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.

EXPERIMENTAL PROCEDURES

Materials

Plasmids, Bacterial Strains, and Growth Conditions

DNA Techniques

Transformation of E. coli Cells

Kdo Transferase Assay

Plasmid Construction

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.

Construction of a Mutant with an Insertion in the Chromosomal Copy of kdtA

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.

Phospholipid Analysis

RESULTS

Temperature-sensitive Growth of Strain CJB26

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.

Loss of Kdo Transferase Activity at 44 °C

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.''

Effect of Plasmid Loss on Lipid IV Accumulation in CJB26

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).

Rescue of the kdtA::kan Mutation of CJB26 by the C. trachomatis gseA Gene

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.

Specific Binding of an Antibody Directed Against the Genus-specific Epitope by NEB1

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.

DISCUSSION

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 IVper 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.

FOOTNOTES

*

This research was supported in part by National Institutes of Health Grant GM-51310 (to C. R. H. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Present address: Dept. of Inflammation and Autoimmune Diseases, Hoffmann-La Roche, 340 Kingsland St., Nutley, NJ 07110.

¶

Supported by a National Institutes of Health training grant in biological chemistry to Duke University.

**

Present address: Dept. of Biochemistry, Duke University Medical Center, Durham, NC 27710. To whom correspondence should be addressed.

()

The abbreviations used are: Kdo, 3-deoxy-D-manno-octulosonic acid; LPS, lipopolysaccharide; kb, kilobase(s).

ACKNOWLEDGEMENTS

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