From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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Heptosyltransferase I, encoded by the
rfaC(waaC) gene of Escherichia
coli, is thought to add
L-glycero-D-manno-heptose
to the inner 3-deoxy-D-manno-octulosonic acid
(Kdo) residue of the lipopolysaccharide core. Lipopolysaccharide
isolated from mutants defective in rfaC lack heptose and
all other sugars distal to heptose. The putative donor,
ADP-L-glycero-D-manno-heptose,
has never been fully characterized and is not readily available. In cell extracts, the analog ADP-mannose can serve as an alternative donor
for RfaC-catalyzed glycosylation of the acceptor,
Kdo2-lipid IVA. Using a T7 promoter construct
that overexpresses RfaC ~15,000-fold, the enzyme has been purified to
near homogeneity. NH2-terminal sequencing confirms that the
purified enzyme is the rfaC gene product. The subunit
molecular mass is 36 kDa. Enzymatic activity is dependent upon the
presence of Triton X-100 and is maximal at pH 7.5. The apparent
Km (determined at near saturating concentrations of
the second substrate) is 1.5 mM for ADP-mannose and 4.5 µM for Kdo2-lipid IVA. Chemical
hydrolysis of the RfaC reaction product at 100 °C in the presence of
sodium acetate and 1% sodium dodecyl sulfate generates fragments
consistent with the inner Kdo residue of Kdo2-lipid
IVA as the site of mannosylation. The analog, Kdo-lipid
IVA, functions as an acceptor, but is mannosylated at less
than 1% the rate of Kdo2-lipid IVA. The
purified enzyme displays no activity with ADP-glucose, GDP-mannose,
UDP-glucose, or UDP-galactose. Mannosylation of Kdo2-lipid
IVA catalyzed by RfaC proceeds in high yield and may be
useful for the synthesis of lipopolysaccharide analogs. Pure RfaC can
also be used together with Kdo2-[4-32P]lipid
IVA to assay for the physiological donor (presumably
ADP-L-glycero-D-manno-heptose) in a crude, low molecular weight fraction isolated from wild type cells.
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INTRODUCTION |
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Lipopolysaccharide (LPS)1 is a major component of the outer leaflet of the outer membranes of Gram-negative bacteria (1-4). It is composed of three domains (Fig. 1): 1) a hydrophobic anchor, known as lipid A, that consists of an acylated disaccharide of glucosamine; 2) a non-repeating oligosaccharide, designated the core, that serves as a barrier to many antibiotics; and 3) the O-antigen, that extends outwards from the core and is comprised of a distinct repeating oligosaccharide. All components of LPS are required for the virulence of Gram-negative bacteria (1, 3, 5). The O-antigen and many of the core sugars are not required for viability (1, 3, 6-8), but the lipid A and Kdo residues of the inner core are essential for growth of Escherichia coli and related organisms (9-13).
Most of the genes of core oligosaccharide biosynthesis are contained in the rfa(waa) cluster near minute 82 on the E. coli chromosome (1, 3, 14-16). The functions of these genes have been deduced from genetic studies, in conjunction with partial physical and chemical characterizations of isolated LPS (1, 3). Direct enzymatic studies of E. coli core biosynthesis beyond Kdo have been limited (1) because the structure of the core is not fully established. Consequently, the acceptor substrates of most of the enzymes involved in core glycosylation and the products generated by these enzymes are not fully characterized (1, 17-19). In vitro assays dependent upon time and protein have not generally been developed (1, 17, 20), and key synthetic donors and acceptors are not available (1).
The inner core of E. coli contains 2-3 Kdo residues, 2-3 heptose residues, and several other substoichiometric substituents (Fig. 1) (1-4, 20). Mutants that lack heptose are viable but display a deep rough phenotype (3). They are sensitive to detergents, hydrophobic antibiotics, and rough-specific bacteriophages (3, 7). The incorporation of the first heptose residue into LPS is thought to be catalyzed by the rfaC(waaC) gene product (1, 3), designated heptosyltransferase I (17, 21).
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Previous studies of E. coli and Salmonella heptosyltransferase I suffered from some of the above mentioned limitations (17). Since synthetic ADP-L-glycero-D-manno-heptose was not available, partially purified preparations of ADP-heptose, isolated from cells of Shigella sonnei (22), were utilized. However, the heptosyl acceptor employed, Kdo2-lipid IVA, was well characterized (23). Kdo2-lipid IVA is thought to be capable of acquiring a complete core in vivo (24). Even so, the products generated by this in vitro system could only be isolated in radiochemical amounts insufficient for physical analysis (17).
Recently, we have described a new assay for heptosyltransferase I of
E. coli, using commercially available ADP-mannose as an
alternative donor in place of
ADP-L-glycero-D-manno-heptose (21), as shown in Fig. 2. ADP-mannose is a naturally occurring sugar
nucleotide found in corn (25, 26). Here, we report the first
characterization of the catalytic properties of heptosyltransferase I,
using ADP-mannose as the donor and
Kdo2-[4-32P]lipid IVA as the
acceptor. We have purified RfaC to near homogeneity using this
optimized assay system, and we have characterized the product as
mannosyl-Kdo2-lipid IVA (proposed structure
shown in Fig. 2). We have also devised a
new procedure for the isolation of a crude (low molecular weight) sugar
nucleotide-containing fraction from various strains of E. coli and Salmonella. Assays utilizing these sugar
nucleotide preparations in place of ADP-mannose demonstrate that pure
RfaC is capable of glycosylating
Kdo2-[4
-32P]lipid IVA with a
single sugar presumed to be
ADP-L-glycero-D-manno-heptose. This reaction should serve as a functional assay for the definitive isolation and structural characterization of the elusive endogenous heptosyl donor of LPS biosynthesis.
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EXPERIMENTAL PROCEDURES |
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Materials and Bacterial Strains--
Materials and kits
purchased were: [-32P]ATP (NEN Life Science Products);
Hepes, Mes, Tris, bovine serum albumin (BSA), Reactive Green 19, ADP-mannose, and all other sugar nucleotides (Sigma); Triton X-100 and
bicinchoninic assay reagents (Pierce); silica gel 60 thin layer
chromatography plates (E. Merck); yeast extract and tryptone (Difco);
Wizard Mini-prep kit (Promega); PCR reagents (Stratagene); restriction
enzymes (New England Biolabs); shrimp alkaline phosphatase (U. S.
Biochemical Corp.); custom primers and T4 DNA ligase (Life
Technologies, Inc.); Qiaex II gel extraction kit (Qiagen);
polyacrylamide gel reagents (National Diagnostics); Centricon
centrifugation devices (Amicon); and Immobilon P polyvinyldifluoride membranes (Millipore). All solvents were reagent grade. Radiochemical analysis of thin layer plates was performed with a model 425S Molecular
Dynamics PhosphorImager equipped with ImageQuant software.
Preparation of Radiolabeled Substrates--
The
[4-32P]lipid IVA was generated from
[
-32P]ATP and the tetra-acylated disaccharide
1-phosphate precursor, using the E. coli 4
-kinase from
membranes of strain BR7 (36). The labeled lipid IVA was
converted to either Kdo2-[4
-32P]lipid
IVA using purified E. coli Kdo transferase (23,
37) or to Kdo-[4
-32P]lipid IVA using a
Haemophilus influenzae extract (24, 38). The products were
purified by preparative thin layer chromatography and stored at
20 °C as an aqueous dispersion (23, 37). Prior to each use, these
substrates were subjected to ultrasonic irradiation in a water bath for
60 s.
Assay Conditions--
Unless indicated, reaction mixtures
(10-40 µl) contained 50 mM Hepes, pH 7.5, 0.1% Triton
X-100, 10 µM Kdo2-[4-32P]lipid
IVA at 80,000 cpm/nmol, and 1.0 mM ADP-mannose
(21). The enzyme source, added last to initiate the reaction, contained 1 mg/ml BSA when diluted to less than 1 µg/ml. The enzyme generally comprised 1/10 of the reaction volume. Reactions were incubated at
30 °C for 5-60 min.
Analysis of the Reaction Products by Thin Layer Chromatography-- Reactions were terminated by spotting 5-µl portions of the reaction mixture onto a Silica Gel 60 thin layer plate. After drying in a stream of cold air, plates were developed in chloroform/pyridine/88% formic acid/water (30:70:16:10, v/v). The plate was dried and then exposed to a PhosphorImager screen overnight. The amount of product formed was calculated from the percent conversion of the radioactive substrate (of known specific radioactivity) to product.
General Recombinant DNA Techniques-- Plasmids were prepared using the Promega Wizard Miniprep kit. Restriction endonucleases, shrimp alkaline phosphatase, and T4 DNA ligase were all used according to the manufacturer's instructions. DNA fragments were isolated from agarose gels using a Qiaex II gel extraction kit (Qiagen). All other techniques involving manipulation of nucleic acids were from Ausubel et al. (39). Cells were made competent for electroporation by resuspension in 10% glycerol (39), as described. Transformation of plasmid DNA into competent cells was performed by high voltage electroporation using a Bio-Rad Gene Pulser II at 2.5 kV, 200 ohms, and 25 microfarads.
Placing rfaC under T7 Promoter Control-- The cloning of PCR-generated rfaC DNA into a vector under T7 promoter control is outlined in Fig. 3 (40-42). The forward primer was synthesized with a GC clamp, an NdeI restriction site, and an rfaC coding strand starting at the translation initiation site (primer sequences are shown in legend for Fig. 3). The reverse primer was synthesized with a GC clamp, a BamHI restriction site, and an rfaC anticoding strand that includes the stop site. The PCR was performed using Pfu polymerase, as specified by the manufacturer. The plasmid pLC10-7 (28, 29) was used as the template. Amplification was carried out in a 50-µl reaction mixture containing 100 ng of template, 20 mM Tris-HCl, pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 0.1% Triton X-100, 0.1% BSA, 2 mM MgSO4, 250 µM of each of the dNTPs, 200 ng of each primer, and 1.2 units of Pfu polymerase. The reaction was subjected to 30 cycles of denaturation (1 min, 94 °C), annealing (1 min, 55 °C), and extension (1.5 min, 72 °C) in a DNA thermal cycler. The reaction product was analyzed on a 1% agarose gel, digested with NdeI and BamHI, and ligated into the expression vector pET3a that had been similarly digested. The resulting desired hybrid plasmid (designated pJK1) was transformed into E. coli SURE cells, reisolated and digested again to verify its structure, and finally transformed into cells of strain BLR(DE3)pLysS.
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Growth Conditions and Cell-free Extract Preparation-- BLR(DE3)pLysS/pET3a and BLR(DE3)pLysS/pJK1 were grown from a single colony in 1 liter of LB medium (43) containing ampicillin (50 µg/ml) and chloramphenicol (30 µg/ml) at 37 °C until the A600 reached approximately 0.5. The culture was split into two equal portions, and one portion was induced with 100 µg/ml IPTG. Both cultures were incubated with shaking at 225 rpm for an additional 3 h at 37 °C, the A600 was recorded, and the cells were harvested by centrifugation for 10 min at 6000 × gav at 4 °C. All subsequent steps were performed either on ice or at 4 °C. The cell pellet was resuspended in a minimal volume, typically 10 ml of 50 mM Hepes, pH 7.5, and broken by passage through a 5-ml French pressure cell at 18,000 p.s.i. Unbroken cells and debris were removed by centrifugation for 10 min at 6000 × g. The resulting crude extract supernatant was used to prepare membranes. The crude extract was subjected to ultracentrifugation at 100,000 × gav for 60 min. The membrane pellet was resuspended in 1.5 ml of 50 mM Hepes, pH 7.5. The protein content of each fraction was determined by the bicinchoninic acid (BCA) assay (44) using BSA as the standard.
Making Solubilized Membranes-- A 1 ml portion containing 8-10 mg/ml protein of the BLR(DE3)pLysS/pJK1 membranes was mixed with an equal volume of 2% Triton X-100 and incubated on ice for 2 h with periodic gentle inversion of the tube. The solubilization mixture was then centrifuged at 100,000 × gav for 60 min to remove any unsolubilized proteins. The pellet was resuspended in 750 µl of 50 mM Hepes, pH 7.5, and the protein contents of both the solubilized and unsolubilized fractions were determined by the BCA assay (44).
Reactive Green 19 Column Chromatography of RfaC--
One gram of
Reactive Green 19 resin suspended in 5 ml of water was equilibrated in
a small plastic disposable column with 10 column volumes of
equilibration buffer (50 mM Hepes, pH 7.5, 0.1% Triton
X-100). A 4-mg sample of solubilized membrane proteins (in 1.25 ml) was
diluted 10-fold with 50 mM Hepes pH 7.5, and the material
was then applied to the column at a flow rate of 1 ml/min. Fractions of
2.5 ml were collected throughout. Next, the column was washed with 25 ml of equilibration buffer. Elution was carried out in three stages: 1)
25 ml of equilibration buffer plus 0.5 M NaCl, 2) 25 ml of
equilibration buffer plus 1.0 M NaCl, and, finally, 3) 25 ml of equilibration buffer plus 2.5 M NaCl. The protein
content of each fraction was determined using the BCA assay (44). The
peak of enzyme activity was determined by assaying each fraction in the
linear range under standard conditions for detection of mannose
transfer to Kdo2-[4-32P]lipid
IVA. The protein in certain samples was also visualized by
10% polyacrylamide gel electrophoresis in the presence of SDS, using
the Laemmli buffer system (45) in conjunction with Bio-Rad Mini-Protean
II electrophoresis equipment.
Preparation of the Purified Protein for NH2-terminal Sequencing-- Approximately 20 µg (400 µl) of Green 19 purified protein was concentrated 40-fold on a Microcon 10 device, according to the manufacturer's instructions. The concentrated sample was loaded onto a 10% polyacrylamide SDS gel along with a lane containing prestained standards as a control for transfer. Electrophoresis was carried out at 200 V for 50 min in a Laemmli gel buffer system. The gel was then soaked in 10 mM CAPS, pH 11, for 10 min at 4 °C. A polyvinylidene difluoride membrane was prepared while the electrophoresis was in progress by brief soaking in methanol, rinsing with water, and then soaking in 10 mM CAPS, pH 11. A Bio-Rad SD electroblotter was used according to the manufacturer's directions at 20 V for 40 min. Protein bands transferred to the membrane were visualized by Coomassie staining, and the band of interest was excised. NH2-terminal amino acid sequencing of the intact protein was carried out by Dr. John Leszyk of the Worcester Foundation for Experimental Biology, Shrewsbury, MA.
Sodium Acetate Hydrolysis of Mannosyl-Kdo2-lipid IVA-- Two 10-µl reaction mixtures were prepared containing 50 mM Hepes, pH 7.5, 0.1% Triton X-100, 0.4 µM Kdo2-lipid IVA (1 × 107 cpm/nmol or 40,000 cpm/reaction), and 1 mM ADP-mannose. To only one tube, 0.3 µg/ml purified RfaC was added. Both tubes were incubated for 30 min at 30 °C. Next, 4 µl of 10% SDS and 26 µl of 50 mM sodium acetate pH 4.5 were added (46-48) to both tubes to give a final pH of approximately 5.0, and the tubes were incubated in a boiling water bath. At 0, 1, 2, 5, 10, 20, and 30 min, 5-µl samples were withdrawn and spotted onto a silica TLC plate. The plate was developed and analyzed by PhosphorImager analysis, as described above.
Preparation of Crude (Low Molecular Weight) Sugar Nucleotide Fractions from Living Cells-- Five different strains (see below) were studied. Single colonies of each organism were used to inoculate 250-ml LB broth cultures. These cultures were grown at 37 °C until the A600 reached approximately 1.0. The cells were centrifuged for 10 min at 6000 × g (4 °C). The cell pellets were each then extracted with 5 ml of 50% ethanol at room temperature and incubated on ice for another 30 min with occasional stirring. The precipitates were removed by 10-min centrifugations at 6000 × g (4 °C). The supernatants were collected and placed in a SpeedVac (Savant) for 4 h to reduce the volumes to one half and to remove the most of the ethanol. Next, 500-µl portions of each of these supernatants were applied to Centricon 3 filtration devices and centrifuged at top speed in an Eppendorf microcentrifuge for 30 min at 4 °C. The flow-throughs, consisting of compounds with molecular weights less than 3000, were collected and used as the source of crude sugar nucleotides for studies of purified RfaC specificity.
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RESULTS |
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Overexpression of the rfaC Gene Using the T7 Promoter-- The NdeI restriction site (CATATG) in the multiple cloning cassette of pET3a can be used to insert a piece of foreign DNA with an overlapping transcriptional start site (ATG) (Fig. 3). This positions the inserted gene properly in the context of a strong T7 promoter region and ribosome binding site (41, 42). The inserted DNA in plasmid pJK1 (Fig. 3), generated by PCR as described under "Experimental Procedures," contains such a NdeI site, and in addition, a BamHI site at the opposite end of the gene to ensure unidirectional cohesive end cloning. Because the T7 promoter drives expression of rfaC in pJK1, a host strain that codes for T7 RNA polymerase, such as BLR(DE3)pLysS, is required to obtain expression.
Extracts were prepared from cells of BLR(DE3)pLysS containing either pJK1 or vector alone (pET3a). Duplicate cultures of each were first grown to A600 = 0.5, and then for another 3 h either with or without IPTG. The extracts were assayed for RfaC activity using ADP-mannose as the sugar donor. As shown in Table I, the specific activity of the transferase in extracts of BLR(DE3)pLysS/pJK1 grown without IPTG was over 200-fold higher than in extracts of the vector control strain. The presence of IPTG in the growth medium enhanced the expression of the transferase another 50-fold in BLR(DE3)pLysS/pJK1, as judged by assaying the activity (Table I) and analysis by gel electrophoresis (data not shown).
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Purification of the Overproduced Transferase-- It was observed previously that the rfaC-encoded transferase of wild type E. coli was membrane-associated (21). We therefore used membranes from the overproducing strain BLR(DE3)pLysS/pJK1 for the purification. Only about one quarter of the total activity present in the crude extract of BLR(DE3)pLysS/pJK1 was recovered in the membranes; nevertheless, a 2-fold increase in the specific activity was observed (Table II). Upon solubilization of the membranes with 1% Triton X-100, approximately 40% of the transferase activity was recovered with an additional slight increase in specific activity (Table II). At this stage, as shown by gel electrophoresis (Fig. 4), the transferase comprised a large fraction of the protein present in the solubilized sample, as judged by the presence of an overproduced band at ~36 kDa. It was therefore possible to employ only one chromatography step to obtain a homogeneous protein.
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Amino-terminal Sequence of the Purified Protein-- The predominant protein band present in the purified enzyme preparation (Fig. 4) was transferred to a polyvinylidene difluoride membrane, and subjected to amino acid sequencing. The sole NH2-terminal amino acid sequence found was MRVLIVKTSSMGDVL. This matches exactly the amino acid sequence predicted from the nucleotide sequence of E. coli rfaC (4, 17).
Effect of pH and Detergent on Transferase Activity-- Mannosyltransferase activity was measured using purified RfaC in the range of pH 5.5 to 8.9, as shown in Fig. 6A. The pH optimum for this reaction is centered around 7.5. Fig. 6B shows that the detergent Triton X-100 is required for activity. Under standard assay conditions with 10 µM Kdo2-lipid IVA, no activity is detected without detergent. Since Kdo2-lipid IVA probably does not form a true solution in water, the detergent likely interacts with the lipid substrate to generate mixed micelles, allowing the enzyme better access to the substrate. At concentrations greater than 0.1% Triton X-100, however, the transferase activity is inhibited, probably because of surface dilution effects (49).
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Kinetic Properties of the Purified Enzyme-- As seen in Fig. 7, the mannosyltransferase activity of RfaC is linearly dependent upon both time and protein concentration. The reaction is well behaved, and the enzyme can catalyze the quantitative mannosylation of Kdo2-lipid IVA (data not shown).
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Specificity of the Transferase for Its Sugar Nucleotide Donor Substrate-- The proposed physiological sugar nucleotide substrate for this reaction, ADP-L-glycero-D-manno-heptose (22), is not readily available for use in in vitro assays. Because we were able to substitute ADP-mannose in the assay, we also examined the question of whether or not other sugar nucleotides could function as alternate donors. ADP-mannose, GDP-mannose, ADP-glucose, UDP-glucose, and UDP-galactose were all tested at 1 mM under otherwise standard assay conditions. As seen in Fig. 9, transferase activity was only detectable with the ADP-mannose substrate. Even at enzyme levels that were 50 times higher than those used in the standard assay, these other sugar nucleotides could not serve as donors. These results imply that the transferase recognizes the axial OH at the C-2 position of the pyranose ring in the donor sugar. The structure of the nucleotide is also very important for the functioning of E. coli RfaC, since GDP-mannose did not substitute for ADP-mannose.
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Specificity of E. coli RfaC for Lipid Acceptors--
As shown in
Fig. 10, several lipid acceptors were
tested as substrates for purified RfaC. Each was
4-32P-labeled and present in otherwise standard assay
conditions at 10 µM. Kdo2-lipid
IVA was by far the best substrate of those tested, supporting a specific activity of 2280 nmol/min/mg. Under the conditions of Fig. 10, 50-fold less enzyme was used with
Kdo2-lipid IVA than with the other acceptors.
Nevertheless, measurable glycosylation of Kdo-lipid IVA was
detected at a rate that was approximately 170 times less (13.5 nmol/min/mg) than with Kdo2-lipid IVA. No activity was detected using lipid IVA as the substrate.
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Sodium Acetate Hydrolysis of Mannosyl-Kdo2-lipid
IVA Generated by E. coli RfaC--
Hydrolysis of LPS or
its Kdo containing precursors at 100 °C in pH 4.5 sodium acetate
buffer containing 1% SDS specifically cleaves all glycosidic linkages
involving the anomeric carbon of Kdo (47, 48). The half-life of such
linkages under these conditions is 5-10 min. The glycosidic linkages
between the glucosamine moieties of lipid A and the phosphomonoester
groups at positions 1 and 4 of lipid A are not disturbed. As seen in
Fig. 11A, hydrolysis of
Kdo2-[4
-32P]lipid IVA under
similar conditions transiently produces Kdo-[4
-32P]lipid
IVA. At later times (30 min), [4
-32P]lipid
IVA becomes the predominant hydrolysis product. The time course of the products generated by the hydrolysis of
mannosyl-Kdo2-[4
-32P]lipid IVA
(Fig. 11B) is slightly more complicated because the substrate used for the hydrolysis experiment was enzymatically generated in a reaction that went to only 95% completion (Fig. 11B, time 0). It is nevertheless clear from the
migration of the hydrolysis products that the main species formed with
time are both mannosyl-Kdo-[4
-32P]lipid IVA
and [4
-32P]lipid IVA (in approximately equal
amounts during the first 10 min of the reaction). This result
demonstrates that mannose must be attached to the inner Kdo (consistent
with the acceptor specificity results of Fig. 10), since
mannosyl-Kdo-[4
-32P]lipid IVA would not be
generated if mannose were attached to the outer Kdo residue.
Furthermore, virtually no Kdo-[4
-32P]lipid
IVA was seen during hydrolysis of
mannosyl-Kdo2-[4
-32P]lipid IVA
(Fig. 11B), further excluding the possibility that a
significant fraction of the molecules are derivatized with mannose on
the outer Kdo.
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Purified RfaC Utilizes Low Molecular Weight Substance(s) Extracted
from Cells to Modify Kdo2-lipid IVA--
To
demonstrate that pure RfaC can utilize the putative in vivo
donor substrate,
ADP-L-glycero-D-manno-heptose,
a crude sugar nucleotide fraction was isolated from a number of
different strains for use as a co-substrate in the RfaC-catalyzed
modification of Kdo2-[4-32P]lipid
IVA. As shown in Fig. 12
(lane 1), in a control reaction with excess pure RfaC and
Kdo2-[4
-32P]lipid IVA,
ADP-mannose produced the expected band shift. A low molecular weight
fraction from the Salmonella typhimurium rfaE mutant SL1102
(Fig. 12, lane 6), which is defective in the biosynthesis of
ADP-L-glycero-D-manno-heptose
(1, 4), did not support any RfaC-catalyzed modification of
Kdo2-[4
-32P]lipid IVA. In
lanes 2-5, various other crude sugar nucleotide containing
fractions were utilized. Lane 2 contains the low molecular weight extract from the wild type E. coli strain, D21. The
observed band shift with the D21 derived donor indicates that
ADP-L-glycero-D-manno-heptose (or something like it) is indeed present in this organism. Lanes 3 and 4 contain the low molecular weight isolates from
two rfaC
strains, which can supposedly
synthesize
ADP-L-glycero-D-manno-heptose but
cannot transfer it to Kdo2-[4
-32P]lipid
IVA (1, 4). D21f2 is an E. coli mutant,
and SA1377 (Fig. 12) is a S. typhimurium strain. These
organisms appear to accumulate considerable amounts of
ADP-L-glycero-D-manno-heptose like material, as evidenced by the massive band shifts (lanes 3 and 4) supported by the low molecular weight
fractions isolated from these strains. Lane 5 shows the
results obtained with a low molecular weight fraction from SL3600 of
S. typhimurium, a mutant that is defective in the epimerase
(RfaD) that is believed to convert
ADP-D-glycero-D-manno-heptose
to
ADP-L-glycero-D-manno-heptose (54). The low molecular weight material from SL3600 does not support an
efficient band shift of Kdo2-[4
-32P]lipid
IVA when incubated together with pure RfaC, consistent with
the absence of heptose in the LPS of SL3600 (1, 4). A subtle, but
reproducible, observation is that the crude nucleotides isolated from
the E. coli rfaC mutant D21f2 generate a product that
migrates slightly more rapidly (lane 3) than that formed with the corresponding nucleotides of the S. typhimurium
rfaC mutant SA1377 (lane 4). This finding suggests that
there may be more than one molecular species of heptose donor in living
cells. Whatever the explanation, the results of Fig. 12 provide a
simple new assay for the isolation and definitive characterization of these elusive sugar donors.
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DISCUSSION |
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Recently, we reported that it is possible to assay
heptosyltransferase I (RfaC) of E. coli in crude cell
extracts using ADP-mannose in place of
ADP-L-glycero-D-manno-heptose
(21) as the donor. The lack of available
ADP-L-glycero-D-manno-heptose
has prevented the characterization of RfaC activity, even though the
gene that encodes RfaC has been known for some time (17). ADP-mannose and
ADP-L-glycero-D-manno-heptose
are very similar in structure (21). All chiral centers that they have
in common are identical, but heptose contains one additional
CH2OH group (Fig. 2). Based on the composition and
structure of the E. coli core (1, 4), the linkage formed by
RfaC in vitro is proposed to be ,1-5 to the inner Kdo.
The observation that the mannose residue is indeed attached to the
inner Kdo (Fig. 11) and that Kdo-[4
-32P]lipid
IVA functions as an alternative, albeit slow, substrate (Fig. 10) supports the proposed structure (Fig. 2). Additional studies
will be necessary to confirm the
,1-5 linkage.
By using our ADP-mannose assay to follow activity and by constructing a strain that overproduces transferase activity by 15,000-fold (Table I), we were able to develop a facile purification scheme for RfaC (Table II). Only a 14.5-fold purification of the overexpressed protein was necessary to achieve homogeneity. The pure protein displays a specific activity that is 220,000 times higher than that of wild type crude extracts. NH2-terminal sequencing of the purified protein confirmed that RfaC and the mannosyltransferase activity are indeed identical.
To determine the catalytic properties of RfaC, purified protein was used. The pH optimum for the reaction is 7.5, and a non-ionic detergent, such as Triton X-100, is required for activity (Fig. 6). Bovine serum albumin was used in all assays involving purified protein at <1 µg/ml to prevent inconsistencies due to enzyme adsorption to the sides of the reaction tubes. Inclusion of BSA in these assays typically improved the rate of conversion of substrate to product by 10%. The apparent Km for Kdo2-lipid IVA in this reaction was calculated as 4.53 µM (Fig. 8). This is similar to what is observed for the related GDP-mannose-dependent mannosyltransferase of Rhizobium leguminosarum, which has an apparent Km of 6.25 µM for Kdo2-lipid IVA (21). However, the apparent Km of E. coli RfaC for ADP-mannose is 1.47 mM (Fig. 8). This is nearly 3 orders of magnitude higher than the Km for GDP-mannose in the Rhizobium system (21). This difference may reflect the fact that ADP-mannose is not the physiological substrate for E. coli RfaC. Kinetic studies of RfaC using the putative physiological substrate, ADP-L-glycero-D-manno-heptose, were not attempted, because this material is not available.
ADP-mannose is an unusual sugar nucleotide found in corn (25, 26), but it has not been described in E. coli. GDP-mannose, which could be synthesized by appropriately re-engineered strains of E. coli, does not serve as a substrate in the E. coli RfaC-catalyzed reaction (Fig. 9). The core sugar composition of E. coli K12 LPS does not include detectable mannose (1, 4). It would be of great interest to express the R. leguminosarum mannosyltransferase in E. coli constructs able to generate GDP-mannose, but lacking their own rfaC gene. Such mutants should contain mannose in place of the inner heptose normally found in the LPS core. The consequences of this modification on further core extension and outer membrane protein assembly would be of considerable interest. Toward this end, we have recently identified a R. leguminosarum clone that appears to encode the GDP-mannose-dependent transferase and several additional enzymes of R. leguminosarum core assembly (50).
As shown in Fig. 10, lipid IVA is not a substrate for the RfaC-catalyzed reaction. Lipid IVA lacks the Kdo moiety to which the mannose is attached (Fig. 11). Surprisingly, Kdo-lipid IVA is a relatively poor mannose acceptor (Fig. 10) despite the fact that it possesses the Kdo residue to which the mannose is linked (Fig. 11). Kdo-lipid IVA does not accumulate and is not available as an acceptor in wild type E. coli cells because it is rapidly converted to Kdo2-lipid IVA by the bifunctional Kdo transferase (37). The relative inactivity of Kdo-lipid IVA as a substrate is not without precedent. The late E. coli acyltransferase, HtrB, similarly catalyzes efficient addition of laurate to Kdo2-lipid IVA but not to Kdo-lipid IVA (51).
The synthesis of Kdo2-lipid IVA proceeds in a defined, linear sequence of seven enzymatic reactions (1). After this point in the biosynthetic pathway, the late acyltransferases can function, the core can be built up from the proximal heptoses to the more distal sugars, and other substoichiometric modifications can be made (1). It has not been determined in what order these diverse reactions take place in living cells, or even if these reactions are ordered in vivo, since they can occur independently of each other in vitro. It should now be possible to examine the kinetics RfaC using lauroyl-Kdo2-lipid IVA and Kdo2-lipid A (which contains a myristate in addition to the laurate) as acceptors for mannose in place of Kdo2-lipid IVA. Previous studies with crude ADP-heptose isomers (22) isolated from S. sonnei suggested that Kdo2-lipid A might be a much better acceptor than Kdo2-lipid IVA, but this data could not be quantified due to lack of the purified donor (52).
Under the conditions utilized in our experiments, heptosyltransferase I behaves as a peripheral membrane protein. In some studies, the activity was found to be membrane-associated in wild type cells (21), but in other studies with different buffers the activity partitioned mainly into the cytosol (17). In the overproducer, much of the activity was cytosolic (Table II), but purification of the cytosolic material was not attempted. The data are consistent with the proposal that the enzymes of core assembly function as peripheral membrane proteins at the cytoplasmic face of the inner membrane (4) where they have access to both their cytosolic sugar nucleotide substrates and their lipid acceptors. If a purification of RfaC in the absence of detergent could be devised, x-ray crystallography might be feasible. The further study of the cytosolic RfaC is of considerable importance, as very little structural information is available for glycosyltransferases, most of which are integral membrane proteins.
In previous work, when concentrated crude cytosols and partially purified ADP-heptose preparations (22) were used for the in vitro glycosylation of Kdo2-lipid IVA, two more hydrophilic products were generated, as judged by thin layer analysis. These likely correspond to the products of heptosyltransferase I (RfaC) and heptosyltransferase II (RfaF) (17). It appears that RfaF cannot efficiently utilize mannosyl-Kdo2-lipid IVA, ADP-mannose, or both as substrates, since only one mannose residue is incorporated into Kdo2-lipid IVA in crude cell extracts when ADP-mannose is used as the donor (Fig. 9).
At present, RfaC is the last step in the E. coli LPS pathway that can be assayed quantitatively. However, we have now also shown that unfractionated low molecular weight extracts of various bacterial strains do support the modification of Kdo2-lipid IVA catalyzed by pure RfaC in what appears to be a single glycosylation (Fig. 12). Wild type E. coli and S. typhimurium cells appear to contain ADP-L-glycero-D-manno-heptose (or something like it), as do the rfaC mutants, D21f2 and SA1377 (Fig. 12). Mutants in rfaC cannot transfer the heptose to the lipid A acceptor, and accordingly they appear to accumulate much more of the donor substrate than wild type (Fig. 12, lanes 3 and 4). Mutants in the biosynthetic pathway for the formation of the putative ADP-L-glycero-D-manno-heptose, such as SL3600 (rfaD) and SL1102 (rfaE), do not contain a competent donor pool (Fig. 12, lanes 5 and 6). The actual function of RfaE in the biosynthesis of ADP-L-glycero-D-manno-heptose is unknown (1, 4). Mutants in rfaD should accumulate ADP-D-glycero-D-manno-heptose (54), which apparently is a poor substrate for RfaC (Fig. 12, lane 5), consistent with the heptose-deficient LPS found in such strains (1, 4). The band shift assays shown in Fig. 12 will now finally permit the functional isolation and definitive structural characterization of the heptose donor(s) required for LPS assembly. The availability of these donors should enable to development of qunatitative new assays for other putative heptosyltransferases, such as RfaF, RfaQ, and RfaK (1, 4).
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM-51796 (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 Pharmacology Training
Program 5T32GM07105 at Duke University.
§ To whom correspondence should be addressed. Tel.: 919-684-5326; E-mail: raetz{at}biochem.duke.edu.
1
The abbreviations used are: LPS,
lipopolysaccharide; BSA, bovine serum albumin; PCR, polymerase chain
reaction; IPTG, isopropyl-1-thio--D-galactopyranoside; Kdo, 3-deoxy-D-manno-octulosonic acid; CAPS,
3-(cyclohexylamino)propanesulfonic acid; Mes,
4-morpholine-ethanesulfonic acid.
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