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J. Biol. Chem., Vol. 275, Issue 45, 34954-34962, November 10, 2000

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3-Deoxy-D-manno-oct-2-ulosonic Acid (Kdo) Transferase (WaaA) and Kdo Kinase (KdkA) of Haemophilus influenzae Are Both Required to Complement a waaA Knockout Mutation of Escherichia coli^*

Werner Brabetz, Sven Müller-Loennies, and Helmut Brade

From the Division of Medical and Biochemical Microbiology, Research Center Borstel, Center for Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany

Received for publication, June 15, 2000, and in revised form, August 21, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The lipopolysaccharide (LPS) of the deep rough mutant Haemophilus influenzae I69 consists of lipid A and a single 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residue substituted with one phosphate at position 4 or 5 (Helander, I. M., Lindner, B., Brade, H., Altmann, K., Lindberg, A. A., Rietschel, E. T., and Zähringer, U. (1988) Eur. J. Biochem. 177, 483-492). The waaA gene encoding the essential LPS-specific Kdo transferase was cloned from this strain, and its nucleotide sequence was identical to H. influenzae DSM11121. The gene was expressed in the Gram-positive host Corynebacterium glutamicum and characterized in vitro to encode a monofunctional Kdo transferase. waaA of H. influenzae could not complement a knockout mutation in the corresponding gene of an Re-type Escherichia coli strain. However, complementation was possible by coexpressing the recombinant waaA together with the LPS-specific Kdo kinase gene (kdkA) of H. influenzae DSM11121 or I69, respectively. The sequences of both kdkA genes were determined and differed in 25 nucleotides, giving rise to six amino acid exchanges between the deduced proteins. Both E. coli strains which expressed waaA and kdkA from H. influenzae synthesized an LPS containing a single Kdo residue that was exclusively phosphorylated at position 4. The structure was determined by nuclear magnetic resonance spectroscopy of deacylated LPS. Therefore, the reaction products of both cloned Kdo kinases represent only one of the two chemical structures synthesized by H. influenzae I69.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Haemophilus influenzae is a nonenteric Gram-negative bacterium that is found in the human respiratory tract and may cause severe diseases, in particular septicemia and meningitis in children. One major virulence factor of this pathogen is the type b capsular polysaccharide, which is also the basis of the presently used vaccine (1). In addition, lipopolysaccharides (LPS)¹ play a crucial role in the interaction of this microorganism with the host's immune system. LPS contribute to each stage of pathogenesis of H. influenzae infection including colonization of the upper respiratory tract, systemic dissemination, and the invasion of the central nervous system (2). During these processes, several surface exposed epitopes of the molecule are the subject of high frequency phase variation.

LPS are the major amphiphilic constituents of the outer leaflet of the outer membrane of Gram-negative bacteria. They share a common architecture composed of a membrane-anchored phosphorylated and acylated (16) linked glucosamine disaccharide, termed lipid A, to which a carbohydrate moiety of varying size is attached. The latter always contains 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) linked to lipid A. Mutants that are defective in biosynthetic enzymes of the Kdo-lipid A region are conditionally thermosensitive, indicating that this minimal structure is absolutely required for the integrity of the outer membrane and microbial cell growth (3, 4). Based on these findings, the corresponding enzymes evoked increasing interest as potential targets for new antibiotics against Gram-negative bacteria (5). The lipid A-linked Kdo residue is often substituted with a second Kdo, forming the disaccharide -Kdo-(24)--Kdo, which also has been identified as the minimal core structure in enterobacterial deep rough mutants of the Re-type. Further core sugars are always attached to position 5 of the inner Kdo in wild-type bacteria. In contrast, LPS from H. influenzae contain only a single Kdo residue substituted with phosphate or 2-aminoethanol pyrophosphate in position 4 (6). A deep rough mutant, termed I69, has been obtained from H. influenzae Rd/b⁺ (7), and chemical analyses of its LPS revealed two molecules composed of lipid A and a single Kdo residue phosphorylated either at position 4 or 5 (8, 9). Immunofluorescence with monoclonal antibodies (mAbs) identified both molecules in living bacteria of this strain (10).

Kdo transferases (WaaA) have been described as multifunctional enzymes that are able to transfer several Kdo residues from CMP-Kdo to different precursor molecules forming different linkages, which has not been observed for other glycosyltransferases (11). Bifunctional enzymes capable of synthesizing an (24)-linked Kdo disaccharide have been cloned and characterized from Escherichia coli (12), Acinetobacter baumannii, and Acinetobacter haemolyticus (13). Furthermore, in Chlamydiaceae, even tri- and tetrafunctional WaaA have been identified that are responsible for the biosynthesis of a family-specific surface epitope comprising an -Kdo-(28)--Kdo-(24)--Kdo trisaccharide of diagnostic value (12, 14, 15). In contrast, a monofunctional Kdo transferase activity has been demonstrated together with an LPS-specific, ATP-dependent Kdo kinase from membrane extracts of H. influenzae (16). These data have been recently confirmed by the cloning and in vitro characterization of a Kdo kinase gene (kdkA) (17). However, due to limiting amounts of the in vitro reaction products of KdkA, the position to which phosphate had been transferred has not been determined.

We have established a cloning system based on a defined Re-type E. coli strain that is devoid of the host's Kdo transferase activity and additionally harbors a waaCF mutation within the heptosyltransferase I and II genes involved in the consecutive transfer of L-glycero-D-manno-heptose residues to Kdo. This strategy allowed us to characterize LPS that were synthesized in vivo by cloned Kdo transferases without interfering activity of the essential host-specific enzyme (18). Using this approach, we now show that the cloned monofunctional waaA from H. influenzae is not able to complement a knockout mutation within the corresponding gene of E. coli; however, cloning of both waaA and kdkA from a wild type or the I69 strain of H. influenzae, respectively, we are able to complement the mutation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- [-³²P]ATP (4 × 10¹⁵ Bq/mol) was obtained from ICN Biochemicals. Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs. CTP, NAD, isopropyl--D-thiogalactoside, hemin, and antibiotics were obtained from Sigma. Silica Gel 60 thin layer chromatography (TLC) plates were from Merck. Kdo and synthetic tetraacyl lipid A precursor compound 405 (1-monophosphoryl) and 406 (bisphosphoryl) (19) were gifts of P. Kosma (University of Agricultural Sciences, Vienna, Austria) and S. Kusumoto (Osaka University, Osaka, Japan), respectively. CMP-Kdo synthetase was partially purified from Corynebacterium glutamicum R163/pJKB14 as described (13), and lipid A 4'-kinase was prepared from E. coli BLR(DE3)/pLysS/pJK2 according to Garrett et al. (20).

Plasmids, Bacterial Strain, and Growth Conditions-- Plasmids and bacterial strains used in this study are listed in Table I. H. influenzae strains were cultivated at 37 °C in brain-heart-infusion (Life Technologies, Inc.) supplemented with 10 mg/liter hemin and 10 mg/liter NAD as described (21). E. coli and C. glutamicum strains were cultivated at 37 and 30 °C, respectively, in Luria-Bertani medium (10 g/liter casein peptone, 5 g/liter yeast extract, 5 g/liter NaCl, pH 7.2; compounds were from Life Technologies) supplemented with the appropriate antibiotics (both strains: 20 mg/liter kanamycin sulfate; E. coli only: 50 mg/liter streptomycin sulfate, 12.5 mg/liter tetracycline/HCl, 100 mg/liter ampicillin). Isopropyl--D-thiogalactoside (1 mM) was added to induce recombinant genes that had been cloned under transcriptional control of the tac or trc promoter.

General Cloning Techniques and Sequence Analysis-- Most DNA procedures were done according to standard techniques (22). All polymerase chain reactions (PCRs) used for cloning were performed with Pfu DNA polymerase (Stratagene), which exhibits proofreading activity. The digoxygenin-11-dUTP system (Roche Diagnostics) was used for DNA labeling and detection in Southern experiments according to the instructions of the manufacturer. DNA sequence analysis of both strands of the cloned genes was done by cycle sequencing with fluorescent dye terminators and sequence-specific primers on an ABI 377 sequencing automat (Perkin-Elmer). The computer programs genworks (Intelligenetics), custalx1.8 (23), genedoc (K. B. Nicholas and H. B. Nicholas, Jr.),² and blast (servers at the National Center for Biotechnology Information (NCBI)) were used to analyze DNA and deduced amino acid sequences.

Plasmid Constructions-- The primers HinfI (5'-ATATGGATCCGAATTTCTTTATGTGGCG-3', BamHI site underlined and start codon in boldface type) and HinfII (5'-ATATCTGCAGCGTCATACATTGCGCTCC-3', PstI site underlined and stop codon in boldface type) were used to amplify by PCR a 1297-bp waaA-encoding fragment from chromosomal DNA of H. influenzae I69. The amplificate was cut with BamHI and PstI and ligated with pCB20 (13), which had been linearized with the same restriction enzymes. The resultant construct, termed pCB23, encoded the Kdo transferase under transcriptional control of the tac promoter (Fig. 1A). The expression cassette with the tac promoter, waaA of H. influenzae I69, and the kanamycin resistance gene (kan) of pCB20 was amplified from pCB23 with the primers W151 (5'-TTTTTCGTCGACGGTACCCGG-3', SalI site underlined) and W152 (5'-AGAAAGTGGTCGACCCACGGTTGATG-3', SalI site underlined), cut with SalI, and ligated into the SalI site of pJSC2 (4). The construct with the orientation of the inset shown in Fig. 1B was selected and termed pJKB49. The recombinant strain was cultivated at 30 °C due to the temperature-sensitive origin of replication of pJSC2 (4). The kdkA genes were amplified by PCR from chromosomal DNA of H. influenzae DSM11121 and H. influenzae I69 using the primers HIK3 (5'-ATAACAACGTCATGACCCACCAATTCCAACAAG-3', BspHI site underlined and start codon in boldface type) and HIK4 (5'-TCATAATTAGGTACCTTATTGATGATAAGCTGACGT-3', KpnI site underlined and stop codon in boldface type). The 755-bp amplificates were cut with BspHI and KpnI and ligated with pCF-TET (25), which had been linearized with NcoI and KpnI. The resultant plasmids that encoded the kdkA genes of H. influenzae DSM11121 or H. influenzae I69 under transcriptional control of the trc promoter were termed pJKB113 and pJKB113A, respectively (Fig. 1C). In addition, PCR fragments that encoded waaA or kdkA of H. influenzae DSM11121 and H. influenzae I69 with additional sequences around the open reading frames were amplified from chromosomal DNA and sequenced to determine the 5'- and 3'-ends of the genes including the binding sites of the cloning primers. For this purpose, the primer pairs HIWAAA1 (5'-AATGACCGTTCGTAATCGG-3') and HIWAAA2 (5'-ATTGCAACGCTAATTGTAGGC-3') were used in the case of waaA, and HIKDKA1 (5'-CTTCTGGGCTTTCAATGCC-3') and HIKDKA2 (5'-TAAGGCATGACAGACATCGC-3') were used in the case of kdkA.

Construction and Genetic Characterization of Chromosomal Knockout Mutations-- The plasmids pJKB113 and pJKB113A were linearized with PvuII and ScaI and transformed into E. coli JC7623 (26), which is recBC sbcBC and, thus, could be used to transfer the expression cassettes with the kdkA genes of the H. influenzae strains DSM11121 and I69 together with the tetracycline resistance gene of pCF-TET to the chromosomal waaCF locus by homologous recombination in one step (27). The corresponding strains derived from pJKB113 and pJKB113A were termed E. coli WBB21 and E. coli WBB32, respectively. Both recombinant strains were further transformed with pJKB49 that had been linearized with EcoRI and AflII to integrate the waaA gene from H. influenzae together with the kanamycin resistance gene (kan) into the chromosomal waaA locus. The corresponding derivatives of E. coli WBB21 and E. coli WBB32 were termed E. coli WBB22 and E. coli WBB34, respectively. E. coli WBB01 (25), which is a waaCF derivative of strain E. coli JC7623, was used as a control recipient of linearized pJKB49 DNA. All chromosomal knockout mutants were characterized by Southern experiments, PCR, and DNA sequence analysis of the recombinant genes reamplified from the chromosomes. For this purpose, the primers LACP (5'-CGGCTCGTATAATGTGTGGA-3') and WSB10R (5'-CGATTGTAAAACAGGCTGGC-3') were used to amplify by PCR an 824-bp kdkA encoding fragment from chromosomal DNA of the recombinant strains that was not present in the wild type strain. The primers EC49A (5'-TGATCTGGATACGGCTCTGG-3') and EC49B (5'-GCAGCAATCAGCGTAATACG-3') were used to amplify from E. coli K-12 wild type DNA a 499-bp fragment around the single SalI site that is located within the waaA gene (see Fig. 1B). The same PCR revealed a single product of 3057 bp in the cases of E. coli WBB22 and E. coli WBB34.

Analysis of Kdo Transferase Activity-- The preparation of cell extracts from recombinant strains and standard conditions for the Kdo transferase assay were performed as described (13). The protein content in cell extracts was determined using a commercial reagent (Bradford kit; Bio-Rad) and bovine serum albumin as a standard. The enzyme reaction mixture contained in a total volume of 20 µl of 50 mM Tris/HCl, pH 7.5, 10 mM MgCl₂, 3.2 mM Triton X-100, 2 mM Kdo, 5 mM CTP, 0.1 mM synthetic bisphosphorylated tetraacyl lipid A precursor 406 (18), 1.67 picokatal of CMP-Kdo synthetase and cell extracts from recombinant C. glutamicum strains (40 µg of protein). The in vitro tests were incubated up to 60 min at 37 °C and stopped by spotting 5 µl onto a TLC plate. Kinetic experiments were performed with 40-µl reaction mixtures from which 5-µl samples were withdrawn at different time points and stopped with 10 µl ice-cold ethanol before spotting onto TLC plates. TLC plates were developed with chloroform/pyridine/88% formic acid/water in a ratio of 30:70:16:10 (by volume). Radioactive [4'-³²P]406 was synthesized from monophosphorylated lipid A precursor 405 as described (27) using recombinant lipid A 4'-kinase prepared from E. coli BLR(DE3)/pLysS/pJK2 (19). The specific activity was adjusted with unlabeled 406 to approximately 15,000-20,000 cpm/nmol. Radioactive products were detected and quantified with a PhosphorImager equipped with the software ImageQuant (Molecular Dynamics).

Preparation of Compound Kdo-406-- A standard enzyme reaction with radioactively labeled acceptor 406 and cell extract of C. glutamicum R163/pCB23 was scaled up to 800 µl and incubated for 1 h at 37 °C. The reaction products were separated by TLC as described above. The product Kdo-406 was identified by autoradiography and isolated from the TLC plate according to Brozek et al. (28). The yield of Kdo-406 (39.7%) was calculated from its glucosamine content, which was determined according to published procedures (29).

SDS Polyacrylamide Gel Electrophoresis and Immunological Detection for LPS-- Protein-free whole cell lysates were prepared as described (25). The samples were separated by SDS polyacrylamide gel electrophoresis (15% total acrylamide, 3.3% cross-linker) and silver-stained according to Tsai and Frasch (30). Immunostaining of LPS after blotting onto nitrocellulose membranes was done according to Löbau et al. (15). Colony immunoblots were performed as described (25). The specificities of mAbs used in this study were described elsewhere (mAb A20 (31); mAbs S42-16 and S42-21 (10)) and are indicated in the legend to Fig. 5 and under "Results."

Preparation and Chemical Analysis of Bacterial LPS-- E. coli WBB22 was cultivated for 24 h at 30 °C in 6 liters of LB supplemented with tetracycline and kanamycin sulfate. The cells were centrifuged at 4 °C; washed 10 min with 200 ml of ice-cold 0.9% NaCl, 16 h with 200 ml of 96% ethanol, and twice for 3 h with 200 ml of acetone; and air-dried (yield: 1.1 g of dry cells/liter). Bacterial LPS was obtained by extraction of the dried bacteria with phenol-chloroform-petrolether as described (32) (yield: 4.1%). Analyses of GlcN, Kdo, and phosphate were performed as described (29). LPS (50 mg, containing 30 µmol of GlcN) was de-O-acylated by mild hydrazinolysis as described (33) (yield: 34 mg, 21 µmol of GlcN, 70%). De-O-acylated LPS (30 mg) was subjected to de-N-acylation by strong alkaline treatment as described (33). After neutralization with hydrochloric acid and desalting on Sephacryl G-10 (Amersham Pharmacia Biotech) in pyridinium actetate, pH 4.0, a single fraction was obtained, which was analyzed by analytical high performance anion exchange chromatography (HPAEC) on a Dionex DX 300 chromatography system equipped with a Dionex CarboPac PA 1 column (4.5 × 250 mm), eluted at 1 ml/min using eluents A (water) and B (1 M sodium acetate, pH 6.0) and a linear gradient of 0-60% B in 70 min.

NMR Spectroscopy-- NMR spectra were recorded on a solution of 5 mg of oligosaccharide (in 500 µl of D₂O, 99.99%; Sigma) at pD 2.8 after three deuterium exchanges by evaporation at reduced pressure. All spectra were recorded at a temperature of 300 K using standard Bruker pulse programs. ¹H NMR and ¹H,¹³C COSY spectra were recorded on a Bruker DRX600 spectrometer (600.13 MHz, measured relative to acetone, 2.225 ppm) equipped with a 5-mm multinuclear inverse probe head with Z-gradient. One-dimensional ¹³C NMR (90.6 MHz, relative to acetone, 31.07 ppm) and ³¹P spectra (145.8 MHz, relative to 85% H₃PO₄, 0.00 ppm) were recorded on a Bruker DPX360 spectrometer equipped with a 5-mm multinuclear inverse Z-gradient probe head. ¹³C and ³¹P NMR chemical shift assignments were achieved by inverse heteronuclear multiple quantum coherence (HMQC) experiments (34) in phase-sensitive mode using states-time proportional phase incrementation (35). The ¹H,¹³C HMQC spectrum was recorded by sampling 2048 data points in t2 and 256 increments of 32 scans in t1. Garp decoupling was applied during t2. The spectral width was 110 ppm in F1 and 10 ppm in F2. Prior to Fourier transformation, a qsine window function was applied in F2, and a sine bell window function was applied in F1. The data matrix was zero-filled in F1 to 512 data points. The ¹H,³¹P HMQC spectrum was recorded over a spectral width of 10 ppm in F2 and 14 ppm in F1. 256 increments of 40 scans each were collected acquiring 2048 data points in F2. ¹H,¹H COSY was performed in phase-sensitive mode as double quantum filtered (DQF)-COSY using the cosydqfprtp pulse program acquiring 4096 data points in F2 and 512 increments in F1 over a spectral width of 4496 Hz. Prior to Fourier transformation, data were multiplied with a squared sine bell window function. Coupling constants were determined on a first order basis. The data matrix was zero-filled in the F1 dimension to give a matrix of 4096 × 1024 data points. The ROESY experiment was performed using the roesyprtp pulse program with a mixing time of 250 ms collecting 2048 data points in the F2 dimension and 512 t1 increments in the F1 dimension over a spectral width of 6009 Hz.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cloning and Sequence Analysis of waaA from H. influenzae I69-- The waaA gene was amplified by PCR from chromosomal DNA of H. influenzae I69, ligated downstream of the tac promoter into the E. coli/C. glutamicum shuttle vector pCB20 and transformed into E. coli XL-1Blue to give the plasmid pCB23 (Fig. 1A; for details, see "Experimental Procedures"). The nucleotide sequence of the cloned waaA gene was determined and shown to be identical to the corresponding DNA region of H. influenzae DSM11121 (identical to strain ATCC 51907, which has been used to sequence the whole genome (36)).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Plasmid constructs. A, the shuttle plasmid pCB23 in which the Kdo transferase gene from H. influenzae I69 (waaAHI, horizontal hatched bar) was cloned downstream of the plasmid-encoded tac promoter (Ptac) was used for gene expression in C. glutamicum. The kanamycin resistance gene (kan, open box), and the origins of replication for E. coli (oriEc1, thin black line) and C. glutamicum (oriCg, vertically hatched box) are indicated. B, the plasmid pJKB49 harbors the gene expression cassette (shown above) with the tac promoter (Ptac), the Kdo transferase gene from H. influenzae (waaAHI, horizontal hatched bar), and the kanamycin resistance gene (kan, open box) integrated into the single SalI site within the waaA gene of E. coli, which was encoded by the plasmid pJSC2 (shown below). Chromosomal DNA fragments of E. coli with the 5'-end of waaQ (heptosyltransferase III gene), waaA, coaD (phosphopantetheine adenylyltransferase gene), and the 3'-end of fpg (formamidopyrimidine-DNA glycosylase gene) are drawn as a black box. A thin line represents the vector part with the chloramphenicol resistance gene (cat) and the temperature-sensitive origin of replication for E. coli (oriEcts). (C) The plasmids pJKB113 and pJKB113A contain, as shown at the top, the tetracycline resistance gene (tet) and the gene expression cassette with the trc promoter (Ptrc), the Kdo kinase genes from H. influenzae DSM11121 (pJKB113) or I69 (pJKB113A) (kdkA, hatched box), respectively, inserted into the plasmid-encoded heptosyltransferase I and II genes (waaC and waaF, respectively) of E. coli. An 849-bp DNA fragment that included the 3'-part of waaF and the 5'-part of waaC was deleted in these constructs (indicated by ). A chromosomal DNA fragment from E. coli (black box), which encoded gmhD (ADP-L-glycero-D-manno-heptose-4-epimerase gene), waaF, waaC, and waaL (unassigned gene), and the vector part (thin line) with the origin of replication for E. coli (oriEc2) and the ampicillin resistance gene (bla) are indicated. All restriction sites mentioned in this paper are shown; those that were inactivated during the constructions are depicted in parenthesis.

In Vitro Characterization of the Cloned Kdo Transferase from H. influenzae I69-- The plasmid pCB23 was transformed into the Gram-positive host C. glutamicum R163 from which cell extracts were prepared and subjected to in vitro assays. The enzyme was able to transfer one Kdo residue to the acceptor 406 (Fig. 2, lane 3), and its activity depended on the presence of Kdo and CMP-Kdo synthetase (Fig. 2, lanes 3-6). CTP could be provided, but in limiting amounts, from cell extracts of recombinant C. glutamicum (Fig. 2, lane 7). A negative control with a cell extract from C. glutamicum R163/pCB20, which harbored the cloning vector without insert revealed no conversion of the (4'-³²P]-radiolabeled compound 406 (data not shown). A small amount of an additional compound that had the same Rf value as the reaction product Kdo₂-406 formed by the recombinant WaaA of E. coli (cell extract from C. glutamicum R163/pJKB16) could be detected (Fig. 2, lanes 3 and 4). Therefore, Kdo-406 was isolated from a scaled up reaction mixture that had been performed with the cell extract of C. glutamicum R163/pCB23 (Fig. 3, lane 2), quantified, and used as an acceptor for the recombinant Kdo transferase of H. influenzae (Fig. 3, lanes 4-8). Almost no transfer of Kdo could be observed using Kdo-406 concentrations of 10 µM (Fig. 3, lane 4) or 100 µM (Fig. 3, lane 5). In the absence of a CMP-Kdo-generating system, even small amounts of 406 were liberated in an enzyme-dependent manner from Kdo-406 (Fig. 2B, lanes 6 and 8). A linear increase of Kdo-406 was observed within the first 20 min at 37 °C using acceptor 406 and the recombinant Kdo transferase from H. influenzae (Fig. 4, closed circle). The specific activity of the enzyme within the cell extract of C. glutamicum R163/pCB23 was calculated to 1.2 nmol min¹ mg of protein¹. Small amounts of Kdo-406 were also formed in vitro by the recombinant WaaA of E. coli (Fig. 4, open circle) under the same reaction conditions. However, Kdo₂-406 was identified as the major in vitro product of this enzyme (Fig. 4, open squares), which is known to be bifunctional (12). The specific activity of the Kdo₂-406 formation within the cell extract of C. glutamicum R163/pJKB16 was calculated to 6.7 nmol min¹ mg of protein¹. Thus, we concluded that the cloned waaA from H. influenzae encoded a monofunctional Kdo transferase, which was in contrast to the bifunctional activity of the corresponding enzyme from E. coli but in agreement with data published for membrane preparations from another strain of H. influenzae (16).

View larger version (52K): [in this window] [in a new window]   Fig. 2.   In vitro activity of the cloned Kdo transferase from H. influenzae I69 with bisphosphorylated tetraacyl lipid A precursor 406 as an acceptor. The enzyme reactions were performed in 50 mM Tris/HCl, pH 7.5, with cell extracts from C. glutamicum R163/pCB23 (133 µg of protein) and incubated for 1 h at 37 °C. The products were separated by TLC and recorded with a PhosphorImager. Lane 1, isolated compound 406; lane 2, isolated compound Kdo-406; lane 3, complete reaction mixture with cell lysate from C. glutamicum R163/pJKB16 (waaA from E. coli); lane 4, complete reaction mixture with cell lysate from C. glutamicum R163/pCB23 (waaA from H. influenzae); lane 5, as lane 4 but without Kdo; lane 6, as lane 4 but without CTP; lane 7, as lane 4 but without CMP-Kdo synthetase.

View larger version (59K): [in this window] [in a new window]   Fig. 3.   In vitro activity of the cloned Kdo transferase from H. influenzae I69 with isolated Kdo-406 as an acceptor. The enzyme reactions were performed in 50 mM Tris/HCl, pH 7.5, with cell extracts from C. glutamicum R163/pCB23 (133 µg of protein) and incubated for 1 h at 37 °C. The products were separated by TLC and recorded with a PhosphorImager. Lane 1, isolated compound 406; lane 2, isolated compound Kdo-406, lane 3, complete reaction mixture with cell lysate from C. glutamicum R163/pJKB16 (waaA from E. coli); lane 4, complete reaction mixture with cell lysate from C. glutamicum R163/pCB23 (waaA from H. influenzae) and 10 µM Kdo-406; lane 5, as lane 4 but 100 µM Kdo-406; lane 6, as lane 5 but without Kdo; lane 7, as lane 5 but without CTP; lane 8, as lane 5 but without CMP-Kdo synthetase.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Kdo transferase activities in cell extracts of recombinant C. glutamicum strains. Enzyme reactions were performed in 50 mM Tris/HCl, pH 7.5, at 37 °C using 100 µM radiolabeled compound 406 as an acceptor. Samples were withdrawn at different time points, stopped with ice-cold ethanol, developed by TLC, and quantified with a PhosphorImager. Extracts from C. glutamicum R163/pCB23 (133 µg of protein; WaaA from H. influenzae I69 (, Kdo-406)) or C. glutamicum R163/pJKB16 (200 µg of protein, WaaA from E. coli (, Kdo-406; , Kdo2-406)) were used.

Cloning and Sequence Analysis of kdkA from H. influenzae DSM11121 and H. influenzae I69-- The kdkA genes (17) encoding LPS-specific Kdo kinase were amplified from chromosomal DNA of H. influenzae DSM11121 and I69, and both DNA fragments were cloned into the plasmid pCF-TET (25) under transcriptional control of the trc promoter (Fig. 1C; for details, see "Experimental Procedures"). The corresponding derivatives of this plasmid with the kdkA genes were termed pJKB113 (H. influenzae DSM11121) and pJKB113A (H. influenzae I69). The nucleotide sequences of both kdkA genes were determined; that from H. influenzae DSM11121 was identical to the published one (36), whereas kdkA from H. influenzae I69 revealed 25 different nucleotides, six of which gave rise to altered amino acids between the deduced protein sequences (data not shown).

Construction of Deep Rough E. coli Strains That Express kdkA and waaA of H. influenzae-- The plasmid pJKB49 was constructed (Fig. 1B; for details, see "Experimental Procedures") to address the question of whether the monofunctional Kdo transferase of H. influenzae could complement a waaA knockout mutation within the deep rough E. coli WBB01. The vector contained the expression cassette with the tac promoter, the waaA gene of H. influenzae, and the kanamycin resistance gene amplified from pCB23 and integrated into the single SalI site within the waaA gene of E. coli, which was encoded by the insert of plasmid pJSC2. Several attempts to move by homologous recombination (27) the defective waaA allele with the inserted Kdo transferase gene of H. influenzae and the kanamycin resistance gene to the chromosomal waaA locus of E. coli failed, although different incubation temperatures between room temperature and 37 °C were applied or isopropyl--D-thiogalactoside was added to the agar plates to induce the recombinant waaA gene. Thus, we concluded that waaA of H. influenzae was not able to complement a knockout mutation within the corresponding chromosomal gene of the Re-type E. coli WBB01.

The plasmids pJKB113 and pJKB113A, which harbored the expression cassettes with the tetracycline gene, the trc promoter, and the kdkA genes from H. influenzae inserted into the plasmid-encoded waaCF genes were then linearized with PvuII and ScaI (see Fig. 1C) and transformed into E. coli JC7623 (recBC sbcBC; parent of E. coli WBB01) to move the defective allele together with the kdkA genes to the chromosomal waaCF locus by homologous recombination. The resultant strains E. coli WBB21 and WBB32 exhibited the waaCF::tet6 mutation of E. coli WBB01 but contained in addition the kdkA genes of H. influenzae DSM11121 and I69, respectively, integrated into the chromosomes. The genotypes of these strains were confirmed by PCR and Southern hybridization (data not shown). In contrast to E. coli WBB01, competent cells of E. coli WBB21 and WBB32 could be successfully transformed with linearized pJKB49 DNA at 30 °C, resulting in the strains E. coli WBB22 and WBB34, respectively. The presence of the waaA gene of H. influenzae as well as the absence of an intact copy of the waaA gene from E. coli within the chromosomes of these strains was confirmed by PCR and Southern hybridization (data not shown). These results showed that both genes, waaA and kdkA, of H. influenzae are essential but also sufficient to complement a waaA knockout mutation within the deep rough E. coli WBB01.

The growth of the recombinant strains that expressed genes of H. influenzae was tested in comparison with E. coli WBB01. All strains were nonmotile and showed cell division defects forming cell filaments due to their recBC sbcBC (37) and waaCF (25) genotype. The strains E. coli WBB21 and WBB32 revealed the same growth characteristics as WBB01, which displayed growth reduction above 37 °C (data not shown). The living cell counts (colony-forming units) of growing cultures (A₆₀₀ = 0.6-0.7) were compared at different temperatures. E. coli WBB01, WBB21, and WBB32 revealed smaller and approximately 10-18% fewer colonies after 24 h at 42 °C than at 30-37 °C. In contrast, growth reduction was more pronounced in the case of E. coli WBB22 and WBB34, which only expressed waaA from H. influenzae together with the kdkA genes. The latter strains formed 70-85% fewer colonies at 37 °C as compared with 30 °C, and no visible colonies were detected after 24 h at 42 °C.

Characterization of LPS from Deep Rough E. coli Strains That Express kdkA and waaA of H. influenzae Using mAbs-- LPS were analyzed in protein-free cell lysates of the bacteria by SDS polyacrylamide gel electrophoresis and silver staining (Fig. 5A). The LPS of all recombinant strains possessed similar electrophoretic mobilities as compared with the Re-type E. coli WBB01 (Fig. 5A, lane 2) or the deep rough H. influenzae I69 (Fig. 5A, lane 3). A Western blot analysis with mAb A20 (31) (Fig. 5B), which recognizes a single Kdo residue, gave positive results with LPS of E. coli WBB01, WBB21, and WBB32 but not with the samples of H. influenzae I69 and E. coli WBB22 and WBB34. In contrast, LPS from the recombinant strains E. coli WBB22 and WBB34 reacted with mAb S42-21 (Fig. 5C), which recognizes -Kdo-4-phosphate, but not with mAb S42-16 (Fig. 5D), which is specific for -Kdo-5-phosphate (10). The specificity and sensitivity of mAb S42-16 was confirmed by the positive staining of LPS from H. influenzae I69 (Fig. 5D, lane 3).

View larger version (55K): [in this window] [in a new window]   Fig. 5.   Characterization of LPS from recombinant E. coli strains that express waaA and kdkA from H. influenzae DSM11121 and I69 using monoclonal antibodies. LPS from protein-free cell lysates of strain E. coli JC7623 (lane 1), E. coli WBB01 (lane 2), H. influenzae I69 (lane 3), E. coli WBB21 (lane 4), E. coli WBB32 (lane 5), E. coli WBB22 (lane 6), and E. coli WBB34 (lane 7) were separated by SDS polyacrylamide gel electrophoresis and stained with alkaline silver nitrate (A) or blotted onto nitrocellulose membranes and developed with monoclonal antibody A20 (B; recognizing a single Kdo residue), S42 21 (C; recognizing -Kdo-4P), or S42-16 (D; recognizing -Kdo-5P).

Chemical Characterization of LPS from E. coli WBB22 and WBB34-- LPS were extracted from E. coli WBB22 and WBB34 and subjected to successive de-O- and de-N-acylation. After gel permeation chromatography, one LPS fraction was obtained for both recombinant strains, which consisted of a single oligosaccharide eluting at 41.1 min in analytical HPAEC (Fig. 6, B and C). In contrast, LPS from H. influenzae I69 revealed two fractions by HPAEC, which eluted at 41.1 (I in Fig. 6A) and 46.2 min (II in Fig. 6A), respectively, and could be assigned by NMR analyses to -Kdo-4P-(26)-GlcN-4P-(16)-GlcN-1P (I) and -Kdo-5P-(26)-GlcN-4P-(16)-GlcN-1P (II) (data not shown). The oligosaccharide obtained from the LPS of strain WBB22 was further characterized by nuclear magnetic resonance spectroscopy. ¹H NMR spectra contained two signals of anomeric protons at 5.722 and 4.854 ppm belonging to two GlcN residues (Fig. 7, A and B, respectively), as shown by full assignment of proton and carbon signals in two-dimensional ¹H,¹H COSY and ¹H,¹³C HMQC spectra (Tables II and III). In addition, two signals of deoxy protons of a single -Kdo residue (Fig. 7C) at 2.028 and 2.204 ppm (axial and equatorial H3) were present. The ³¹P NMR spectrum contained three signals at 1.5, 0.45, and 0.53 ppm. Glycosidic phosphorylation of residue A was evident from an additional coupling of its anomeric proton (J_H,P, 6 Hz), and couplings of its anomeric carbon (J_C,P, 5.2 Hz) and carbon C-2 (J_C,P, 8.8 Hz). Couplings of C-4 (J_C,P, 5.2 Hz) and C-5 (J_C,P, 7.0 Hz) of residue B and a downfield shift of carbon C-4 indicated phosphorylation at this position of residue B. Likewise, signals of carbons 4 (J_C,P, 4.6 Hz) and 5 (J_C,P, 3.0 Hz) of residue C (Kdo) were split due to phosphorylation at C-4, leading to far downfield shifts of signals of C-4 and in particular of H-4 (Fig. 7). In DQF-COSY (Fig. 8), there was no signal of Kdo H-4 at higher field as would be expected for a Kdo residue phosphorylated in position 5. Therefore, position 4 was quantitatively phosphorylated, and phosphate at position 5 was not present. Sites of phosphorylation were confirmed by ¹H,³¹P HMQC, which showed cross-correlation signals to protons at phosphorylated sites. These results thus confirmed the analytical HPAEC analysis of crude deacylated LPS, which showed a single peak corresponding to the 4'-phosphorylated molecule (Fig. 6). ROESY showed ROE contacts between H-1 of residue B and H-6a (weak) and H-6b (strong) of residue A. Thus, residue B was linked to position 6 of residue A. Since Kdo is a ketose lacking an anomeric proton, no ROE contacts were observed between residues C (Kdo) and B. Comparison of chemical shift values with published data of substances that possess the same sugar sequence proved the substitution at position 6 of the -GlcN residue (B), which is in accordance with all other bacterial lipopolysaccharides investigated so far. Since the results of Western blot and HPAEC analysis did not provide any evidence that the LPS of strain WBB34 differed from that of WBB22, the NMR analyses of the phosphorylated and deacylated carbohydrate backbone from LPS of strain WBB34 was not repeated.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Analytical HPAE-chromatograms of deacylated LPS obtained from H. influenzae I69 (A), E. coli WBB22 (B), and E. coli WBB34 (C). I, -Kdo-4P-(26)-GlcN-4P-(16)-GlcN-1P; II, -Kdo-5P-(26)-GlcN-4P-(16)-GlcN-1P.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   1H NMR spectrum recorded at 600 MHz and chemical structure of deacylated LPS of E. coli WBB22. Assignments are shown for characteristic 1H reporter signals.

View larger version (11K): [in this window] [in a new window]   Fig. 8.   1H,1H DQF-COSY spectrum of the deacylated LPS of E. coli WBB22. The far downfield shift of H-4 of residue C (Kdo) is indicated, proving the phosphorylation exclusively at this position and not at position 5.

In summary, E. coli WBB22 and WBB34 produced LPS composed of a single terminal -Kdo-residue, which is exclusively phosphorylated at position 4 and is linked to the 6'-position of the lipid A backbone (GlcN-4P-(16)-GlcN-1P). It therefore represents only one of the two chemical structures known to be present in H. influenzae I69 LPS (Fig. 6A) (9, 10).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The waaA and kdkA genes of the Rd strain H. influenzae DSM11121 and the deep rough mutant I69 were cloned and sequenced. Both Kdo transferase genes had identical nucleotide sequences, whereas differences could be observed between the two Kdo kinases with respect to their gene and deduced protein sequences. A TBLASTN search with both KdkA amino acid sequences was performed at NCBI and revealed positive results (E < 10¹⁸) with data from H. influenzae and the unfinished genomes of Acinetobacillus actinomycetemcomitans, Pasteurella multocida, Vibrio cholerae, Shewanella putrefaciens and Bordetella pertussis. These bacteria belong to the families Pasteurellaceae or Vibrionaceae, members of which are known to possess LPS with single Kdo residues phosphorylated at position 4 (6). An amino acid sequence alignment of KdkA revealed a conserved amino acid sequence motif within the C-terminal half of all proteins, which closely resembles the consensus pattern of active sites from bacterial aminoglycoside phosphotransferases (38) as well as pro- and eukaryotic protein kinases (39) (Fig. 9A). The proposed consensus pattern for KdkA (Fig. 9B, 1) only differs from that of serine/threonine-specific (Fig. 9B, 2; prosite PS00108) or tyrosine-specific (Fig. 9B, 3; prosite PS00109) protein kinases in a conserved arginine residue separated by one amino acid from the catalytically active aspartate. All different amino acids of the two proteins from H. influenzae matched into nonconserved regions of all aligned sequences (data not shown), although two of them were located 13 residues upstream of the putative active site motif (Fig. 9A).

View larger version (40K): [in this window] [in a new window]   Fig. 9.   Amino acid sequence motif of the active site of KdkA proteins. A, amino acid sequence alignment of the putative active site of KdkA proteins. The single letter code for amino acids is used. Identical amino acids are shaded. Different amino acids between the two sequences from H. influenzae are boxed. HinRd, H. influenzae DSM11121; HinI69, H. influenzae I69; Aac, A. actinomycetemcomitans; Pmu, P. multocida; Vch, V. cholerae; Spu, S. putrefaciens; Bpe, B. pertussis. B, proposed consensus pattern of the active site of KdkA. 1, KdkA pattern; 2, serine/threonine-specific protein kinases (prosite PS00108); 3, tyrosine-specific protein kinases (prosite PS00109); 4, aminoglycoside phosphotransferases. The aspartate residue that is located at position 165 of the H. influenzae sequence and to which the -phosphate residue from ATP may be bound in the catalytically active state is marked with an asterisk.

The recombinant waaA gene was expressed in C. glutamicum. This Gram-positive cloning host is devoid of LPS and Kdo, and thus, the activity of the cloned Kdo transferase could be studied without interference by host cell enzymes (13, 15). The cloned WaaA from H. influenzae was characterized in vitro as a monofunctional enzyme (Figs. 3 and 4), confirming the published data that have been obtained with the purified protein from a nontypeable H. influenzae strain (16). Differences in the activity of the enzyme to the bifunctional WaaA from E. coli (Fig. 4) were further evident from the observation that waaA from H. influenzae could not complement a knockout mutation within the corresponding gene of a deep rough E. coli strain. In contrast, this was possible by coexpressing waaA and kdkA from H. influenzae, and the resultant recombinant E. coli strains WBB22 and WBB34 displayed deep rough LPS containing a single phosphorylated Kdo residue (Figs. 5-8). Thus, the presence of at least two negatively charged groups within the inner core region seems to be a fundamental prerequisite for the functional integrity of the outer membrane in E. coli. This has been also suggested for the deep rough H. influenzae I69 (7-9). However, an isogenic mutant derived from a wild type H. influenzae has been constructed (40) and later was shown to harbor a kdkA knockout (17). This strain still allows the attachment of further core sugars and is viable under laboratory conditions but displays reduced virulence in vivo (40). Although detailed structural data on the LPS of this strain are lacking, a phosphorylated Kdo region seems not to be absolutely required for growth and multiplication of H. influenzae. Recently, Isobe et al. (41) reported on the complementation of an E. coli waaA::kan mutation with the monofunctional Kdo transferase of B. pertussis resulting in a complex LPS phenotype. In this case, two additional genes of the donor strain, waaC and baf, were introduced in high copy numbers, which may also contribute together with heptosyltransferases and other LPS-specific enzymes of the cloning host to the observed complementation. Although these findings suggest that a monofunctional Kdo transferase might be able to substitute for WaaA of E. coli in the absence of KdkA in vivo, our results clearly showed that this is not the case for the isolated gene and its defined biosynthetic step. The strains E. coli WBB22 and WBB34 exhibited a thermosensitive phenotype in comparison with the Re-type E. coli WBB01, indicating that the expression of a Kdo-4-phosphate core is not compatible with the cell physiology of the recipient at higher temperatures. In this context, it is noted that the recipients E. coli JC7623 and WBB01 also exhibit defects in the segregation of chromosomes and cell division due to their recBC sbcBC (37) and waaCF (25) genotype.

LPS representing exclusively reaction products of the recombinant WaaA and KdkA from H. influenzae could be prepared from the E. coli WBB22 and WBB34. The reactivity of LPS from these strains with mAb (Fig. 5) as well as extensive chemical and NMR analyses (Figs. 6-8) unequivocally proved that Kdo-4-phosphate is the single, in vivo synthesized product of both Kdo kinases when expressed in E. coli. This represents only one of the two chemical structures known from H. influenzae I69 LPS (Fig. 6A, I and II) (8-10). Thus, catalytical properties of the cloned KdkA from H. influenzae I69 due to differences in the amino acid sequence as compared with the enzyme from the Rd strain DSM11121 cannot account for the additional formation of Kdo-5-phosphate in the native donor strain. Therefore, additional biochemical factors or enzymatic activities must be discussed for the biosynthesis of this structure in the mutant H. influenzae I69, which is derived from the type b strain RM4066 (42).

LPS with a single phosphorylated Kdo residue are characteristic for many Gram-negative pathogens like representatives of the genus Haemophilus, Bordetella, and Vibrio. Our results show that it is possible to construct a deep rough enterobacterial strain expressing a Kdo-4-phosphate core that is the basic structural element of such LPS. This recombinant E. coli strain can be used as a versatile cloning host and as a source of structurally defined acceptor molecules for the biochemical characterization of further glycosyltransferases involved in the biosynthesis of the inner core LPS.

	ACKNOWLEDGEMENTS

We thank R. Moxon (University of Oxford, Oxford, UK) for strain H. influenzae I69 and C. R. H Raetz (Duke University, Durham, NC) for providing the strains E. coli CJB26 and E. coli BLR(DE3)/pLysS/pJK2. Kdo was a gift of P. Kosma. Compounds 405 and 406 were provided by S. Kusumoto. The technical assistance of A. Denzin, R. Engel and V. Susott is gratefully acknowledged.

	FOOTNOTES

^* This work was supported by Deutsche Forschungsgemeinschaft SFB470/Grant A1 (to H. B.).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.

The nucleotide sequences reported in this paper have been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession numbers AJ277814, AJ277816, AJ277815, and AJ277817.

To whom correspondence should be addressed. Tel.: 49-4537188488; Fax: 49-4537188686; E-mail: wbrabetz@fz-borstel.de.

Published, JBC Papers in Press, August 21, 2000, DOI 10.1074/jbc.M005204200

² Genedoc: Analysis and Visualization of Genetic Variation is available on the World Wide Web.

	ABBREVIATIONS

The abbreviations used are: LPS, lipopolysaccharide(s); Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; mAb, monoclonal antibody; COSY, correlation spectroscopy; DQF, double quantum-filtered; ROE, rotating frame Overhauser effect; ROESY, rotating frame Overhauser effect spectroscopy; HMQC, heteronuclear multiple quantum coherence; HPAEC, high performance anion exchange chromatography; PCR, polymerase chain reaction; bp, base pair(s).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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