From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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The lpxK gene has been proposed to
encode the lipid A 4'-kinase in Escherichia coli (Garrett,
T. A., Kadrmas, J. L., and Raetz, C. R. H. (1997)
J. Biol. Chem. 272, 21855-21864). In cell extracts, the kinase phosphorylates the 4'-position of a tetraacyldisaccharide 1-phosphate precursor (DS-1-P) of lipid A, but the enzyme has not yet
been purified because of instability. lpxK is
co-transcribed with an essential upstream gene, msbA, with
strong homology to mammalian Mdr proteins and ABC transporters.
msbA may be involved in the transport of newly made lipid A
from the inner surface of the inner membrane to the outer membrane.
Insertion of an -chloramphenicol cassette into msbA also
halts transcription of lpxK. We have now constructed a
strain in which only the lpxK gene is inactivated by
inserting a kanamycin cassette into the chromosomal copy of lpxK. This mutation is complemented at 30 °C by a hybrid
plasmid with a temperature-sensitive origin of replication carrying
lpxK+. When this strain (designated TG1/pTAG1)
is grown at 44 °C, the plasmid bearing the
lpxK+ is lost, and the phenotype of an
lpxK knock-out mutation is unmasked. The growth of
TG1/pTAG1 was inhibited after several hours at 44 °C, consistent
with lpxK being an essential gene. Furthermore, 4'-kinase activity in extracts made from these cells was barely detectable. In
accordance with the proposed biosynthetic pathway for lipid A, DS-1-P
(the 4'-kinase substrate) accumulated in TG1/pTAG1 cells grown at
44 °C. The DS-1-P from TG1/pTAG1 was isolated, and its structure was
verified by 1H NMR spectroscopy. DS-1-P had not been
isolated previously from bacterial cells. Its accumulation in TG1/pTAG1
provides additional support for the pathway of lipid A biosynthesis in
E. coli. Homologs of lpxK are present in the
genomes of other Gram-negative bacteria.
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INTRODUCTION |
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Lipopolysaccharide (LPS)1 is an essential glycolipid of Gram-negative bacteria (1-5). It is a complex molecule that forms the outer leaflet of the outer membrane and is important in forming an effective permeability barrier (3, 6, 7). The lipid A portion of LPS is required for bacterial viability and is a potent immunostimulant (1-5). Indeed, Gram-negative sepsis is thought to be mediated by over-stimulation of the immune system by bacterially derived lipid A (1-5). In Escherichia coli K12, lipid A is a disaccharide of glucosamine that is phosphorylated at the 1- and 4'-positions and acylated at the 2-, 3-, 2'-, and 3'-positions with (R)-3-hydroxymyristate (Fig. 1) (1-5). Two additional fatty acyl chains are esterified to the 2'- and 3'-hydroxymyristoyl chains to form acyloxyacyl moieties characteristic of lipid A (1-5).
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The biosynthetic pathway for making lipid A in E. coli is
well understood (1-3). Nine enzymes are required to synthesize Kdo2-lipid A (Fig. 1) (1-3). With the recent
identification of the gene encoding the lipid A 4'-kinase (8), the
genes encoding 8 of the 9 enzymes required for the biosynthesis of
Kdo2-lipid A have been identified (3). The lipid A
4'-kinase catalyzes the transfer of the -phosphate from ATP to the
4'-position of tetraacyldisaccharide 1-phosphate (DS-1-P) to form
tetraacyldisaccharide 1,4'-bis-phosphate (lipid
IVA) (Fig. 1) (9). Phosphorylation of the 4'-OH group is
necessary for the action of distal biosynthetic enzymes, such as the
Kdo transferase (10, 11), and for recognition of lipid A by mammalian
cells during endotoxin stimulation (5).
The lipid A 4'-kinase gene was recently identified as orfE
(a previously reported open reading frame of unknown function) (12),
and it is now referred to as lpxK (8). lpxK forms
an operon with an essential upstream gene, called msbA,
which has homology to ABC transporters and mammalian Mdr proteins (12, 13). msbA has been implicated in the transport of lipid A
from its site of biosynthesis on the inner surface of the inner
membrane to the outer membrane (12, 13, 43). Georgopoulos and
co-workers (12, 13) constructed a strain with an -cam cassette
inserted in the msbA gene. Because lpxK is
co-transcribed with msbA (12, 13), this insertion stops
expression of both msbA and lpxK. Complementation
analysis showed that both msbA and lpxK were
required for growth (12). It has also been found that
glucosamine-labeled LPS precursors accumulate in the inner membrane of
msbA/lpxK knock-outs (13). This phenomenon was
attributed to the loss of the putative transport protein, MsbA.
However, given the fact that lpxK plays an integral role in
the biosynthesis of lipid A (8), the apparent accumulation of LPS in
the inner membrane might be due to the build up of lipid A
precursor(s), which could accumulate when the 4'-kinase is inactivated.
These precursors may not be efficiently transported to the outer
membrane by the putative lipid A transport machinery and may even
inhibit transport of lipid A. Direct evidence for the function of MsbA
as a lipid A transporter in strains bearing extra copies of
lpxK is presented in the accompanying manuscript (43).
In the present work, we have constructed a strain with an insertion mutation in only the lpxK gene. In strain TG1/pTAG1 the chromosomal copy of lpxK is inactivated by insertion of a kanamycin resistance cassette into the center of the lpxK gene. This insertion mutation is complemented by a plasmid carrying lpxK+ and a temperature-sensitive origin of replication. Thus, the lpxK knock-out genotype can be induced by growth at 44 °C. At this temperature the hybrid plasmid carrying lpxK+ is no longer maintained in the cells, and both the lpxK gene and its product are gradually lost. Under these conditions, the phenotype of cells with no lipid A 4'-kinase can be examined. We now show that the 4'-kinase (LpxK) is indeed required for growth, and upon its depletion, the expected precursor, DS-1-P, accumulates in cells.
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EXPERIMENTAL PROCEDURES |
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Materials-- 32Pi was obtained from NEN Life Science Products; 0.25-mm glass-backed Silica Gel 60 thin layer chromatography plates and high performance thin layer chromatography (HPTLC) plates were from E. Merck; yeast extract and tryptone were from Difco; restriction enzymes and Klenow DNA polymerase (large fragment) were from New England Biolabs; T4 DNA ligase was from Life Technologies, Inc.; shrimp alkaline phosphatase was from U. S. Biochemical Corp.; DEAE-cellulose (DE52) was from Whatman; and octadecylsilane (C18) silica was from Baker. All solvents were reagent grade from Malinckrodt. CDCl3 and CD3OD were purchased from Aldrich.
Bacterial Strains, Growth Conditions, and DNA
Techniques--
Table I lists the E. coli K-12 strains used
in this study. Cells were cultured at 30, 37, or 44 °C in Luria
Broth (LB) consisting of 10 g of tryptone, 5 g of yeast
extract, and 10 g of NaCl per liter (14). Antibiotics were added
as necessary at 50 µg/ml for ampicillin, 12 µg/ml for tetracycline,
30 µg/ml for chloramphenicol, and 30 µg/ml for kanamycin. Mini
preparations of plasmid DNA were made using the Qiaprep Spin Miniprep
Kit (Qiagen). Large scale preparations of plasmid DNA were made using
the Bigger Prep kit from 5 Prime 3 Prime, Inc., Boulder, CO. DNA
fragments were isolated from agarose gels using the Qiagen Qiaex II gel
extraction kit. Restriction enzymes, T4 DNA ligase, and Klenow DNA
polymerase were used according to the manufacturers' instructions.
Transformation of E. coli with plasmid DNA was done using
salt-competent cells (14).
Plasmid Constructions-- Table I lists all of the plasmids used in this study. pTAG1 contains the lpxK gene cloned into pMAK705, a vector with a temperature-sensitive origin of replication (15). pJK2 (8) and pMAK705 were digested with XbaI and BamHI. The 1-kb lpxK gene from pJK2 and the 6-kb linearized pMAK705 were gel-purified from a 1% agarose gel. The lpxK gene was ligated into pMAK705. A portion of the ligation mixture was transformed into competent E. coli XL1-Blue (Stratagene), and colonies resistant to chloramphenicol were selected. Plasmid DNA was isolated from chloramphenicol-resistant clones and digested with XbaI and BamHI to identify those constructs with the desired insert. This plasmid is called pTAG1. This plasmid was tested for its ability to promote LpxK expression.
A plasmid analogous to pTAG1 was constructed with a kanamycin cassette inserted into the NsiI site of lpxK gene. pJK2 was digested with NsiI, and pUC-4K (Amersham Pharmacia Biotech) was digested with PstI. The 5.5-kb linearized pJK2 and the 1.2-kb kanamycin cassette from pUC-4K were gel-purified and ligated together. A portion of the ligation was transformed into E. coli XL1-Blue, and colonies resistant to ampicillin were selected. Plasmids were isolated from ampicillin-resistant colonies and digested with NdeI and BamHI to verify the presence of the correct 2.2-kb insert. The lpxK::kan construct described above was digested with XbaI and BamHI and cloned into pMAK705 exactly as for pTAG1, yielding pTAG2. pNGH1-amp was constructed from pNGH1 (16). pNGH1 was digested with BamHI and SalI yielding 3.9- and 1.6-kb fragments. pACYC177 was digested with BamHI and XhoI yielding 2.5- and 1.4-kb fragments. The 2.5-kb pACYC177 fragment which contains theConstruction of TG1/pTAG1, a Mutant with an Insertion in the
Chromosomal Copy of lpxK--
TG1/pTAG1 was constructed following the
method of Hamilton et al. (15) (Fig.
2). Competent MC1061 cells (18) were
transformed with pTAG2 and grown at 30 °C to an
A600 of 0.6. Next, 1 × 105
cells were plated on prewarmed LB plates containing 30 µg/ml chloramphenicol and incubated at 44 °C. This selects for cells in
which pTAG2 has integrated into the genome. A single colony was used to
inoculate 1 ml of LB containing chloramphenicol and grown at 30 °C
to stationary phase. A portion of the culture was diluted 1:1000 into
fresh LB containing chloramphenicol and again grown at 30 °C to
stationary phase. The above outgrowth was repeated once more. During
this outgrowth, the integrated plasmid will occasionally excise
carrying either the wild type lpxK or the lpxK::kan allele (Fig. 2) (15). The cells were
plated on LB containing chloramphenicol at 30 °C. Cells in which the
plasmid had excised were identified by their inability to grow at
44 °C in the presence of chloramphenicol. Plasmids were then
isolated from 14 such temperature-sensitive strains and digested with
XbaI and BamHI. Of the 14 colonies, 11 contained
the pTAG2 insert. Three, however, had the pTAG1 insert, indicating that
the lpxK::kan allele had replaced the wild type
lpxK gene on the chromosome (15). One of these strains was
made recA by P1 transduction using
BLR(DE3)pLysS (Novagen) as the donor. The presence of the
recA
phenotype was verified by the strain's sensitivity
to UV light (19). This strain is designated TG1/pTAG1.
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Construction of TG1/pTAG6-- Strain TG1/pTAG6 was made by transforming TG1/pTAG1 with pTAG6 and selecting for transformants at 30 °C on LB plates containing ampicillin. Single colonies were then streaked to LB plates containing ampicillin to 44 °C. pTAG1 is then lost, and pTAG6 (which is not temperature-sensitive for replication) provides wild type lpxK. TG1/pTAG8 was constructed in a similar manner.
Preparation of Cell-free Extracts and Assay for 4'-Kinase Activity-- Cell-free extracts for determination of 4'-kinase activity in cells grown at 44 °C were prepared as follows. Single colonies of TG1/pTAG1 and TG1/pTAG6 were inoculated into 5 ml of LB medium containing the appropriate antibiotics and grown at 30 °C overnight. Portions of the overnight cultures were diluted into 200 ml of LB broth (containing no antibiotics) to A600 of 0.01, and the growth temperature was shifted to 44 °C. When the cultures reached A600 of 0.2-0.3, portions were diluted 10-fold into fresh pre-warmed medium, and the growth cycles were continued. The cells from the remaining cultures (about 180 ml) were collected by centrifugation at 10,000 × g for 15 min at 4 °C. The cell pellets were washed with 200 ml of 50 mM HEPES, pH 7.4; the centrifugation was repeated, and the final cell pellets were resuspended in 1 ml of the same buffer. Meanwhile, the growth cycles of the diluted cultures were continued as above, diluting further as necessary to maintain logarithmic growth. The volumes of the cultures were adjusted to provide 180-200 ml of cell culture at A600 of about 0.3 for preparation of cell-free extracts at the indicated times after the shift to 44 °C.
To prepare cell-free extracts, cells were broken in a French pressure cell at 20,000 p.s.i., and unbroken cells were removed by centrifugation at 3500 × g. The protein concentrations were determined using the Bio-Rad protein assay kit with bovine serum albumin as a standard. 4'-Kinase activity was assayed as described previously (8). Typically, 100 µM DS-1-32P (1000 cpm/nmol), 1 mg/ml cardiolipin, 50 mM Tris chloride, pH 8.5, 5 mM ATP, 1% Nonidet P-40, and 5 mM MgCl2 were mixed with 2 to 0.05 mg/ml protein fractions and incubated at 30 °C for various times. Reactions were stopped by spotting a portion onto a Silica Gel 60 thin layer chromatography plate. Plates were developed in chloroform/methanol/water/acetic acid (25:15:4:2, v:v), dried, and exposed to a Molecular Dynamics PhosphorImager screen. Conversion of DS-1-32P to 1-32P-lipid IVA was quantified using ImageQuant software (Molecular Dynamics). DS-1-32P and carrier DS-1-P were prepared as described previously (20).Analysis of the Lipid A to Glycerophospholipid Ratio--
Single
colonies of TG1/pTAG1 and TG1/pTAG6 were inoculated into separate 3-ml
cultures of LB medium containing the appropriate antibiotics and grown
overnight at 30 °C. Each overnight culture was then diluted into two
25-ml portions of fresh LB medium (containing no antibiotics) to an
A600 of 0.01. One was grown at 30 °C and the
other at 44 °C. The cells were labeled with
32Pi for about two doubling times.
32Pi (5 µCi/ml) was added to the cultures
grown at 30 °C when the A600 reached 0.15 and
grown to an A600 of 0.5. The cultures grown at
44 °C were diluted 10-fold into fresh prewarmed medium whenever the
A600 reached ~0.2. When the cumulative growth
yield was 13.4 for TG1/pTAG1 (the point at which the growth of
TG1/pTAG1 begins to slow down) and 27.5 for TG1/pTAG6, both cultures
were labeled with 5 µCi/ml 32Pi. TG1/pTAG1
was labeled for 3 h, and TG1/pTAG6 was labeled for 1.5 h.
Each culture was split into three tubes (~8 ml per tube). The cells
were collected by centrifugation at 3000 × g, and the pellets were frozen at 20 °C for further analysis. Using one tube
of each labeled culture, the lipid A to glycerophospholipid ratio was
then determined as described previously (16, 21) with the following
modification. Samples were analyzed by thin layer chromatography in a
system containing chloroform/methanol/water/ammonia (40:25:4:2, v/v).
HPTLC plates were used for rapid chromatography in this solvent system
because the formation of an ammonia-catalyzed deacylation product was
minimized.
Extraction and Detection of a Lipid A Precursor That Accumulates at 44 °C in TG1/pTAG1-- A large batch of TG1/pTAG1 that had been shifted to 44 °C was prepared as follows. Overnight cultures of TG1/pTAG1 and TG1/pTAG6 grown at 30 °C were used to inoculate LB medium to an A600 of 0.01. Cultures were then grown at 44 °C and were diluted 10-fold as necessary to keep the optical density below 0.3 for 10.5 h. TG1/pTAG1 cultures were diluted into successively larger volumes to a final volume of 3 liters. A TG1/pTAG6 culture was maintained at 50 ml. Cells were harvested by centrifugation at 10,000 × g for 15 min at 4 °C, washed once with PBS (1 liter for TG1/pTAG1 and 10 ml for TG1/pTAG6), and resuspended in PBS (30 ml for TG1/pTAG1 and 2 ml for TG/pTAG6).
Lipid A precursor accumulation was examined in the non-labeled cells of TG1/pTAG1 and TG1/pTAG6 shifted to 44 °C for 10.5 h. TG1/pTAG1 cells (200 µl of the above 30-ml suspension) were brought to a volume of 2 ml by the addition of PBS. These TG1/pTAG1 cells and the entire 2-ml suspension of TG1/pTAG6 (as prepared above) were then extracted with a neutral single phase Bligh Dyer system (chloroform/methanol/PBS, 1:2:0.8) (22, 23) (9.5 ml total volume). Cell debris was removed by centrifugation at 3000 × g for 10 min. The supernatant was converted to a two-phase Bligh-Dyer system by the addition of chloroform and PBS to make the final solvent proportions 2:2:1.8 (chloroform/methanol/PBS) (22, 23). The phases were resolved by centrifugation, and the lower phase was washed with fresh pre-equilibrated upper phase. The final lower phase was dried under nitrogen and redissolved in 50 µl of chloroform/methanol (4:1). About 100 µg of total extracted lipid was loaded onto each lane of the HPTLC plate, which was developed in chloroform/methanol/water/ammonia (40:25:4:2,v/v). The lipids were detected by charring with 20% sulfuric acid in ethanol.Purification of the Accumulated Lipid A Precursor from TG1/pTAG1
Shifted to 44 °C--
TG1/pTAG1 cell pellets (7.5 ml) from cultures
that had been grown at 44 °C for 10.5 h, as described above,
were used to prepare the accumulated lipid A precursor. PBS was added
to bring the volume to 80 ml, and the suspension was distributed into
four 150-ml Corning glass centrifuge bottles equipped with Teflon-lined lids. A single phase Bligh-Dyer system (22, 23) was made by adding 25 ml of chloroform and 50 ml of methanol to each bottle. The suspension
was dispersed with ultrasound in a bath sonicator for 2 min and then
centrifuged at 2500 × g for 20 min. The supernatant was divided equally into four fresh glass bottles, and 25 ml of chloroform and 25 ml of PBS were added to each bottle to form a
two-phase Bligh-Dyer system. After thorough mixing, the suspensions were centrifuged at 2500 × g for 15 min, and the upper
phases were removed. Each of the lower phases was washed twice with 20 ml of fresh pre-equilibrated upper phase. The centrifugation was repeated to resolve the phases after each wash. The lower phases were
collected, and the solvent was removed by rotary evaporation. The dried
lower phase material was re-dissolved in 5 ml of
chloroform/methanol/water (2:3:1, v/v) and loaded onto a 1-ml
DEAE-cellulose column (Whatman DE52), pre-equilibrated in the same
solvent (20). The column was washed with 3 ml of
chloroform/methanol/water (2:3:1, v/v). Unexpectedly, the sample flowed
through the column in the initial chloroform/methanol/water (2:3:1,
v/v) wash. We attributed this behavior to residual salts in the
concentrated lower phases. Therefore, the run-through fractions (5.2 ml) were pooled and converted into a two-phase Bligh-Dyer system by
changing the solvent ratios to 2:2:1.8 (chloroform/methanol/PBS, v/v).
The lower phase was then washed again with fresh pre-equilibrated upper
phase (5 ml) and dried under N2. The resulting material was
redissolved in 1 ml of chloroform/methanol/water (2:3:1, v/v), and it
was loaded again onto a 1-ml pre-equilibrated DEAE-cellulose column.
This time the unknown lipid that accumulates at 44 °C in the
TG1/pTAG1 was retained by the column. The column was washed with 3 ml
of chloroform/methanol/water (2:3:1, v/v), then with 6 ml of
chloroform/methanol, 60 mM ammonium acetate (2:3:1, v/v),
and 2 ml each of chloroform/methanol/aqueous ammonium acetate (2:3:1,
v/v) containing sequentially 70, 80, 90, 100, 120, and 480 mM ammonium acetate in the water component. Fractions (1 ml) were collected, and a portion of each was spotted onto an HPTLC
plate. The plate was developed in chloroform/methanol/water/ammonia (40:25:4:2, v/v) and charred, as described above. The compound that
accumulated in cells of TG1/pTAG1 grown at 44 °C eluted with 60 mM ammonium acetate as the aqueous component, and it was
almost pure, as judged by thin layer chromatography and charring. The pooled fractions (4 ml) were then converted to a two-phase Bligh-Dyer system by changing the solvent proportions to 2:2:1.8
(chloroform/methanol/water, v/v). The phases were resolved by
centrifugation at 3000 × g for 10 min. The lower phase
was washed once with 3 ml of fresh pre-equilibrated upper phase, and
the centrifugation was repeated. The final lower phase was dried down
with N2 and stored at 20 °C.
Analysis of the Accumulated Lipid as DS-1-P by 1H NMR Spectroscopy-- Approximately 1 mg of purified material was dissolved in 0.6 ml of CDCl3/CD3OD (4:1, v/v), and its 1H NMR spectrum was recorded on a Varian 500 Unity spectrometer using a 5001.3-Hz spectral window with the 5-mm probe set at 20 °C. Chemical shifts were referenced to the methyl protons of internal tetramethylsilane (0.00 ppm). A line broadening of 0.05 Hz before Fourier transformation was used to process the data.
Two-dimensional 1H correlation (COSY) spectra were recorded in the absolute value mode over the same spectral region used in the one-dimensional 1H NMR spectrum. Two hundred fifty six time increments were collected and zero-filled to 2048 points with sine-bell weighting along both dimensions. One hundred eighty scans were collected per increment, and the relaxation delay was 1 s. ![]() |
RESULTS |
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Temperature Sensitivity of TG1/pTAG1 on Plates-- Strain TG1/pTAG1 is a mutant with a kanamycin cassette inserted into the chromosomal copy of lpxK, constructed by homologous recombination (Fig. 2 and Table I) (15). The mutation is covered by a plasmid, pTAG1, bearing lpxK+ and a temperature-sensitive origin of replication. Strain TG1/pTAG6 is similar to TG1/pTAG1, except that lpxK+ is on a plasmid with a non-temperature-sensitive origin of replication (Table I). Strains TG1/pTAG1 and TG1/pTAG6 were tested for their ability to grow at 44 °C. A single colony of each was streaked onto two LB plates containing kanamycin and tetracycline. One plate was incubated at 30 °C and the other at 44 °C. TG1/pTAG1 is able to grow and form single colonies at 30 °C but not at 44 °C, indicating that loss of the lpxK gene product is lethal (data not shown). Strain TG1/pTAG6 is able to grow and form single colonies at 30 and 44 °C (data not shown). This result is consistent with the finding by Karow and Georgopoulos (12) that orfE/lpxK is an essential gene.
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Temperature-sensitive Growth of TG1/pTAG1 in Liquid Medium-- To quantify the effects of lpxK inactivation on lipid A biosynthesis, cells were studied in shaking culture at 44 °C. Overnight cultures of MC1061/pTAG1, MC1061/pTAG6, TG1/pTAG1, and TG1/pTAG6 were first grown at 30 °C in the presence of the appropriate antibiotics (Table I). The cultures were diluted into 25 ml of LB without antibiotics to a final A600 of 0.01. The temperature was then shifted to 44 °C and growth was continued with shaking at 250 rpm. To maintain logarithmic growth, the cultures were diluted 10-fold whenever the A600 reached 0.2-0.3. The results of one such experiment are shown in Fig. 3, in which A600 is the cumulative growth yield corrected for dilution. MC1061 containing either lpxK on a temperature-sensitive or a non-temperature-sensitive plasmid grows logarithmically at 44 °C for the duration of the experiment (10 h). TG1/pTAG6, a strain with the insertion mutation in the chromosomal copy of lpxK covered by a non-temperature-sensitive plasmid bearing lpxK+, grows nearly as well as MC1061/pTAG1 or MC1061/pTAG6. However, growth of TG1/pTAG1 slows after about 4.5 h at 44 °C and stops altogether after 10 h. This result is consistent with lpxK being required for growth.
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Assays of 4'-Kinase in Extracts Prepared from TG1/pTAG1 Grown at 44 °C-- We next wanted to determine if loss of the lpxK gene leads to loss of lipid A 4'-kinase activity in cell extracts. Cultures of TG1/pTAG1 and TG1/pTAG6 were grown logarithmically at 44 °C as in Fig. 3. At regular intervals, portions of the cells were harvested. Cell-free extracts were prepared and assayed for 4'-kinase (Fig. 5). Extracts from TG1/pTAG6 contained measurable 4'-kinase after prolonged growth at 44 °C. However, the specific activity was 3-fold lower than in extracts of 30 °C grown TG1/pTAG6 (0-min time point in Fig. 5) but still 7-fold higher than in extracts of wild type cells (not shown). Extracts of TG1/pTAG1 have high 4'-kinase levels when cells are grown at 30 °C (0 min time point in Fig. 5). However, extracts of TG1/pTAG1 cells grown for 5 h at 44 °C display drastically lower 4'-kinase activity. After 10 h of growth at 44 °C, the kinase is barely detectable. The finding that loss of lpxK leads to loss of 4'-kinase activity supports the view that lpxK is the structural gene for the enzyme.
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Lipid Composition of TG1/pTAG1 and TG1/pTAG6 Grown at 30 and 44 °C-- The lipid A to glycerophospholipid ratio of wild type E. coli is 0.10-0.17, depending on the strain and growth conditions (16, 21, 25). This ratio reflects the necessary balance between the biosynthesis of LPS and glycerophospholipids in the cell. Over- or under-production of these biomolecules affects membrane biogenesis and cell viability (16, 21, 25). One would expect lipid A biosynthesis to be compromised in cells grown under conditions in which the 4'-kinase is depleted. In addition, the lack of the 4'-kinase activity might lead to the accumulation of the kinase substrate, DS-1-P (Fig. 1), a metabolite that has not been isolated previously from cells because of its low abundance.2
TG1/pTAG1 and TG1/pTAG6 were grown at both 30 and 44 °C and labeled for several hours with 32Pi, as described under "Experimental Procedures." The lipid A to glycerophospholipid ratio was determined (Table II) to be 0.1 in TG1/pTAG1 grown at 44 °C, as compared with 0.16 in the control TG1/pTAG6 grown at the same temperature. This analysis suggests that lipid A biosynthesis is slightly compromised under the labeling conditions employed.
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Isolation and 1H NMR Spectroscopy of the Compound That Accumulates in TG1/pTAG1-- While migration of the radiolabeled unknown with a DS-1-P standard upon thin layer chromatography (Fig. 6) is suggestive, further structural analysis was needed to confirm that the accumulated metabolite was indeed DS-1-P. Unlabeled TG1/pTAG1 and TG1/pTAG6 cells were therefore grown at 44 °C on a larger scale, and the cell pellets were extracted, as described under "Experimental Procedures." The crude lipids were first separated by chromatography on a high performance thin layer plate and charred with sulfuric acid (Fig. 7). Lanes 1 and 4 show the positions DS-1-P standards, prepared enzymatically from lipid X and UDP-diacylglucosamine (20). Lanes 2 and 3 show the total lipids extracted from TG1/pTAG1 and TG1/pTAG6, respectively, grown at 44 °C. A substance migrating with about the same RF as the authentic DS-1-P standard was detected by charring the lipids of TG1/pTAG1, but not of TG1/pTAG6, confirming the findings with the 32P-labeled cells (Fig. 6).
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Accumulated Lipid Isolated from TG1/pTAG1 Is a Substrate for the 4'-Kinase-- If DS-1-P is indeed accumulating in TG1/pTAG1 grown at 44 °C then, when purified, it should serve as a substrate for the 4'-kinase in vitro. We attempted to phosphorylate the material isolated from TG1/pTAG1 with membranes of strain BLR(DE3)pLysS/pJK2, a strain that overexpresses 4'-kinase activity about 3000-fold compared with wild type (8). Like synthetic DS-1-P (data not shown), the material isolated from TG1/pTAG1 serves as an excellent substrate for the 4'-kinase reaction (Fig. 9, lane 4). Quantitative conversion of the isolated material to a substance migrating like lipid IVA is possible, as judged by sulfuric acid charring following thin layer chromatography (Fig. 9). The reaction is dependent upon the presence of ATP (Fig. 9, lane 3 versus 4). In lane 5, authentic lipid IVA, isolated from a Kdo-deficient mutant of Salmonella (28, 29), was spotted as a standard.
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Rescue of the lpxK::kan Insertion Mutation in TG1/pTAG1 with the lpxK Homolog of F. novicida-- F. novicida is a facultative intracellular bacterial pathogen (17). Mdluli et al. (17) identified a locus of F. novicida that is required for virulence of the bacteria in mice. The locus, called valAB, is homologous to the msbA/lpxK locus in E. coli (17). valB is 66.8% similar and 41.4% identical to lpxK (8) and may encode the F. novicida lipid A 4'-kinase. To test this hypothesis, extracts of XL1-Blue cells harboring pKEM14-5, a plasmid containing valAB and a portion of polA (17), were assayed for 4'-kinase activity. These extracts possess about 10 times more 4'-kinase activity than crude extracts of XL1-Blue cells containing vector alone (17) (data not shown). To determine whether valB can rescue TG1/pTAG1 grown at 44 °C, pTAG8, which contains valB and about one-third of polA (but no valA), was constructed. pTAG8 and pACYC177 were transformed separately into competent TG1/pTAG1, and transformants were selected on LB plates at 30 °C containing ampicillin. Single colonies were then streaked to LB plates containing ampicillin and grown at 30 and 44 °C. Cells that contained pACYC177 were able to grow and form single colonies at 30 but not 44 °C. Cells that contained pTAG8 were able to grow and form single colonies at both 30 and 44 °C. The latter cells were ampicillin-resistant but chloramphenicol-sensitive, showing that pTAG1 had been lost. These findings indicate that valB can provides the necessary 4'-kinase activity to promote growth of E. coli. A growth experiment in liquid medium was also performed, as in Fig. 3. TG1/pTAG8 was able to grow at 44 °C, exactly like TG1/pTAG6 (data not shown).
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DISCUSSION |
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We have constructed and characterized an E. coli strain (TG1/pTAG1) with an insertion mutation in the structural gene (lpxK) (8) encoding the lipid A 4'-kinase. TG1/pTAG1 has a kanamycin cassette inserted into the chromosomal copy of lpxK. This lpxK::kan allele is complemented by a hybrid plasmid containing lpxK+ and a temperature-sensitive origin of replication (15).
By using TG1/pTAG1, we have confirmed the essential nature of lpxK, originally suggested by Karow and Georgopoulos (12) prior to the identification of the function of the gene (8). Once the lpxK gene and its product are depleted, cell growth and viability are reduced (Figs. 3 and 4), and 4'-kinase activity is lost (Fig. 5). The lipid A to glycerophospholipid ratio decreases (Table II), indicating gradual inhibition of lipid A biosynthesis. Furthermore, when TG1/pTAG1 cells are grown at 44 °C, an additional substance accumulates to high levels in the lipid fraction (Figs. 6-9) that we have identified unequivocally as DS-1-P. This is the first demonstration of DS-1-P as a natural product, given its low abundance in wild type cells (3, 26).2 In our earlier work, DS-1-P was characterized only after enzymatic synthesis in vitro by lipid A disaccharide synthase (the lpxB gene product) (20, 27). However, other key lipid A precursors, such as 2,3-diacylglucosamine (lipid X) (30, 31), UDP-2,3-diacylglucosamine (UDP-DAG) (31), and tetraacyldisaccharide 1,4-bis-phosphate (lipid IVA) (28, 29, 32), have been isolated from various strains of E. coli and Salmonella.
The accumulation of DS-1-P in cells lacking the 4'-kinase strongly supports the hypothesis that DS-1-P is the physiological lipid acceptor for the 4'-kinase (9). Until this work, other schemes for the incorporation of the 4'-phosphate into lipid A could not be excluded. For instance, one alternative possibility was the 4'-phosphorylation of UDP-DAG. Given the massive accumulation of DS-1-P in vivo in TG1/pTAG1 at 44 °C and the fact that UDP-DAG does not serve as a substrate for the cloned 4'-kinase in vitro (8), this possibility is now rendered very unlikely. The NMR spectrum of the accumulated lipid isolated from TG1/pTAG1 (Fig. 8) clearly shows that DS-1-P, not UDP-DAG, accumulates in vivo in the absence of 4'-kinase.
TG1/pTAG1 can be used to assess the function of lpxK variants from other bacteria. In this work, we have shown that valB, the F. novicida lpxK homolog (17), is able to substitute for lpxK in E. coli. TG1/pTAG8, a strain in which the lpxK::kan insertion is covered by valB on a non-temperature-sensitive plasmid, is able to grow at 44 °C as well as TG1/pTAG6, an analogous strain with the E. coli lpxK+ on a non-temperature-sensitive plasmid (data not shown). Rescue of TG1/pTAG1 at 44 °C will also be useful in evaluating the function of lpxK truncations (for instance those lacking the hydrophobic N-terminal domain of LpxK) in the search for an active, soluble form of the kinase. Likewise, expression of His-tagged variants of lpxK in the TG1/pTAG1 background could be used to determine if His-tagged LpxK is active and is tightly bound to other proteins in the absence of competing wild type LpxK.
One protein that may interact with LpxK is MsbA. msbA and lpxK are co-transcribed (12, 13). Although it appears that MsbA plays an important role in lipid A transport (43), a role for LpxK in transport (in addition to its enzymatic function as the 4'-kinase) cannot yet be excluded. For instance, LpxK might form part of the putative membrane channel through which MsbA mediates lipid A flip-flop (43). Other heterodimeric membrane channels, such as the CydCD ABC transporter for periplasmic cytochrome c assembly, have the expression of their protein components tightly linked in operons (33), like msbA and lpxK. It is difficult to ask these questions at present because the one available mutation in the kinase is an insertion (Table I), which would also likely impair its putative role in a transport complex with MsbA. If MsbA and LpxK do form a complex, it might be possible to immunoprecipitate MsbA together with LpxK or to isolate lpxK point mutations that retain 4'-kinase activity but do not effectively interact with MsbA.
The identification of second site suppressors that might allow cells to grow without a 4'-kinase could be useful in further understanding lipid A biosynthesis, transport, and function. One possible class of second site suppressors might be mutations in the kdtA gene (11, 34). A mutation in KdtA that allows efficient transfer of Kdo from CMP-Kdo to DS-1-P (rather than the preferred substrate lipid IVA) (10, 11, 34) might allow growth in the absence of 4'-kinase. Interestingly, the 4'-phosphate is not found in the lipid As of all Gram-negative bacteria (35-38). In Rhizobium leguminosarum, this phenomenon is attributed to the presence of a specific membrane-bound 4'-phosphatase that removes the 4'-phosphate after the 4'-kinase and Kdo transferase have generated Kdo2-lipid IVA (39).
The recent surge in microbial genomics is greatly facilitating the study of lipid A biosynthesis in a broad range of Gram-negative bacteria besides E. coli. The completed genomes of Hemophilus influenzae (40) and Helicobacter pylori (41) contain variants of all the known genes of the E. coli lipid A pathway, including lpxK. However, the genome of Synechocystis is very peculiar in that it contains only the genes encoding the first five enzymes of lipid A biosynthesis, leading to the formation of DS-1-P (42). Synechocystis lacks the genes for the 4'-kinase, the Kdo transferase, the late acyltransferases, and the enzymes required for generating CMP-Kdo (42). Although biochemical studies are very limited, LPS isolated from Synechocystis lacks the 4'-phosphate on its lipid A and does not contain Kdo (35), consistent with the genomics. How DS-1-P would be processed in the absence of a 4'-kinase and a Kdo transferase in Synechocystis is unclear. The identification of the unique genes and enzymes that make distinct lipid A variants in diverse bacteria will facilitate the modification of lipid A-like molecules in living cells and may provide insights into why lipid A is necessary for the viability of E. coli.
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FOOTNOTES |
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* This work was supported in part 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 predoctoral fellowship DGE092-53851 from the National
Science Foundation.
§ To whom correspondence should be addressed. Tel.: 919-684-5326; E-mail: raetz{at}biochem.duke.edu.
1 The abbreviations used are: LPS, lipopolysaccharide; DS-1-P, tetraacyldisaccharide 1-phosphate; HPTLC, high performance thin layer chromatography; kb, kilobase pair(s); PBS, phosphate-buffered saline; COSY, two-dimensional 1H correlation spectroscopy; Kdo, 3-deoxy-D-manno-octulosonic acid; UDP-DAG, UDP-2,3-diacylglucosamine.
2 C. E. Bulawa and C. R. H. Raetz, unpublished observations.
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