Identification of the Structural Gene for the TDP-Fuc4NAc:Lipid II Fuc4NAc Transferase Involved in Synthesis of Enterobacterial Common Antigen in Escherichia coliK-12

Bacterial strains and growth conditions.The bacterial strains used in this study are listed in Table1. Where indicated, transductions were carried out by using P1 vir as described by Silhavy et al. (30). Cultures of E. coli were grown in Luria-Bertani (LB) broth (23) or on LB agar at 37°C. Cultures of Pseudomonas aeruginosa were grown at 32°C in a salts medium containing K₂HPO₄ (7 g/liter), KH₂PO₄ (3 g/liter), MgSO₄ · 7H₂O (0.1 g/liter), and (NH₄)₂SO₄(1.0 g/liter), as well as 3% glycerol as the sole source of carbon. The following antibiotics were added to media when appropriate at the indicated final concentrations: ampicillin (100 μg/ml), chloramphenicol (30 μg/ml), and tetracycline (20 μg/ml).

Radiochemicals, chemicals, and reagents. d-[1-³H]glucose (15 Ci/mmol), UDP-N-acetyl-d-[6-³H](N)glucosamine (20.4 Ci/mmol), and UDP-N-acetyl-d-[U-¹⁴C]glucosamine (283 mCi/mmol) were purchased from New England Nuclear Corp. UDP-N-acetyl-d-[U-¹⁴C]mannosaminuronic acid (283 mCi/mmol) and unlabeled UDP-N-acetyl-d-mannosaminuronic acid were enzymatically synthesized from UDP-N-acetyl-d-[U-¹⁴C]glucosamine (283 mCi/mmol) and UDP-N-acetyl-d-glucosamine, respectively (4).d-[1-³H]glucose-6-phosphate was enzymatically synthesized fromd-[1-³H]glucose (15 Ci/mmol) by using yeast hexokinase. TDP-d-[1-³H]glucose (TDP-[³H]glucose) was enzymatically synthesized fromd-[1-³H]glucose-6-phosphate by using partially purified TDP-glucose pyrophosphorylase prepared from extracts of P. aeruginosa 7700 as described by Kornfeld and Glaser (11). TDP-4-acetamido-4,6-dideoxy-d-[1-³H]galactose (TDP-[³H]Fuc4NAc) and TDP-Fuc4NAc were synthesized from TDP-[³H]glucose and TDP-d-glucose, respectively, as described by Matsuhashi and Strominger (18). All other chemicals and reagents were purchased from standard commercial sources.

Plasmid constructions.The plasmids used in this study are listed in Table 1. Primers used for PCR amplification of nucleotide sequences are listed in Table 2. PCR amplifications were carried out by using Taq polymerase (Sigma Chemicals) according to standard protocols. Plasmid pRL150 was constructed by PCR amplification of the DNA sequence from bp 7067 to 8519 (GenBank accession no. AE000455 ) by using primers F1 and F8 and genomic DNA from strain AB1133 as a template. The amplified sequence included o359 in addition to 375 bp immediately upstream of the translational start site, and it was cloned into the TA cloning site of the pBAD-TOPO vector (Invitrogen, Inc.). One of the resulting constructs contained the C-terminal nucleotide sequence ofo359 in-frame with the polyhistidine (His₆) encoding nucleotide sequence of the vector. Plasmid pRL151 was constructed by PCR amplification of the insert fragment of pRL150 containing the entireo359-C-terminal His₆ tag fusion by using primers F1 and BAD1. The EcoRI- andBamHI-cleaved PCR product was subcloned into pBluescript II KS(+) (Stratagene) that was restricted by the same enzymes. Plasmids pRL153, pRL154, pRL155, pRL156, and pRL160 were constructed by PCR amplification of the DNA sequences from bp 7067 to 9948, 7160 to 9948, 7160 to 8683, 7463 to 8683, and 7691 to 9948 (GenBank accession no.AE000455 ) by using primer pairs F1-F7, F2-F7, F2-F6, F3-F6, and F4-F7 and genomic DNA from strain AB1133 as a template, respectively. The PCR products were cloned into the TA-cloning site of the pCR 2.1-TOPO cloning vector. T4 DNA ligase (Invitrogen) and restriction enzymes were used in accordance with the manufacturer's recommendations.

In vitro synthesis of lipid III.Cell envelopes were prepared as previously described (4). Reaction mixtures for the in vitro synthesis of lipid III contained the following in a final volume of 55 μl: 50 mM Tris-HCl (pH 8.2), 30 mM MgCl₂, 5 mM 2-mercaptoethanol, 0.14 μM TDP-[³H]Fuc4NAc (2.56 × 10⁷ dpm/μmol), and cell envelope membranes (700 to 850 μg of protein). Partially purified native His₆-tagged o359 gene product (6.75 μg of protein) and 0.24 μM UDP-[¹⁴C]ManNAcA (6.3 × 10⁵ dpm/μmol) were added to reaction mixtures where indicated. Reactions were incubated at 37°C for 30 min and then terminated by the addition of 1.0 ml of chloroform-methanol (3:2, by volume). The radioactive products were extracted from reaction mixtures as described previously (3), and they were analyzed directly by ascending paper chromatography with SG-81 filter paper (Whatman, Inc.). Protein was determined by using the BCA Protein Assay Reagent Kit (Pierce) according to the instructions provided by the manufacturer.

In vivo assay for lipid II accumulation.The incorporation of [³H]GlcNAc into lipid II was determined as previously described (28). Briefly, bacteria were grown with vigorous aeration at 37°C in 60 ml of LB medium supplemented with glucose (0.2%, final concentration) to anA₆₀₀ of 0.4. The cells were then harvested by centrifugation, resuspended in fresh medium (6 ml), and incubated at 37°C with [³H]GlcNAc (75 μCi, 8.2 Ci/mmol) for 30 min. The labeled cells were then poured over crushed ice, harvested by centrifugation, and washed with cold 0.9% saline. The washed cells were then successively extracted with 95% ethanol (6 ml) and acetone (6 ml) and dried in vacuo. The dried cells were extracted with 1.5 ml of chloroform-methanol (3:2, by volume), and 75 μl of the resulting extract was analyzed by ascending paper chromatography on EDTA-treated SG-81 paper by using chloroform-methanol-water-concentrated ammonium hydroxide (88:48:10:1, by volume) as the developing solvent. The amount of radioactivity in the region of the chromatogram corresponding to lipid II was then determined as described below.

Partial purification of C-terminal histidine-taggedo359 gene product.Strain PND788F was grown in 200 ml of LB-glucose medium at 37°C. IPTG (isopropyl-β-d-thiogalactopyranoside) was added atA₆₀₀ = 0.2 to give a final concentration of 1 mM, and the culture was incubated overnight with vigorous aeration. The cells were harvested by centrifugation and lysed with 10 ml of B-PER (Pierce) according to the manufacturer's recommendation. The suspension was then briefly sonicated, and the insoluble material was then separated from the soluble fraction (B-PER extract) by centrifugation at 12,000 × g. The pellet was resuspended in 5 ml of a solution containing 8 M urea, 0.1 M NaH₂PO₄, and 0.01 M Tris-HCl (pH 8.0; final concentrations) and then incubated at room temperature for 60 min with gentle stirring. The urea-soluble fraction was next separated from cell debris by centrifugation at 12,000 ×g for 20 min. The His-tagged o359 gene product present in the B-PER extract and the urea-soluble fraction were then partially purified by affinity chromatography by using nickel-nitrilotriacetic acid (Ni⁺-NTA) agarose resin (Qiagen, Inc.) under native and denaturing conditions, respectively. Briefly, for partial purification by using native conditions, imidazole was added to 4 ml of crude B-PER extract to give a final concentration of 10 mM. Then, 1 ml of a slurry containing 50% Ni⁺-NTA agarose resin was added, and the mixture was incubated with gentle shaking at 4°C for 60 min. The mixture was poured into a small column and, after the resin settled, the liquid fraction was allowed to flow out of the column. The resin was then washed twice with 4 ml of wash buffer (50 mM NaH₂PO₄, pH 8.0; 300 mM NaCl; 20 mM imidazole). His-tagged protein was then eluted by four successive 0.5-ml washes of the resin with elution buffer (50 mM NaH₂PO₄, pH 8.0; 300 mM NaCl, 250 mM imidazole), and fractions were analyzed by Western blot analyses by using mouse anti-Penta-His antibody (Qiagen, Inc.) and horseradish peroxidase-conjugated anti-mouse immunoglobulin G (IgG) (Amersham). Fractions containing C-terminal His-tagged o359were used for in vitro assay of Fuc4NAc transferase activity.

For partial purification of the C-terminal His-tagged o359gene product using denaturing conditions, 4 ml of the urea-soluble extract was mixed with 1 ml of a slurry containing 50% Ni⁺-NTA agarose resin and incubated with gentle shaking for 60 min at room temperature. The mixture was poured into a small column and, after the resin settled, the liquid fraction was allowed to flow out of the column. The resin was then washed twice with 4 ml of buffer B (8 M urea, 0.1 M NaH₂PO₄, 0.01 M Tris-hydrochloride; pH 6.3). His-tagged protein was then eluted with four successive 0.5-ml washes of the resin with buffer C (8 M urea, 0.1 M NaH₂PO₄, 0.01 M Tris-hydrochloride; pH 5.9), followed by four successive 0.5-ml washes with buffer D (8 M urea, 0.1 M NaH₂PO₄, 0.01 M Tris-hydrochloride; pH 4.5). The fractions were analyzed by Western blot analyses by using mouse anti-Penta-His antibody (Qiagen) and horseradish peroxidase-conjugated anti-mouse IgG (Amersham). Increased yields of more highly purified urea-soluble His-tagged protein were obtained by combining sonicated B-PER extracts with the urea-soluble fraction, followed by batch purification by using a modification of the denaturing conditions as described above. Briefly, the material that eluted from the column with buffer C was concentrated by centrifugation by using a Microcon YM-30 centrifugal filtration device (Amicon Bioseparations) according to the manufacturer's recommendations and then reapplied to a new column containing Ni⁺-NTA agarose resin. The column was then eluted successively with buffers B and C as described above. The material that eluted after development of the column with buffer C was detected as a single band by Western blot analyses.

Cellular localization of C-terminal histidine-taggedo359 gene product.The cellular localization of C-terminal His₆-tagged o359 gene product was determined by using a modification of the microprocedure described by Carlone et al. (6). Strain PND788F was grown with vigorous aeration in 500 ml of LB broth at 37°C. IPTG was added at A₆₀₀ = 0.2 to give a final concentration of 1 mM, and the culture was incubated overnight with vigorous aeration. The cells were then harvested by centrifugation, suspended in 5 ml of ice-cold 20 mM Tris-HCl–10 mM EDTA (pH 7.5) (Tris-EDTA) buffer, and disrupted by three passages through a French pressure cell (10,000 lb/in², high ratio) at 0 to 4°C. Unbroken cells and cellular debris were removed by centrifugation at 20,000 × g for 20 min. Cell envelope membranes were isolated by centrifugation of the turbid supernatant solution at 200,000 × g for 2 h. The resulting pellet was resuspended in 1 ml of Tris-EDTA buffer, and the total cell envelope protein concentration was determined by using the BCA Protein Assay Reagent Kit (Pierce) according to the instructions provided by the manufacturer. The suspension was then diluted with Tris-EDTA buffer to give a protein concentration of 4.4 mg/ml, and 1/10 volume of a 10% (wt/vol) solution of Sarkosyl (N-lauroylsarcosine, sodium salt) (Sigma) was subsequently added with gentle mixing. The mixture was then incubated in an ice bath for 1 h, followed by centrifugation at 200,000 × g (2 h, 4°C). The supernatant solution was removed, and the pellet was resuspended in 0.5 ml of Tris-EDTA buffer and once again extracted with 1/10 volume of Sarkosyl, followed by centrifugation at 200,000 × g (2 h, 4°C) as described above. The quantity of protein in the Sarkosyl-insoluble fraction and pooled Sarkosyl-soluble fractions was determined. The proteins in these fractions were then precipitated by the addition of 8 volumes of cold acetone. The precipitates were resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer.

Determination of the nucleotide sequence in the region betweeno416 and wecGThe nucleotide sequence of the wec gene cluster in the region betweeno416 and wecG (2,882 bp) contained in the cloned fragment of pRL153 was determined in both directions with BigDye Terminator Cycle Sequencing Ready Reaction kits (Applied Biosystems) and an ABI377 sequencer (Applied Biosystems). The data from these experiments were compared with the corresponding nucleotide sequence from E. coli (GenBank accession number AE000455 ). The nucleotide sequence of the same region of the chromosome of the mutant strain PND788 was also determined in order to confirm the position of the Tn10::cam insertion.

Passive hemagglutination assay.The presence of ECA on the surface of bacterial strains was determined by passive hemagglutination assay with polyclonal rabbit anti-ECA antiserum as previously described (26).

Western blot analyses.Western blot analyses were carried out as previously described (26), except that SDS-PAGE was conducted by using 10% polyacrylamide gels, and C-terminal His₆-tagged WecF was detected by using mouse anti-tetra-His antibody (Qiagen), horseradish peroxidase-conjugated anti-mouse IgG (Amersham), and Western blot Chemiluminescence Reagent Plus (NEN Life Science Products).

Chromatographic and electrophoretic procedures.Paper chromatography was carried out by using SG-81 paper (Whatman) that was prepared as previously described (28). Samples were spotted onto the paper, and the chromatogram was developed with a solvent mixture containing chloroform-methanol-water-concentrated ammonium hydroxide (88:48:10:1, by volume). Lanes of the air-dried chromatogram were cut into 1-cm sections, and the sections were soaked in 0.5 ml of 1.25% SDS for 6 to 8 h at 42°C. The samples were then analyzed for radioactivity by liquid scintillation counting.

Nucleotide sequence accession number.The revised nucleotide sequence of the region of the wec gene cluster betweeno416 and wecG has been deposited in the GenBank database under accession number AF375882 .