All possible combinations of insertion mutations in the three genes encoding the acyl carrier protein-dependent late acyltransferases of lipid A biosynthesis, designated lpxL(htrB), lpxM(msbB), and lpxP, were generated in Escherichia coli K12 W3110. Mutants defective in lpxM synthesize penta-acylated lipid A molecules and grow normally. Strains lacking lpxP fail to incorporate palmitoleate into their lipid A at 12°C but make normal amounts of hexa-acylated lipid A and are viable. Although lpxL mutants and lpxL lpxM double mutants grow slowly on minimal medium at all temperatures, they do not grow on nutrient broth above 32°C. Such mutants retain the ability to synthesize some penta-and hexa-acylated lipid A molecules because of limited induction of lpxP at 30°C but not above 32°C. MKV15, an E. coli lpxL lpxM lpxP triple mutant, likewise grows slowly on minimal medium at all temperatures but not on nutrient broth at any temperature. MKV15 synthesizes a lipid A molecule containing only the four primary (R)-3-hydroxymyristoyl chains. The outer membrane localization and content of lipid A are nearly normal in MKV15, as is the glycerophospholipid and membrane protein composition. However, the rate at which the tetra-acylated lipid A of MKV15 is exported to the outer membrane is reduced compared with wild type. The integrity of the outer membrane of MKV15 is compromised, as judged by antibiotic hypersensitivity, and MKV15 undergoes lysis following centrifugation. MKV15 may prove useful as a host strain for expressing late acyltransferase genes from other Gram-negative bacteria, facilitating the re-engineering of lipid A structure in living cells and the design of novel vaccines.

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...

Dominant drug resistance markers offer experimental flexibility in the study of Saccharomyces cerevisiae by eliminating the dependence on auxotrophic mutations and, because they are phenotypically neutral, avoid the deleterious effects of auxotrophic mutations. We have developed a new dominant resistance marker, dsdAMX4, for use in the genetic manipulation of S. cerevisiae. The dsdA gene, which is derived from Escherichia coli and encodes a D-serine deaminase, confers to S. cerevisiae resistance to D-serine and the ability to use D-serine as a nitrogen source. Here we describe the construction of a dsdAMX4 cassette, capable of expression in S. cerevisiae, and the characterization of this new marker for use in chromosomal gene disruption. The unique selection properties of the dsdAMX4 cassette make it an important addition to the existing array of S. cerevisiae genetic tools.