(Received for publication, January 21, 1997)
From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
Overexpression of the Escherichia coli msbB gene on high copy plasmids suppresses the temperature-sensitive growth associated with mutations in the htrB gene. htrB encodes the lauroyl transferase of lipid A biosynthesis that acylates the intermediate (Kdo)2-lipid IVA (Brozek, K. A., and Raetz, C. R. H. (1990) J. Biol. Chem. 265, 15410-15417). Since msbB displays 27.5% identity and 42.2% similarity to htrB, we explored the possibility that msbB encodes a related acyltransferase. In contrast to htrB, extracts of strains with insertion mutations in msbB are not defective in transferring laurate from lauroyl acyl carrier protein to (Kdo)2-lipid IVA. However, extracts of msbB mutants do not efficiently acylate the product formed by HtrB, designated (Kdo)2-(lauroyl)-lipid IVA. Extracts of strains harboring msbB+ bearing plasmids acylate (Kdo)2-(lauroyl)-lipid IVA very rapidly compared with wild type. We solubilized and partially purified MsbB from an overproducing strain, lacking HtrB. MsbB transfers myristate or laurate, activated on ACP, to (Kdo)2-(lauroyl)-lipid IVA. Decanoyl, palmitoyl, palmitoleoyl, and (R)-3-hydroxymyristoyl-ACP are poor acyl donors. MsbB acylates (Kdo)2-(lauroyl)-lipid IVA about 100 times faster than (Kdo)2-lipid IVA. The slow, but measurable, rate whereby MsbB acts on (Kdo)2-lipid IVA may explain why overexpression of MsbB suppresses the temperature-sensitive phenotype of htrB mutations. Presumably, the acyloxyacyl group generated by excess MsbB substitutes for the one normally formed by HtrB.
The htrB gene was first described by Karow and Georgopoulos (1, 2) as essential for rapid growth of Escherichia coli on nutrient broth above 33 °C. At elevated temperatures, peculiar morphological changes are observed in htrB-deficient strains, including bulging of the cell surface and filamentation (1-3). At permissive temperatures, htrB mutants display increased resistance to bile salts (3). Based on these phenotypes, Karow and Georgopoulos (1, 3-5) suggested function(s) for htrB in cell envelope assembly, including possible roles in peptidoglycan, lipopolysaccharide, and fatty acid biosynthesis.
We have recently demonstrated (6) that the htrB gene of
E. coli encodes the lauroyl transferase that acylates the
key lipid A biosynthesis intermediate
(Kdo1)2-lipid IVA
(7) (Fig. 1). This conclusion is based on assays of extracts prepared
from htrB-deficient mutants, in which lauroyl transferase
activity is undetectable (6). Conversely, cells overexpressing the
htrB gene on hybrid plasmids overproduce the transferase
several hundredfold (6). We have purified the overproduced enzyme to
homogeneity and have shown by N-terminal sequencing that
htrB is indeed the structural gene for the lauroyl
transferase.2 Identification of htrB
as the lauroyl transferase is further supported by fatty acid analyses
of lipopolysaccharide isolated from htrB-deficient strains
of E. coli, in which the amount of laurate is reduced (4).
Lipid A from htrB-deficient Hemophilus influenzae
is also under-acylated (8).
Karow and Georgopoulos (3, 5) identified several genes that, when introduced on hybrid plasmids, suppress the temperature-sensitive growth associated with htrB mutations. The msbA suppressor encodes a putative transport protein with a remarkable similarity to the mammalian mdr genes (5). Most Mdr proteins pump hydrophobic drugs out of animal cells, but some are involved in secretion of phospholipids into bile (9). Although the biochemical function of msbA is unknown, the sequence similarity of msbA to mdr suggests that MsbA might be involved in a translocation process, such as the movement of newly made lipid A from the cytoplasmic surface of the inner membrane to the periplasm (10-12). Whatever its role, the msbA gene is essential for growth (5).
A second suppressor gene, designated msbB, was found to have 27.5% sequence identity and 42.2% similarity to htrB (3) suggesting a biochemical function related to htrB. MsbB reverses the temperature sensitivity associated with htrB mutations when introduced on plasmids that are maintained at high copy number (3). The msbB gene itself is not essential for growth (3). MsbB knockouts greatly reduce the amount of myristate attached lipid A, but they do not affect the laurate content (13). Lipopolysaccharide isolated from msbB mutants contains penta-acylated lipid A (designated (Kdo)2-(lauroyl)-lipid IVA in Fig. 1) (13). MsbB was discovered independently by Somerville et al. (13), who found that whole E. coli cells harboring msbB insertions are orders of magnitude less immunostimulatory than are wild-type cells.
Using direct enzymatic assays, we now demonstrate that the msbB gene of E. coli encodes a distinct, late-functioning acyltransferase of lipid A assembly (7). MsbB functions optimally after laurate incorporation by HtrB has taken place (Fig. 1). The slow, but significant, rate at which MsbB acylates (Kdo)2-lipid IVA explains why msbB+ works only as a high multi-copy suppressor of htrB mutations (3). Our findings support the view that at least one acyloxyacyl residue must be present on a significant fraction of the lipid A moieties of E. coli to allow rapid growth above 33 °C. A preliminary abstract of our findings has appeared (14).
[-32P]ATP was obtained from
DuPont NEN. Pyridine, chloroform, methanol, and 88% formic acid were
from Fisher. All detergents were of high quality grade (peroxide- and
carbonyl-free). Triton X-100 was from Pierce, and Thesit was from
Sigma. Acyl carrier protein was purchased from Sigma. Other items were
obtained from the following companies: 0.25-mm glass-backed silica gel
60 thin layer chromatography plates (E. Merck), yeast extract and
Tryptone (Difco), and DEAE-Sepharose CL-6B (Pharmacia Biotech
Inc.).
Strains used in this study are derivatives of E. coli K12, and their genotypes are listed in Table I. Cultures were grown in Luria broth, consisting of 5 g of NaCl, 5 g of yeast extract, and 10 g of Tryptone per liter (15). Antibiotics were added, when required, at 100 µg/ml for ampicillin, 12 µg/ml for tetracycline, and 10 µg/ml for chloramphenicol.
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Plasmid DNAs were isolated using the Wizard miniprep kit (Promega). Other recombinant DNA techniques were performed as described previously (16).
Isolation and Preparation of SubstratesLipid
IVA (17), (Kdo)2lipid IVA (18, 19),
[4-32P]lipid IVA (20, 21), and
(Kdo)2-[4
-32P]lipid IVA (18, 19)
were prepared as described previously. (Kdo)2-(lauroyl)-[4
-32P]lipid
IVA was synthesized from lauroyl-ACP and
(Kdo)2-[4
-32P]lipid IVA using a
DEAE-Sepharose purified HtrB that was isolated from an
msbB-deficient strain (MLK1067) containing the
htrB overexpressing plasmid, pKS12 (6). The preparative
reaction contained 50 mM HEPES, pH 7.5, 0.2% Triton X-100,
50 µM (Kdo)2-[4
-32P]lipid
IVA (2 × 103 dpm/nmol), 50 µM lauroyl-ACP, 5 mM MgCl2, 50 mM NaCl, and 8 µg of lauroyl transferase (specific
activity, 2200 nmol × min
1 × mg
1)
per ml. The total reaction volume was 150 µl. After incubation at
30 °C for 30 min, the reaction mixture was applied as a single line
onto a 20 × 20-cm Silica Gel 60 thin layer chromatography plate.
After air drying, the plate was developed to the top in the solvent
chloroform, pyridine, 88% formic acid, water (30:70:16:10, v/v). The
product was detected by a brief autoradiography, and the region of the
silica plate containing the desired, more rapidly migrating product was
scraped off into a scintered glass funnel. The silica chips were washed
once with 4 ml of chloroform. The product,
(Kdo)2-(lauroyl)-[4
-32P]lipid
IVA, was then eluted with three 3.8-ml portions of a single phase, acidic Bligh and Dyer solvent mixture consisting of chloroform, methanol, 0.1 M HCl (1:2:0.8, v/v) (22). The eluted
material (12 ml) was converted to a two-phase, acidic Bligh and Dyer
system by the addition of 3 ml each of chloroform and 0.1 M
HCl. The tube was mixed vigorously, and the phases were separated by a brief centrifugation. The lower phase was transferred to a new glass
tube, and 18 drops of pyridine were added before the solvent was
evaporated under a stream of nitrogen. The dried lipid was resuspended
in 10 mM Tris chloride, pH 7.5, containing 1 mM
EDTA. It was stored at
80 °C and was resuspended by brief (1 min)
sonic irradiation prior to use.
Acyl-ACPs containing various acyl chains were synthesized from the corresponding free fatty acids and acyl carrier protein using solubilized membranes from the acyl-ACP synthase overproducing strain, E. coli LCH109/pLCH5/pGP1-2, as described previously (6).
Preparation of Cell-free Extracts, Membranes, and Soluble FractionsCrude cell-free extracts were made from 1-2 liters of
logarithmically growing cultures. After harvesting by low speed
centrifugation at 2 °C, cells were washed once in 30 mM
HEPES, pH 7.5, containing 1 mM EDTA and 1 mM
EGTA (half the volume of the original culture). The washed cell pellet
was resuspended in 30 mM HEPES, pH 7.5, containing 1 mM EDTA and 1 mM EGTA (a volume approximately
equal to the volume of the cell pellet). Cells were broken using an ice-cold French pressure cell (SLM Instruments, Urbana, IL) at 20,000 psi. The broken cell suspension was adjusted to 10 mM
MgSO4, and DNase I was added to 1 µg/ml. After a brief
sonic irradiation on an ice water bath to decrease the viscosity, the
suspension was incubated for 30 min at 30 °C. Unbroken cells were
removed by centrifugation at 1,000 × g for 10 min.
Membranes and soluble fractions were separated by centrifugation at
150,000 × g for 60 min. The supernatant was
centrifuged a second time to remove residual contaminating membranes.
The membrane pellet was resuspended in 25 ml of 30 mM
HEPES, pH 7.5, containing 1 mM EDTA and 1 mM EGTA, and it was centrifuged again as above to generate the final, washed membrane fraction (~40 mg/ml). The membrane suspension was
stored at 80 °C.
Protein concentrations were determined with the bicinchoninic assay (Pierce), using bovine serum albumin as the standard (23).
Assay for Acyl-ACP-dependent Acylation of Kdo-containing PrecursorsHtrB-catalyzed acylation was assayed
using Method I described earlier (6). In most cases, the reaction
mixture contained 50 mM HEPES, pH 7.5, 0.1% Triton X-100,
25 µM (Kdo)2-[4-32P]lipid
IVA (~2 × 103 dpm/nmol), 25 µM lauroyl-ACP, 0.1 mg/ml bovine serum albumin, and
0.1-1000 µg/ml enzyme at 30 °C in a final volume of 10-20 µl.
With highly purified preparations of HtrB, enzyme stability is
increased by including 50 mM NaCl and 5 mM
MgCl2 in the assay mixture (6). The MsbB-catalyzed
acylation of the product generated by HtrB, designated
(Kdo)2-(lauroyl)-[4
-32P]-lipid
IVA, was assayed in a reaction mixture containing 50 mM HEPES, pH 7.5, 0.1% Triton X-100, and 0.1 mg/ml bovine
serum albumin. Unless otherwise indicated, 25 µM
(Kdo)2-(lauroyl)-[4
-32P]lipid
IVA (~2 × 103 dpm/nmol) was used as the
acceptor, and 25 µM myristoyl-ACP was the donor. Under
these conditions, MsbB displays a slight kinetic preference for
myristoyl-ACP over lauroyl-ACP. MsbB functions about 100-fold more
rapidly with (Kdo)2-(lauroyl)-[4
-32P]lipid
IVA than with
(Kdo)2-[4
-32P]lipid IVA as the
acceptor. Since studies using partially purified MsbB showed a
stabilizing effect of sodium chloride, 50 mM NaCl was also
included in the reaction in some experiments. MgCl2
inhibited MsbB slightly. Reactions were stopped by spotting a 4-5-µl
sample onto a Silica Gel 60 thin layer chromatography plate. After air drying and developing the thin layer chromatography plate in the solvent chloroform, pyridine, 88% formic acid, water (30:70:16:10, v/v), the plates were exposed to a PhosphorImager screen. The amount of
radioactivity present in the labeled spots corresponding to substrate
and product were quantitated using a Molecular Dynamics Imager. The
specific activity of both HtrB and MsbB was expressed in terms of
nmol/min/mg protein.
Cells were grown and
labeled by the method of Galloway and Raetz (24), with several
modifications. Briefly, bacteria from 30 °C overnight cultures were
inoculated into 20 ml of fresh LB broth and grown for several hours at
30 °C until A600 had reached ~0.1. Next,
two 5.0-ml portions of each culture were transferred into two new
culture tubes, and 32Pi (5 µCi/ml) was added
to both. One was grown for 3 h at 30 °C, and the other was
grown for 3 h at 42 °C. 32P-Labeled cells were
washed twice with 5.0 ml of phosphate-buffered saline, pH 7.4, and
resuspended in 0.8 ml of the same buffer. Washed cells were extracted
at room temperature for 60 min with a single phase Bligh and Dyer
mixture, formed by the addition of 2 ml of methanol and 1 ml of
chloroform. After centrifugation in a clinical centrifuge at top speed
for 20 min, the pellets were recovered and washed once with 5.0 ml of a
single phase of Bligh and Dyer mixture, consisting of
chloroform/methanol/water (1:2:0.8, v/v). The pellets were resuspended
in 3.6 ml of 0.2 M HCl by sonic irradiation in a bath. The
resuspended pellets were incubated at 100 °C for 90 min. The
acid-hydrolyzed material was converted to a two-phase Bligh and Dyer
system by addition of 4 ml of chloroform and 4 ml of methanol. After
centrifugation at low speed, the upper phases were removed, and the
lower phases were washed twice with 4.0 ml of pre-equilibrated acidic
upper phase of a two-phase Bligh and Dyer system, generated by mixing chloroform, methanol, 0.2 M HCl (2:2:1.8, v/v). The washed
lower phases were dried under a nitrogen stream. The lipid A
4-monophosphates were redissolved in 100 µl of chloroform/methanol
(4:1, v/v), and approximately 1000 cpm of the samples was applied to
each lane of a Silica Gel 60 thin layer chromatography plate. The plate was developed in the solvent of chloroform, pyridine, 88% formic acid,
water (50:50:16:5, v/v). The plate was dried and exposed overnight to a
PhosphorImager screen.
Extracts of strains bearing
htrB mutations do not catalyze
lauroyl-ACP-dependent acylation of
(Kdo)2-[4-32P]lipid IVA, as
shown in Fig. 2 by the absence of
(Kdo)2-(lauroyl)-lipid IVA formation (product
a in lane 2). The transfer of laurate from
lauroyl-ACP to (Kdo)2-[4
-32P]lipid
IVA was efficient in extracts of strains harboring an insertion in msbB (Fig. 2, lane 6), as in the
wild type (Fig. 2, lane 4). A similar pattern was observed
when myristoyl-ACP was substituted for lauroyl-ACP (not shown), except
that the initial rates of
(Kdo)2-[4
-32P]lipid IVA
acylation were 5-10-fold slower with myristoyl-ACP, given the
selectivity of HtrB for laurate (6).
Acylation of (Kdo)2-[4
In crude extracts or
membrane preparations from wild-type cells one observes two distinct
acylations of (Kdo)2-[4-32P]lipid
IVA (7). With lauroyl-ACP as the donor, the first acylation is relatively rapid compared with the second (7). With myristoyl-ACP, the first acylation is 5-10-fold slower than with lauroyl-ACP, but the
second acylation is slightly faster than with lauroyl-ACP (7). Given
that purified HtrB catalyzes only one rapid acylation of
(Kdo)2-[4
-32P]lipid IVA (6), it
seemed plausible that MsbB might encode the second acyltransferase
(Fig. 1) that we previously observed in cell extracts (7).
To approach this problem, we initially incubated membranes of wild-type
cells that overexpress either htrB+ or
msbB+ with
(Kdo)2-[4-32P]lipid IVA and
lauroyl-ACP. As shown in Fig. 3, panel B,
overexpression of htrB+ in the presence of
msbB+ on the chromosome resulted in very rapid
formation and accumulation of a monoacylated product at the relatively
low extract concentrations employed (4 µg/ml). Alternatively,
overexpression of msbB+ in the presence of
htrB+ on the chromosome (Fig. 3, panel
A) did not increase the overall extent of
(Kdo)2-[4
-32P]lipid IVA
acylation compared with wild-type (hence the need to use 100 µg/ml
membranes), but it did result in the accumulation of the diacylated
material (product b) at the expense of the monoacylated
product a. The difference between HtrB and MsbB
overproduction in a wild-type background is especially apparent when
comparing the product distribution at the 40-min time point in
panel A of Fig. 3 (excess MsbB) to the 10-min time point in
panel B of Fig. 3 (excess HtrB). In both cases the
conversion of substrate to product is similar (~30%), but diacylated
material is generated in strains overproducing MsbB, whereas
monoacylated product is formed when HtrB is overproduced. The results
of Fig. 3 provide support for the idea that MsbB catalyzes the second acylation and can use lauroyl-ACP as the donor under the in
vitro assay conditions employed.
Acylation by MsbB Follows HtrB-catalyzed Acylation of (Kdo)2-[4
To evaluate further the function of MsbB and its relationship to HtrB, we prepared membranes from strains of msbB or htrB-deficient cells overexpressing htrB and msbB, respectively (6). In this way, we could study cell extracts that contained only one or the other enzyme.
As shown in Fig. 4, panel A, membranes of
strain MLK53/pBS233 (containing only msbB+) did
not catalyze any measurable acylation of
(Kdo)2-[4-32P]lipid IVA under
the conditions employed. Membranes of cells containing only HtrB (Fig.
4, panel B) catalyzed mainly the formation of product
a. In Fig. 4, panel A, the
(Kdo)2-[4
-32P]lipid IVA and
acyl-ACP were first preincubated for 60 min with 10 µg/ml membranes
of MLK53/pBS233. At time 0, a second 60-min incubation was started.
Samples were then withdrawn during the second incubation period for
product analysis at the indicated times. In Fig. 4, panels B
and C, membranes of strain MLK1067/pKS12 (containing only
htrB+) were preincubated for 60 min with
(Kdo)2-[4
-32P]lipid IVA and
acyl-ACP to generate significant amounts of
(Kdo)2-(lauroyl)-[4
-32P]lipid
IVA (product a). At time 0, only a small volume of buffer was added to the reaction mixture in panel B. In
panel C, a 10 µg/ml portion of membranes of MLK53/pBS233
(containing only msbB+) was added at time 0. The
incubations were continued for another 60 min and analyzed as in
panel A. The system containing only HtrB (panel
B) catalyzed the formation of more product a, whereas
the system containing both HtrB and MsbB (panel C) resulted in very rapid accumulation of the diacylated product b derived from product a. These findings show that MsbB
functions efficiently only after HtrB-catalyzed acylation of
(Kdo)2-[4
-32P]lipid IVA has
taken place (Fig. 4, panel A compared with panel C).
Specific Activity of MsbB in Extracts of Mutants and Plasmid-bearing Strains
A direct assay for MsbB was developed
using 25 µM
(Kdo)2-(lauroyl)-[4-32P]lipid
IVA as the acceptor and 25 µM acyl-ACP as the
donor (see "Experimental Procedures"). Acylation was detected by
thin layer chromatography and PhosphorImager analysis as in Fig. 4.
With the direct assay, the specific activity of MsbB in extracts of wild-type cells was ~0.3 nmol/min/mg, but in extracts of
msbB-deficient mutants, the activity was below the limits of
detection (~0.03 nmol/min/mg). Overexpression of
msbB+ on multi-copy hybrid plasmids resulted in
~100-fold higher specific activity of MsbB in cell extracts or
membranes compared with wild-type, as shown in Table II.
The large size of the observed effects on MsbB-specific activity
supports the proposal that msbB is the structural gene
encoding the MsbB acyltransferase.
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Washed
membranes (5 ml) from E. coli MLK53/pBS233 were diluted in
20 mM Tris chloride, pH 7.8, containing 1 mM
EDTA and 1 mM EGTA to a final protein concentration of
~17 mg/ml protein (208 mg of protein in a total volume of 12 ml). The
membrane suspension was mixed with an equal volume of solubilization
buffer, giving final concentrations of 2.5% Triton X-100, 20 mM Tris chloride, pH 7.8, 1 mM EDTA, 1 mM EGTA, 100 mM sodium phosphate, 150 mM NaCl, and 10% glycerol. The solubilization mixture was
incubated with slow stirring at 5 °C for 2 h. Insoluble
material was removed by centrifugation for 60 min at 170,000 × g at 5 °C. The supernatant was transferred to a new tube
and diluted to a final volume of 72 ml with 20 mM Tris
chloride, pH 7.8, 1 mM EDTA, and 1 mM EGTA to
reduce the NaCl concentration. The solubilized proteins were loaded
onto a DEAE-Sepharose CL-6B column (45-ml packed bed-volume) equilibrated in 0.2% Thesit, 20 mM Tris chloride, pH 7.8, 1 mM EDTA, 1 mM EGTA, 50 mM NaCl,
and 10% glycerol. After washing the column with 140 ml of
equilibration buffer, bound proteins were eluted with a 400-ml linear
gradient of 50-500 mM NaCl in 0.2% Thesit, 20 mM Tris chloride, pH 7.8, 1 mM EDTA, 1 mM EGTA, and 10% glycerol. The absorbance at 280 nm of
each fraction was measured (Fig. 5, open
circles), and the four peak fractions (Fig. 5, shaded area) of MsbB activity (filled circles) were pooled.
The combined fractions (20 ml) were diluted 4-fold in 20 mM
Tris chloride, pH 7.8, 1 mM EDTA, and 1 mM
EGTA, and the material was loaded onto a 1.8-ml DEAE-Sepharose CL-6B
column, equilibrated as above, to concentrate the sample. The total
protein was then eluted into a total volume of 1.5 ml of 0.05% Triton
X-100, 20 mM Tris chloride, pH 7.8, 1 mM EDTA,
1 mM EGTA, 200 mM NaCl, and 10% glycerol. The fractionated MsbB was stored in aliquots at 80 °C.
Stability and Assay of Purified MsbB
Using
DEAE-Sepharose-purified MsbB, we examined the stability and time
dependence of product formation, using enzymatically synthesized
(Kdo)2-(lauroyl)-[4-32P]lipid
IVA and myristoyl-ACP as the substrates
(Fig. 6). In contrast to the significant activation
previously observed with HtrB (6), we found that MsbB was slightly
inhibited by the presence of 5 mM MgCl2 (Fig.
6). However, 50 mM NaCl was beneficial (Fig. 6), as in the
case of HtrB (6), and MsbB was stable to preincubation under assay
conditions for 30 min in the presence of NaCl (not shown). The MsbB
assay carried out in the presence of 50 mM NaCl displayed
excellent linearity with respect to time (Fig. 6) and protein
concentration (not shown).
Using the improved conditions, the fractions generated in the course of solubilizing and purifying MsbB were reassayed. Table II shows that the final MsbB preparation is purified about 24-fold relative to washed membranes from the overproducing strain employed, and about 2400-fold relative to crude extracts of wild-type cells.
Selectivity of MsbB for Acyl Chain LengthAs shown in
Fig. 7, DEAE-Sepharose-purified MsbB displays a slight
kinetic preference for myristoyl-ACP over lauroyl-ACP. Other acyl
donors, including decanoyl-, palmitoyl-, palmitoleoyl-, and
(R)-3-hydroxymyristoyl-ACP are inactive as acyl donors when (Kdo)2-(lauroyl)-[4-32P]lipid
IVA is employed as the acceptor (Fig. 7). The virtual absence of activity with decanoyl-ACP is interesting. HtrB does utilize
decanoyl-ACP as a donor in vitro at about one-third of the
rate of lauroyl-ACP. Given the fact that we have tested the same
acyl-ACP preparations with both HtrB (6) and MsbB (Fig. 7), we conclude
that the optimal activity of MsbB with various acyl-ACPs under our
assay conditions is set approximately two carbons longer than that of
HtrB. In living cells, however, the selectivity of HtrB for laurate and
of MsbB for myristate must be higher. No decanoate is found in the
position normally occupied by laurate on lipid A under ordinary growth
conditions in living cells (Fig. 1), and very little laurate is
incorporated in place of myristate (11, 25-28).
Selectivity of MsbB for Penta-acylated Lipid A Precursors
The
DEAE-Sepharose-purified preparation of MsbB was used to confirm the
specificity of the enzyme for the penta-acylated acceptor, first
suggested by the results of Fig. 4. As shown in Fig. 8, we compared [4-32P]lipid IVA,
(Kdo)2-[4
-32P]lipid IVA, and
(Kdo)2-(lauroyl)-[4
-32P]lipid
IVA (each 25 µM) under matched conditions as
myristate acceptors. The reactions in the odd-numbered lanes
contained myristoyl-ACP, whereas the ones in the even lanes
did not. All observed acylations were dependent upon added
myristoyl-ACP. Using a concentration of MsbB (0.1 µg/ml) that
resulted in 90% acylation of
(Kdo)2-(lauroyl)-[4
-32P]lipid
IVA to generate
(Kdo)2-[4
-32P]lipid A (i.e.
product b) in 60 min (Fig. 8, lane 5), we
observed only 1-2% acylation of
(Kdo)2-[4
-32P]lipid IVA (Fig. 8,
lane 3) (product a
) and of
[4
-32P]lipid IVA (Fig. 8, lane 1)
(product c). The slow but measurable acylation of the
tetra-acylated acceptor, (Kdo)2-[4
-32P]lipid
IVA (Fig. 8, lane 3), with myristate to generate
(Kdo)2-[myristoyl]-[4
-32P]lipid
IVA (i.e. product a
) (proposed
structure in Fig. 1) probably explains why overexpression of
msbB+ suppresses the temperature sensitivity of
strains defective in htrB (3).
State of Lipid A Acylation in msbB- and htrB-deficient Mutants and the Effect of Plasmids Bearing msbB+
In previous
studies (24), we described a simple, radiochemical method for
determining the lipid A content of E. coli. Our procedure
involves the labeling of cells with 32Pi,
followed by Bligh-Dyer extraction of the glycerophospholipids (24).
Next, the cell residue is treated with O.2 M HCl at
100 °C to release all the lipid A moieties of lipopolysaccharide as
a series of 4-monophosphates (24). These compounds are separated into
hexa-acylated, penta-acylated, and tetra-acylated species, using silica
gel thin layer chromatography (24), and they can be detected by
autoradiography or PhosphorImager analysis.
As shown in Fig. 9 (lanes 2 and
3), the lipid A-derived 4-monophosphates of wild-type
cells, labeled for 3 h at either 30 or 42 °C in LB broth (15)
with 32Pi, consist mostly of hexa-acylated,
some penta-acylated, and a few tetra-acylated forms. The relative
abundance of the hexa-acylated species may be underestimated slightly
by our procedure because of partial deacylation caused by the acid
hydrolysis (24). However, strains defective in msbB yield
mostly penta-acylated 4
-monophosphates with only minor amounts of the
tetra-acylated species at both 30 or 42 °C (Fig. 9, lanes
4 and 5), consistent with the proposed function of MsbB
(Fig. 1). Under nonpermissive conditions (42 °C), strains defective
in htrB or in both htrB and msbB (Fig.
9, lanes 7 and 9, respectively) yield mostly
tetra-acylated lipid A 4
-monophosphates. Interestingly, when grown at
30 °C, these mutants do generate significant amounts of
penta-acylated and even some hexa-acylated forms (Fig. 9, lanes
6 and 8), the origin of which is not entirely clear
(see "Discussion"). However, when msbB+ is
overexpressed in htrB-deficient cells, growth and
considerable amounts of additional penta-acylated 4
-monophosphates are
restored at 42 °C (Fig. 9, lane 11 versus lane 7). These
observations support the view that high level overexpression of MsbB
bypasses the need for HtrB action prior to MsbB-catalyzed acylation.
Rapid cell growth apparently requires that a critical fraction of the
lipid A moieties of lipopolysaccharide be at least penta-acylated. The enzymatic source of these acylations seems not to be as important.
As shown by the results of Figs. 2, 3, 4, 8, and 9, msbB encodes a distinct acyltransferase of lipid A assembly that functions optimally after laurate incorporation by HtrB has occurred. The proposed intermediates generated by MsbB and HtrB are shown in Fig. 1. These structural proposals are based on what is known about lipid A released from E. coli lipopolysaccharide by acid hydrolysis (11, 25-28). The proposed structures also are in accord with the molecular weights of (Kdo)2-lipid IVA acylation products generated previously with crude cell extracts (7). The lauroyl and myristoyl groups are attached by HtrB and MsbB to the lipid IVA moiety of (Kdo)2-lipid IVA (7). However, the structures of the acylated derivatives of (Kdo)2-lipid IVA generated using homogeneous preparations of HtrB and MsbB remain to be established unequivocally.
Besides HtrB and MsbB, there are at least two additional enzymes that may be able to acylate (Kdo)2-lipid IVA in E. coli extracts. One of these transfers palmitate from the 1-position of a glycerophospholipid to the (R)-3-hydroxy moiety of the N-linked (R)-3-hydroxymyristate of the proximal glucosamine unit (20). The palmitoyltransferase has not been purified, and the gene encoding it is unknown. In addition, we have recently obtained evidence for a cold shock-induced enzyme that transfers palmitoleate from palmitoleoyl-ACP to (Kdo)2-lipid IVA.3 In living cells of E. coli and Salmonella typhimurium, cold shock causes the incorporation of an equivalent of palmitoleate into lipid A at the expense of laurate (29, 30). Palmitoleate is not incorporated into (Kdo)2-lipid IVA by purified HtrB (6).
As shown in Fig. 9, E. coli mutants defective in
htrB or in both htrB and msbB contain
lipid A moieties that are mainly tetra-acylated when cells are grown at
42 °C for 3 h. The presence of excess msbB+ on hybrid plasmids maintained at high copy
number restores some penta-acylated lipid A species (Fig. 9) and
suppresses the temperature-sensitive growth phenotype associated with
htrB mutations (3). These findings suggest that the slow
rate of acylation of (Kdo)2-lipid IVA catalyzed
by MsbB (Fig. 8, product a) may be sufficient to generate a
significant number of acyloxyacyl moieties when MsbB is overproduced in
htrB-deficient cells. We presume that (Kdo)2-(myristoyl)-lipid IVA, having the
proposed structure shown at the bottom of Fig. 1, is being generated
when the htrB mutation is suppressed by overexpression of
msbB+.
E. coli mutants defective in htrB or in both htrB and msbB do contain some penta- and hexa-acylated lipid A species when grown at 30 °C (Fig. 9). These species might arise by action of the palmitoyltransferase (20) or the cold-induced palmitoleoyltransferase noted above. To determine the origin of these more highly acylated lipid A species present in mutants defective in both htrB and msbB, it will be necessary to carry out additional chemical analyses. Preliminary results with laser desorption mass spectrometry4 indicate that lipid A isolated from mutants defective in both htrB and msbB grown at 30 °C consists of a mixture of tetra-acylated and penta-acylated forms. Both forms contain four hydroxymyristoyl chains, but the penta-acylated species contains an additional palmitate or palmitoleate.
The effects of the msbA multi-copy suppressor (5) on the
extent of acylation of the lipid A moieties present in mutants defective in both htrB and msbB remain to be
examined. Recently, Polissi and Georgopoulos (31) have found that
lipopolysaccharide accumulates in the inner membranes of
htrB deficient mutants. Introduction of both
msbA+ and orfE+ (an
essential downstream gene of unknown function) on hybrid plasmids
partially restored-deficient translocation of lipopolysaccharide to the
outer membrane in htrB mutants (31). Insertion mutations that conditionally inactivate both msbA and orfE
also appear to accumulate lipopolysaccharide in their inner membranes
(31). If msbA is really involved in lipid A export, as
anticipated because of its sequence similarity to mammalian Mdr
proteins, one should see no restoration of lipid A acylation upon
suppression of
htrB
msbB
mutants by
msbA+. If msbA, like msbB, encodes a
distinct acyltransferase, however, then the extent of lipid A acylation
would be increased when mutants defective in both htrB and
msbB are complemented by msbA.
A search of genome data bases indicates that H. influenzae contains htrB and msbB homologues (8). Based on mass spectrometry of lipid A isolated from htrB mutants of H. influenzae (8), it is likely that HtrB function is conserved in this organism. All known HtrB and MsbB sequences contain the peptide LCFP in the vicinity of residue 70.
The identification of the genes that are responsible for the early and late acylations of lipid A should facilitate the construction of E. coli strains possessing greatly reduced or altered endotoxin activities. The msbB insertion mutations described by Somerville et al. (13) reduce the ability of intact E. coli to activate macrophages by several orders of magnitude. By further modifying the structure and composition of E. coli lipid A, it may be possible to create strains with virtually no endotoxin activity. For instance, substitution of E. coli lpxA with Pseudomonas lpxA (32), coupled with deletion of msbB, would result in the generation of a lipid A with considerable similarity to that of Rhodobacter sphaeroides (33, 34). Lipid A of R. sphaeroides actually antagonizes the activities of wild-type E. coli endotoxin (33, 34). Such E. coli mutants, if viable, might be very useful for the expression and preparation of proteins intended for injection into animals, since quantitative removal of lipopolysaccharide would not be as critical. Modification of the structure of lipid A in living bacterial cells might also provide new insights into the functions of lipid A in outer membrane biogenesis and during infection of animals.
We thank Dr. Margaret Karow for providing the htrB- and msbB-related strains listed in Table I and for many helpful discussions.