Ca2+-induced Phosphoethanolamine Transfer to the Outer 3-Deoxy-D-manno-octulosonic Acid Moiety of Escherichia coli Lipopolysaccharide

A NOVEL MEMBRANE ENZYME DEPENDENT UPON PHOSPHATIDYLETHANOLAMINE*

Margaret I. KanipesDagger , Shanhua Lin§, Robert J. Cotter§, and Christian R. H. RaetzDagger

From the Dagger  Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 and the § Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received for publication, October 3, 2000, and in revised form, October 19, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Certain strains of Escherichia coli and Salmonella contain lipopolysaccharide (LPS) modified with a phosphoethanolamine (pEtN) group at position 7 of the outer 3-deoxy-D-manno-octulosonic acid (Kdo) residue. Using the heptose-deficient E. coli mutant WBB06 (Brabetz, W., Muller-Loennies, S., Holst, O., and Brade, H. (1997) Eur. J. Biochem. 247, 716-724), we now demonstrate that the critical parameter determining the presence or absence of pEtN is the concentration of CaCl2 in the medium. As judged by mass spectrometry, half the LPS in WBB06, grown on nutrient broth with 5 mM CaCl2, is derivatized with a pEtN group, whereas LPS from WBB06 grown without supplemental CaCl2 is not. Membranes from E. coli WBB06 or wild-type W3110 grown on 5-50 mM CaCl2 contain a novel pEtN transferase that uses the precursor Kdo2-[4'-32P]lipid IVA as an acceptor. Transferase is not present in membranes of E. coli grown with 5 mM MgCl2, BaCl2, or ZnCl2. Hydrolysis of the in vitro reaction product, pEtN-Kdo2-[4'-32P]lipid IVA, at pH 4.5 shows that the pEtN substituent is located on the outer Kdo moiety. Membranes from an E. coli pss knockout mutant grown on 50 mM CaCl2, which lack phosphatidylethanolamine, do not contain measurable transferase activity unless exogenous phosphatidylethanolamine is added back to the assay system. The induction of the pEtN transferase by 5-50 mM CaCl2 suggests possible role(s) in establishing transformation competence or resisting environmental stress, and represents the first example of a regulated covalent modification of the inner core of E. coli LPS.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Lipopolysaccharide (LPS)1 is the major constituent of the outer leaflet of the outer membranes of Gram-negative bacteria (1-5). LPS consists of three covalently linked domains. These are: 1) the lipid A moiety, a glucosamine-based phospholipid that serves as the hydrophobic membrane anchor of LPS; 2) the core region, a nonrepeating oligosaccharide decorated with several phosphate-containing substituents; and 3) the O-antigen, a distal repeating oligosaccharide (Fig. 1). The lipid A and the 3-deoxy-D-manno-octulosonic acid (Kdo) portions of the LPS core are essential for the growth of Escherichia coli and most other Gram-negative bacteria (1-5). Mutants lacking LPS sugars distal to Kdo are usually viable, but often show reduced virulence and sensitivity to killing by serum (5).

The inner core of E. coli K-12 LPS consists of two Kdo residues and two (or three) L-glycero-D-manno-heptose moieties (Fig. 1) (2, 6). The latter are usually derivatized with phosphate and ethanolamine pyrophosphate groups (Fig. 1) (5). Mutations in the biosynthesis or incorporation of heptose, including ones that eliminate the phosphate-containing substituents, reduce the stability of the outer membrane (2, 4, 7, 8). The resulting "deep rough" phenotype includes hypersensitivity to certain antibiotics, decreased levels of outer membrane proteins, and resistance to bacteriophages that utilize LPS core sugars as receptors (2, 4, 7, 8).

Recently, Brabetz and co-workers (9) constructed an E. coli deletion mutant (WBB06) that lack both heptosyltransferase I (WaaC/RfaC) and heptosyltransferase II (WaaF/RfaF), resulting in the production of a truncated LPS missing all sugars distal to Kdo (Fig. 1). Intriguingly, the authors noted that deletion of both heptosyltransferases was associated with the formation of an unusual LPS structural variant in WBB06 (Fig. 1), characterized by a high degree of pEtN substitution at position 7 of the outer Kdo residue (9). Modification of Kdo with pEtN had not been observed in earlier studies of similarly truncated LPS molecules (10), isolated from heptosyltransferase-deficient point mutants of E. coli. We therefore re-examined the structure of intact LPS from WBB06, using newer purification and MALDI-TOF mass spectrometry methods (11). We found no evidence for a pEtN substituent on this LPS when the cells were grown in standard LB broth (12). However, supplementation of the medium with high concentrations of CaCl2 (5-50 mM) resulted in the appearance of a Kdo-linked pEtN moiety, as reported by Brabetz et al. (9), who had routinely included 5 mM CaCl2 in their media to increase the growth rate of WBB06.



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Fig. 1.   Schematic molecular structure of E. coli K-12 LPS. The heptose units of the inner core are constitutively modified with phosphate and ethanolamine pyrophosphate groups (5, 6, 8). In certain strains of E. coli and Salmonella, however, a pEtN moiety has also been found on the outer Kdo residue (5, 9). As shown in the present study, the presence of the pEtN unit on the outer Kdo of E. coli WBB06 is dependent on 5-50 mM CaCl2 in the growth medium.

We have now discovered a novel CaCl2-induced enzyme that modifies the outer Kdo moiety of E. coli LPS with a pEtN group. We have developed an assay for this enzyme using membranes from cells grown in the presence of 5-50 mM CaCl2. The LPS precursor, Kdo2-[4'-32P]lipid IVA (13), functions as an acceptor of the pEtN moiety in our in vitro system (Fig. 2). The conditions that induce the pEtN transferase share some similarities with the treatments that make E. coli cells competent for DNA-mediated transformation (14, 15). The mechanism of pEtN transferase induction by CaCl2 may provide new insights into the functions of the LPS core. We are not aware of any previous reports of CaCl2-induced enzymes in E. coli.



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Fig. 2.   Proposed reaction catalyzed by the calcium-induced pEtN transferase. The position of the pEtN substituent on the outer Kdo moiety of the putative in vitro reaction product is not yet established. It is assumed to be attached at position 7 based on the work of Brabetz et al. (9) with LPS isolated from WBB06 (9).



    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- [gamma -32P]ATP was purchased from Perkin Elmer Life Science. Kdo, Hepes, CaCl2, and phospholipids were obtained from Sigma. Triton X-100 was purchased from Pierce. Yeast extract and tryptone were obtained from Difco. The Silica Gel 60 thin layer chromatography plates were from Merck. Pyridine, chloroform, and methanol were of reagent grade and were purchased from VWR.

Bacterial Strains and Growth Conditions-- The E. coli deep rough mutant strain WBB06, harboring a deletion that removes portions of both the waaC and waaF genes, was kindly provided by Dr. Werner Brabetz (9). E. coli strains W3110 and W3899 were obtained from the E. coli Genetic Stock Center, Yale University. Strains were generally grown in LB broth (10 g of NaCl, 10 g of tryptone, and 5 g of yeast extract per liter) (12). When necessary, the cultures were supplemented with tetracycline (12 µg/ml). In some experiments, CaCl2 was added to the medium at final concentrations ranging from 5 µM to 50 mM, as indicated. E. coli strain AD90 was obtained from Dr. William Dowhan at the University of Texas, Houston (16). AD90 harbors an insertion mutation in the pss gene, but is able to grow without the pss+ covering plasmid when 20-50 mM MgCl2 or CaCl2 is added to the medium. Under these conditions, the membranes of AD90 lack detectable phosphatidylethanolamine.

Preparation of E. coli Membranes-- Typically, cell-free extracts were prepared by growing a 100-ml LB broth culture, supplemented with or without various divalent cations, at 37 °C to A600 = 1.7. Cells were harvested by centrifugation at 4,000 × g for 10 min. Cells were resuspended in 4 ml of 50 mM Hepes, pH 7.5, and broken by a single passage through a French pressure cell at 18,000 psi. Cellular debris was removed by centrifugation at 4000 × g for 15 min. Membranes were prepared by ultracentrifugation at 100,000 × g for 60 min. The membranes were resuspended in 8 ml of Hepes, pH 7.5, and the ultracentrifugation was repeated a second time at 100,000 × g to remove any contaminating cytosol. The final membrane pellet was resuspended in 1 ml 50 mM Hepes, pH 7.5, and stored at -80 °C until further use. Protein concentrations were determined by the bicinchoninic acid method (Pierce) (17).

In Vitro Assay for the CaCl2-induced pEtN Transferase-- The calcium-induced phosphoethanolamine transferase was assayed in cell-free extracts or membrane preparations using the LPS precursor, Kdo2-[4'-32P]lipid IVA, which was prepared enzymatically and purified as described previously (13, 18). The radiolabeled substrate was stored at -20 °C as a frozen aqueous dispersion at ~60,000 cpm/µl in 10 mM Tris HCl, pH 7.9, with 1 mM EDTA and 1 mM EGTA. Prior to each use, the radiolabeled substrate was subjected to ultrasonic irradiation in a water bath sonicator for 1 min. The pEtN transferase was assayed in a reaction mixture (10 µl final volume) containing 50 mM Hepes, pH 7.5, 0.1% Triton X-100, 1.25 mM dithiothreitol, and 2.5 µM Kdo2-[4'-32P]lipid IVA (~250,000 cpm/nmol). The reactions were started by addition of enzyme, and were incubated for the indicated time periods at 30 °C. The reactions were stopped by application of 4-µl samples directly onto a silica gel TLC plate. After drying at room temperature, the plate was developed in the solvent chloroform, pyridine, 88% formic acid, water (30:70:16:10, v/v). After drying, the plate was analyzed and the percent conversion to product quantified using a Molecular Dynamics PhosphorImager (STORM 840), equipped with ImageQuant software.

Characterization of the in Vitro pEtN Transferase Reaction Product by Hydrolysis at pH 4.5-- Two 10-µl reaction tubes were prepared containing 50 mM Hepes, pH 7.5, 0.1% Triton X-100, 2.5 µM Kdo2-[4'-32P]lipid IVA (8,000 cpm/tube) or 2.5 µM pEtN-Kdo2-[4'-32P]lipid IVA (8,000 cpm/tube). The pEtN-Kdo2-[4'-32P]lipid IVA sample was generated using the standard pEtN transferase assay conditions, described above, and was purified by thin layer chromatography, as reported for the substrate Kdo2-[4'-32P]lipid IVA (13, 18). Next, 4 µl of 10% SDS and 26 µl of 50 mM sodium acetate, pH 4.5, were added to each tube. The tubes were then incubated in a boiling water bath. At various times, 4-µl samples were withdrawn and spotted onto a silica TLC plate. The plate was developed in the solvent chloroform, pyridine, 88% formic acid, water (30:70:16:10, v/v), and analyzed with a PhosphorImager, as described above for the assay.

Extraction and Purification of LPS from the Heptose-deficient Mutant WBB06-- To determine what fraction of the LPS molecules in WBB06 are covalently modified with pEtN, 100 ml of LB broth cultures of WBB06 were grown in the presence or absence of 5 mM CaCl2 to A600 = 1.7. Cells were harvested by centrifugation at 4,000 × g for 10 min and washed once with 40 ml of phosphate-buffered saline (19). The final cell pellets were resuspended in 40 ml of phosphate-buffered saline. Glycerophospholipids and LPS were extracted for 1 h at room temperature with a single phase Bligh/Dyer mixture (20), generated by adding 100 ml of methanol and 50 ml of chloroform to the cell suspension. This method is applicable to truncated "Re" LPS (2) made by mutants defective in heptose biosynthesis, which is soluble in this mixture, but not to more extensively glycosylated LPS molecules. The precipitated proteins and nucleic acids were removed by centrifugation at 4,000 × g for 15 min. The supernatant was converted to a two-phase Bligh/Dyer system, which consists of chloroform/methanol/water (2:2:1.8, v/v), by adding appropriate volumes of chloroform and water. The phases were separated by centrifugation at 4,000 × g for 15 min. The lower phase was dried by rotary evaporation, re-dissolved in 5 ml of chloroform/methanol/water (2:3:1, v/v), and applied to a 1-ml DEAE cellulose column (acetate form) equilibrated with the same solvent mixture (11, 21, 22). Following sample loading, the column was washed with 10 column volumes of chloroform/methanol/water (2:3:1, v/v). The bound phospholipids and LPS were eluted with 5-ml steps of chloroform/methanol/aqueous ammonium acetate (2:3:1, v/v), with ammonium acetate concentrations of 60, 120, 240, and 480 mM successively in the aqueous component. For each step elution, 1-ml fractions were collected. The appearance of the phospholipids and the LPS was determined by spotting 10-µl portions of each fraction onto a silica TLC plate. The plate was developed in the solvent chloroform, pyridine, 88% formic acid, water (30:70:16:10, v/v). After drying, the substances were visualized by spraying the plate with 10% sulfuric acid in ethanol and charring on a hot plate. The Re LPS emerged at the very end of the chloroform, methanol, 240 mM aqueous ammonium acetate wash and at the beginning of the chloroform, methanol, 480 mM aqueous ammonium acetate step. All fractions containing LPS were pooled and converted to a two-phase Bligh/Dyer mixture by addition of appropriate amounts of chloroform and water. The lower phase was dried under a stream of N2 and stored at -20 °C.

Mass Spectrometry of Intact LPS from WBB06-- Spectra were acquired in either the positive-ion or the negative-ion mode on a Kratos Analytical (Manchester, United Kingdom) time of flight matrix-assisted laser desorption-ionization (MALDI) mass spectrometer, equipped with a 337-nm nitrogen laser, a 20 kV extraction voltage, and time-delayed extraction (11). Each spectrum was the average of 50 shots. The matrix was a mixture of saturated 6-aza-2-thiothymine in 50% aqueous acetonitrile and 10% tribasic ammonium citrate (9:1, v/v) for the negative mode. WBB06 LPS samples were first dissolved in a mixture of chloroform/methanol (4:1, v/v) before being mixed with the matrix (1:1, v/v) on a slide (0.6 µl final volume). The sample/matrix mixture was allowed to dry at room temperature prior to mass analysis. Hexa-acylated lipid A 1,4'-bis-phosphate from E. coli, used to calibrate the spectrometer, was purchased from Sigma.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

MALDI-TOF Mass Spectrometry of LPS Isolated from E. coli WBB06-- Re LPS from WBB06, a heptose-deficient E. coli mutant with a deletion that inactivates the adjacent waaC(rfaC) and the waaF(rfaF) genes, was previously reported to contain a pEtN substituent attached to its outer Kdo residue (9). However, in our initial experiments, MALDI-TOF mass spectrometry of LPS isolated from WBB06 cells grown on LB broth did not detect any pEtN-containing species (Fig. 3, panel A). The negative mode spectrum showed major ions at m/z 2317.2, 2237.8, 1876.2, and 1796.8 atomic mass units. The signal at m/z 2317.2 can be attributed to [M-H]- of a hexa-acylated (Kdo)2-lipid A variant with a 1-pyrophosphate substituent (Fig. 3, panel A, inset) (11, 23). The signal at m/z 2237.8 atomic mass units is interpreted as [M-H]- of the predominant hexa-acylated (Kdo)2-lipid A 1,4'-bis-phosphate molecule (Fig. 3, panel A), characteristic of E. coli K-12. The peak at m/z 1876.2 atomic mass units could arise by loss of the Kdo disaccharide during mass spectrometry from the 1-pyrophosphate variant, while the peak at m/z 1796.8 atomic mass units is explained by loss of the Kdo disaccharide from the 1,4'-bis-phosphate species. There is no evidence for peaks indicative of LPS molecules derivatized with an additional pEtN moiety on their outer Kdo moiety (or elsewhere), as would be expected around 2360 or 2440 atomic mass units for the bis-phosphate and the 1-pyrophosphate variant, respectively.



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Fig. 3.   MALDI-TOF mass spectra of intact LPS from E. coli WBB06 grown in the absence or presence of added CaCl2. Spectra were acquired in the negative mode. Proposed structures corresponding to the major molecular species present under each growth condition are shown. Panel A, LPS isolated from cells grown without CaCl2 supplementation. Panel B, LPS isolated from cells grown with 5 mM CaCl2 in the growth medium. Arrows indicate the structural origins of the largest observed molecular ions.

Effects of Supplementation with 5 mM CaCl2 in the Growth Medium on the Structure of WBB06 LPS-- The absence of a molecular species with a pEtN substituent in the spectrum of Fig. 3, panel A, was explained by the unanticipated effect of CaCl2 on the structure of E. coli LPS. Brabetz et al. (9) had noted that the growth rate of WBB06 was increased to that of wild type E. coli by the inclusion of 5 mM CaCl2 in the medium. Therefore, their experiments were carried out with LPS isolated from calcium-supplemented cells. When we grew WBB06 in LB broth in the presence of 5 mM added CaCl2, MALDI-TOF analyses revealed that a pEtN unit was indeed present in about half of the LPS molecules (Fig. 3, panel B). The proposed structure of this pEtN-(Kdo)2-lipid A 1,4'-bis-phosphate species is shown in Fig. 3B, inset. The negative mode spectrum showed major ions at m/z 2359.6, 2237.3, and 1797.4 atomic mass units. The peaks at m/z 2359.6 and 2237.3 atomic mass units are attributed to [M-H]- of pEtN-(Kdo)2-lipid A and (Kdo)2-lipid A, respectively, both in the form of the 1,4'-bis-phosphate species (Fig. 3, panel B). (The 1-pyrophosphate variant of lipid A seen in Fig. 3, panel A, is apparently suppressed by the inclusion of 5 mM CaCl2 in the growth medium.) The peak at m/z 1797.4 could arise during mass spectrometry by cleavage of the inner Kdo-lipid A linkage of either the m/z 2359.6 or the m/z 2237.3 species. Whether the pEtN is attached to the outer or the inner Kdo residue cannot be determined from the results shown in Fig. 3. However, the spectra demonstrate unequivocally that the addition of the pEtN moiety to the Kdo region of Re LPS is absolutely dependent upon the presence of CaCl2 in the growth medium. Addition of the pEtN substituent to the lipid A moiety is excluded by the absence of a significant peak at m/z 1920 atomic mass units (Fig. 3, panel B) (11). Almost all of the minor peaks in Fig. 3 (not labeled) are explained by the presence of a small amounts of penta-acylated lipid A species in this particular LPS preparation (i.e. missing the myristoyl chain that is incorporated by MsbB) (24, 25).

Calcium-induced Expression of a Novel pEtN Transferase in E. coli Membranes-- Washed membranes, obtained from WBB06 or wild-type W3110 grown with or without 5 mM CaCl2, were assayed for pEtN transferase using Kdo2-[4'-32P]lipid IVA as the acceptor substrate. The addition of a pEtN unit to Kdo2-[4'-32P]lipid IVA generates a more hydrophilic lipid product that migrates slower than Kdo2-[4'-32P]lipid IVA. As shown in Fig.4, membranes of E. coli W3110 and WBB06 that had been grown in 5 mM CaCl2 (lanes 3 and 4) supported the conversion of Kdo2-[4'-32P]lipid IVA to a new polar product with the expected Rf. However, membranes from the same strains grown in the absence of added CaCl2 did not (Fig. 4, lanes 1 and 2). All subsequent enzymatic experiments were done using membranes from strain WBB06 grown in the presence of CaCl2, since membranes of this strain displayed somewhat higher pEtN transferase activity than membranes of W3110 (Fig. 4, lanes 4 and 3, respectively).



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Fig. 4.   Ca2+-induced expression of a novel pEtN transferase in E. coli membranes. Membranes of wild-type (W3110) or the heptose-deficient mutant (WBB06) were assayed for pEtN transferase activity using standard conditions, as described under "Experimental Procedures." A final protein concentration of 1 mg/ml was employed, and the reaction tubes were incubated for 60 min at 30 °C. Lane 1, W3110 grown without CaCl2; lane 2, WBB06 grown without CaCl2; lane 3, W3110 grown with 5 mM CaCl2; lane 4, WBB06 grown with 5 mM CaCl2; lane 5, no membranes.

A quantitative assay was developed for the calcium-induced pEtN transferase using Kdo2-[4'-32P]lipid IVA as substrate (Fig. 2). As expected for an enzyme that modifies the Kdo region, [4'-32P]lipid IVA does not serve as a substrate (not shown). The reaction has a pH optimum around 7.5 and is dependent upon the presence of the non-ionic detergent Triton X-100 (0.05-0.1%). Dithiothreitol (1.25 mM) is slightly stimulatory (not shown). EDTA added to the assay at 1 mM is strongly inhibitory (not shown), but this inhibition is reversed by inclusion of 1-2 mM MgCl2 or CaCl2. In the absence of added EDTA, however, supplementation of the assay mixture with 1-10 mM MgCl2 or CaCl2 is not stimulatory (not shown), suggesting that relatively low concentrations of these divalent cations are sufficient. The final optimized reaction mixture consisted of 50 mM Hepes, pH 7.5, 0.1% Triton X-100, 1.25 mM dithiothreitol, and 2.5 µM Kdo2-[4'-32P]lipid IVA. Product formation was proportional to time and protein concentration (Fig. 5, panels A and B). The calculated specific activity of the pEtN transferase in WBB06 membranes assayed at 1.25 mg/ml was about 8 pmol/min/mg. No activity was present in a membrane-free high-speed supernatant.



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Fig. 5.   An assay for the Ca2+-induced pEtN transferase. Panel A, product formation at 0.75 mg/ml membrane protein was detected and quantified by PhosphorImager analysis. Panel B, product formation was linear with time and protein concentration. The assay system (30 µl) contained 50 mM Hepes, pH 7.5, 1.25 mM dithiothreitol, 0.1% Triton X-100, and 2.5 µM Kdo2-[4'-32P]lipid IVA with either 1.25 or 0.75 mg/ml WBB06 membranes from cells grown in the presence of 5 mM CaCl2. At the indicated times, 4-µl portions were withdrawn and spotted onto a thin layer plate, which was developed and analyzed as described under "Experimental Procedures."

Effect of Various Divalent Cations in the Growth Medium on the Expression of pEtN Transferase Activity-- E. coli mutant WBB06 was grown on LB broth in the presence of supplemental concentrations of CaCl2 ranging from 5 µM to 50 mM. Transferase activity was the highest when the membranes were obtained from cells grown in 50 mM CaCl2 (Fig. 6, lane 7). No activity was observed in membranes from cells grown with 0.5 mM supplemental CaCl2 or less (Fig. 6). WBB06 was also grown in the presence of other divalent cations to determine whether or not the induction effect is specific for calcium. Membranes were isolated from WBB06 cells grown with supplemental 5 mM MgCl2, BaCl2, or ZnCl2, and were assayed for transferase activity. None of these alternative divalent cations could substitute for CaCl2 (Fig. 7).



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Fig. 6.   Effect of added CaCl2 in the growth medium on the activity of the pEtN transferase. Membranes were isolated from E. coli WBB06 grown on LB broth with various added supplemental concentrations of CaCl2. Reactions were carried out under standard assay conditions with Kdo2-[4'-32P]lipid IVA as the acceptor substrate using 1 mg/ml washed membranes. Lane 1, no enzyme; lane 2, without added CaCl2; lane 3, with 5 µM CaCl2; lane 4, with 50 µM CaCl2; lane 5, with 500 µM CaCl2; lane 6, with 5 mM CaCl2; lane 7, with 50 mM CaCl2.



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Fig. 7.   No effect of divalent cations other than Ca2+ on the expression of the pEtN transferase. Washed membranes were obtained from WBB06 cells grown on LB broth in the presence of various supplemental divalent cations. Transferase reactions were carried out under standard conditions with 1 mg/ml washed membranes in the assay system. Lane 1, no enzyme; lane 2, grown with 5 mM CaCl2; lane 3, with 50 mM CaCl2; lane 4, with 5 mM MgCl2; lane 5, with 50 mM MgCl2; lane 6, with 5 mM BaCl2; lane 7, with 5 mM ZnCl2.

Location of the pEtN Substituent in the Reaction Product as Judged by pH 4.5 Hydrolysis at 100 °C-- Hydrolysis in sodium acetate buffer, pH 4.5, at 100 °C was used to determine the location of the hydrophilic modification present in the presumed in vitro product, pEtN-Kdo2-[4'-32P]lipid IVA. Hydrolysis at pH 4.5 under these conditions cleaves only those glycosidic linkages involving the anomeric carbons of Kdo, thereby randomly splitting off the outer and the inner Kdo moieties of pEtN-Kdo2-[4'-32P]lipid IVA at comparable rates (26, 27).

Following prolonged incubation of the substrate Kdo2-[4'-32P]lipid IVA at 100 °C, [4'-32P]lipid IVA eventually emerges as the sole hydrolysis product (Fig. 8, panel A), but with transient appearance of equivalent amounts of Kdo-[4'-32P]lipid IVA and [4'-32P]lipid IVA at early time points. Hydrolysis of the putative pEtN-Kdo2-[4'-32P]lipid IVA (Fig. 8, panel B) shows the same time course and pattern of product formation. If the hydrophilic pEtN substituent were attached to the inner Kdo residue, a different pattern of radioactive lipids would have been observed, since release of the outer Kdo residue would have yielded an intermediate lipid species containing a single Kdo still derivatized with the pEtN moiety (27). The latter hydrolysis intermediate would have migrated more slowly on silica TLC than the intermediate Kdo-[4'-32P]lipid IVA (27). The nearly identical time courses and patterns of fragments obtained with Kdo2-[4'-32P]lipid IVA and pEtN-Kdo2-[4'-32P]lipid IVA under these hydrolysis conditions reveal that the calcium-induced pEtN transferase modifies the outer Kdo residue.



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Fig. 8.   Time course of hydrolysis at pH 4.5 of Kdo2-[4'-32P]lipid IVA and the in vitro product, pEtN-Kdo2-[4'-32P]lipid IVA. Panel A shows the hydrolysis at pH 4.5 (100 °C) of Kdo2-[4'-32P]lipid IVA as the control. Panel B shows the hydrolysis of the pEtN-Kdo2 [4'-32P]lipid IVA, which was first purified by thin layer chromatography. At the indicated times, 4-µl portions of each hydrolysis mixture were spotted onto a silica gel thin layer chromatography plate, which was developed and imaged as described under "Experimental Procedures."

Demonstration that Phosphatidylethanolamine Is the Donor Substrate for the pEtN Transferase-- E. coli cells with a null mutation in the pss gene, which encodes phosphatidylserine synthase, are unable to generate phosphatidylethanolamine (16). Such mutants are able to grow slowly in the presence of 50 mM CaCl2. Membranes of the pss null mutants AD90 grown with 50 mM CaCl2 were prepared and assayed for pEtN transferase activity with Kdo2-[4'-32P]lipid IVA as the acceptor. As shown in Fig. 9 (panel A, lane 3), the absence of phosphatidylethanolamine in E. coli membranes completely abolishes transferase activity. However, membranes from the calcium-induced parental wild-type strain W3899 from which AD90 was derived (16) possess robust pEtN transferase activity (Fig. 9, panel A, lane 2). The results strongly suggest that phosphatidylethanolamine is the donor substrate for the transferase.



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Fig. 9.   Membranes of an E. coli pss mutant lack pEtN transferase unless supplemented with phosphatidylethanolamine. Reactions were carried out under standard assay conditions with 1 mg/ml membranes. After 60 min, 4-µl portions were withdrawn, spotted, and analyzed by thin layer chromatography. All strains were grown in LB medium supplemented with 50 mM CaCl2. The pss null mutants AD90 was first cured of its temperature-sensitive covering plasmids, as described previously (16). Panel A: lane 1, no enzyme; lane 2, W3899 membranes, parent of AD90; lane 3, AD90 membranes; lane 4, AD90 membranes supplemented with 1 mM 1-oleoyl-2-palmityol-phosphatidylethanolamine; lane 5, AD90 membranes supplemented with 1 mM 1-oleoyl-2-palmityol-phosphatidylglycerol; lane 6, AD90 membranes supplemented with 1 mM 1-oleoyl-2-palmityol-phosphatidylcholine. Panel B, lane 1, no enzyme; lane 2, W3899; lane 3, AD90 membranes with dilauroyl phosphatidylethanolamine; lane 4, AD90 membranes with dimyristoyl phosphatidylethanolamine; lane 5, AD90 membranes with dipalmitoyl phosphatidylethanolamine; lane 6, AD90 membranes with distearoyl phosphatidylethanolamine; lane 7, AD90 membranes with E. coli phosphatidylethanolamine; lane 8, AD90 membranes with 1-oleoyl-2-palmityol phosphatidylethanolamine; lane 9, AD90 membranes with dioleoyl phosphatidylethanolamine (all lipids at 1 mM).

To confirm this conclusion, various commercially available phospholipid preparations were added back to the membranes of the phosphatidylethanolamine-deficient strain AD90 in an attempt to reconstitute pEtN transferase activity. As shown in Fig. 9A, lane 4, only phosphatidylethanolamine (1-oleoyl,2-palmitoyl) resulted in partial restoration of enzymatic activity, whereas phosphatidylglycerol and phosphatidylcholine with the same fatty acid distributions (Fig. 9A, lanes 5 and 6) did not. Phosphatidylethanolamine isolated from E. coli was the most effective (Fig. 9B, lane 7), although dilauroyl phosphatidylethanolamine (Fig. 9B, lane 3) and various unsaturated phosphatidylethanolamine preparations (Fig. 9B, lanes 8 and 9) were also very effective. Interestingly, both dipalmitoyl and distearoyl phosphatidylethanolamine were completely inactive (Fig. 9B, lanes 5 and 6). Taken together, these findings strongly support the view that phosphatidylethanolamine is the physiologically relevant pEtN donor in our enzymatic system.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Brabetz et al. (9) recently reported that the E. coli mutant WBB06, which harbors a deletion spanning the waaC(rfaC) and waaF(rfaF) genes, contains Re LPS that is decorated with a pEtN moiety at position 7 of the outer Kdo sugar. Previous investigators, using Re LPS isolated from E. coli strains harboring waaC point mutations (10), had not observed this modification. We have now resolved this discrepancy by demonstrating that this particular modification of LPS with pEtN in living cells is the consequence of growth in the presence of 5-50 mM CaCl2 (Fig. 3), and is unrelated to the deletion of the heptosyltransferase genes in WBB06. The earlier studies with point mutants had been carried out with cells grown in the absence of CaCl2, whereas Brabetz et al. (9) included 5 mM CaCl2 in their media to enhance the growth rate of their mutant strain.

We have found for the first time conditions for assaying an E. coli enzyme that modifies the outer Kdo sugar of the LPS core with a pEtN moiety. Membranes from wild-type W3110 and mutant WBB06 grown in the presence of 5-50 mM CaCl2 (Figs. 4 and 6) both contain a novel activity that utilizes the intermediate Kdo2-[4'-32P]lipid IVA as an acceptor substrate. The enzyme is not detected in membranes from cells grown with other divalent cations (Fig. 7). We suggest that the enzyme modifies the outer Kdo sugar with pEtN (Fig. 2) based on the following lines of evidence. First, the in vitro activity that we detect in membranes from E. coli cells grown in the presence of 5-50 mM CaCl2 parallels the in vivo modification of LPS with pEtN (Figs. 3 and 4). Second, the results from the pH 4.5 hydrolysis of the pEtN-Kdo2-[4'-32P]lipid IVA product generated in vitro show that the modification is indeed located on the outer Kdo residue (Fig. 8). Third, the calcium-induced pEtN transferase is not seen in membranes of an E. coli pss null mutant (16), which is completely deficient in the biosynthesis of phosphatidylethanolamine, strongly suggesting that phosphatidylethanolamine is the donor substrate for the pEtN moiety (Figs. 2 and 9). In fact, reconstitution of the assay system with 1 mM phosphatidylethanolamine, but not phosphatidylglycerol or phosphatidylcholine, partially restores enzymatic activity (Fig. 9). The exact location of the pEtN substituent on the outer Kdo residue of our in vitro product remains to be established.

The regulatory mechanisms that high calcium concentrations induce to bring about the modification of the outer Kdo sugar with pEtN are uncertain. In some Gram-negative bacteria, the signal transduction system, PhoP/PhoQ, is activated by low concentrations of Mg2+ or Ca2+ (10 µM) (28). The PhoP/PhoQ system regulates the expression of at least 40 genes, some of which encode enzymes that modify lipid A (29). In the presence of high concentrations of Mg2+ or Ca2+ (10 mM), the PhoP/PhoQ system is shut off (30, 31). Since pEtN transferase activity is observed only when cells are grown in 5-50 mM CaCl2 (Figs. 6 and 7), expression of the enzyme is not likely to be regulated by PhoP/PhoQ, which in contrast to our system, senses both Mg2+ and Ca2+. It will be interesting to determine the regulatory mechanism by which high CaCl2 concentrations induce the pEtN transferase. We consider a transcriptional effect the most likely possibility, but we are not aware of other E. coli enzymes or proteins that are induced by high Ca2+, perhaps only because this phenomenon has received so little attention.

Previous studies have shown that the inner heptose residue of E. coli LPS (Fig. 1) is modified in a constitutive manner with an ethanolamine pyrophosphate group (8, 32). Based on radioactive labeling studies with living cells, phosphatidylethanolamine is thought to be the precursor of the ethanolamine phosphate portion of the ethanolamine pyrophosphate moiety on heptose (33). However, in vitro systems have not been reported for ethanolamine pyrophosphate biosynthesis. It is unlikely that our calcium-induced pEtN transferase plays a role in modification of the heptose region, since the latter is independent of calcium (5, 8, 34). However, the pEtN transferases that modify the Kdo and heptose regions might display sequence or structural homology to each other. Unfortunately, the relevant genes encoding these enzymes are not yet known.

The function of our calcium-induced pEtN transferase is uncertain. It might play a role in rendering cells competent for DNA-mediated transformation, given that the conditions used to make cells competent involve exposure to high levels of CaCl2 (14, 15), similar to what we use to induce the pEtN transferase. Alternatively, the presence of the pEtN moiety on the outer Kdo sugar might ensure resistance of the outer membrane to environmental stresses, such as exposure to certain detergents, antibiotics, or serum. The latter contains about 1 mM Ca2+, which might be sufficient to trigger induction. Future studies will be aimed at cloning the gene encoding the calcium-induced pEtN transferase, since the characterization of a null mutant should reveal the function of this unusual enzyme.


    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM-51310 (to C. R. H. R.) and GM-54882 (to R. J. C.).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.

To whom correspondence should be addressed. Tel.: 919-684-5326; Fax: 919-684-8885; E-mail: raetz@biochem.duke.edu.

Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M009019200


    ABBREVIATIONS

The abbreviations used are: LPS, lipopolysaccharide; MALDI-TOF, matrix-assisted laser desorption-ionization time of flight; Kdo, 3-deoxy-D-manno-octulosonic acid; pEtN, phosphoethanolamine.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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