headgroup specificity may contribute transport inhibition due to its bulky nature 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. ### Competing Interest Statement The authors have declared no competing interest.