From the Department of Biochemistry, Duke University
Medical Center, Durham, North Carolina 27710 and the ¶ Department
of Molecular Biology and Microbiology, Tufts University Health Sciences
Campus, Boston, Massachusetts 02111
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ABSTRACT |
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Palmitoleate is not present in lipid A isolated
from Escherichia coli grown at 30 °C or higher, but it
comprises ~11% of the fatty acyl chains of lipid A in cells grown at
12 °C. The appearance of palmitoleate at 12 °C is accompanied by
a decline in laurate from ~18% to ~5.5%. We now report that
wild-type E. coli shifted from 30 °C to 12 °C acquire
a novel palmitoleoyl-acyl carrier protein (ACP)-dependent
acyltransferase that acts on the key lipid A precursor
Kdo2-lipid IVA. The palmitoleoyl transferase is
induced more than 30-fold upon cold shock, as judged by assaying
extracts of cells shifted to 12 °C. The induced activity is maximal
after 2 h of cold shock, and then gradually declines but does not
disappear. Strains harboring an insertion mutation in the
lpxL(htrB) gene, which encodes the enzyme that
normally transfers laurate from lauroyl-ACP to Kdo2-lipid
IVA (Clementz, T., Bednarski, J. J., and Raetz,
C. R. H. (1996) J. Biol. Chem. 271, 12095-12102) are not defective in the cold-induced palmitoleoyl
transferase. Recently, a gene displaying 54% identity and 73%
similarity at the protein level to lpxL was found in the
genome of E. coli. This lpxL homologue, designated lpxP, encodes the cold shock-induced
palmitoleoyl transferase. Extracts of cells containing lpxP
on the multicopy plasmid pSK57 exhibit a 10-fold increase in the
specific activity of the cold-induced palmitoleoyl transferase compared
with cells lacking the plasmid. The elevated specific activity of the
palmitoleoyl transferase under conditions of cold shock is attributed
to greatly increased levels of lpxP mRNA. The
replacement of laurate with palmitoleate in lipid A may reflect the
desirability of maintaining the optimal outer membrane fluidity at
12 °C.
Lipopolysaccharide
(LPS)1 is a major component
of the outer leaflet of the outer membrane of Escherichia
coli and is a defining feature of Gram-negative bacteria (1-6).
LPS consists of three domains: 1) lipid A, the hydrophobic membrane
anchor (Fig. 1); 2) the core sugar region; and 3) the O-antigen polymer
(1-6). Studies of E. coli mutants in LPS biosynthesis have
demonstrated that the lipid A domain and the Kdo residues of the
proximal core are required for growth (1-3, 7).
In wild-type E. coli grown at 30 °C or above, lipid A
contains six fatty acyl chains (Fig. 1) (3, 4, 8). Four
R-3-hydroxymyristate residues are attached directly to
the glucosamine disaccharide backbone (3, 4, 8). Laurate and myristate
are attached to the R-3-hydroxy groups of the
R-3-hydroxymyristate residues in the distal unit (Fig. 1)
(3, 4, 9). In the later steps of the lipid A biosynthetic pathway (Fig.
1), the key intermediate Kdo2-lipid IVA is
sequentially acylated with laurate and myristate (3, 4, 9-11). The
lauroyl and myristoyl transferases require the presence of the Kdo
disaccharide for optimal activity (9-11). The substrate preferences of
these enzymes are consistent with the accumulation of lipid
IVA, rather than lipid A, in mutants defective in Kdo
biosynthesis (12-15).
As shown by Clementz et al. (10, 11, 16), the
lpxL(htrB) gene, which was initially identified
as being required for growth of E. coli above 32 °C (17,
18), encodes the lauroyl transferase (Fig. 1). We have recently
purified LpxL to homogeneity and confirmed that lpxL is
indeed the structural gene for the lauroyl
transferase.2 The LpxL
reaction product, Kdo2-(lauroyl)-lipid IVA, is
the preferred substrate for the myristoyl transferase (9, 11), which is encoded by the lpxM gene (previously designated
msbB) (Fig. 1) (19). The lpxM(msbB)
gene displays distant, but nevertheless significant, sequence
similarity to lpxL (19). High copy number plasmids
bearing lpxM can suppress the temperature-sensitive growth of mutants defective in lpxL (19), presumably because LpxM
acylates Kdo2-lipid IVA at a slow rate
(11).
In 1979, prior to the elucidation of the structure and biosynthesis of
lipid A, van Alphen et al. studied the fatty acid
composition of lipid A from E. coli grown at 37 °C
versus 12 °C (20). At 37 °C, laurate was present at
0.16 µmol/mg of LPS, but it decreased to 0.05 µmol/mg at 12 °C
(20). The decrease in laurate at 12 °C was counterbalanced by the
appearance of palmitoleate, which was present at 0.10 µmol/mg of LPS
in E. coli cells grown at 12 °C (20). Only trace amounts
of palmitoleate were found in LPS at 37 °C. A similar effect of cold
shock on lipid A composition was reported for Salmonella
typhimurium (21). It is unlikely that palmitoleate incorporation
is catalyzed by LpxL, since palmitoleoyl-acyl carrier protein (ACP) is
not a substrate for LpxL (9, 10).
We now describe the induction of a novel palmitoleoyl transferase in
extracts of E. coli cells subjected to cold shock (Fig. 1).
This enzyme transfers palmitoleate from palmitoleoyl-ACP to Kdo2-lipid IVA, which is also the acceptor for
the lpxL-encoded lauroyl transferase (Fig. 1). An inducible
palmitoleoyl transferase would account for the appearance of
palmitoleate in lipid A of cells grown at 12 °C. The palmitoleoyl
transferase is not encoded by lpxL, but appears to be the
product of another lpxL homologue found in E. coli (22) that is now designated lpxP. The palmitoleoyl transferase is induced within minutes after cold shock and is accompanied by a massive increase in the levels of lpxP
mRNA. A cold shock-induced acyltransferase has not been reported previously.
Materials--
[ Bacterial Strains and Growth Conditions--
Strains used in
this study are derivatives of E. coli K-12 wild-type W3110,
obtained from the E. coli Genetic Stock Center, Yale
University. Strain MLK53 harbors a Tn10 insertion in the lpxL(htrB) gene (18, 19). In some experiments
strain MC1000 (lpxL+lpxM+lpxP+)
(72) was used as the host for the vector pACYC184 (camr
tetr p15A replicon) (73), or for the hybrid plasmid pSK57,
which harbors lpxP+ on a 5.6-kilobase pair
EcoRI fragment derived from Kohara Isolation and Preparation of Lipid Substrates--
Lipid
IVA and (Kdo)2-lipid IVA were
isolated and purified as described (14, 24, 25).
(Kdo)2-[4'-32P]lipid IVA was
prepared from [4'-32P]lipid IVA as described
previously (25-27), except that membranes of the 4'-kinase
overproducing strain BLR(DE3)pLysS/pJK2 (28) were used to increase the
yield of [4'-32P]lipid IVA. Lauroyl,
myristoyl, palmitoyl, R-3-hydroxymyristoyl, and
palmitoleoyl-ACP were synthesized from the corresponding fatty acids
and commercial acyl carrier protein, using solubilized membranes from
the acyl-ACP synthase overproducing strain E. coli
LCH109/pLCH5/pGP1-2, as described previously (10, 29).
Assay for Palmitoleoyl Transferase Activity--
Cell-free
extracts were assayed for palmitoleoyl transferase under conditions
similar to those used for the lauroyl transferase (9, 10). The reaction
mixture (typically 20 µl) contained 50 mM Hepes, pH 7.5, 0.1% Triton X-100, 5 mM MgCl2, 250 mM NaCl, and 6 µM
(Kdo)2-[4'-32P]lipid IVA (32,000 cpm/nmol). Palmitoleoyl-ACP was included as the acyl donor at 12 µM, and the final concentration of enzyme was 0.1 mg/ml
of crude cell free extract. The tubes were incubated at 12 °C for
5-60 min for MC1000/pSK57(lpxP+) or for 10-20
h for W3110 (wild-type) or MLK53 (lpxL Assay for Lauroyl Transferase Activity--
The crude cell-free
extracts were assayed for lauroyl transferase activity using
lauroyl-ACP and (Kdo)2-[4'-32P]lipid
IVA as substrates, as described previously (10). The assay
temperature was 30 °C for the lauroyl transferase, unless otherwise indicated.
Conditions for Palmitoleoyl Transferase Induction and Preparation
of Cell-free Extracts--
Cultures (800 ml) were grown in
Luria-Bertani broth at 30 °C to mid-log phase
(A600 ~ 0.4). Half of the culture was shifted to 12 °C. Samples (50 ml) were collected from the 12 °C and
30 °C cultures at the time of shift and at various times after the shift, as indicated. At each time point, the cells were immediately harvested by centrifugation at 4200 × g for 15 min at
room temperature. The pellets were washed with 25 ml of 30 mM Hepes, pH 7.5, containing 1 mM EDTA and 1 mM EGTA, and harvested as above. The pellets were resuspended in 1 ml of 30 mM Hepes, pH 7.5, containing 1 mM EDTA and 1 mM EGTA. The cells were broken
using an ice-cold French pressure cell (SLM Instruments, Urbana, IL) at
10,000 p.s.i. To remove unbroken cells and large debris, the extracts
were centrifuged for 15 min at 4200 × g at 4 °C.
The supernatant (referred to as the cell extract) was divided into
aliquots and stored at RNA Isolation--
Cultures (typically 10 ml) were grown at
30 °C to mid-log phase (A600 ~ 0.4). A
portion of the culture (5 ml) was shifted to 12 °C. At 1 h
after the shift, 2-ml samples from the 30 °C and the 12 °C
cultures were transferred to glass tubes at 100 °C, containing 0.5 ml 2.5% SDS, 1 M NaCl, and 50 mM EDTA, pH 8, to ensure rapid lysis (31). The samples were rapidly mixed and were
boiled in a water bath for another 2 min. After cooling to room
temperature, the RNA was isolated by phenol extraction using Phase lock
gel from 5Prime Northern Blot Analysis--
The Northern blot was done as
described (34). The 32P-labeled DNA probe for the
lpxP mRNA was constructed by digesting pSK57 with
SalI and EcoRI to yield a 1.2-kilobase pair
product containing lpxP. This fragment was isolated by
agarose gel electrophoresis, and it was purified using the QIAEX Gel
extraction kit (QIAGEN). Then, 25 ng of the DNA fragment was used as
the template for the Prime-It II random primer labeling kit
(Stratagene). The labeling reaction was done with
[ A Novel Palmitoleoyl Transferase in Extracts of Cold-shocked Cells
of E. coli--
In E. coli or S. typhimurium
cells cultured below 15 °C, a palmitoleate residue replaces most of
the laurate that is normally linked to lipid A in cells grown at
30-42 °C (20, 21). Conversely, no palmitoleate is detected in lipid
A of cells grown above 30 °C (8, 20, 21). We therefore assayed
extracts of cells that had been shifted from 30 °C to 12 °C for
2 h during mid-log phase for the induction of a
palmitoleoyl-ACP-dependent acyltransferase capable of using
the key precursor (Kdo)2-lipid IVA as the
substrate (Fig. 1). The latter was
previously shown to function as the acceptor for a laurate residue
(Fig. 1) in the reaction catalyzed by the Kdo-dependent
acyltransferase LpxL(HtrB) in extracts of cells grown at 30 °C or
higher (10). As demonstrated in Fig. 2,
extracts (0.1 mg/ml) of wild-type E. coli shifted to
12 °C for 2 h converted (Kdo)2-[4'-32P]lipid IVA to a
more rapidly migrating product in the presence of palmitoleoyl-ACP, as
judged by TLC analysis (Fig. 2), indicative of palmitoleate transfer.
However, no acylated product was formed in the absence of
palmitoleoyl-ACP in such cold-shocked extracts. Very little
palmitoleoyl-ACP-dependent acylation of
(Kdo)2-[4'-32P]lipid IVA was
detected in extracts of the 30 °C grown control cells (Fig. 2).
The low assay temperature (12 °C) was necessary for demonstrating
the induction of the palmitoleoyl transferase because of its
instability at higher temperatures. In addition, the presence at
30 °C of an unrelated, interfering enzyme that transfers a palmitate
residue from the 1-position of phosphatidylethanolamine or
phosphatidylglycerol to (Kdo)2-[4'-32P]lipid
IVA or other lipid A precursors
(35)3 obscures the
palmitoleoyl-ACP-dependent acylation activity (data not
shown). The ACP-independent palmitoyl transferase reaction is
suppressed by carrying out the assays at 12 °C (data not shown).
The Palmitoleoyl Transferase Is Not Encoded by the lpxL
Gene--
To determine if the cold-induced activity was dependent upon
the lpxL gene, we assayed the palmitoleoyl transferase
activity in extracts of cold-shocked cells of strain MLK53 (17), in
which the lpxL gene is inactivated by an insertion mutation.
As in the isogenic wild-type strain,
palmitoleoyl-ACP-dependent acylation of
(Kdo)2-[4'-32P]lipid IVA was
induced in MLK53 cells grown at 12 °C (Fig.
3A). Very little
palmitoleoyl-ACP-dependent acylation was observed in assays
of extracts made from MLK53 cells grown at 30 °C, but the low level
of activity seen in such extracts does appear to be slightly higher
than the wild-type controls (Fig. 3A). The absence of the
lauroyl transferase in extracts of MLK53 cells grown either at 12 °C
or 30 °C was confirmed (Fig. 3B), consistent with
previous studies (10). Therefore, the cold-induced palmitoleoyl transferase activity is independent of the lpxL gene. Cold
shock also had very little effect on the activity of the lauroyl
transferase in extracts of wild-type cells, which were assayed at
30 °C with lauroyl-ACP and
(Kdo)2-[4'-32P]lipid IVA as
substrates (Fig. 3B).
Multiple Copies of lpxP, an E. coli Homologue of lpxL, Direct the
Overexpression of the Cold-induced Palmitoleoyl
Transferase--
E. coli contains two genes that display
sequence similarity to lpxL. One of these, previously
designated msbB (now termed lpxM as shown in Fig.
1), encodes the myristoyl transferase that is required for the final
acylation of lipid A in wild-type cells (11, 19). The other
lpxL homologue, which was found in the E. coli
genome (22) and is now designated lpxP (Fig. 1), codes for a
protein that displays 54% identity and 73% similarity to LpxL (Fig.
4) over the entire 306-amino acid residue
length of LpxL. To determine if lpxP encodes the cold
shock-induced palmitoleoyl transferase activity, we assayed the
palmitoleoyl transferase in extracts of
MC1000(pSK57/lpxP+), which contains
lpxP behind its native promoter on a pACYC184 vector, after
shifting cells from 30 °C to 12 °C for 2 h. As shown in Fig.
3A, the palmitoleoyl transferase specific activity was about
7-fold higher in extracts of MC1000(pSK57/lpxP+)
than in wild-type. However, the specific activity of the lauroyl transferase (LpxL) in extracts of
MC1000(pSK57/lpxP+) was about the same as that
in wild-type extracts (Fig. 3B), and the lauroyl transferase
was not induced by cold shock. Given the sequence similarity of LpxL
and LpxP, together with the striking increase in the cold-induced
palmitoleoyl transferase (Fig. 3A) in
MC1000(pSK57/lpxP+) extracts, we propose that
lpxP is the structural gene for the palmitoleoyl
transferase.
A Quantitative Assay for LpxP in MC1000(pSK57/lpxP+)
Extracts--
As shown in Fig. 5, the
formation of
(Kdo)2-[4'-32P](palmitoleoyl)-lipid
IVA was linear with time and protein concentration in
extracts of strain MC1000(pSK57/lpxP+), despite
the unusual assay conditions (12 °C). Furthermore, nearly complete
conversion of substrate to product was achieved upon prolonged
incubation with a 2-fold excess of palmitoleoyl-ACP over
(Kdo)2-[4'-32P]lipid IVA. The
palmitoleoyl transferase did not acylate [4'-32P]lipid
IVA (Fig. 6), demonstrating
that the presence of the Kdo disaccharide in the substrate is required
for activity, as in the case of LpxL (9, 10).
Selectivity of the Cold-induced Acyltransferase for
Palmitoleoyl-ACP--
MLK53 (which lacks the lauroyl transferase) (10)
was grown either at 30 °C or 12 °C, and extracts were then
assayed with various substrates at 12 °C to determine the acyl chain
specificity of the cold-induced palmitoleoyl transferase. As shown in
Fig. 7, palmitoleoyl-ACP was the only
acyl donor capable of supporting robust acylation of
(Kdo)2-[4'-32P]lipid IVA.
Lauroyl-ACP, myristoyl-ACP, palmitoyl-ACP,
R-3-hydroxymyristoyl-ACP, palmitoleoyl-coenzyme A, and
palmitoyl-coenzyme A were virtually inactive as substrates (Fig. 7). A
small amount of acylation was seen with myristoyl-ACP (Fig. 7), but
this was not cold shock-induced, and might be explained by the direct
acylation of (Kdo)2-[4'-32P]lipid
IVA by LpxM. LpxM normally prefers
(Kdo)2-[4'-32P](lauroyl)-lipid
IVA as the acceptor (Fig. 1), but it can also function with
(Kdo)2-[4'-32P]lipid IVA at a
slow rate (11). The LpxM gene is intact in strain MLK53 (11). Taken
together, the results clearly demonstrate that the cold-induced
palmitoleoyl transferase is highly selective for palmitoleoyl-ACP,
consistent with the effects of cold shock on the fatty acid composition
of lipid A (Fig. 1).
Time Course of Palmitoleoyl Transferase Induction following Cold
Shock--
Cells were grown at 30 °C to mid-log phase, and were
then divided into two equal portions, so that parallel cultures could be studied at 12 °C and 30 °C, as shown in Figs.
8 and 9.
Samples were then removed from the cultures growing at 30 °C or
12 °C at 0.25, 0.5, 1, 2, 4, and 6 h. Over this time frame, the
cold-shocked cells did not increase in density, as shown for the
wild-type cells in Fig. 8, but slow growth did resume after about
8 h at 12 °C (data not shown). Extracts of the samples taken
from both the 30 °C and the 12 °C cells were assayed for
palmitoleoyl-ACP-dependent acylation of
(Kdo)2-[4'-32P]lipid IVA, as
shown in Fig. 9. Strains W3110 (wild-type), MLK53 (harboring an
insertion in lpxL), and
MC1000(pSK57/lpxP+) were analyzed in this
manner. In every case, the induction of the palmitoleoyl transferase
was detected after only 15 min of incubation at 12 °C, despite the
absence of a measurable increase in the optical density (Fig. 8). The
specific activity of the palmitoleoyl transferase peaked in all three
strains after 2 h at 12 °C (Fig. 9). In W3110 and MLK53,
however, the specific activity of the enzyme decreased slightly after
3 h of growth at 12 °C.
Very little palmitoleoyl transferase was seen in extracts of the
cultures held at 30 °C (Fig. 9), which gradually entered stationary
phase over the course of the experiment. However, a small amount of
palmitoleoyl transferase above background was observed in extracts of
log phase MLK53 cells at 30 °C (Fig. 9B). This trace of
palmitoleoyl transferase activity may account for the fact that MLK53
does actually synthesize some penta- and even some hexa-acylated lipid
A species (despite the absence of LpxL) at 30 °C, as noted
previously in studies of lipid A composition (11, 36). Mass
spectrometry (data not shown) indicates that palmitoleate indeed
accounts for the presence of the penta-acylated lipid A species in
MLK53 grown at 30 °C. It may be that when LpxL is missing the
lpxP gene is switched on at higher temperatures than in
wild-type cells.
lpxP mRNA Is Measurable in Cells Grown at 12 °C, but Not at
30 °C--
To determine if an increase in lpxP mRNA
levels could account for the induction of the palmitoleoyl transferase
during cold shock, Northern blotting was used to compare the
lpxP mRNA levels extracted from cells grown at 30 °C
or 12 °C. A blot of 10-µg RNA samples from both W3110 and MLK53,
grown at either 30 °C or 12 °C, is shown in Fig.
10. A heavy band is detected at the
position expected for lpxP mRNA (~1000 nucleotides) in
the total RNA from the cultures grown at 12 °C. There is no
lpxP band in the RNA samples extracted from the cultures
grown at 30 °C. The accumulation of lpxP mRNA in the
cultures grown only at 12 °C (Fig. 10) and the time course of
palmitoleoyl transferase induction at 12 °C (Fig. 9) suggest that
lpxP is a typical cold shock-induced gene, probably
regulated at the level of transcription and/or message stability (37,
38).
As demonstrated previously in our laboratory, the enzyme
LpxL(HtrB) catalyzes the transfer of laurate from lauroyl-ACP to the
lipid A precursor (Kdo)2-lipid IVA in extracts
of cells grown at 30 °C or above (Fig. 1) (10). We now have
discovered an additional Kdo-dependent acyltransferase
(LpxP) in E. coli that is required for the biosynthesis of a
distinct molecular species of lipid A, present only in cells grown at
low temperatures (Fig. 1) (20, 21). LpxP transfers palmitoleate from
palmitoleoyl-ACP to (Kdo)2-lipid IVA, and it is
induced within 15 min in log phase cells shifted from 30 °C to
12 °C (Fig. 9). The palmitoleoyl transferase is a distinct enzyme,
not an additional activity associated with the lauroyl transferase, as
demonstrated by the fact that the palmitoleoyl transferase is induced
normally (Fig. 3) in mutant cells (MLK53) (10) harboring an insertion
in the lpxL gene. Furthermore, the palmitoleoyl transferase
is greatly overproduced in strains harboring multiple copies of the
lpxP gene expressed from its own promoter (Fig. 3). LpxL and
LpxP share 54% identity and 73% similarity, with one gap over a
segment of 301 out of 306 amino acids (Fig. 4), as determined by BLASTp
analysis (39). In contrast, LpxL and LpxM (Fig. 1) show only 29%
identity and 46% similarity, with 21 gaps over a sequence of 309 amino
acids out of the 323 residues that comprise LpxM (39).
Extracts of wild-type E. coli grown at 12 °C contain both
the lauroyl and the palmitoleoyl transferase activities (Fig. 3). The
mechanisms by which the cells determine the amount of palmitoleate versus laurate that is transferred to
(Kdo)2-lipid IVA at 12 °C in vivo
may be quite interesting and will require further study. Based on mass
spectrometry, we estimate that about 80% of the lipid A residues of
cells grown at 12 °C are acylated with palmitoleate rather than
laurate.4
The level of lpxP mRNA increases by several orders of
magnitude in cells grown at 12 °C (Fig. 10). This phenomenon must
surely account for the induction of the palmitoleoyl transferase
activity upon cold shock (Figs. 3 and 9). The time course of LpxP
induction and its gradual decline in wild-type cells after several
hours of growth at 12 °C (Fig. 9) is reminiscent of the response
seen with many cold shock proteins, including the extensively studied RNA-binding protein CspA (37, 38, 40). The mechanisms by which cold
shock induces CspA are still not fully understood (37, 38). Initial
work suggested that increased transcription accounted for elevated CspA
message levels in cold-shocked cells (37, 38). More recent studies
support the view that CspA mRNA stability is also selectively
enhanced at lower growth temperatures, possibly because of temperature
effects on mRNA secondary structure (41, 42). RNase E may play a
direct role in the control of mRNA stability during cold shock
(43). In addition, translation of cold shock mRNA may be
selectively enhanced (41, 42). Given these considerations, it will be
interesting to express lpxP behind a constitutive promoter and to measure LpxP activity in such constructs as a function of
temperature. Expression of a reporter gene like lacZ or
cat at various temperatures behind the native
lpxP promoter might also be informative. Finally, a search
for proteins that control the activity of the lpxP promoter
in vivo might reveal how cells detect cold shock and
readjust their lipid A pathway accordingly.
Among the enzymes of lipid A biosynthesis, LpxP may be unique in its
induction at low temperatures, but it is not the only enzyme of the
pathway that is regulated. The deacetylase (LpxC) that catalyzes the
second, committed step of lipid A biosynthesis is increased about
10-fold in cells treated with the specific deacetylase inhibitor,
L-573,655 (44, 45), or in point mutants with low levels of
lpxA (44, 46). Deacetylase regulation is not accompanied by
significant changes in lpxC mRNA (44). Instead, LpxC
protein levels may be controlled by the membrane-associated protease
FtsH (47).
Additional regulation of lipid A biosynthesis has recently been
discovered by Miller and co-workers (48-50). These investigators have
shown that the PhoP/PhoQ two component system is required for the
modification of S. typhimurium lipid A with palmitate, L-4-aminoarabinose, and/or 2-hydroxymyristate (48-50). While these modifications are not required for cell growth, they are critical for
intracellular survival and resistance to basic antibacterial peptides
(48-50). The PhoP/PhoQ system exerts its actions by regulating a
second two-component system, known as PmrA/PmrB (48, 49), which in turn
is thought to control the expression of the putative enzymes that
modify S. typhimurium lipid A. The PhoP/PhoQ system is
switched on by low pH, as might be encountered by bacteria within
endosomes, and by low magnesium ion concentrations (48). Whether or not
the PhoP/PhoQ system is involved in the cold shock-induced modification
of lipid A remains to be determined.
Both procaryotic (51-55) and eucaryotic (55-57) organisms increase
the degree of unsaturation of their glycerophospholipid acyl chains at
low temperatures in a process termed homeoviscous adaptation (53),
presumably to maintain the optimal membrane fluidity. The acylation of
lipid A with palmitoleate instead of laurate (Fig. 1) might therefore
function to adjust outer membrane fluidity in E. coli cells
shifted to low temperatures, as would occur during prolonged survival
outside of an animal host. Gram-negative bacteria like
Haemophilus influenzae, which are transmitted from animal to
animal without having to persist in the environment, do not contain a
lpxP homologue (58). The melting point of
cis-9-palmitoleic acid is 0.5 °C, whereas that of lauric
acid is 44.2 °C (59). The liquid-crystalline transition
temperature(s) of lipid A substituted with palmitoleate rather than
laurate would likely decrease in a related manner, but very little is
actually known about the physical properties of lipid A and its
precursors (60-62). We have constructed a mutant in which the E. coli lpxP gene is inactivated. Although this strain can grow at
12 °C, it is extremely hypersensitive to antibiotics at low
temperatures.2
To our knowledge, no one has previously reported an acyltransferase
induced under conditions of cold shock (or other environmental stress)
that selectively incorporates a specific fatty acyl chain into a
membrane lipid precursor. The glycerol-3-phosphate acyltransferases of
E. coli certainly are not regulated in this manner (52) and do not display a high degree of selectivity for their acyl donor substrates (63). There is only a slight difference in the fatty acid
composition of the glycerophospholipids of E. coli cells grown at 27 °C versus 37 °C (54). However, a 2-fold
increase in unsaturation (mainly increased cis-vaccenate at
the expense of palmitate) is observed in the glycerophospholipids of
E. coli cells grown at 17 °C versus 37 °C
(54). The condensing enzyme (3-ketoacyl-ACP synthase II), encoded by
the fabF gene, is thought to control the unsaturated acyl
chain content of the E. coli glycerophospholipids (52, 64).
This may be mediated by a direct effect of temperature on FabF
activity, which is higher at low temperatures and therefore might
increase the size of the unsaturated fatty acid precursor pool
appropriately (52, 64). In other organisms, including Synechocystis, Bacillus subtilis,
Acanthamoeba, and carp, cold shock induces the transcription
of specific fatty acid desaturases (55, 65, 75), likewise increasing
the pool of available unsaturated fatty acids.
Mutants of E. coli that are defective in the fabA
or the fabB genes require unsaturated fatty acids for growth
(51, 52, 56, 66). Many different unnatural and polyunsaturated fatty acids, including trans-unsaturated compounds, can support
the growth of such auxotrophs (51, 52, 56, 67). Unlike the lipid A
acyltransferases (3), the glycerophospholipid acyltransferases are not
specific for their acyl donor substrates (63), and therefore many
different unnatural fatty acid analogs can be incorporated into the
glycerophospholipids of such auxotrophs. The composition of lipid A in
fatty acid auxotrophs supplemented with analogs has never been
examined, but it should be unaffected, since exogenous fatty acids are
activated as coenzyme A thioesters, and not as acyl-ACPs (1, 3, 66).
However, DiRienzo and Inouye (68) reported that unsaturated fatty acid
auxotrophs supplemented with elaidate (an unnatural
trans-unsaturated fatty acid) do not grow at low
temperatures and accumulate lipid A precursor(s). The chemical structures of these substances were not determined (68). Their characterization might yet provide interesting new insights into the
regulation of lipid A biosynthesis and into the biological significance
of lipid A modification with palmitoleate during cold shock.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP and
[
-32P]dATP were obtained from NEN Life Science
Products. Pyridine, chloroform, methanol, and 88% formic acid were
from Fisher. Triton X-100 was Surfact-Amps grade from Pierce. ACP was
purchased from Sigma. Hybond-N nylon membranes were obtained from
Amersham Pharmacia Biotech. PhosphorImager screens were from Molecular
Dynamics. 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,
Amersham Pharmacia Biotech. Formamide, salmon sperm DNA, and RNA
standards were obtained from Life Technologies, Inc. All other
chemicals were purchased from Sigma.
clone 10D3 (74) in
pACYC184. Cultures were generally grown in Luria-Bertani (LB) broth
consisting of 10 g of NaCl, 5 g of yeast extract, and 10 g of Tryptone per liter (23). Tetracycline was used at a concentration
of 50 µg/ml.
).
Incubation at 12 °C improved the stability of the palmitoleoyl transferase and eliminated an interfering acylation of
(Kdo)2-[4'-32P]lipid IVA in crude
extracts that was not dependent upon the presence of palmitoleoyl-ACP.
The reaction was stopped by spotting 5 µl of the assay mixture onto a
silica gel thin layer plate. After the spots were allowed to dry under
a cool air stream, the plates were developed in the solvent
chloroform/pyridine/88% formic acid/water (30:70:16:10, v/v), dried,
and then exposed to a PhosphorImager screen for about 16 h. The
percent conversion of substrate to product was determined using a
Molecular Dynamics PhosphorImager, and the specific enzymatic activity
was calculated in nanomoles/min/mg based upon the original substrate concentration.
80 °C. The protein concentrations were
determined with the bicinchoninic assay (Pierce) using bovine serum
albumin as the standard (30).
3Prime, Inc. (32, 33). The
A260/A280 ratio for all
of the samples was between 1.7 and 1.9, and 10 µg of each RNA sample
was used for each lane of the Northern blot.
-32P]dATP (150,000 cpm/nmol) as recommended by the
manufacturer. The probe was purified away from contaminating free
nucleotides by using a NucTrap probe purification column (Stratagene).
Commercial RNA standards were analyzed in parallel during
electrophoresis (34) and were stained with methylene blue to estimate
the size of the lpxP transcript. The dried Northern blot was
visualized using a PhosphorImager.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Role of (Kdo)2-lipid
IVA as a precursor of lipid A in normal and cold-shocked
E. coli. The intermediate
(Kdo)2-lipid IVA is a key precursor of lipid A,
conserved in diverse Gram-negative bacteria (3). In cells grown at
30 °C or higher, (Kdo)2-lipid IVA is
acylated by the sequential action of LpxL and LpxM, which incorporate
laurate and myristate, respectively, but do not function on precursors
lacking the Kdo disaccharide (9-11).
Palmitoleoyl-ACP-dependent acylation of
(Kdo)2-lipid IVA, catalyzed by the homologue
LpxP, is observed only in extracts of cells subjected to cold shock
(i.e. transferred from 30 °C to 12 °C for at least 15 min). Mass spectrometry2 demonstrates that ~80% of the
lipid A moieties isolated from 12 °C grown E. coli cells
contain palmitoleate instead of laurate. In 30 °C grown cells no
palmitoleate is detected (20). Prior to elucidation of its function in
lipid A biosynthesis (9-11), the lpxL gene was called
htrB (high temperature requirement for rapid growth above
32 °C) (17, 18), and lpxM was designated msbB
(multicopy suppressor of htrB) (19). The lpxP
gene (which displays even greater sequence homology to lpxL
than does lpxM) was provisionally termed ddg
(sequenced in the GenBankTM/EBI Data Bank with accession number
1872207), because of a proposed dam-dependent
growth phenotype (69), but this idea was not confirmed (K. R. Sreekumar, unpublished data). The lpxL and lpxM
genes have also recently been designated waaM and
waaN by some authors (70, 71). We suggest that the
waa nomenclature be restricted to bacterial
glycosyltransferase genes (71), and that lpx be used for
genes encoding enzymes of lipid A biosynthesis (3).
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Fig. 2.
Palmitoleoyl-ACP-dependent
acylation of (Kdo)2-[4'-32P]lipid
IVA in extracts of E. coli grown at
12 °C. Crude cell extracts were prepared from mid-log phase
cells of strain W3110, shifted from 30 °C to 12 °C for 2 h.
Palmitoleoyl transferase activity was assayed using 6 µM
(Kdo)2-[4'-32P]lipid IVA and 12 µM palmitoleoyl-ACP, as indicated. The more hydrophobic
product is presumably
(Kdo)2-[4'-32P](palmitoleoyl)-lipid
IVA based on its RF.
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Fig. 3.
Specific activities of the palmitoleoyl
(LpxP) and the lauroyl (LpxL) transferases in extracts of E. coli strains grown at 30 or 12 °C. Mid-log phase
cells of wild-type W3110, lpxL mutant MLK53, or MC1000
(bearing multiple copies of pSK57/lpxP+) were
shifted from 30 °C to 12 °C for 2 h. Extracts were then
assayed at 12 °C for the palmitoleoyl transferase (A) or
at 30 °C for the lauroyl transferase (B), using
(Kdo)2-[4'-32P]lipid IVA as the
acceptor.
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Fig. 4.
Sequence alignment of the
lpxL(htrB) and the
lpxP(ddg) gene products. LpxL
and LpxP are each 306 amino acid residues long. The numbering refers to
the sequence of LpxL. LpxL and LpxP share 54% identity and 73%
similarity, with one gap over a segment of 301 amino acids, as
determined by BLASTp analysis (39).
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Fig. 5.
An assay for the cold shock-induced
palmitoleoyl transferase. The time course (A) at 0.08 mg/ml crude extract protein and the protein concentration dependence
(B) at 30 min of palmitoleoyl transfer from palmitoleoyl-ACP
to (Kdo)2-[4'-32P]lipid IVA at
12 °C were determined using extracts of cold-shocked cells of
MC1000(pSK57/lpxP+), prepared as in Fig.
3.
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Fig. 6.
Kdo dependence of the cold shock-induced
palmitoleoyl transferase. Crude extracts of
MC1000(pSK57/lpxP+), grown to mid-log phase and
shifted for 2 h to 12 °C as in Fig. 3, were assayed at 0.1 mg/ml for palmitoleate transfer to the indicated acceptor under the
standard assay conditions for 5 h at 12 °C. The acyl acceptor
was either 6 µM
(Kdo)2-[4'-32P]lipid IVA or 6 µM [4'-32P]lipid IVA, as
indicated.
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Fig. 7.
Acyl donor specificity of the cold
shock-induced acyltransferase. Crude extracts of MLK53
(lpxL ), grown to mid-log phase at 30 °C or
shifted for 2 h to 12 °C as in Fig. 3, were assayed at 0.1 mg/ml for acylation of (Kdo)2-[4'-32P]lipid
IVA under the palmitoleoyl transferase conditions for
11 h at 12 °C with the indicated acyl donors. The abbreviations
are as follows: 12:0 ACP, lauroyl-ACP; 14:0 ACP,
myristoyl-ACP; 16:0 ACP, palmitoyl-ACP; 16:1 ACP,
palmitoleoyl-ACP; OH14:0 ACP,
R-3-hydroxymyristoyl-ACP; 16:1 CoA, palmitoleoyl
coenzyme A; 16:0 CoA, palmitoyl coenzyme A.
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Fig. 8.
Growth of the wild-type E. coli
strain W3110 at 30 °C and after a shift to 12 °C. The
cells were grown to A600 = 0.4 at 30 °C, and
the culture was split into two equal portions at time 0. Closed circles indicate the
A600 measurements for the culture held at
30 °C, and open symbols are the
A600 measurements for the cells shifted to
12 °C at time 0.
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Fig. 9.
Specific activity of the palmitoleoyl
transferase in crude extracts made at various times after shifting
cells from 30 to 12 °C. Three strains of E. coli were grown on LB broth in a rotary shaker at 30 °C (250 rpm) to mid-log phase, as in Fig. 8. At time 0, half of each culture
was shifted to 12 °C, and growth was allowed to continue. Cell
extracts were then prepared at the indicated times both from the
30 °C and the 12 °C cultures. The extracts were assayed at
12 °C to determine the specific activity of the palmitoleoyl
transferase. The incubation times were 11 h for the extracts of
W3110 and MLK53, and 15-30 min for
MC1000(pSK57/lpxP+). Closed
symbols are used for the 12 °C cultures and
open symbols for the 30 °C grown cells.
Panel A, wild-type W3110; panel
B, MLK53(lpxL ); panel
C, MC1000(pSK57/lpxP+).
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Fig. 10.
Northern blot analysis of lpxP
RNA isolated from 30 °C and 12 °C grown cells. The
total RNA was rapidly extracted from W3110 and MLK53 cells grown either
at 30 °C or 12 °C. The RNA samples (10 µg each) were separated
by gel electrophoresis in lanes adjacent to commercial size standards,
and the gels were analyzed by Northern blotting using a
32P-labeled lpxP DNA probe. The probe was used
at 5 × 105 cpm/ml of hybridization solution. The
final nitrocellulose membrane was exposed overnight to a PhosphorImager
screen.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported 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 National Institutes of Health Training Grant GM08558 in Biological Chemistry to Duke University.
Supported at Tufts University by GM-34123 (to M. Schaechter)
and GM-38035 (to A. Wright).
** To whom correspondence should be addressed. Tel.: 919-684-5326; Fax: 919-684-8885; E-mail: Raetz{at}biochem.duke.edu.
2 S. M. Carty and C. R. H. Raetz, manuscript in preparation.
3 R. Bishop and C. R. H. Raetz, unpublished data.
4 S. M. Carty and C. R. H. Raetz, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: LPS, lipopolysaccharide; ACP, acyl carrier protein.
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