Effect of Cold Shock on Lipid A Biosynthesis in Escherichia coli
INDUCTION AT 12 °C OF AN ACYLTRANSFERASE SPECIFIC FOR PALMITOLEOYL-ACYL CARRIER PROTEIN*

Sherry M. CartyDagger §, Kodangattil R. Sreekumarparallel , and Christian R. H. RaetzDagger **

From the Dagger  Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 and the  Department of Molecular Biology and Microbiology, Tufts University Health Sciences Campus, Boston, Massachusetts 02111

    ABSTRACT
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EXPERIMENTAL PROCEDURES
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Palmitoleate is not present in lipid A isolated from Escherichia coli grown at 30 °C or higher, but it comprises ~11% of the fatty acyl chains of lipid A in cells grown at 12 °C. The appearance of palmitoleate at 12 °C is accompanied by a decline in laurate from ~18% to ~5.5%. We now report that wild-type E. coli shifted from 30 °C to 12 °C acquire a novel palmitoleoyl-acyl carrier protein (ACP)-dependent acyltransferase that acts on the key lipid A precursor Kdo2-lipid IVA. The palmitoleoyl transferase is induced more than 30-fold upon cold shock, as judged by assaying extracts of cells shifted to 12 °C. The induced activity is maximal after 2 h of cold shock, and then gradually declines but does not disappear. Strains harboring an insertion mutation in the lpxL(htrB) gene, which encodes the enzyme that normally transfers laurate from lauroyl-ACP to Kdo2-lipid IVA (Clementz, T., Bednarski, J. J., and Raetz, C. R. H. (1996) J. Biol. Chem. 271, 12095-12102) are not defective in the cold-induced palmitoleoyl transferase. Recently, a gene displaying 54% identity and 73% similarity at the protein level to lpxL was found in the genome of E. coli. This lpxL homologue, designated lpxP, encodes the cold shock-induced palmitoleoyl transferase. Extracts of cells containing lpxP on the multicopy plasmid pSK57 exhibit a 10-fold increase in the specific activity of the cold-induced palmitoleoyl transferase compared with cells lacking the plasmid. The elevated specific activity of the palmitoleoyl transferase under conditions of cold shock is attributed to greatly increased levels of lpxP mRNA. The replacement of laurate with palmitoleate in lipid A may reflect the desirability of maintaining the optimal outer membrane fluidity at 12 °C.

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Lipopolysaccharide (LPS)1 is a major component of the outer leaflet of the outer membrane of Escherichia coli and is a defining feature of Gram-negative bacteria (1-6). LPS consists of three domains: 1) lipid A, the hydrophobic membrane anchor (Fig. 1); 2) the core sugar region; and 3) the O-antigen polymer (1-6). Studies of E. coli mutants in LPS biosynthesis have demonstrated that the lipid A domain and the Kdo residues of the proximal core are required for growth (1-3, 7).

In wild-type E. coli grown at 30 °C or above, lipid A contains six fatty acyl chains (Fig. 1) (3, 4, 8). Four R-3-hydroxymyristate residues are attached directly to the glucosamine disaccharide backbone (3, 4, 8). Laurate and myristate are attached to the R-3-hydroxy groups of the R-3-hydroxymyristate residues in the distal unit (Fig. 1) (3, 4, 9). In the later steps of the lipid A biosynthetic pathway (Fig. 1), the key intermediate Kdo2-lipid IVA is sequentially acylated with laurate and myristate (3, 4, 9-11). The lauroyl and myristoyl transferases require the presence of the Kdo disaccharide for optimal activity (9-11). The substrate preferences of these enzymes are consistent with the accumulation of lipid IVA, rather than lipid A, in mutants defective in Kdo biosynthesis (12-15).

As shown by Clementz et al. (10, 11, 16), the lpxL(htrB) gene, which was initially identified as being required for growth of E. coli above 32 °C (17, 18), encodes the lauroyl transferase (Fig. 1). We have recently purified LpxL to homogeneity and confirmed that lpxL is indeed the structural gene for the lauroyl transferase.2 The LpxL reaction product, Kdo2-(lauroyl)-lipid IVA, is the preferred substrate for the myristoyl transferase (9, 11), which is encoded by the lpxM gene (previously designated msbB) (Fig. 1) (19). The lpxM(msbB) gene displays distant, but nevertheless significant, sequence similarity to lpxL (19). High copy number plasmids bearing lpxM can suppress the temperature-sensitive growth of mutants defective in lpxL (19), presumably because LpxM acylates Kdo2-lipid IVA at a slow rate (11).

In 1979, prior to the elucidation of the structure and biosynthesis of lipid A, van Alphen et al. studied the fatty acid composition of lipid A from E. coli grown at 37 °C versus 12 °C (20). At 37 °C, laurate was present at 0.16 µmol/mg of LPS, but it decreased to 0.05 µmol/mg at 12 °C (20). The decrease in laurate at 12 °C was counterbalanced by the appearance of palmitoleate, which was present at 0.10 µmol/mg of LPS in E. coli cells grown at 12 °C (20). Only trace amounts of palmitoleate were found in LPS at 37 °C. A similar effect of cold shock on lipid A composition was reported for Salmonella typhimurium (21). It is unlikely that palmitoleate incorporation is catalyzed by LpxL, since palmitoleoyl-acyl carrier protein (ACP) is not a substrate for LpxL (9, 10).

We now describe the induction of a novel palmitoleoyl transferase in extracts of E. coli cells subjected to cold shock (Fig. 1). This enzyme transfers palmitoleate from palmitoleoyl-ACP to Kdo2-lipid IVA, which is also the acceptor for the lpxL-encoded lauroyl transferase (Fig. 1). An inducible palmitoleoyl transferase would account for the appearance of palmitoleate in lipid A of cells grown at 12 °C. The palmitoleoyl transferase is not encoded by lpxL, but appears to be the product of another lpxL homologue found in E. coli (22) that is now designated lpxP. The palmitoleoyl transferase is induced within minutes after cold shock and is accompanied by a massive increase in the levels of lpxP mRNA. A cold shock-induced acyltransferase has not been reported previously.

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Materials-- [gamma -32P]ATP and [alpha -32P]dATP were obtained from NEN Life Science Products. Pyridine, chloroform, methanol, and 88% formic acid were from Fisher. Triton X-100 was Surfact-Amps grade from Pierce. ACP was purchased from Sigma. Hybond-N nylon membranes were obtained from Amersham Pharmacia Biotech. PhosphorImager screens were from Molecular Dynamics. Other items were obtained from the following companies: 0.25-mm glass-backed Silica Gel 60 thin-layer chromatography plates, E. Merck; yeast extract and Tryptone, Difco; and DEAE-Sepharose CL-6B, Amersham Pharmacia Biotech. Formamide, salmon sperm DNA, and RNA standards were obtained from Life Technologies, Inc. All other chemicals were purchased from Sigma.

Bacterial Strains and Growth Conditions-- Strains used in this study are derivatives of E. coli K-12 wild-type W3110, obtained from the E. coli Genetic Stock Center, Yale University. Strain MLK53 harbors a Tn10 insertion in the lpxL(htrB) gene (18, 19). In some experiments strain MC1000 (lpxL+lpxM+lpxP+) (72) was used as the host for the vector pACYC184 (camr tetr p15A replicon) (73), or for the hybrid plasmid pSK57, which harbors lpxP+ on a 5.6-kilobase pair EcoRI fragment derived from Kohara lambda  clone 10D3 (74) in pACYC184. Cultures were generally grown in Luria-Bertani (LB) broth consisting of 10 g of NaCl, 5 g of yeast extract, and 10 g of Tryptone per liter (23). Tetracycline was used at a concentration of 50 µg/ml.

Isolation and Preparation of Lipid Substrates-- Lipid IVA and (Kdo)2-lipid IVA were isolated and purified as described (14, 24, 25). (Kdo)2-[4'-32P]lipid IVA was prepared from [4'-32P]lipid IVA as described previously (25-27), except that membranes of the 4'-kinase overproducing strain BLR(DE3)pLysS/pJK2 (28) were used to increase the yield of [4'-32P]lipid IVA. Lauroyl, myristoyl, palmitoyl, R-3-hydroxymyristoyl, and palmitoleoyl-ACP were synthesized from the corresponding fatty acids and commercial acyl carrier protein, using solubilized membranes from the acyl-ACP synthase overproducing strain E. coli LCH109/pLCH5/pGP1-2, as described previously (10, 29).

Assay for Palmitoleoyl Transferase Activity-- Cell-free extracts were assayed for palmitoleoyl transferase under conditions similar to those used for the lauroyl transferase (9, 10). The reaction mixture (typically 20 µl) contained 50 mM Hepes, pH 7.5, 0.1% Triton X-100, 5 mM MgCl2, 250 mM NaCl, and 6 µM (Kdo)2-[4'-32P]lipid IVA (32,000 cpm/nmol). Palmitoleoyl-ACP was included as the acyl donor at 12 µM, and the final concentration of enzyme was 0.1 mg/ml of crude cell free extract. The tubes were incubated at 12 °C for 5-60 min for MC1000/pSK57(lpxP+) or for 10-20 h for W3110 (wild-type) or MLK53 (lpxL-). Incubation at 12 °C improved the stability of the palmitoleoyl transferase and eliminated an interfering acylation of (Kdo)2-[4'-32P]lipid IVA in crude extracts that was not dependent upon the presence of palmitoleoyl-ACP. The reaction was stopped by spotting 5 µl of the assay mixture onto a silica gel thin layer plate. After the spots were allowed to dry under a cool air stream, the plates were developed in the solvent chloroform/pyridine/88% formic acid/water (30:70:16:10, v/v), dried, and then exposed to a PhosphorImager screen for about 16 h. The percent conversion of substrate to product was determined using a Molecular Dynamics PhosphorImager, and the specific enzymatic activity was calculated in nanomoles/min/mg based upon the original substrate concentration.

Assay for Lauroyl Transferase Activity-- The crude cell-free extracts were assayed for lauroyl transferase activity using lauroyl-ACP and (Kdo)2-[4'-32P]lipid IVA as substrates, as described previously (10). The assay temperature was 30 °C for the lauroyl transferase, unless otherwise indicated.

Conditions for Palmitoleoyl Transferase Induction and Preparation of Cell-free Extracts-- Cultures (800 ml) were grown in Luria-Bertani broth at 30 °C to mid-log phase (A600 ~ 0.4). Half of the culture was shifted to 12 °C. Samples (50 ml) were collected from the 12 °C and 30 °C cultures at the time of shift and at various times after the shift, as indicated. At each time point, the cells were immediately harvested by centrifugation at 4200 × g for 15 min at room temperature. The pellets were washed with 25 ml of 30 mM Hepes, pH 7.5, containing 1 mM EDTA and 1 mM EGTA, and harvested as above. The pellets were resuspended in 1 ml of 30 mM Hepes, pH 7.5, containing 1 mM EDTA and 1 mM EGTA. The cells were broken using an ice-cold French pressure cell (SLM Instruments, Urbana, IL) at 10,000 p.s.i. To remove unbroken cells and large debris, the extracts were centrifuged for 15 min at 4200 × g at 4 °C. The supernatant (referred to as the cell extract) was divided into aliquots and stored at -80 °C. The protein concentrations were determined with the bicinchoninic assay (Pierce) using bovine serum albumin as the standard (30).

RNA Isolation-- Cultures (typically 10 ml) were grown at 30 °C to mid-log phase (A600 ~ 0.4). A portion of the culture (5 ml) was shifted to 12 °C. At 1 h after the shift, 2-ml samples from the 30 °C and the 12 °C cultures were transferred to glass tubes at 100 °C, containing 0.5 ml 2.5% SDS, 1 M NaCl, and 50 mM EDTA, pH 8, to ensure rapid lysis (31). The samples were rapidly mixed and were boiled in a water bath for another 2 min. After cooling to room temperature, the RNA was isolated by phenol extraction using Phase lock gel from 5Prime right-arrow 3Prime, Inc. (32, 33). The A260/A280 ratio for all of the samples was between 1.7 and 1.9, and 10 µg of each RNA sample was used for each lane of the Northern blot.

Northern Blot Analysis-- The Northern blot was done as described (34). The 32P-labeled DNA probe for the lpxP mRNA was constructed by digesting pSK57 with SalI and EcoRI to yield a 1.2-kilobase pair product containing lpxP. This fragment was isolated by agarose gel electrophoresis, and it was purified using the QIAEX Gel extraction kit (QIAGEN). Then, 25 ng of the DNA fragment was used as the template for the Prime-It II random primer labeling kit (Stratagene). The labeling reaction was done with [alpha -32P]dATP (150,000 cpm/nmol) as recommended by the manufacturer. The probe was purified away from contaminating free nucleotides by using a NucTrap probe purification column (Stratagene). Commercial RNA standards were analyzed in parallel during electrophoresis (34) and were stained with methylene blue to estimate the size of the lpxP transcript. The dried Northern blot was visualized using a PhosphorImager.

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A Novel Palmitoleoyl Transferase in Extracts of Cold-shocked Cells of E. coli-- In E. coli or S. typhimurium cells cultured below 15 °C, a palmitoleate residue replaces most of the laurate that is normally linked to lipid A in cells grown at 30-42 °C (20, 21). Conversely, no palmitoleate is detected in lipid A of cells grown above 30 °C (8, 20, 21). We therefore assayed extracts of cells that had been shifted from 30 °C to 12 °C for 2 h during mid-log phase for the induction of a palmitoleoyl-ACP-dependent acyltransferase capable of using the key precursor (Kdo)2-lipid IVA as the substrate (Fig. 1). The latter was previously shown to function as the acceptor for a laurate residue (Fig. 1) in the reaction catalyzed by the Kdo-dependent acyltransferase LpxL(HtrB) in extracts of cells grown at 30 °C or higher (10). As demonstrated in Fig. 2, extracts (0.1 mg/ml) of wild-type E. coli shifted to 12 °C for 2 h converted (Kdo)2-[4'-32P]lipid IVA to a more rapidly migrating product in the presence of palmitoleoyl-ACP, as judged by TLC analysis (Fig. 2), indicative of palmitoleate transfer. However, no acylated product was formed in the absence of palmitoleoyl-ACP in such cold-shocked extracts. Very little palmitoleoyl-ACP-dependent acylation of (Kdo)2-[4'-32P]lipid IVA was detected in extracts of the 30 °C grown control cells (Fig. 2).


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Fig. 1.   Role of (Kdo)2-lipid IVA as a precursor of lipid A in normal and cold-shocked E. coli. The intermediate (Kdo)2-lipid IVA is a key precursor of lipid A, conserved in diverse Gram-negative bacteria (3). In cells grown at 30 °C or higher, (Kdo)2-lipid IVA is acylated by the sequential action of LpxL and LpxM, which incorporate laurate and myristate, respectively, but do not function on precursors lacking the Kdo disaccharide (9-11). Palmitoleoyl-ACP-dependent acylation of (Kdo)2-lipid IVA, catalyzed by the homologue LpxP, is observed only in extracts of cells subjected to cold shock (i.e. transferred from 30 °C to 12 °C for at least 15 min). Mass spectrometry2 demonstrates that ~80% of the lipid A moieties isolated from 12 °C grown E. coli cells contain palmitoleate instead of laurate. In 30 °C grown cells no palmitoleate is detected (20). Prior to elucidation of its function in lipid A biosynthesis (9-11), the lpxL gene was called htrB (high temperature requirement for rapid growth above 32 °C) (17, 18), and lpxM was designated msbB (multicopy suppressor of htrB) (19). The lpxP gene (which displays even greater sequence homology to lpxL than does lpxM) was provisionally termed ddg (sequenced in the GenBankTM/EBI Data Bank with accession number 1872207), because of a proposed dam-dependent growth phenotype (69), but this idea was not confirmed (K. R. Sreekumar, unpublished data). The lpxL and lpxM genes have also recently been designated waaM and waaN by some authors (70, 71). We suggest that the waa nomenclature be restricted to bacterial glycosyltransferase genes (71), and that lpx be used for genes encoding enzymes of lipid A biosynthesis (3).


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Fig. 2.   Palmitoleoyl-ACP-dependent acylation of (Kdo)2-[4'-32P]lipid IVA in extracts of E. coli grown at 12 °C. Crude cell extracts were prepared from mid-log phase cells of strain W3110, shifted from 30 °C to 12 °C for 2 h. Palmitoleoyl transferase activity was assayed using 6 µM (Kdo)2-[4'-32P]lipid IVA and 12 µM palmitoleoyl-ACP, as indicated. The more hydrophobic product is presumably (Kdo)2-[4'-32P](palmitoleoyl)-lipid IVA based on its RF.

The low assay temperature (12 °C) was necessary for demonstrating the induction of the palmitoleoyl transferase because of its instability at higher temperatures. In addition, the presence at 30 °C of an unrelated, interfering enzyme that transfers a palmitate residue from the 1-position of phosphatidylethanolamine or phosphatidylglycerol to (Kdo)2-[4'-32P]lipid IVA or other lipid A precursors (35)3 obscures the palmitoleoyl-ACP-dependent acylation activity (data not shown). The ACP-independent palmitoyl transferase reaction is suppressed by carrying out the assays at 12 °C (data not shown).

The Palmitoleoyl Transferase Is Not Encoded by the lpxL Gene-- To determine if the cold-induced activity was dependent upon the lpxL gene, we assayed the palmitoleoyl transferase activity in extracts of cold-shocked cells of strain MLK53 (17), in which the lpxL gene is inactivated by an insertion mutation. As in the isogenic wild-type strain, palmitoleoyl-ACP-dependent acylation of (Kdo)2-[4'-32P]lipid IVA was induced in MLK53 cells grown at 12 °C (Fig. 3A). Very little palmitoleoyl-ACP-dependent acylation was observed in assays of extracts made from MLK53 cells grown at 30 °C, but the low level of activity seen in such extracts does appear to be slightly higher than the wild-type controls (Fig. 3A). The absence of the lauroyl transferase in extracts of MLK53 cells grown either at 12 °C or 30 °C was confirmed (Fig. 3B), consistent with previous studies (10). Therefore, the cold-induced palmitoleoyl transferase activity is independent of the lpxL gene. Cold shock also had very little effect on the activity of the lauroyl transferase in extracts of wild-type cells, which were assayed at 30 °C with lauroyl-ACP and (Kdo)2-[4'-32P]lipid IVA as substrates (Fig. 3B).


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Fig. 3.   Specific activities of the palmitoleoyl (LpxP) and the lauroyl (LpxL) transferases in extracts of E. coli strains grown at 30 or 12 °C. Mid-log phase cells of wild-type W3110, lpxL mutant MLK53, or MC1000 (bearing multiple copies of pSK57/lpxP+) were shifted from 30 °C to 12 °C for 2 h. Extracts were then assayed at 12 °C for the palmitoleoyl transferase (A) or at 30 °C for the lauroyl transferase (B), using (Kdo)2-[4'-32P]lipid IVA as the acceptor.

Multiple Copies of lpxP, an E. coli Homologue of lpxL, Direct the Overexpression of the Cold-induced Palmitoleoyl Transferase-- E. coli contains two genes that display sequence similarity to lpxL. One of these, previously designated msbB (now termed lpxM as shown in Fig. 1), encodes the myristoyl transferase that is required for the final acylation of lipid A in wild-type cells (11, 19). The other lpxL homologue, which was found in the E. coli genome (22) and is now designated lpxP (Fig. 1), codes for a protein that displays 54% identity and 73% similarity to LpxL (Fig. 4) over the entire 306-amino acid residue length of LpxL. To determine if lpxP encodes the cold shock-induced palmitoleoyl transferase activity, we assayed the palmitoleoyl transferase in extracts of MC1000(pSK57/lpxP+), which contains lpxP behind its native promoter on a pACYC184 vector, after shifting cells from 30 °C to 12 °C for 2 h. As shown in Fig. 3A, the palmitoleoyl transferase specific activity was about 7-fold higher in extracts of MC1000(pSK57/lpxP+) than in wild-type. However, the specific activity of the lauroyl transferase (LpxL) in extracts of MC1000(pSK57/lpxP+) was about the same as that in wild-type extracts (Fig. 3B), and the lauroyl transferase was not induced by cold shock. Given the sequence similarity of LpxL and LpxP, together with the striking increase in the cold-induced palmitoleoyl transferase (Fig. 3A) in MC1000(pSK57/lpxP+) extracts, we propose that lpxP is the structural gene for the palmitoleoyl transferase.


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Fig. 4.   Sequence alignment of the lpxL(htrB) and the lpxP(ddg) gene products. LpxL and LpxP are each 306 amino acid residues long. The numbering refers to the sequence of LpxL. LpxL and LpxP share 54% identity and 73% similarity, with one gap over a segment of 301 amino acids, as determined by BLASTp analysis (39).

A Quantitative Assay for LpxP in MC1000(pSK57/lpxP+) Extracts-- As shown in Fig. 5, the formation of (Kdo)2-[4'-32P](palmitoleoyl)-lipid IVA was linear with time and protein concentration in extracts of strain MC1000(pSK57/lpxP+), despite the unusual assay conditions (12 °C). Furthermore, nearly complete conversion of substrate to product was achieved upon prolonged incubation with a 2-fold excess of palmitoleoyl-ACP over (Kdo)2-[4'-32P]lipid IVA. The palmitoleoyl transferase did not acylate [4'-32P]lipid IVA (Fig. 6), demonstrating that the presence of the Kdo disaccharide in the substrate is required for activity, as in the case of LpxL (9, 10).


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Fig. 5.   An assay for the cold shock-induced palmitoleoyl transferase. The time course (A) at 0.08 mg/ml crude extract protein and the protein concentration dependence (B) at 30 min of palmitoleoyl transfer from palmitoleoyl-ACP to (Kdo)2-[4'-32P]lipid IVA at 12 °C were determined using extracts of cold-shocked cells of MC1000(pSK57/lpxP+), prepared as in Fig. 3.


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Fig. 6.   Kdo dependence of the cold shock-induced palmitoleoyl transferase. Crude extracts of MC1000(pSK57/lpxP+), grown to mid-log phase and shifted for 2 h to 12 °C as in Fig. 3, were assayed at 0.1 mg/ml for palmitoleate transfer to the indicated acceptor under the standard assay conditions for 5 h at 12 °C. The acyl acceptor was either 6 µM (Kdo)2-[4'-32P]lipid IVA or 6 µM [4'-32P]lipid IVA, as indicated.

Selectivity of the Cold-induced Acyltransferase for Palmitoleoyl-ACP-- MLK53 (which lacks the lauroyl transferase) (10) was grown either at 30 °C or 12 °C, and extracts were then assayed with various substrates at 12 °C to determine the acyl chain specificity of the cold-induced palmitoleoyl transferase. As shown in Fig. 7, palmitoleoyl-ACP was the only acyl donor capable of supporting robust acylation of (Kdo)2-[4'-32P]lipid IVA. Lauroyl-ACP, myristoyl-ACP, palmitoyl-ACP, R-3-hydroxymyristoyl-ACP, palmitoleoyl-coenzyme A, and palmitoyl-coenzyme A were virtually inactive as substrates (Fig. 7). A small amount of acylation was seen with myristoyl-ACP (Fig. 7), but this was not cold shock-induced, and might be explained by the direct acylation of (Kdo)2-[4'-32P]lipid IVA by LpxM. LpxM normally prefers (Kdo)2-[4'-32P](lauroyl)-lipid IVA as the acceptor (Fig. 1), but it can also function with (Kdo)2-[4'-32P]lipid IVA at a slow rate (11). The LpxM gene is intact in strain MLK53 (11). Taken together, the results clearly demonstrate that the cold-induced palmitoleoyl transferase is highly selective for palmitoleoyl-ACP, consistent with the effects of cold shock on the fatty acid composition of lipid A (Fig. 1).


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Fig. 7.   Acyl donor specificity of the cold shock-induced acyltransferase. Crude extracts of MLK53 (lpxL-), grown to mid-log phase at 30 °C or shifted for 2 h to 12 °C as in Fig. 3, were assayed at 0.1 mg/ml for acylation of (Kdo)2-[4'-32P]lipid IVA under the palmitoleoyl transferase conditions for 11 h at 12 °C with the indicated acyl donors. The abbreviations are as follows: 12:0 ACP, lauroyl-ACP; 14:0 ACP, myristoyl-ACP; 16:0 ACP, palmitoyl-ACP; 16:1 ACP, palmitoleoyl-ACP; OH14:0 ACP, R-3-hydroxymyristoyl-ACP; 16:1 CoA, palmitoleoyl coenzyme A; 16:0 CoA, palmitoyl coenzyme A.

Time Course of Palmitoleoyl Transferase Induction following Cold Shock-- Cells were grown at 30 °C to mid-log phase, and were then divided into two equal portions, so that parallel cultures could be studied at 12 °C and 30 °C, as shown in Figs. 8 and 9. Samples were then removed from the cultures growing at 30 °C or 12 °C at 0.25, 0.5, 1, 2, 4, and 6 h. Over this time frame, the cold-shocked cells did not increase in density, as shown for the wild-type cells in Fig. 8, but slow growth did resume after about 8 h at 12 °C (data not shown). Extracts of the samples taken from both the 30 °C and the 12 °C cells were assayed for palmitoleoyl-ACP-dependent acylation of (Kdo)2-[4'-32P]lipid IVA, as shown in Fig. 9. Strains W3110 (wild-type), MLK53 (harboring an insertion in lpxL), and MC1000(pSK57/lpxP+) were analyzed in this manner. In every case, the induction of the palmitoleoyl transferase was detected after only 15 min of incubation at 12 °C, despite the absence of a measurable increase in the optical density (Fig. 8). The specific activity of the palmitoleoyl transferase peaked in all three strains after 2 h at 12 °C (Fig. 9). In W3110 and MLK53, however, the specific activity of the enzyme decreased slightly after 3 h of growth at 12 °C.


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Fig. 8.   Growth of the wild-type E. coli strain W3110 at 30 °C and after a shift to 12 °C. The cells were grown to A600 = 0.4 at 30 °C, and the culture was split into two equal portions at time 0. Closed circles indicate the A600 measurements for the culture held at 30 °C, and open symbols are the A600 measurements for the cells shifted to 12 °C at time 0.


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Fig. 9.   Specific activity of the palmitoleoyl transferase in crude extracts made at various times after shifting cells from 30 to 12 °C. Three strains of E. coli were grown on LB broth in a rotary shaker at 30 °C (250 rpm) to mid-log phase, as in Fig. 8. At time 0, half of each culture was shifted to 12 °C, and growth was allowed to continue. Cell extracts were then prepared at the indicated times both from the 30 °C and the 12 °C cultures. The extracts were assayed at 12 °C to determine the specific activity of the palmitoleoyl transferase. The incubation times were 11 h for the extracts of W3110 and MLK53, and 15-30 min for MC1000(pSK57/lpxP+). Closed symbols are used for the 12 °C cultures and open symbols for the 30 °C grown cells. Panel A, wild-type W3110; panel B, MLK53(lpxL-); panel C, MC1000(pSK57/lpxP+).

Very little palmitoleoyl transferase was seen in extracts of the cultures held at 30 °C (Fig. 9), which gradually entered stationary phase over the course of the experiment. However, a small amount of palmitoleoyl transferase above background was observed in extracts of log phase MLK53 cells at 30 °C (Fig. 9B). This trace of palmitoleoyl transferase activity may account for the fact that MLK53 does actually synthesize some penta- and even some hexa-acylated lipid A species (despite the absence of LpxL) at 30 °C, as noted previously in studies of lipid A composition (11, 36). Mass spectrometry (data not shown) indicates that palmitoleate indeed accounts for the presence of the penta-acylated lipid A species in MLK53 grown at 30 °C. It may be that when LpxL is missing the lpxP gene is switched on at higher temperatures than in wild-type cells.

lpxP mRNA Is Measurable in Cells Grown at 12 °C, but Not at 30 °C-- To determine if an increase in lpxP mRNA levels could account for the induction of the palmitoleoyl transferase during cold shock, Northern blotting was used to compare the lpxP mRNA levels extracted from cells grown at 30 °C or 12 °C. A blot of 10-µg RNA samples from both W3110 and MLK53, grown at either 30 °C or 12 °C, is shown in Fig. 10. A heavy band is detected at the position expected for lpxP mRNA (~1000 nucleotides) in the total RNA from the cultures grown at 12 °C. There is no lpxP band in the RNA samples extracted from the cultures grown at 30 °C. The accumulation of lpxP mRNA in the cultures grown only at 12 °C (Fig. 10) and the time course of palmitoleoyl transferase induction at 12 °C (Fig. 9) suggest that lpxP is a typical cold shock-induced gene, probably regulated at the level of transcription and/or message stability (37, 38).


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Fig. 10.   Northern blot analysis of lpxP RNA isolated from 30 °C and 12 °C grown cells. The total RNA was rapidly extracted from W3110 and MLK53 cells grown either at 30 °C or 12 °C. The RNA samples (10 µg each) were separated by gel electrophoresis in lanes adjacent to commercial size standards, and the gels were analyzed by Northern blotting using a 32P-labeled lpxP DNA probe. The probe was used at 5 × 105 cpm/ml of hybridization solution. The final nitrocellulose membrane was exposed overnight to a PhosphorImager screen.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

As demonstrated previously in our laboratory, the enzyme LpxL(HtrB) catalyzes the transfer of laurate from lauroyl-ACP to the lipid A precursor (Kdo)2-lipid IVA in extracts of cells grown at 30 °C or above (Fig. 1) (10). We now have discovered an additional Kdo-dependent acyltransferase (LpxP) in E. coli that is required for the biosynthesis of a distinct molecular species of lipid A, present only in cells grown at low temperatures (Fig. 1) (20, 21). LpxP transfers palmitoleate from palmitoleoyl-ACP to (Kdo)2-lipid IVA, and it is induced within 15 min in log phase cells shifted from 30 °C to 12 °C (Fig. 9). The palmitoleoyl transferase is a distinct enzyme, not an additional activity associated with the lauroyl transferase, as demonstrated by the fact that the palmitoleoyl transferase is induced normally (Fig. 3) in mutant cells (MLK53) (10) harboring an insertion in the lpxL gene. Furthermore, the palmitoleoyl transferase is greatly overproduced in strains harboring multiple copies of the lpxP gene expressed from its own promoter (Fig. 3). LpxL and LpxP share 54% identity and 73% similarity, with one gap over a segment of 301 out of 306 amino acids (Fig. 4), as determined by BLASTp analysis (39). In contrast, LpxL and LpxM (Fig. 1) show only 29% identity and 46% similarity, with 21 gaps over a sequence of 309 amino acids out of the 323 residues that comprise LpxM (39).

Extracts of wild-type E. coli grown at 12 °C contain both the lauroyl and the palmitoleoyl transferase activities (Fig. 3). The mechanisms by which the cells determine the amount of palmitoleate versus laurate that is transferred to (Kdo)2-lipid IVA at 12 °C in vivo may be quite interesting and will require further study. Based on mass spectrometry, we estimate that about 80% of the lipid A residues of cells grown at 12 °C are acylated with palmitoleate rather than laurate.4

The level of lpxP mRNA increases by several orders of magnitude in cells grown at 12 °C (Fig. 10). This phenomenon must surely account for the induction of the palmitoleoyl transferase activity upon cold shock (Figs. 3 and 9). The time course of LpxP induction and its gradual decline in wild-type cells after several hours of growth at 12 °C (Fig. 9) is reminiscent of the response seen with many cold shock proteins, including the extensively studied RNA-binding protein CspA (37, 38, 40). The mechanisms by which cold shock induces CspA are still not fully understood (37, 38). Initial work suggested that increased transcription accounted for elevated CspA message levels in cold-shocked cells (37, 38). More recent studies support the view that CspA mRNA stability is also selectively enhanced at lower growth temperatures, possibly because of temperature effects on mRNA secondary structure (41, 42). RNase E may play a direct role in the control of mRNA stability during cold shock (43). In addition, translation of cold shock mRNA may be selectively enhanced (41, 42). Given these considerations, it will be interesting to express lpxP behind a constitutive promoter and to measure LpxP activity in such constructs as a function of temperature. Expression of a reporter gene like lacZ or cat at various temperatures behind the native lpxP promoter might also be informative. Finally, a search for proteins that control the activity of the lpxP promoter in vivo might reveal how cells detect cold shock and readjust their lipid A pathway accordingly.

Among the enzymes of lipid A biosynthesis, LpxP may be unique in its induction at low temperatures, but it is not the only enzyme of the pathway that is regulated. The deacetylase (LpxC) that catalyzes the second, committed step of lipid A biosynthesis is increased about 10-fold in cells treated with the specific deacetylase inhibitor, L-573,655 (44, 45), or in point mutants with low levels of lpxA (44, 46). Deacetylase regulation is not accompanied by significant changes in lpxC mRNA (44). Instead, LpxC protein levels may be controlled by the membrane-associated protease FtsH (47).

Additional regulation of lipid A biosynthesis has recently been discovered by Miller and co-workers (48-50). These investigators have shown that the PhoP/PhoQ two component system is required for the modification of S. typhimurium lipid A with palmitate, L-4-aminoarabinose, and/or 2-hydroxymyristate (48-50). While these modifications are not required for cell growth, they are critical for intracellular survival and resistance to basic antibacterial peptides (48-50). The PhoP/PhoQ system exerts its actions by regulating a second two-component system, known as PmrA/PmrB (48, 49), which in turn is thought to control the expression of the putative enzymes that modify S. typhimurium lipid A. The PhoP/PhoQ system is switched on by low pH, as might be encountered by bacteria within endosomes, and by low magnesium ion concentrations (48). Whether or not the PhoP/PhoQ system is involved in the cold shock-induced modification of lipid A remains to be determined.

Both procaryotic (51-55) and eucaryotic (55-57) organisms increase the degree of unsaturation of their glycerophospholipid acyl chains at low temperatures in a process termed homeoviscous adaptation (53), presumably to maintain the optimal membrane fluidity. The acylation of lipid A with palmitoleate instead of laurate (Fig. 1) might therefore function to adjust outer membrane fluidity in E. coli cells shifted to low temperatures, as would occur during prolonged survival outside of an animal host. Gram-negative bacteria like Haemophilus influenzae, which are transmitted from animal to animal without having to persist in the environment, do not contain a lpxP homologue (58). The melting point of cis-9-palmitoleic acid is 0.5 °C, whereas that of lauric acid is 44.2 °C (59). The liquid-crystalline transition temperature(s) of lipid A substituted with palmitoleate rather than laurate would likely decrease in a related manner, but very little is actually known about the physical properties of lipid A and its precursors (60-62). We have constructed a mutant in which the E. coli lpxP gene is inactivated. Although this strain can grow at 12 °C, it is extremely hypersensitive to antibiotics at low temperatures.2

To our knowledge, no one has previously reported an acyltransferase induced under conditions of cold shock (or other environmental stress) that selectively incorporates a specific fatty acyl chain into a membrane lipid precursor. The glycerol-3-phosphate acyltransferases of E. coli certainly are not regulated in this manner (52) and do not display a high degree of selectivity for their acyl donor substrates (63). There is only a slight difference in the fatty acid composition of the glycerophospholipids of E. coli cells grown at 27 °C versus 37 °C (54). However, a 2-fold increase in unsaturation (mainly increased cis-vaccenate at the expense of palmitate) is observed in the glycerophospholipids of E. coli cells grown at 17 °C versus 37 °C (54). The condensing enzyme (3-ketoacyl-ACP synthase II), encoded by the fabF gene, is thought to control the unsaturated acyl chain content of the E. coli glycerophospholipids (52, 64). This may be mediated by a direct effect of temperature on FabF activity, which is higher at low temperatures and therefore might increase the size of the unsaturated fatty acid precursor pool appropriately (52, 64). In other organisms, including Synechocystis, Bacillus subtilis, Acanthamoeba, and carp, cold shock induces the transcription of specific fatty acid desaturases (55, 65, 75), likewise increasing the pool of available unsaturated fatty acids.

Mutants of E. coli that are defective in the fabA or the fabB genes require unsaturated fatty acids for growth (51, 52, 56, 66). Many different unnatural and polyunsaturated fatty acids, including trans-unsaturated compounds, can support the growth of such auxotrophs (51, 52, 56, 67). Unlike the lipid A acyltransferases (3), the glycerophospholipid acyltransferases are not specific for their acyl donor substrates (63), and therefore many different unnatural fatty acid analogs can be incorporated into the glycerophospholipids of such auxotrophs. The composition of lipid A in fatty acid auxotrophs supplemented with analogs has never been examined, but it should be unaffected, since exogenous fatty acids are activated as coenzyme A thioesters, and not as acyl-ACPs (1, 3, 66). However, DiRienzo and Inouye (68) reported that unsaturated fatty acid auxotrophs supplemented with elaidate (an unnatural trans-unsaturated fatty acid) do not grow at low temperatures and accumulate lipid A precursor(s). The chemical structures of these substances were not determined (68). Their characterization might yet provide interesting new insights into the regulation of lipid A biosynthesis and into the biological significance of lipid A modification with palmitoleate during cold shock.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM-51310 (to C. R. H. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by National Institutes of Health Training Grant GM08558 in Biological Chemistry to Duke University.

parallel Supported at Tufts University by GM-34123 (to M. Schaechter) and GM-38035 (to A. Wright).

** To whom correspondence should be addressed. Tel.: 919-684-5326; Fax: 919-684-8885; E-mail: Raetz{at}biochem.duke.edu.

2 S. M. Carty and C. R. H. Raetz, manuscript in preparation.

3 R. Bishop and C. R. H. Raetz, unpublished data.

4 S. M. Carty and C. R. H. Raetz, unpublished data.

    ABBREVIATIONS

The abbreviations used are: LPS, lipopolysaccharide; ACP, acyl carrier protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Raetz, C. R. H. (1990) Annu. Rev. Biochem. 59, 129-170[CrossRef][Medline] [Order article via Infotrieve]
  2. Raetz, C. R. H. (1993) J. Bacteriol. 175, 5745-5753[Medline] [Order article via Infotrieve]
  3. Raetz, C. R. H. (1996) in Escherichia coli and Salmonella: Cellular and Molecular Biology (Neidhardt, F. C., ed), 2nd Ed., Vol. 1, pp. 1035-1063, American Society for Microbiology, Washington, DC
  4. Rietschel, E. T., Kirikae, T., Schade, F. U., Mamat, U., Schmidt, G., Loppnow, H., Ulmer, A. J., Zähringer, U., Seydel, U., Di Padova, F., Schreier, M., and Brade, H. (1994) FASEB J. 8, 217-225[Abstract/Free Full Text]
  5. Schnaitman, C. A., and Klena, J. D. (1993) Microbiol. Rev. 57, 655-682[Abstract]
  6. Morrison, D. C., and Ryan, J. L. (eds) (1992) Bacterial Endotoxic Lipopolysaccharides: Molecular Biochemistry and Cellular Biology, Vol. I, CRC Press, Boca Raton, FL
  7. Raetz, C. R. H. (1998) Prog. Clin. Biol. Res. 397, 1-14
  8. Odegaard, T. J., Kaltashov, I. A., Cotter, R. J., Steeghs, L., van der Ley, P., Khan, S., Maskell, D. J., and Raetz, C. R. H. (1997) J. Biol. Chem. 272, 19688-19696[Abstract/Free Full Text]
  9. Brozek, K. A., and Raetz, C. R. H. (1990) J. Biol. Chem. 265, 15410-15417[Abstract/Free Full Text]
  10. Clementz, T., Bednarski, J. J., and Raetz, C. R. H. (1996) J. Biol. Chem. 271, 12095-12102[Abstract/Free Full Text]
  11. Clementz, T., Zhou, Z., and Raetz, C. R. H. (1997) J. Biol. Chem. 272, 10353-10360[Abstract/Free Full Text]
  12. Rick, P. D., and Osborn, M. J. (1977) J. Biol. Chem. 252, 4895-4903[Medline] [Order article via Infotrieve]
  13. Rick, P. D., Fung, L. W.-M., Ho, C., and Osborn, M. J. (1977) J. Biol. Chem. 252, 4904-4912[Abstract]
  14. Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16088[Abstract/Free Full Text]
  15. Strain, S. M., Armitage, I. M., Anderson, L., Takayama, K., Qureshi, N., and Raetz, C. R. H. (1985) J. Biol. Chem. 260, 16089-16098[Abstract/Free Full Text]
  16. Clementz, T., Bednarski, J., and Raetz, C. R. H. (1995) FASEB J. 9, A1311
  17. Karow, M., Fayet, O., Cegielska, A., Ziegelhoffer, T., and Georgopoulos, C. (1991) J. Bacteriol. 173, 741-750[Medline] [Order article via Infotrieve]
  18. Karow, M., and Georgopoulos, C. (1991) Mol. Microbiol. 5, 2285-2292[Medline] [Order article via Infotrieve]
  19. Karow, M., and Georgopoulos, C. (1992) J. Bacteriol. 174, 702-710[Abstract]
  20. Van Alphen, L., Lugtenberg, B., Rietschel, E. T., and Mombers, C. (1979) Eur. J. Biochem. 101, 571-579[Medline] [Order article via Infotrieve]
  21. Wollenweber, H.-W., Schlecht, S., Lüderitz, O., and Rietschel, E. T. (1983) Eur. J. Biochem. 130, 167-171[Abstract]
  22. Blattner, F. R., Plunkett, G., III, Bloch, C. A., Perna, N. T., Burland, V., Riley, M., Collado-Vides, J., Glasner, J. D., Rode, C. K., Mayhew, G. F., Gregor, J., Davis, N. W., Kirkpatrick, H. A., Goeden, M. A., Rose, D. J., Mau, B., and Shao, Y. (1997) Science 277, 1453-1474[Abstract/Free Full Text]
  23. Miller, J. R. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  24. Hampton, R. Y., Golenbock, D. T., and Raetz, C. R. H. (1988) J. Biol. Chem. 263, 14802-14807[Abstract/Free Full Text]
  25. Brozek, K. A., Hosaka, K., Robertson, A. D., and Raetz, C. R. H. (1989) J. Biol. Chem. 264, 6956-6966[Abstract/Free Full Text]
  26. Belunis, C. J., and Raetz, C. R. H. (1992) J. Biol. Chem. 267, 9988-9997[Abstract/Free Full Text]
  27. Kadrmas, J. L., Allaway, D., Studholme, R. E., Sullivan, J. T., Ronson, C. W., Poole, P. S., and Raetz, C. R. H. (1998) J. Biol. Chem. 273, 26432-26440[Abstract/Free Full Text]
  28. Garrett, T. A., Kadrmas, J. L., and Raetz, C. R. H. (1997) J. Biol. Chem. 272, 21855-21864[Abstract/Free Full Text]
  29. Kelly, T. M., Stachula, S. A., Raetz, C. R. H., and Anderson, M. S. (1993) J. Biol. Chem. 268, 19866-19874[Abstract/Free Full Text]
  30. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochem. 150, 76-85[Medline] [Order article via Infotrieve]
  31. Sarmientos, P., Sylvester, J. E., Contente, S., and Cashel, M. (1983) Cell 32, 1337-1346[Medline] [Order article via Infotrieve]
  32. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159[CrossRef][Medline] [Order article via Infotrieve]
  33. Murphy, N. R., and Hellwig, R. J. (1996) BioTechniques 21, 934-939[Medline] [Order article via Infotrieve]
  34. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York
  35. Brozek, K. A., Bulawa, C. E., and Raetz, C. R. H. (1987) J. Biol. Chem. 262, 5170-5179[Abstract/Free Full Text]
  36. Zhou, Z., White, K. A., Polissi, A., Georgopoulos, C., and Raetz, C. R. H. (1998) J. Biol. Chem. 273, 12466-75[Abstract/Free Full Text]
  37. Yamanaka, K., Fang, L., and Inouye, M. (1998) Mol. Microbiol. 27, 247-255[CrossRef][Medline] [Order article via Infotrieve]
  38. Jones, P. G., and Inouye, M. (1994) Mol. Microbiol. 11, 811-818[Medline] [Order article via Infotrieve]
  39. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997) Nucleic Acids Res. 25, 3389-3402[Abstract/Free Full Text]
  40. Thieringer, H. A., Jones, P. G., and Inouye, M. (1998) Bioessays 20, 49-57[CrossRef][Medline] [Order article via Infotrieve]
  41. Goldenberg, D., Azar, I., Oppenheim, A. B., Brandi, A., Pon, C. L., and Gualerzi, C. O. (1997) Mol. Gen. Genet. 256, 282-290[CrossRef][Medline] [Order article via Infotrieve]
  42. Goldenberg, D., Azar, I., and Oppenheim, A. B. (1996) Mol. Microbiol. 19, 241-248[Medline] [Order article via Infotrieve]
  43. Fang, L., Jiang, W., Bae, W., and Inouye, M. (1997) Mol. Microbiol. 23, 355-364[Medline] [Order article via Infotrieve]
  44. Sorensen, P. G., Lutkenhaus, J., Young, K., Eveland, S. S., Anderson, M. S., and Raetz, C. R. H. (1996) J. Biol. Chem. 271, 25898-25905[Abstract/Free Full Text]
  45. Onishi, H. R., Pelak, B. A., Gerckens, L. S., Silver, L. L., Kahan, F. M., Chen, M. H., Patchett, A. A., Galloway, S. M., Hyland, S. A., Anderson, M. S., and Raetz, C. R. H. (1996) Science 274, 980-982[Abstract/Free Full Text]
  46. Anderson, M. S., Bull, H. S., Galloway, S. M., Kelly, T. M., Mohan, S., Radika, K., and Raetz, C. R. H. (1993) J. Biol. Chem. 268, 19858-19865[Abstract/Free Full Text]
  47. Ogura, T., Inoue, K., Tatsuta, T., Suzaki, T., Karata, K., Young, K., Su, L.-H., Fierke, C. A., Jackman, J. E., Raetz, C. R. H., Coleman, J., Tomoyasu, T., and Matsuzawa, H. (1999) Mol. Microbiol. 31, 833-844[CrossRef][Medline] [Order article via Infotrieve]
  48. Guo, L., Lim, K. B., Gunn, J. S., Bainbridge, B., Darveau, R. P., Hackett, M., and Miller, S. I. (1997) Science 276, 250-253[Abstract/Free Full Text]
  49. Gunn, J. S., Lim, K. B., Krueger, J., Kim, K., Guo, L., Hackett, M., and Miller, S. I. (1998) Mol. Microbiol. 27, 1171-1182[CrossRef][Medline] [Order article via Infotrieve]
  50. Guo, L., Lim, K. B., Poduje, C. M., Daniel, M., Gunn, J. S., Hackett, M., and Miller, S. I. (1998) Cell 95, 189-198[Medline] [Order article via Infotrieve]
  51. Cronan, J. E. J., and Gelmann, E. P. (1975) Bacteriol. Rev. 39, 232-256[Medline] [Order article via Infotrieve]
  52. Cronan, J. E., Jr., and Rock, C. O. (1987) in Escherichia coli and Salmonella typhimurium (Neidhardt, F. C., ed), Vol. I, pp. 474-497, ASM Publications, Washington, DC
  53. Sinensky, M. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 522-525[Abstract]
  54. Morein, S., Andersson, A.-S., Rilfors, L., and Lindblom, G. (1996) J. Biol. Chem. 271, 6801-6809[Abstract/Free Full Text]
  55. Vigh, L., Maresca, B., and Harwood, J. L. (1998) Trends Biochem. Sci. 23, 369-374[CrossRef][Medline] [Order article via Infotrieve]
  56. Silbert, D. F. (1975) Annu. Rev. Biochem. 44, 315-339[CrossRef][Medline] [Order article via Infotrieve]
  57. Quinn, P. J., and Chapman, D. (1980) CRC Crit. Rev. Biochem. 8, 1-117[Medline] [Order article via Infotrieve]
  58. Fleischmann, R. D., Adams, M. D., White, O., Clayton, R. A., Kirkness, E. F., Kerlavage, A. R., Bult, C. J., Tomb, J.-F., Dougherty, B. A., Merrick, J. M., McKenney, K., Sutton, G., FitzHugh, W., Fields, C., Gocayne, J. D., Scott, J., Shirley, R., Liu, L.-I., Glodek, A., Kelley, J. M., Weidman, J. F., Phillips, C. A., Spriggs, T., Hedblom, E., Cotton, M. D., Utterback, T. R., Hanna, M. C., Nguyen, D. T., Saudek, D. M., Brandon, R. C., Fine, L. D., Fritchman, J. L., Furhmann, J. L., Geohagen, N. S. M., Gnehm, C. L., McDonald, L. A., Small, K. V., Fraser, C. M., Smith, H. O., and Venter, J. C. (1995) Science 269, 496-512[Medline] [Order article via Infotrieve]
  59. Small, D. M. (1986) in The Physical Chemistry of Lipids: Handbook of Lipid Research (Hanahan, D. J., ed), Vol. 4, Plenum Press, New York
  60. Lipka, G., Demel, R. A., and Hauser, H. (1988) Chem. Phys. Lipids 48, 267-280[Medline] [Order article via Infotrieve]
  61. Hofer, M., Hampton, R. Y., Raetz, C. R. H., and Yu, H. (1991) Chem. Phys. Lipids 59, 167-181[Medline] [Order article via Infotrieve]
  62. Din, Z. Z., Mukerjee, P., Kastowsky, M., and Takayama, K. (1993) Biochemistry 32, 4579-4586[Medline] [Order article via Infotrieve]
  63. Green, P. R., Merrill, A. H., Jr., and Bell, R. M. (1981) J. Biol. Chem. 256, 11151-11159[Free Full Text]
  64. Garwin, J. L., Klages, A. L., and Cronan, J. E., Jr. (1980) J. Biol. Chem. 255, 3263-3265[Abstract/Free Full Text]
  65. Tiku, P. E., Gracey, A. Y., Macartney, A. I., Beynon, R. J., and Cossins, A. R. (1996) Science 271, 815-818[Abstract]
  66. Magnuson, K., Jackowski, S., Rock, C. O., and Cronan, J. E., Jr. (1993) Microbiol. Rev. 57, 522-542[Abstract]
  67. Lands, W. E. (1988) Prog. Clin. Biol. Res. 282, 11-28[Medline] [Order article via Infotrieve]
  68. DiRienzo, J. M., and Inouye, M. (1983) Eur. J. Biochem. 135, 351-357[Abstract]
  69. Carty, S. M., Sreekumar, K., and Raetz, C. R. H. (1997) FASEB J. 11, A1423
  70. Khan, S. A., Everest, P., Servos, S., Foxwell, N., Zahringer, U., Brade, H., Rietschel, E. T., Dougan, G., Charles, I. G., and Maskell, D. J. (1998) Mol. Microbiol. 29, 571-579[CrossRef][Medline] [Order article via Infotrieve]
  71. Reeves, P. R., Hobbs, M., Valvano, M. A., Skurnik, M., Whitfield, C., Coplin, D., Kido, N., Klena, J., Maskell, D., Raetz, C. R. H., and Rick, P. D. (1996) Trends Microbiol. 4, 495-503[CrossRef][Medline] [Order article via Infotrieve]
  72. Casadaban, M. J., and Cohen, S. N. (1980) J. Mol. Biol. 138, 179-207[Medline] [Order article via Infotrieve]
  73. Chang, A. C., and Cohen, S. N. (1978) J. Bacteriol. 134, 1141-1156[Medline] [Order article via Infotrieve]
  74. Kohara, Y., Akiyama, K., and Isono, K. (1987) Cell 50, 495-508[Medline] [Order article via Infotrieve]
  75. Aguilar, P. S., Cronan, J. E., Jr., and de Mendoza, D. (1998) J. Bacteriol. 180, 2194-2200[Abstract/Free Full Text]


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