(Received for publication, February 8, 1996)
From the
By assaying lysates of Escherichia coli generated with
the hybrid bacteriophages of an ordered library (Kohara, Y.,
Akiyama, K., and Isono, K.(1987) Cell 50, 495-508), we
identified two clones (
232 and
233) capable of overexpressing
the lauroyl transferase that functions after
3-deoxy-D-manno-octulosonic acid (Kdo) addition in
lipid A biosynthesis (Brozek, K. A., and Raetz, C. R. H.(1990) J.
Biol. Chem. 265, 15410-15417). The E. coli DNA
inserts in
232 and
233 suggested that a known gene (htrB) required for rapid growth above 33 °C might encode
the lauroyl transferase. Using the intermediate (Kdo)
-lipid
IV
as the laurate acceptor, extracts of strains with
transposon insertions in htrB were found to contain no lauroyl
transferase activity. Cells harboring hybrid htrB
plasmids overproduced transferase activity 100-200-fold.
The overproduced transferase was solubilized with a non-ionic detergent
and purified further by DEAE-Sepharose chromatography. With lauroyl
acyl carrier protein as the donor, the purified enzyme rapidly
incorporated one laurate residue into (Kdo)
-lipid
IV
. The rate of laurate incorporation was reduced by
several orders of magnitude when either one or both Kdos were absent in
the acceptor. With a matched set of acyl-acyl carrier proteins, the
enzyme incorporated laurate 3-8 times faster than decanoate or
myristate, respectively. Transfer of palmitate, palmitoleate, or R-3-hydroxymyristate was very slow. Taken together with
previous studies, our findings indicate that htrB encodes a
key, late functioning acyltransferase of lipid A biosynthesis.
Lipid A, the hydrophobic anchor of the outer membrane lipopolysaccharide of Escherichia coli(1, 2, 3, 4, 5) , consists of a glucosamine disaccharide that is phosphorylated at positions 1 and 4`, and is acylated with R-3-hydroxymyristate at positions 2, 3, 2`, and 3` (Fig. 1). Lipid A of wild-type E. coli cells contains two additional fatty acyl chains, primarily laurate and myristate(3, 6, 7, 8) . The latter are esterified to the R-3-hydroxy groups of the distal glucosamine residue (Fig. 1), forming the acyloxyacyl moieties that are characteristic of lipid A (1, 3, 6, 7, 8) . Variations in the composition and location of the non-hydroxylated acyl chains can occur(1, 8) . For instance, minor lipid A species are observed in E. coli in which the myristate residue is missing entirely (8) or is replaced with laurate (not shown in Fig. 1)(9) . An additional palmitate residue may also sometimes be present in acyloxyacyl linkage on the proximal glucosamine unit (not shown in Fig. 1), either with or without the myristate on the distal glucosamine(1, 4, 8) . The laurate residue that is attached to the distal N-linked R-3-hydroxymyristate (Fig. 1) is found in almost every lipid A moiety of wildtype cells (8, 9) .
Figure 1: Pathway for the late stages of lipid A biosynthesis in E. coli. Evidence for the complete scheme has recently been reviewed(2, 3, 5) .
In
previous studies, we described the existence of novel
acyltransferase(s) in extracts of E. coli that can incorporate
one or two laurate (or myristate) residues into lipid A
precursors(10) . The lauroyl/myristoyl transferase(s) require
lauroyl- or myristoyl acyl carrier proteins (ACP) ()as
donors(10) . They appear to function at a late stage of lipid A
assembly, since they recognize lipid A disaccharide precursors that are
glycosylated with Kdo as substrates (Fig. 1)(10) . Their
extraordinary Kdo dependence explains why lipid IV
(Fig. 1), rather than fully acylated lipid A, accumulates
in living cells when the biosynthesis of Kdo is
interrupted(11, 12, 13, 14) .
Prior to the present work, no late acyltransferase had been purified (10) , and gene(s) encoding these enzyme(s) were not
known(3) . The issue of whether one or more distinct
acyltransferases were required for acyloxyacyl group formation was not
resolved(10) . A priori, it seemed reasonable that a
separate enzyme would be needed for the generation of each acyloxyacyl
moiety. In cell extracts, two laurates, two myristates, or one of each
can be transferred efficiently to (Kdo)-lipid
IV
(10) , despite the distinct composition of the
dominant lipid A molecular species made in wild-type cells (Fig. 1)(7, 8) .
To identify a gene encoding
a Kdo-dependent late acyltransferase, we have assayed individual E.
coli lysates generated with each of the hybrid
bacteriophages of the Kohara library (15, 16) for
overproduction of the lauroyl transferase that acts on
(Kdo)
-lipid IV
(Fig. 1)(10) . In
this manner, we have discovered that a known gene (htrB),
described by Karow and Georgopoulos (17, 18, 19, 20, 21) as
required for growth on rich media above 33 °C, encodes the lauroyl
transferase (Fig. 1). This finding explains why lipid A isolated
from htrB deficient mutants contains very little
laurate(20) . The msbB gene(19) , a high
multi-copy suppressor of the temperature sensitivity associated with htrB mutations, encodes a separate late acyltransferase with a
strong kinetic preference for the penta-acylated lipid generated (Fig. 1) by HtrB (22) . The envelope-related alterations
associated with mutations in the htrB gene(17, 19) , such as bulging of the cell
surface and deoxycholate resistance, can now be explored in a
biochemical context. A preliminary communication of our results has
appeared in abstract form(22) .
For the purpose of screening the entire library, lysates
(0.5 µl of a 1:5 dilution) were assayed for lauroyl transferase
activity in a 10-µl screening assay mixture, containing 50 mM Hepes pH 7.5, 0.1% Triton X-100, 10 µM (Kdo)-[4`-
P]lipid IV
(1
10
dpm/nmol), 25 µM lauroyl-ACP, and 0.2 mg/ml bovine serum albumin. Reactions were
incubated for 10 min at 30 °C. A 5-µl portion of each reaction
mixture was spotted onto a Silica Gel 60 thin layer chromatography
plate, which was developed and subjected to PhosphorImager analysis to
determine the extent of acylation catalyzed by each extract (see
below).
In the course of purifying the lauroyl
transferase, an improved assay was developed (Method II). The latter
was identical to Method I, except that 5 mM MgCl and 50 mM NaCl were included in the reaction mixture to
stabilize the enzyme.
Protein concentrations were determined with the bicinchoninic assay (Pierce), using bovine serum albumin as the standard(25) .
Various acyl-ACPs were synthesized from the corresponding fatty acids and commercial acyl carrier protein, as described previously(31) , except that the immobilized acyl-ACP synthase was replaced with 80 µg/ml solubilized membrane protein from the acyl-ACP synthase overproducing strain, E. coli LCH109pLCH5/pGP1-2(32) .
To obtain the acyl-ACP
synthetase, LCH109/pLCH5/pGP1-2 membranes were solubilized using
a modification of the procedure of Rock and Cronan (32, 33) . Two 1-liter cultures of
LCH109/pLCH5/pGP1-2 were grown at 30 °C until the cultures
reached an A of approximately 0.4. The cultures
were then shifted to 42 °C for 30 min. Next, the cultures were
allowed to continue growing for 90 min at 37 °C. To harvest, the
cells were centrifuged at 4500
g for 10 min and
resuspended in 15 ml of 50 mM Tris-HCl, pH 8.0. A French
pressure cell at 18,000 p.s.i. was used to disrupt the cells, and
unbroken cells were removed by centrifuging at 1000
g for 10 min. The supernatant was made 10 mM in MgCl
by adding 1 M MgCl
. Then, the extract was
centrifuged for 1 h at 150,000
g. The pellet was
resuspended in 5 ml of 50 mM Tris-HCl, pH 8.0. Next, a 5-ml
solution of 50 mM Tris-HCl, pH 8.0, containing 4% Triton X-100
and 20 mM MgCl
was added to solubilize the inner
membrane proteins. The suspension was stirred on ice for 30 min. The
solubilized membranes were centrifuged a second time at 150,000
g for 1 h to remove outer membrane proteins, which were not
extracted efficiently under these conditions. The supernatant was
stored in aliquots at -80 °C.
The enzymatic acylation of
ACP with laurate, myristate, palmitate, decanoate, palmitoleate, or R-3-hydroxymyristate was carried out as follows. ACP (1 mg)
and 8.6 mM dithiothreitol were incubated in 500 µl of 40
mM Tris-HCl, pH 8.0, in a sealed tube at 37 °C for 1 h.
Next, a 320-µl solution consisting of 0.7 M LiCl, 40
mM MgCl, 20 mM ATP, pH 8.0, 750
µM fatty acid, 2.7% Triton X-100, and 540 mM Tris-HCl, pH 8.0, was added to the tube with the ACP. Last, 400
µl of 0.25 mg/ml LCH109/pLCH5/pGP1-2 solubilized membranes
was added, and the acylation reaction was allowed to proceed at room
temperature for 1-2 h. The extent of acylation was determined by
analyzing 5-µl portions of the reaction mixture on a
polyacrylamide/urea gel system(34) .
To isolate the product,
the reaction mixture was diluted 10-fold with water and loaded onto a
1-ml column of DEAE-Sepharose equilibrated with 10 mM bis-tris, pH 6.0. The column was washed with 5 bed volumes of 10
mM bis-tris, pH 6.0, 5 volumes of 10 mM bis-tris, pH
6.0, containing 50% isopropyl alcohol, and 5 volumes of 10 mM bis-tris, pH 6.0. The column was eluted with 3 volumes of 10
mM bis-tris, pH 6.0, containing 0.2 M LiCl and 3
volumes of 10 mM bis-tris, pH 6.0, containing 0.6 M LiCl. Fractions of 1 ml were collected. The acyl-ACPs eluted in
the second 0.6 M LiCl fraction. This fraction was concentrated
and exchanged into distilled HO using a Centricon-3
membrane (Amicon). The acyl-ACPs were about 90% pure, as judged by
electrophoresis in the polyacrylamide/urea gel system and staining with
Coomassie Blue (34) .
Figure 2:
Detection of clones in the Kohara
library that direct overexpression of lauroyl transfer to
(Kdo)
-[4`-
P]lipid IV
.
The hybrid
bacteriophages of the Kohara library were used to
infect and generate new lysates on E. coli W3110. Following
their identification with the initial screening assay (see
``Experimental Procedures''), selected lysates (2 µl of a
1:10 dilution) were re-assayed to confirm overproduction of lauroyl
transferase activity in a 20-µl assay mixture, containing 50 mM Hepes pH 7.5, 0.1% Triton X-100, 10 µM (Kdo)
-[4`-
P]lipid IV
(1
10
dpm/nmol), 25 µM lauroyl-ACP, and 0.2 mg/ml bovine serum albumin. After 10 min at
30 °C, the reactions were stopped by spotting 5 µl onto Silica
Gel 60 thin-layer chromatography plates. The plates were developed in
chloroform, pyridine, 88% formic acid, water (30:70:16:10, v/v), and
radioactive spots were detected and analyzed with a PhosphorImager. Arrows indicate the positions of the substrate
(Kdo)
-[4`-
P]lipid IV
and
the monoacylated product a after chromatography. The origin is at the
bottom.
Figure 3:
Lauroyl transferase activity in extracts
of E. coli W3110 compared to the htrB-deficient
strain MLK53. Cell-free extracts (1.0 mg/ml) from strain MLK53 (lanes 1 and 2) and W3110 (lanes 3 and 4) were assayed in a reaction mixture containing 50 mM Hepes pH 7.5, 0.1% Triton X-100, and 25 µM (Kdo)-[4`-
P]lipid IV
(2
10
dpm/nmol). Lanes 1 and 3 also contained 25 µM lauroyl-ACP. Lanes 2 and 4 contained no added lauroyl-ACP. After incubation
for 30 min at 37 °C, 5-µl portions of each reaction were
spotted onto a Silica Gel 60 thin-layer chromatography plate, which was
developed in CHCl
, pyridine, 88% formic acid, water
(30:70:16:10, v/v) and analyzed with a PhosphorImager (Molecular
Dynamics), as in Fig. 2.
Karow
and Georgopoulos (19) identified and isolated another gene of
unknown function, designated msbB, that displays significant
sequence homology to htrB. When present on hybrid plasmids
maintained at very high copy numbers, msbB suppresses the temperature-sensitive growth phenotype of
MLK53(19) . Unlike htrB, however, msbB is not
essential for growth at any temperature(19) . Membranes of
strain MLK1067(19) , harboring an insertion in msbB,
contain normal amounts of lauroyl transferase (Table 2).
Overexpression of msbB
on hybrid plasmids
does not result in increased levels of lauroyl transferase (data not
shown). When pKS12 (htrB
) is transformed into E. coli MLK1067 (msbB1::
Cam)(19) , lauroyl transferase
activity is overproduced, just as when pKS12 is introduced into
wild-type strains, like XL1-Blue (Table 2). These findings are
consistent with the hypothesis that htrB and msbB encode distinct enzymes, and are not likely to be regulatory genes
affecting the same enzyme.
Myristoyl-ACP could replace lauroyl-ACP
as the acyl donor in acylations catalyzed by crude extracts or isolated
membranes from E. coli W3110, XL1-Blue/pKS12, or MLK1067/pKS12
(data not shown). However, the reaction rate with myristoyl-ACP as the
donor and (Kdo)-[4`-
P]lipid IV
as the acceptor was 5-10-fold slower than with lauroyl-ACP.
Extracts of MLK53 were unable to acylate
(Kdo)
-[4`-
P]lipid IV
when myristoyl-ACP was employed as the substrate (data not
shown), as observed with lauroyl-ACP ( Fig. 3and Table 2).
Figure 4: Fractionation of the solubilized lauroyl transferase on DEAE-Sepharose CL-6B. Thesit solubilized membrane proteins (20 mg) were applied onto a 45-ml column as described in the text in detail. Proteins were eluted with a linear gradient from 50 to 500 mM NaCl. Absorbance at 280 nm (open circles) was used as a measure of total protein in each fraction. Lauroyl transferase activity (solid circles) was assayed with Method I using 5 µl of a 1:10 dilution of each fraction. Dotted line, calculated NaCl concentration. Gray area, active fractions that were pooled and concentrated.
Given these findings, the assay conditions were
re-examined in more detail with the partially purified enzyme ( Fig. 5and 6). In the presence of 1 or 5 mM MgCl, the lauroyl transferase showed a dependence on
added detergent that is typical of enzymes using lipid substrates (Fig. 5A). With Triton X-100, the highest activity was
seen at concentrations between 0.1 and 0.2% (w/v). At higher Triton
X-100 concentrations, the activity dropped off, presumably because of
surface dilution of the substrate in mixed micelles. At 5 mM MgCl
, the Triton X-100 optimum was considerably
broader than at 1 mM (Fig. 5A), and therefore
5 mM MgCl
was included in all subsequent assays.
Figure 5:
Effects of Triton X-100 and NaCl on
lauroyl transferase activity and stability. Panel A,
DEAE-Sepharose fractionated HtrB was assayed for 5 min at 30 °C at
a protein concentration of 0.25 µg/ml under conditions similar to
those described in the legend to Fig. 3, except that the
concentration of Triton X-100 was varied, as indicated, and 1 mM MgCl (solid circles) or 5 mM MgCl
(open circles) was also included. Panel B, DEAE-Sepharose purified HtrB (0.25 µg/ml) was
preincubated without lauroyl-acyl carrier protein for 30 min at 30
°C under assay conditions similar to those of Fig. 3, but
with 5 mM MgCl
included. Reactions were started by
addition of 25 µM lauroyl-acyl carrier protein, and the
reaction mixture was incubated at 30 °C for the times indicated. Open squares, preincubation with 50 mM NaCl in
addition to the above components; open triangles,
preincubation without added NaCl; open circles, no
preincubation and no added NaCl.
Even in the presence of 5 mM MgCl, the
DEAE-purified lauroyl transferase was unstable when preincubated under
assay conditions (Fig. 5B, triangles). The addition of
50 mM NaCl together with 5 mM MgCl
to the
reaction mixture was found to stabilize the enzyme to preincubation (Fig. 5B, open squares), resulting in a constant rate
of acylation for 40-60 min at 30 °C and excellent linearity
with respect to added protein (Fig. 6). All subsequent assays
were therefore performed with both 5 mM MgCl
and
50 mM NaCl supplementation (assay Method II). The key assays
of various membrane preparations and purified fractions shown in Table 2and Table 3were all performed using Method II. The
effects of MgCl
and NaCl were less pronounced when crude
extracts or membrane preparations were assayed (data not shown).
Figure 6: Dependence of lauroyl transfer on time and protein concentration under optimized conditions. DEAE-Sepharose purified HtrB was assayed at 30 °C using Method II (see ``Experimental Procedures'') with varying protein concentrations for 15 min (Panel A) or with varying incubation times at 0.2 µg/ml (Panel B).
Figure 7:
Acyl chain length specificity of the
lauroyl transferase. The relative initial rates of acylation of 25
µM (Kdo)-[4`-
P]lipid
IV
were determined at 30 °C using different acyl-acyl
carrier proteins as donors at 25 µM. Transferase activity
was measured using Method II with 0.4 µg/ml of DEAE-Sepharose
purified enzyme. The rates of acylation were normalized to that
observed with 25 µM lauroyl-acyl carrier protein (12:0),
which was defined as 1.0. The other acyl chains tested were: 10:0,
decanoate; 14:0, myristate; HO14:0, R-3-hydroxymyristate;
16:0, palmitate; and 16:1c, palmitoleate.
The specificity of the
purified enzyme for various acceptors was examined using assay Method
II (Fig. 8). At 20 µg/ml of the DEAE-purified enzyme,
complete conversion of 25 µM (Kdo)-[4`-
P]lipid IV
to the mono-acylated product (designated ``a'') was
achieved in 30 min (Fig. 8, lane 3+). In contrast,
less than 1% of [4`-
P]lipid IV
or of
Kdo-[4`-
P]lipid IV
(both at 25
µM) was acylated under otherwise identical conditions (lanes 1+ and 2+, respectively). The
results show that the purified lauroyl transferase functions at least 2
orders of magnitude more rapidly when both Kdo residues are present in
the acceptor.
Figure 8:
Lipid acceptor specificity of the htrB encoded acyl transferase. DEAE-Sepharose purified lauroyl
transferase was used at 20 µg/ml (a large excess as shown by the
data in Fig. 6) in reaction mixtures containing 50 mM Hepes pH 7.5, 0.1% Triton X-100, 5 mM MgCl,
50 mM NaCl, and 25 µM of one of the following
lipids: lanes 1, [4`-
P]lipid IV
(2
10
dpm/nmol); lanes 2,
Kdo-[4`-
P]lipid IV
(2
10
dpm/nmol); lanes 3,
(Kdo)
-[4`-
P]lipid IV
(1
10
dpm/nmol). The reactions were incubated at 30
°C for 30 min with (+) or without(-) 25 µM lauroyl-ACP. Arrows indicate the positions of migration
of the substrates and of product a, the predominant monoacylated
derivative generated from lauroyl-ACP and
(Kdo)
-[4`-
P]lipid
IV
.
At 20 µg/ml enzyme, a trace of acylation is
observed with both [4`-P]lipid IV
and Kdo-[4`-
P]lipid IV
(faint
bands in lanes 1+ and 2+ of Fig. 8just ahead of the major component). These minor products
are not seen in the absence of lauroyl-ACP (Fig. 8, lanes
1- and 2-). A trace of what may be a
diacylated derivative of
(Kdo)
-[4`-
P]lipid IV
is
also observed in Fig. 8(lane 3+), just ahead of
product a. Whether these minor products represent alternative
acylations catalyzed by HtrB itself or are the result of other
contaminating acyltransferases will have to be re-evaluated with HtrB
preparations purified to homogeneity. In crude extracts of wild-type
cells, a rapid second acylation of
(Kdo)
-[4`-
P]lipid IV
is
observed(10) , but this is catalyzed by the msbB gene
product(22) . (
)MsbB is absent in the strain
employed here for the overexpression and partial purification of HtrB.
Although previous studies of htrB have strongly suggested that this gene has a role in envelope assembly(17, 18, 19, 20, 21) , the current findings offer the first direct evidence that HtrB is an enzyme of lipid A biosynthesis. Given the pleiotropic nature of the htrB mutant phenotype, Karow and co-workers considered several possibilities for HtrB function, including roles in peptidoglycan(17) , fatty acid(20) , and lipopolysaccharide biogensis (19, 20, 21) . Although they did not propose a primary defect in the Kdo-dependent lauroyl transferase documented in the present work, several earlier observations support our enzymatic data. Specifically, the striking reduction in the laurate content of the lipopolysaccharide isolated from htrB deficient E. coli(20) and the reduced number of acyl chains attached to the lipid A of htrB deficient H. influenzae(38) are in accordance with our findings.
MsbB and htrB display significant sequence similarity to each
other(19) , consistent with related catalytic roles (22) .MsbB and htrB have no
homology to lpxA(39, 40) and lpxD(31) , the products of which are the R-3-hydroxymyristoyl transferases that function in the initial
stages of lipid A assembly(3, 5) . All four enzymes
are absolutely dependent upon acyl chain activation by ACP, since
coenzyme A thioesters do not serve as
substrates(10, 31, 39) . Consequently, they
may share some common structural motifs. This issue is currently of
considerable interest, since the x-ray crystal structure of LpxA (the
first enzyme of the lipid A pathway) has recently been solved at 2.6
Å(41) . A unique feature of LpxA is its unusual
left-handed parallel
helix domain and trimeric subunit
structure(41) . HtrB and MsbB do not possess the sequence
repeat associated with the left-handed parallel
helix
region(41, 42, 43) , but this does not
necessarily rule out the possibility of a
helical domain.
Imperfect, parallel right-handed
helices (with no obvious
associated sequence repeats) have been reported(44) .
HtrB and MsbB, unlike LpxA (45) and LpxD(31) , are membrane proteins. X-ray crystallography may not be feasible. In previous studies, however, we reported that half of the lauroyl transferase activity was in the cytoplasmic fraction(10) . Different E. coli strains and buffers were employed. Solubilization and purification of HtrB without detergents deserves further exploration.
Both htrB and msbB are present in the Haemophilus genome and show 70% identity with their E. coli counterparts(46) . HtrB and msbB display no sequence homology to other well characterized
acyltransferases, such as glycerol-3-phosphate acyltransferases (46) or N-myristoyl transferases(47) . The
eucaryotic acyloxyacyl hydrolase (48) that removes the acyl
chains incorporated into lipid A by HtrB and MsbB is also unrelated, as
judged by its sequence.
The significance of the phenotypes
associated with insertions in htrB(17, 19) and the possible functions of multi-copy
suppressors of htrB mutations (19, 21) must
be re-evaluated in light of our findings. The increased resistance of
these mutants to deoxycholate and the stimulation of their growth at
non-permissive temperatures by low levels of cationic detergents (19) may be attributed to changes in lipid A structure. The
morphological changes at elevated
temperatures(17, 19) , such as the bulging of the cell
surface that resembles the response to certain -lactams, are more
difficult to explain. One intriguing scenario is that underacylated
lipid A precursors might accumulate in the inner membrane and inhibit
the export of peptidoglycan precursors, preventing peptidoglycan
assembly. The msbA multi-copy suppressor of htrB (a
putative transporter resembling mammalian mdr proteins) (21) might be the proposed lipid A flippase of the inner
membrane(2, 5, 49, 50) . The notion
of a lipid A translocation function for MsbA is supported by the recent
discovery that the mdr-2 protein of the mouse is required for
phosphatidylcholine secretion into bile(51) . It is conceivable
that the overexpression of msbA in htrB-deficient
mutants might remove excess, underacylated lipid A precursors by
accelerating their export to the outer membrane.
Alternatively, MsbA could be an inner membrane translocase specific for peptidoglycan precursors(52) . Its overexpression in the setting of htrB mutations might overcome any inhibitory effects of underacylated lipid A precursors on peptidoglycan export. Further characterization of MsbA function is of great interest, since neither the lipid A nor the peptidoglycan translocases have been identified by biochemical or genetic criteria.
The function of lipid A in Gram-negative bacteria is unknown(1, 2, 5) . Strains with insertion mutations in the genes encoding the early steps of lipid A assembly cannot be grown under any condition(53, 54) . Wild-type E. coli lipid A contains two normal fatty acids in acyloxyacyl linkage (Fig. 1). Lipid As of some other Gram-negative bacteria possess only one acyloxyacyl unit(1, 2, 4) . An important implication of the present studies is that the usual acyloxyacyl moieties of E. coli lipid A are not absolutely essential for growth. Strains with htrB insertion mutations can grow slowly on nutrient media below 33 °C or at all temperatures on minimal media(17, 19) . Cells with insertion mutations in msbB show myristate-deficient lipid A and are not conditionally lethal(20, 22, 55) . Such mutants still possess the laurate-containing acyloxyacyl group generated by HtrB.
To evaluate the significance of acyloxyacyl moieties more thoroughly, a detailed analysis of the molecular species of lipid A that remain in growing htrB mutants of E. coli and in the presence of various suppressors will be of considerable interest. The msbB high multi-copy suppressor of htrB(19) may be functioning by allowing the synthesis of the myristate-containing acyloxyacyl unit that is not made in wild-type cells prior to HtrB catalyzed laurate incorporation (Fig. 1). In addition, palmitate-containing acyloxyacyl moieties can be generated by transacylation of palmitate from the 1 position of a glycerophospholipid to lipid A precursors(28) . The latter pathway is of minimal significance in wild-type cells. It is stimulated under conditions of limited lipid A biosynthesis(5, 13, 56) . It is possible that a deficiency in laurate incorporation due to an htrB mutation is compensated for by increased palmitate transfer. The further biochemical characterization of htrB mutations and their various suppressors should greatly enhance our understanding of lipid A biogenesis and function.