An acyltransferase induced by cold shock in Escherichia coli, designated LpxP, incorporates a palmitoleoyl moiety into nascent lipid A in place of the secondary laurate chain normally added by LpxL(HtrB) (Carty, S. M., Sreekumar, K. R., and Raetz, C. R. H. (1999) J. Biol. Chem. 274, 9677-9685). To determine whether the palmitoleoyl residue alters the properties of the outer membrane and imparts physiological benefits at low growth temperatures, we constructed a chromosomal insertion mutation in lpxP, the structural gene for the transferase. Membranes from the lpxP mutant MKV11 grown at 12°C lacked the cold-induced palmitoleoyltransferase present in membranes of cold-shocked wild type cells but retained normal levels of the constitutive lauroyltransferase encoded by lpxL. When examined by mass spectrometry, about two-thirds of the lipid A molecules isolated from wild type E. coli grown at 12°C contained palmitoleate in place of laurate, whereas the lipid A of cold-adapted MKV11 contained only laurate in amounts comparable with those seen in wild type cells grown at 30°C or above. To probe the integrity of the outer membrane, MKV11 and an isogenic wild type strain were grown at 30 or 12°C and then tested for their susceptibility to antibiotics. MKV11 exhibited a 10-fold increase in sensitivity to rifampicin and vancomycin at 12°C compared with wild type cells but showed identical resistance when grown at 30°C. We suggest that the palmitoleoyltransferase may confer a selective advantage upon E. coli cells growing at lower temperatures by making the outer membrane a more effective barrier to harmful chemicals.A unique glycolipid known as lipopolysaccharide is the major component of the outer surface of the outer membranes of Gram-negative bacteria and forms a barrier around the cell (1-3). Compounds with molecular weights of less than 600 penetrate the outer membrane via porins, but larger, potentially harmful agents, including many antibiotics, are excluded (2). Lipid A is the hydrophobic anchor of the lipopolysaccharide molecule (1, 4 -6). In wild type Escherichia coli grown at 30°C or above, lipid A consists of a ,1Ј-6-linked disaccharide of glucosamine that is phosphorylated at the 1-and 4Ј-positions ( Fig. 1) (1, 4 -6). E. coli lipid A usually contains six acyl chains (1, 4). (R)-3-Hydroxymyristoyl groups are located at positions 2, 3, 2Ј, and 3Ј of the glucosamine disaccharide, and two short saturated acyl chains are attached to the (R)-3-hydroxymyristoyl groups of the distal unit, forming acyloxyacyl moieties (Fig. 1, "Normal" lipid A) (1, 4 -6). Laurate is linked to the (R)-3-hydroxymyristoyl residue at position 2Ј, and myristate is similarly attached at position 3Ј (1, 4 -6). These so-called "secondary" acyl chains are the same in wild type E. coli and Salmonella.Two inner membrane enzymes, LpxL(HtrB) and LpxM(MsbB), which act late in the lipid A pathway (Fig. 1), are responsible for the incorporation of laurate and myristate, respectively (7-9). These enzymes require the presence of a 3-deoxy-D-manno-oct...