(Received for publication, July 18, 1995; and in revised form, September 11, 1995)
From the
The enzyme 3-deoxy-D-manno-octulosonic acid
(Kdo) transferase is encoded by the kdtA gene in Escherichia coli. The enzyme is a single polypeptide that
catalyzes the transfer of two Kdo residues to a
tetraacyldisaccharide-1,4`-bisphosphate precursor of lipid A,
designated lipid IV (Belunis, C. J., and Raetz, C. R.
H.(1992) J. Biol. Chem. 267, 9988-9997). To determine if
Kdo transfer to lipid IV
is required for growth, we
constructed a strain of E. coli with a chromosomal kdtA::kan insertion mutation. In mutants carrying the kdtA::kan allele on the chromosome, cell growth and
Kdo transferase activity were dependent upon a copy of the intact kdtA gene on a plasmid. When the kdtA-bearing plasmid
was itself temperature sensitive for replication, the growth of these
strains was inhibited after several hours at 44 °C, and Kdo
transferase activity in extracts became undetectable. Concomitantly,
the cells accumulated massive amounts of lipid IV
, the
precursor of (Kdo)
-lipid IV
. The kdtA::kan mutation could also be complemented by
hybrid plasmids bearing the gseA gene of Chlamydia
trachomatis. gseA specifies a distinct Kdo transferase that adds
three Kdo moieties to lipid IV
. Lipopolysaccharide from E. coli kdtA::kan constructs complemented by gseA reacts strongly with antibodies directed against the
genus-specific epitope of Chlamydia, whereas
lipopolysaccharide from parental E. coli K-12 does not. Our
studies prove that Kdo attachment during lipid A biosynthesis is
essential for cell growth and accounts for the conditional lethality
associated with mutations in Kdo biosynthesis.
The sugar 3-deoxy-D-manno-octulosonic acid
(Kdo) ()appears to be an essential component of the
lipopolysaccharide (LPS) of Escherichia coli and other
Gram-negative
bacteria(1, 2, 3, 4, 5, 6) .
The enzyme Kdo transferase catalyzes the sequential addition of two Kdo
sugars onto a molecule of lipid IV
(7, 8) ,
a key precursor of lipid A (Fig. 1). Prior to its incorporation,
Kdo is activated to CMP-Kdo by the enzyme CMP-Kdo
synthase(9, 10, 11) . Mutants of Salmonella typhimurium defective in Kdo biosynthesis are
temperature sensitive for growth and accumulate several underacylated
precursors of lipid A(12, 13, 14) , the most
abundant of which is lipid IV
(15, 16) .
Pharmacological inhibition of CMP-Kdo synthase in living cells has
similar effects on LPS biogenesis (17, 18, 19, 20) .
Figure 1:
Kdo transfer and the completion of
lipid A assembly in E. coli. The six enzymatic reactions
leading to the formation of lipid IV have been reviewed
elsewhere(2) . The chromosomal kdtA gene of E.
coli is a bifunctional enzyme catalyzing both the first and the
second Kdo transfers, as indicated(8, 21) . The Kdo
transferase encoded by the gseA gene of C. trachomatis (not shown) catalyzes an additional, third Kdo transfer to the 8
position of the outer Kdo
sugar(33) .
Recently,
Clementz and Raetz (21) described a colony screening technique
to identify E. coli mutants with thermolabile Kdo transferase
in cell extracts. Using these mutants, they were able to isolate the
Kdo transferase gene, designated as kdtA(21) .
Although cell extracts of these mutants contain <5% of wild-type Kdo
transferase activity, when assayed at 42 °C, the strains do not
display temperature-sensitive growth, and they do not accumulate lipid
IV at 42 °C(21) . It appears that these mutants
retain enough residual Kdo transferase activity in vivo to
synthesize adequate levels of (Kdo)
-lipid IV
.
Kdo is a component of capsular polysaccharides in some strains of E. coli and related
bacteria(6, 22, 23) . Since Kdo-containing
polymers other than LPS are not found in all Gram-negative
bacteria(6, 22, 23) , it seems unlikely that
the presence of Kdo in these polymers would account for the conditional
lethality of the Kdo biosynthesis mutants. Nevertheless, the question
of whether or not Kdo transfer to lipid IVper se is essential for cell growth could only be tested by examining the
consequences of complete inactivation of the kdtA gene.
In this paper, we demonstrate that the kdtA gene is indeed essential for growth of E. coli by constructing a kdtA::kan insertion mutation, using a gene replacement method(24) . Growth of this strain is absolutely dependent upon the presence of a functional copy of the kdtA gene (or the related gseA gene) carried on a plasmid.
Figure 2: pJSC20 contains a kdtA gene disrupted by a kan insertion. The construction of pJSC20 is described in detail under ``Experimental Procedures.'' The selections for plasmids pJSC2 and pJSC20 were based on chloramphenicol resistance or chloramphenicol and kanamycin resistance, respectively. The chloramphenicol resistance gene and the temperature-sensitive replicon are indicated as cam and rep, respectively.
pJSC2 and pUC-4K were digested with SalI. The kanamycin cassette from pUC-4K was purified by gel electrophoresis and ligated into pJSC2. The ligation mixture was used to transform competent cells of JM109. Plasmid-bearing cells were selected by growth at 30 °C on LB plates containing chloramphenicol and kanamycin. pJSC20 showed the expected restriction sites, and extracts did not express elevated levels of Kdo transferase activity, indicating disruption of the plasmid-born kdtA gene. Cells carrying pJSC20 grew well in the presence of chloramphenicol and kanamycin at 30 °C but not at 44 °C, confirming that the plasmid pJSC20 is temperature sensitive for replication.
Figure 3: Construction of an E. coli strain bearing a chromosomal kdtA gene disrupted by a kan insertion. The construction of CJB26 (Table 1), which is based on homologous recombination at 44 °C followed by resolution of the cointegrate at 30 °C, is described in detail under ``Experimental Procedures.'' Pathway B, leading to CJB26, leaves the inactivated kdtA gene behind on the chromosome. The covering plasmid of CJB26 is identical to pJSC2, as drawn in Fig. 2. The chloramphenicol resistance gene and the temperature-sensitive replicon are indicated as cam and rep, respectively.
Strain NEB1 was constructed by transforming competent cells of CJB26 with pKEM1, which contains the C. trachomatis gseA gene(33) . The kdtA-bearing plasmid of CJB26 was cured by growth at 44 °C in the absence of chloramphenicol but in the presence of ampicillin to select for pKEM1. The presence of pKEM1 as the only plasmid in the surviving colonies was confirmed by rapid plasmid isolation and restriction digestion analysis.
To determine whether or not the kdtA gene
is essential for growth, cells of CJB26 from an overnight culture grown
at 30 °C in the presence of chloramphenicol were inoculated into LB
medium lacking chloramphenicol at A of
0.1 (Fig. 4). Cells were then cultured at 44 °C with
intermittent back dilution to maintain the A
between 0.06 and 0.6. The results are plotted as a cumulative
growth yield at 44 °C (Fig. 4). After about 4 h, the plating
efficiency of CJB26, as judged by growth of single colonies on LB agar
lacking chloramphenicol at 30 °C (Fig. 4), stopped
increasing. However, the A
of CJB26 continued to
rise slowly. The control strain, MC1061/pJSC2, continued to grow
rapidly after the temperature shift, since the plasmid copies of the kdtA gene were not needed in MC1061 for growth in LB medium at
44 °C without chloramphenicol (Fig. 4). The results of Fig. 4demonstrate that the kdtA gene is essential.
Figure 4:
Temperature-sensitive growth of CJB26.
Overnight cultures of CJB26 and MC1061/pJSC2 were grown in LB broth
supplemented with chloramphenicol at 30 °C. Cultures were diluted
into LB broth without chloramphenicol to an absorbance at 600 nm of
0.1, and growth was continued at 44 °C. To maintain exponential
growth, the cultures were diluted 10-fold into fresh prewarmed medium
lacking chloramphenicol every time the A reached
0.6. Samples for the zero time points were taken just prior to the
temperature shift. The A
values for strains
CJB26 and MC1061/pJSC2 were corrected for dilution of the culture and
therefore represent a cumulative growth yield. Viability (also
corrected for dilution) of strains CJB26 and MC1061/pJSC2 was
determined by diluting and plating portions of the cultures onto LB
plates without chloramphenicol at 30 °C. In a separate experiment
(data not shown), the loss of plasmid DNA in MC1061/pJCS2 at 44 °C
was confirmed by determining viable cell count on LB plates containing
chloramphenicol at 30 °C. The number of chloramphenicol-resistant
cells increased for 1-2 h following the temperature shift (data
not shown), but the viable count of MC1061/pJSC2 on chloramphenicol
then decreased by several orders of magnitude as a percentage of the
total number of cells in the culture, indicating the loss of plasmid
DNA. The viability of CJB26 on LB plates containing chloramphenicol at
30 °C (data not shown) was comparable to its viability in the
absence of chloramphenicol, indicating that only cells possessing
recombinant plasmid with a functional copy of kdtA could
grow.
Figure 5: Dilution of Kdo transferase specific activity in extracts of CJB26 and MC1061/pJSC2 grown at 44 °C. Cells were subjected to a temperature shift as in Fig. 4. At the indicated time points, portions of the cells were harvested by centrifugation, and the cells were broken by passage through a cold French pressure cell at 18,000 psi. Cell-free extracts were assayed for protein and Kdo transferase activity as described under ``Experimental Procedures.''
In the experiment of Fig. 6,
cells of mutant STi50 and CJB26 were grown in parallel at 30 °C and
were uniformly labeled with P
. The STi50
culture was shifted to 44 °C when the A
had
reached 0.8. A portion was harvested just before and 2 h after the
temperature shift for analysis of lipid composition. The CJB26 culture
was first diluted to an A
of 0.1 with excess
medium containing
P
, and then it was shifted
to 44 °C. The CJB26 culture was given time to lose its covering
plasmid and Kdo transferase activity by intermittent 10-fold back
dilution in media containing
P
whenever the A
had reached 0.6. Over the course of 9.5 h,
portions of the CJB26 culture were removed, and the phospholipids were
extracted under acidic Bligh-Dyer conditions(34) . Samples of
5
10
cpm of uniformly labeled phospholipids
obtained at each time point were spotted onto a silica gel plate, which
was developed in chloroform/pyridine/88% formic acid/water (40:60:16:5
(v/v)). The plate was analyzed by autoradiography (Fig. 6).
Cells of the kdsA-deficient mutant STi50 accumulated large
amounts of lipid IV
after 2 h at 44 °C (Fig. 6).
Prior to the temperature shift, the lipids of CJB26 consisted mainly of
glycerophospholipids, which migrated rapidly in the solvent system
employed. After 6.5 h at 44 °C, CJB26 also accumulated lipid
IV
(Fig. 6). The timing of lipid IV
accumulation approximately coincided with the complete loss of
measurable Kdo transferase activity (compare Fig. 4and Fig. 6). Wild-type E. coli do not accumulate lipid
IV
at any growth temperature.
Figure 6:
Lipid IV accumulation in CJB26
grown at 44 °C and in a temperature-sensitive mutant of S.
typhimurium defective in Kdo biosynthesis. Cells of the S.
typhimurium mutant STi50 and the E. coli strain CJB26
were grown for three generations at 30 °C on LB broth containing
P
(20 µCi/ml). When the absorbance at 600
nm had reached 0.8, the STi50 cells were shifted to 44 °C, and the
CJB26 culture was diluted to an A
of 0.1 prior
to the temperature shift. To maintain exponential growth, the CJB26
culture was diluted 1:10 whenever the A
reached
0.6. At the time of the temperature shift (designated 0) and
at the indicated times, samples were withdrawn, and the lipids were
extracted under acidic conditions(34) . A portion of the
radioactive lipids was spotted onto a silica gel plate that was
developed in chloroform/pyridine/88% formic acid/water (40:60:16:5
(v/v)). The glycerophospholipids migrated rapidly, whereas lipid
IV
migrated more slowly. The accumulation of lipid IV
is detectable in STi50 after 2 h at 44 °C, and in CJB26,
after 6 h at 44 °C. Lipid IV
is not detectable in STi50
at 30 °C or CJB26 at 30 °C. Wild-type E. coli cells
also do not accumulate lipid IV
at 30 °C or during
prolonged incubations at 44 °C (data not
shown).
Figure 7:
Growth of MC1061 and NEB1 at various
temperatures. Overnight cultures of MC1061 and NEB1 were grown at 30
°C in LB broth. Cultures of NEB1 were also supplemented with
ampicillin at 125 µg/ml. Following dilution into fresh medium,
cells were grown at 30 °C until A was
0.2. Portions of these cultures were then grown at 30, 37, or 42
°C, starting at time 0 as indicated. Intermittent back dilution was
carried out as required to maintain the A
between 0.06 and 0.6. The A
shown on the y axis therefore represents a cumulative growth
yield.
Southern blots of
chromosomal DNA isolated from MC1061 and NEB1 were prepared and probed
with a P-labeled 1.7-kb EcoRV fragment containing
the complete kdtA gene. Restriction digestion of the
chromosomal DNA with EcoRV should produce fragments of 1.7 kb
from cells containing the wild-type kdtA gene (MC1061) and 3
kb from cells containing the kan insertion within the kdtA gene (NEB1). Digestion of DNA isolated from NEB1 with ClaI should produce fragments of approximately 1.8 and 1.2 kb
because of a single ClaI site inside the kan gene.
The predicted fragments were indeed observed (Fig. 8). These
results confirmed that the kan cassette was located within the kdtA gene of NEB1 and, by inference, of CJB26.
Figure 8:
Southern blot of chromosomal DNA from NEB1
and MC1061. Genomic DNA was isolated from E. coli strains NEB1
and MC1061, and it was subjected to restriction enzyme digestion with
either EcoRV (E) or ClaI (C). The
resulting fragments were separated by agarose gel electrophoresis.
Following transfer of the DNA to a nylon membrane, the blot was probed
with a P-labeled, 1.7-kb EcoRV fragment
containing the complete kdtA gene. The positions of standards
of defined molecular weight are indicated.
Extracts of
the kdtA point mutant TC5 (21) contain <5% of
wild-type Kdo transferase activity when assayed at 42 °C. Extracts
of TC5 cells transformed with the gseA-bearing plasmid pKEM1
regain Kdo transferase activity at 42 °C, generating not only
(Kdo)-lipid IV
but also (Kdo)
-lipid
IV
(33) . Extracts of NEB1 were similarly capable of
transferring three Kdos onto lipid IV
(Fig. 9, panel A) in the absence of any residual KdtA activity. The
steady state level of (Kdo)
-lipid IV
(Fig. 9, panel A) is somewhat higher in the
reaction catalyzed by the gseA gene product than by the kdtA-encoded enzyme (Fig. 9, panel B). This
observation suggests that the two Kdo transferases differ not only with
respect to their ability to incorporate the third Kdo residue but also
in the relative rates at which they catalyze the first and second
glycosylations.
Figure 9:
Kdo transferase activity in extracts of
NEB1 and MC1061. The assay contains 50 mM Hepes, pH 7.5, 3.2
mM Triton X-100, 2 mM Kdo, 100 µM 4`-P lipid IV
(1.2
10
cpm/nmol), 5 mM CTP, 2 milliunits of CMP-Kdo synthase,
10 mM MgCl
, and 1.0 mg/ml cell extract. The
reactions were incubated for the indicated times. Panel A, E. coli NEB1. Panel B, E. coli MC1061.
Figure 10: Dot blot analysis of extracts of NEB1 and MC1061 using a monoclonal antibody directed against the genus-specific epitope of C. trachomatis. Cells of MC1061 and NEB1 were grown to late log phase in LB broth. A portion of each culture (1 ml) was centrifuged to recover the cells. The pellets were resuspended in 200 µl of Laemmli loading buffer (50 mM Tris-chloride, pH 6.8, 2% SDS, 0.1% bromphenol blue, 10% glycerol, and 700 mM 2-mercaptoethanol), and the lysed samples were diluted with phosphate-buffered saline to final protein concentrations of 3-15 µg/ml. Next, 1-µl portions containing the amount of protein indicated were spotted onto a nitrocellulose membrane (0.45 micron Hybond-C super from Amersham) and allowed to dry for 5 min at 42 °C. The membrane was blocked for 1 h at room temperature in 30 ml of phosphate-buffered saline containing 6% Kroger nonfat dry milk and 0.02% sodium azide. The primary antibody (a 1 mg/ml stock of CT403.1) was exposed to the membrane at a 1:5000-fold dilution in a fresh 30-ml portion of the above blocking buffer. After 1 h, the membrane was rinsed three times with phosphate-buffered saline. Next, it was incubated for 1 h in another 30-ml portion of the above blocking buffer supplemented with secondary antibody at a 1:1000 dilution from a 1 mg/ml stock of Promega horseradish peroxidase-conjugated goat anti-mouse antibody. After several final washes with phosphate-buffered saline and distilled water, the genus-specific epitope was detected on the membrane by a 1-min incubation in 40 ml of enhanced chemiluminescence reagents (ECL Western blotting detection reagents from Amersham), followed by a 5-min exposure to Kodak X-OMAT x-ray film.
The phospholipid compositions of NEB1
and MC1061, grown in LB broth at 37 °C, were analyzed by P
labeling, as in Fig. 6. No
accumulation of lipid IV
was observed in NEB1 (data not
shown), demonstrating that GseA can function in vivo at a rate
that is comparable to KdtA. The slow growth of NEB1 (Fig. 7)
therefore cannot be attributed to inefficient Kdo transfer and lipid
IV
accumulation.
Previous genetic and pharmacological studies have
demonstrated that the biosynthesis and activation of Kdo are essential
processes in Gram-negative
bacteria(12, 13, 14, 15, 16, 17, 18, 19, 20) .
When the formation of CMP-Kdo is blocked, cells accumulate large
amounts of lipid IV, consistent with the pathway shown in Fig. 1(12, 13, 14, 15, 16, 17, 18, 19, 20) .
However, given that Kdo can be a constituent of other cell surface
molecules(6, 22, 23) , it is unclear whether
the attachment of Kdo to lipid IV
per se is
required for growth. An E. coli gene encoding a bifunctional
Kdo transferase (kdtA) has previously been identified and
sequenced(21) , but no conditional alleles or insertion
mutations of kdtA have been reported. The physiological
consequences of selective inhibition of Kdo transfer to lipid
IV
therefore remained uncertain.
Using homologous recombination, we have now constructed an E. coli strain (CJB26) with a kan element insertion in the chromosomal copy of the kdtA gene (Fig. 3, pathway B). CJB26 retains a functional copy of kdtA on a hybrid plasmid (Fig. 3, pathway B) that is equivalent to pJSC2 (compare Fig. 2and Fig. 3). Since pJSC2 is derived from pMAK705 (24) and harbors a temperature-sensitive pSC101 replicon (Fig. 2), pJSC2 is capable of replicating at 30 °C but not at 44 °C. Cells of CJB26 are expected to survive only at the lower temperature if the kdtA gene is essential but would grow at both temperatures if the kdtA gene is not essential.
As shown by the behavior of CJB26 in the experiments of Fig. 4Fig. 5Fig. 6, the kdtA gene is
indeed essential. Cells of CJB26 grown at 44 °C lose the ability to
synthesize Kdo transferase as the plasmid is cured (Fig. 5). The
decrease in Kdo transferase activity with time precedes the
accumulation of lipid IV in the cells (Fig. 6). As
Kdo transferase activity falls below wild-type levels, growth slows and
eventually ceases (Fig. 4). Only those daughter cells that
retain the functional copy of kdtA on a plasmid can grow,
while the other daughter cells cannot. Viable cell counts on LB plates
both with and without chloramphenicol supplementation at 30 °C are
identical in cultures of CJB26 shifted to 44 °C (data not shown).
The absence of measurable Kdo transferase in extracts of CJB26 after 6
h at 44 °C (Fig. 5) indicates that there are no additional
genes encoding for Kdo transferase isoenzymes in E. coli.
The decrease in the growth rate of CJB26 and the accumulation of
lipid IV during plasmid curing at 44 °C are similar to
what occurs when temperature-sensitive Kdo biosynthesis mutants of S. typhimurium(12, 13, 14, 15, 16) are
shifted to non-permissive conditions or when CMP-Kdo synthase
inhibitors are added to wild-type Gram-negative
bacteria(17, 18, 19, 20) . Our
findings show that inhibition of LPS biosynthesis is sufficient to
explain the antibacterial effects of CMP-Kdo synthase inhibitors and
the conditional lethality of mutations in Kdo biosynthesis. It is still
uncertain whether Kdo
-lipid IV
is actually
required for growth or whether the accumulation of lipid IV
is toxic.
In previous studies(33) , we showed that the gseA gene of C. trachomatis codes for a novel Kdo
transferase. The C. trachomatis Kdo transferase can add at
least one additional Kdo onto Kdo-lipid IV
.
Preliminary results also indicated that GseA might actually recognize
lipid IV
as a substrate (33) , suggesting that GseA
is a trifunctional Kdo transferase. The fact that we were able to
replace the temperature-sensitive covering plasmid (pJSC2) present in
CJB26 with a thermostable plasmid (pKEM1) bearing only gseA(33) provides strong support for the ability of GseA to
use lipid IV
as a substrate in living cells. As expected
from the ability of pKEM1 to rescue CJB26 at 44 °C, extracts of
NEB1 were able to catalyze the formation of Kdo
-,
Kdo
-, and Kdo
-lipid IV
from lipid
IV
(Fig. 9), despite the insertional inactivation of
the kdtA gene (Fig. 8). Why NEB1 cells grow more slowly
than wild type, especially at elevated temperatures (Fig. 7), is
uncertain, but it is not the result of lipid IV
accumulation.
Southern blot analysis (Fig. 8) confirmed the presence of the kan element within the chromosomal kdtA gene in NEB1. NEB1 was chosen as the source of the genomic DNA for Southern blotting, since the recombinant covering plasmid in NEB1 did not contain any kdtA sequences. A hybridizing band on a Southern blot could only be due to kdtA sequences present on the chromosome and not from contaminating plasmid DNA. Because of the latter issue, we did not use CJB26 grown at 44 °C as the source of genomic DNA.
In summary, we have
constructed a strain of E. coli with an insertion mutation in
the kdtA gene. Biosynthesis of Kdo-lipid IV
and growth of the organism are absolutely dependent on the
presence of a functional copy of an intact kdtA gene on a
plasmid. The mutation can also be complemented by the gseA gene from C. trachomatis. The strain NEB1, which contains
the kdtA::kan mutation on the chromosome and gseA on
a plasmid, is viable, and it can synthesize Kdo-containing LPS that is
recognized by an antibody directed against the genus-specific epitope (Fig. 10). It will be interesting to determine the precise
structure of the LPS made by NEB1. Since NEB1 appears to make
Kdo
-containing LPS (Fig. 10), the strain could prove
to be a valuable source of the epitope for diagnostic assays and
structural studies. NEB1 should also prove useful for the isolation of
the trifunctional Kdo transferase, since no bifunctional Kdo
transferase is present to complicate enzymatic assays.