MsbA-dependent Translocation of Lipids across the Inner Membrane of Escherichia coli

Abstract

MsbA is an essential ABC transporter in Escherichia coli required for exporting newly synthesized lipids from the inner to the outer membrane. It remains uncertain whether or not MsbA catalyzes trans-bilayer lipid movement (i.e. flip-flop) within the inner membrane. We now show that newly synthesized lipid A accumulates on the cytoplasmic side of the inner membrane after shifting an E. coli msbA missense mutant to the non-permissive temperature. This conclusion is based on the selective inhibition of periplasmic, but not cytoplasmic, covalent modifications of lipid A that occur in polymyxin-resistant strains of E. coli. The accessibility of newly synthesized phosphatidylethanolamine to membrane impermeable reagents, like 2,4,6-trinitrobenzene sulfonic acid, is also reduced severalfold. Our data showed that MsbA facilitates the rapid translocation of some lipids from the cytoplasmic to the periplasmic side of the inner membrane in living cells.

An Undecaprenyl Phosphate-Aminoarabinose Flippase Required for Polymyxin Resistance in Escherichia coli

“…Patients with sitosterolemia lack the ABC transporters that pump absorbed sterols, including the plant-derived sitosterol, back into the gut or the bile (66). In bacteria, point mutants in the essential ABC transporter MsbA are defective in flipping nascent LPS across the inner membranes at elevated temperatures (35,36,67,68), as judged by accessibility to periplasmic lipid A modification enzymes. Such mutants are also defective in glycerophospholipid export to the outer membrane (35), but this phenomenon might be secondary to LPS accumulation within the inner membrane.…”

“…Membrane-impermeable reagents, such as sulfo-NHS-biotin or 2,4,6-trinitrobenzene sulfonic acid, have previously been utilized to characterize phosphatidylethanolamine flip-flop in whole cells (36,40). We have successfully utilized sulfo-NHSbiotin (Figs.…”

“…Sulfo-NHS-biotin is a hydrophilic, low molecular weight biotinylation reagent that reacts with any primary amine. It is able to diffuse through the outer membrane porins of E. coli to reach the periplasmic space (36), but it cannot enter the cytoplasm because it does not cross the phospholipid bilayer of the inner membrane (40). Accordingly, if undecaprenyl phosphate-␣-L-Ara4N were trapped on the inner surface of the inner membrane in the PmrL-and/or PmrM-deficient strains, sulfo-NHS-biotin labeling of the endogenous undecaprenyl phosphate-␣-L-Ara4N should be reduced, when compared with the parental strain or to a comparable arnT (Fig.…”

“…2A). Further evidence that L-Ara4N transfer to lipid A occurs on the outer surface of the inner membrane is provided by the observation that L-Ara4N modification of lipid A is dependent upon the essential core-lipid A flippase, MsbA (35,36).…”

“…Despite the presence of the same high levels of undecaprenyl phosphate-␣-L-Ara4N in these mutants as in their parent, lipid A modification with L-Ara4N is almost completely abolished. Using the inner membrane-impermeable amine reagent N-hydroxysulfosuccin-imidobiotin (sulfo-NHS-biotin) (36), in conjunction with mass spectrometry, we show that undecaprenyl phosphate-␣-L-Ara4N is 4 -5-fold less accessible to chemical modification in pmrL/pmrA c and pmrM/pmrA c mutants than in the pmrA c parent, presumably reflecting the reduced ability of undecaprenyl phosphate-␣-LAra4N to gain access to the periplasmic side of the inner membrane. Reduced chemical modification of undecaprenyl phosphate-␣-L-Ara4N is not observed in an arnT mutant derived from the same pmrA c parent, in which undecaprenyl phosphate-␣-L-Ara4N levels also remain as high as they are in the parent.…”