An Escherichia coli Mutant Defective in Lipid Export*

Escherichia coli phospholipids and lipopolysaccharide, made on the inner surface of the inner membrane, are rapidly transported to the outer membrane by mechanisms that are not well characterized. We now report a temperature-sensitive mutant (WD2) with an A270T substitution in a trans-membrane region of the ABC transporter MsbA. As shown by32Pi and 14C-acetate labeling, export of all major lipids to the outer membrane is inhibited by ∼90% in WD2 after 30 min at 44 °C. Transport of newly synthesized proteins is not impaired. Electron microscopy shows reduplicated inner membranes in WD2 at 44 °C, consistent with a key role for MsbA in lipid trafficking.

The envelope of Gram-negative bacteria consists of an inner membrane, the peptidoglycan cell wall, and an outer membrane ( Fig. 1A) (1). The latter is an asymmetric bilayer with glycerophospholipids ( Fig. 1B) on its inner surface and lipid A (Fig. 1B), the hydrophobic anchor of lipopolysaccharide (2, 3), on the outside (1). The lipid A moiety is a hexa-acylated disaccharide of glucosamine unique to Gram-negative bacteria (4) and is a potent activator of innate immunity in animals via the receptor TLR-4 (5-7). The enzymes that make phospholipids and lipid A are well characterized in Escherichia coli (3,8,9). They are located in the cytoplasm or inner membrane and are targets for the design of novel antibacterial agents (10,11).
How E. coli lipids cross the inner membrane and are transported to the outer membrane is unknown (Fig. 1A). A clue to lipopolysaccharide transport has recently emerged from studies of E. coli htrB mutants (12) and their suppression by msbA (13)(14)(15). HtrB is a lauroyl transferase that functions late in lipid A biosynthesis (Fig. 1A) (12). Lipopolysaccharides bearing tetra-acylated lipid A species accumulate in the inner membrane of htrB mutants at 44°C, inhibiting growth (15). MsbA is an essential ABC transporter (Fig. 2), closely related to eucaryotic Mdr proteins (13)(14)(15). MsbA overexpression restores growth of htrB mutants at 44°C without restoring laurate incorporation, resulting in export of lipopolysaccharide with tetra-acylated lipid A anchors to the outer membrane (15).
E. coli msbA knockouts are lethal. However, their biochemical analysis is complicated by long times (4 -8 h) needed to dilute out pre-existing MsbA supplied in trans from a temperaturesensitive plasmid (15) and by the fact that the lpxK gene, which is immediately downstream in an operon with MsbA, is also essential for cell growth (16).
We now report the isolation and characterization of a novel temperature-sensitive point mutant of E. coli that harbors a single amino acid substitution in MsbA. Rapid inhibition of MsbA function in vivo following a shift of mid log phase cells from 30 to 44°C results in the immediate arrest of the export of all newly made lipopolysaccharide and phospholipids, demonstrating that MsbA is an essential component of a general lipid transport system in E. coli.

EXPERIMENTAL PROCEDURES
Isolation of Mutant WD2 and Growth Conditions-WD2 was isolated by random localized mutagenesis (17) of msbA near min 21 of the E. coli chromosome. Briefly, strain LCB273 (F'aroA273::Tn10), obtained from the E. coli Genetic Stock Center, Yale University, was treated for 10 min at 37°C with 40 g/ml nitrosoguanidine (17,18). Cells were washed and used to generate a P1 vir lysate (17) that was used to prepare tetracycline-resistant transductants of wild type E. coli W3110 as single colonies (ϳ100/plate) on LB agar (18) containing 12 g/ml tetracycline and 4 mM sodium citrate (17). Temperature-sensitive mutants were identified by replica plating the transductants at 30 and 44°C; 25 candidates were found among ϳ5000 colonies that were screened in this manner. Each temperature-sensitive mutant was repurified and then transformed with the msbA ϩ hybrid plasmid (pZZ34) (15) or with the vector control (pACYC184; New England Biolabs). In 6 of the 25 mutant candidates, growth was restored at 44°C by pZZ34. The A270T lesion was identified by sequencing msbA from polymerase chain reactionamplified WD2 DNA.
Thin Layer Chromatography of Radioactive Lipid A and Phospholipids-Inner and outer membranes of wild type and mutant cells were separated by ultracentrifugation on isopycnic sucrose gradients as described below. 50-l portions of selected sucrose gradient fractions were each treated with 12.5 mM sodium acetate, pH 4.5, in 1% SDS for 30 min at 100°C to release the covalently bound lipid A moiety from lipopolysaccharide under conditions that do not damage the other phospholipids (15). The radioactive lipid A and phospholipids were then recovered from the hydrolysis mixtures by Bligh/Dyer partitioning (15). The lower phases were washed once with water, dried, redissolved in a small volume of chloroform/methanol (1:1) (v/v), and spotted in 10-l portions onto a silica gel 60 thin layer plate (Merck), which was subsequently developed in the solvent, chloroform/pyridine/88% formic acid/H 2 O (50:50:16:5) (v/v). The locations and amounts of labeled phospholipids and lipid A were analyzed with a Molecular Dynamics PhosphorImager.
Electron Microcopy of Wild Type and Mutant Cells-E. coli cells were fixed and stained as described for halobacteria (19), with minor modifications. Cultures of E. coli W3110 and WD2 were grown at 30°C for 2 h to mid log phase, and then shifted to 44°C for 30 min. Controls were kept at 30°C for 30 min. Bacteria were fixed by mixing an equal volume of fixative and culture. The cells were collected by low speed centrifugation. The primary fixative consisted of 3% glutaraldehyde, 0.1% triethylene-thiophosphoramide, and 1% acrolein in 0.1 M sodium cacodylate buffer, pH 7.2. The secondary fixative contained 0.1% ruthenium tetraoxide in 100 mM potassium phosphate buffer, pH 6.1 (19).

RESULTS AND DISCUSSION
Isolation of Mutant WD2-Because Mdr proteins confer resistance to diverse chemotherapeutic agents (20) and are implicated in transport of some sterols (21)(22)(23) and phospholipids (24), we used a genetic approach to validate the function of MsbA in E. coli. Rapidly inactivating temperature-sensitive point mutations in msbA were isolated by localized chemical mutagenesis near min 21 of the chromosome (17). Mutants in which growth at 44°C was restored by msbA ϩ on a hybrid plasmid were examined further. Three strains with single amino acid substitutions in putative trans-membrane regions of MsbA were obtained. Mutant WD2 harbored the A270T substitution (Fig. 2) and stopped growing after ϳ60 min when shifted from 30 to 44°C (Fig. 2). There was no loss of viability for 30 min at 44°C. The reversion rate of WD2 to temperature resistance was ϳ1 ϫ 10 Ϫ5 .
Inhibition of Lipid Export at 44°C in WD2-To evaluate lipid transport, mid log phase cells at 30°C were shifted to 44°C for 30 min and then labeled for 10 min with 32 P i (Fig. 3,  A and B). Cells were chilled, converted to spheroplasts, and broken by sonic irradiation (25,26). Membranes were separated by isopycnic ultracentrifugation on sucrose gradients (25,26). In wild type cells, membrane-bound radioactivity was evenly distributed between inner and outer membranes (Fig.  3A), reflecting rapid export of new phospholipids and lipopolysaccharide (27,28). In mutant WD2 over 90% of the 32 P was retained in the inner membrane (Fig. 3B). Despite profound inhibition of lipid trafficking, the efficiency of 32 P i incorpora- For the 30-s pulse and pulse/chase, cells were cooled by dilution into an equal volume of LB broth at ice-salt temperature. Cells were harvested by centrifugation, and membranes were prepared at 0°C from spheroplasts generated by lysozyme-EDTA treatment, followed by mild sonic irradiation (25,26). Membranes were washed twice and then separated on a 12-ml 30 -60% (w/w) isopycnic sucrose gradient (25,26) at 4°C by ultracentrifugation in a Beckman SW41 Ti rotor at 36,000 rpm for 16 h. Approximately 30 fractions (0.4 ml each) were collected. Outer membrane phospholipase A (x-x), inner membrane NADH oxidase (squares), total protein (ϳ0.3 mg/ml in the peak fractions) (not shown), and radioactivity (black dots) were measured for each 0.4-ml fraction (15,26) and are expressed as a percent of the total or cpm/50 l.

FIG. 1. Proposed export pathway for lipopolysaccharide and phospholipids in E. coli.
A, phospholipids and the lipid A/core portions of lipopolysaccharide are synthesized on the inner surface of the inner membrane (3,8,9). Laurate and myristate addition to lipopolysaccharide precursor molecules are catalyzed by HtrB and MsbB, respectively (3,12). HtrB mutants cannot grow above 33°C but are rescued by multiple copies of MsbA (15), an essential ABC transporter that may catalyze flip-flop (Step 1) and/or further export of nascent lipid molecules (Step 2). Nothing is known about Step 3. ACP, acyl carrier protein. B, structures of Kdo 2 -lipid A, the minimal lipopolysaccharide found in heptose-deficient mutants of E. coli (3,8,9), and PtdEtn, the major structural membrane phospholipid of E. coli. tion was ϳ2-fold higher in WD2 than wild type, indicating that WD2 was not depleted of ATP. Thin layer chromatography (15) of the 32 P i -labeled lipids (Fig. 4, A and B) from membranes of cells shifted to 44°C showed that transport of both lipopolysaccharide (as judged by release of lipid A) and phospholipids was blocked in WD2.
The 32 P i results were confirmed with 14 C-acetate pulse-chase experiments (Fig. 3, C-F). Wild type and WD2 cultures were shifted to 44°C for 30 min and labeled for 30 s with 20 M [1,2-14 C]acetate. In this case labeled lipids were seen mainly in the inner membrane (Fig. 3, C and D) in both strains. When the 30-s pulse was followed by a 10-min chase with 50 mM nonradioactive acetate, half the 14 C lipids shifted to the outer membrane in wild type (Fig. 3E), but Ͼ90% remained in the inner membrane in WD2 (Fig. 3F). As judged by thin layer analysis of the fractions (not shown), most of the radioactive lipid A was recovered in the outer membrane after the chase of the wild type cells, whereas labeled glycerophospholipids were present in both membranes. The latter finding is consistent with earlier studies demonstrating that phospholipids can re-turn from the outer to the inner membrane, whereas lipopolysaccharide cannot (29,30).
Normal Protein Export in WD2 at 44°C-Inhibition of lipid transport in WD2 was accompanied by an increased buoyant density of the outer membrane (Fig. 3), suggesting protein export was continuing. When cells, shifted to 44°C for 30 min, were labeled for 10 min with 35 S-methione (Fig. 3, G and H), no inhibition of bulk protein transport was seen in WD2, confirming that protein synthesis and secretion were largely intact. The pattern and localization of the major 35 S-labeled proteins in WD2 were also normal, as judged by SDS gel electrophoresis and autoradiography (not shown).
Electron Microscopy of WD2 at 44°C-Because inhibition of lipid export in WD2 at 44°C persisted for 30 -60 min before cell density leveled off (Fig. 2), inner membrane surface area was expected to increase more than outer membrane surface area. Thin section electron microscopy (19) of WD2 grown for 30 min at 44°C showed striking inner membrane reduplications (Fig.  5, B and D) and invaginations, consistent with selective inhibition of lipid export (Fig. 3). Membranes of wild type cells processed in parallel appeared normal (Fig. 5, A and C).
Concluding Remarks-The properties of WD2 suggest that MsbA is needed for the export of all major membrane lipids, including lipolpolysaccharide, in E. coli (Fig. 1A). We propose that MsbA has one or more key functions in lipid trafficking. It may facilitate lipid flip-flop within the inner membrane (Fig.  1A, Step 1). Alternatively (or in addition) MsbA may utilize ATP hydrolysis to export lipids from the periplasmic surface of the inner membrane to the outer envelope (Fig. 1A, Step 2), given recent data suggesting that bacterial lipid flip-flop is energy-independent (31,32). Furthermore, MsbA may also be part of a larger molecular machine, like the "chunnel" for hemolysin secretion, which consists of an ABC transporter (HlyB), a periplasmic inner membrane protein (HlyD), and a gated export tube attached to an outer membrane pore (TolC) (33)(34)(35).
Most mammalian (20) and bacterial Mdr proteins (36) characterized to date catalyze phospholipid flip-flop in vitro. In contrast to strain WD2, however, mutants in previously studied Mdr proteins, such as mouse Mdrl or Mdr3, are not conditionally lethal or grossly altered in global intracellular lipid transport (20). The Mdr2 knockout mouse (24) does lack phosphatidylcholine in its bile but is nevertheless viable. The ABC1 transporter of Tangier's disease (21,22,37,38), the ABCG5/ ABCG8 transporters of sitosterolemia (23), and the P-glycoprotein transporters that maintain amino-phospholipid asymmetry (20,39,40) are also quite specialized, when compared with MsbA (41). The ATP binding cassettes of ABC1, ABCG5, and ABCG8 display distant sequence similarity to the ATP cassette of MsbA, but there is no similarity in the membrane-spanning regions. Furthermore, the amino-phospholipid translocases (20,39,40) are not detected at all in BLASTp comparisons with MsbA. Because there are many additional uncharacterized MsbA homologues in procaryotes and eucaryotes, single and multiple mutants need to be constructed in representative organisms to evaluate the functions of these proteins in lipid trafficking. Given that other E. coli proteins cannot compensate for loss of MsbA, WD2 may prove useful for studying the functions of diverse MsbA homologues by enabling complementation of its lipid export defect and temperature-sensitive growth.