The Incorporation of Glucosamine into Enterobacterial Core Lipopolysaccharide

The core lipopolysaccharide (LPS) of Klebsiella pneumoniae is characterized by the presence of disaccharide αGlcN-(1,4)-αGalA attached by an α1,3 linkage to l-glycero-d-manno-heptopyranose II (ld-HeppII). Previously it has been shown that the WabH enzyme catalyzes the incorporation of GlcNAc from UDP-GlcNAc to outer core LPS. The presence of GlcNAc instead of GlcN and the lack of UDP-GlcN in bacteria indicate that an additional enzymatic step is required. In this work we identified a new gene (wabN) in the K. pneumoniae core LPS biosynthetic cluster. Chemical and structural analysis of K. pneumoniae non-polar wabN mutants showed truncated core LPS with GlcNAc instead of GlcN. In vitro assays using LPS truncated at the level of d-galacturonic acid (GalA) and cell-free extract containing WabH and WabN together led to the incorporation of GlcN, whereas none of them alone were able to do it. This result suggests that the later enzyme (WabN) catalyzes the deacetylation of the core LPS containing the GlcNAc residue. Thus, the incorporation of the GlcN residue to core LPS in K. pneumoniae requires two distinct enzymatic steps. WabN homologues are found in Serratia marcescens and some Proteus strains that show the same disaccharide αGlcN-(1,4)-αGalA attached by an α1,3 linkage to ld-HeppII.

Klebsiella pneumoniae is an important nosocomial pathogen (1) causing infections that may occur at almost all body sites with highest incidence in the urinary and the respiratory tracts. The main populations at risk are neonates, immunocompromised hosts, and patients predisposed by surgery, diabetes, malignancy, etc. (1-4). K. pneumoniae typically express both smooth lipopolysaccharide (LPS) 3 and capsule polysaccharide (K-antigen) on its surface, and both antigens (LPS and capsule) contribute to the pathogenesis of this species.
As in other Enterobacteriaceae in the K. pneumoniae LPS three domains are recognized: the highly conserved and hydrophobic lipid A, the hydrophilic and highly variable O-antigen polysaccharide, and the core oligosaccharide (OS) connecting lipid A and O-antigen. The core domain is usually divided into inner and outer core on the basis of sugar composition. The K. pneumoniae core LPS structure has been determined for several O-serotypes. Although the inner core is highly conserved within the Enterobacteriaceae the K. pneumoniae inner core differs from those of Escherichia coli and Salmonella by the lack of L-glycero-D-manno-heptopyranose I and II (LD-HeppI and -II) phosphoryl modifications and by the presence of a D-glucose (Glc) residue linked by a ␤1,4 bond to LD-HeppI (5)(6)(7). Two outer core types (1 and 2) were described in K. pneumoniae, both containing the disaccharide ␣GlcN-(1,4)-␣GalA attached by an ␣1,3 linkage to LD-HeppII (5)(6)(7). Type 1 core contains a 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residue linked by an ␣2,6 bond to the GlcN residue (5,6), whereas type 2 core contains the disaccharide ␤Glc-(1,6)-␣Glc linked by a ␣1,4 bond to the GlcN residue (7) (Fig. 1).
The genes involved in the K. pneumoniae core LPS biosynthesis are clustered in a region (wa) of the K. pneumoniae chromosome, and two different clusters have been identified to be responsible for type 1 and type 2 core biosynthesis (7,8) (Fig. 1). Furthermore the functions in both inner and outer core biosynthesis of most of the transferases encoded by the wa gene products have been elucidated (7)(8)(9)(10)(11)(12) (Fig. 1). Nevertheless the mechanism leading to the incorporation of the outer core GlcN residue remains unknown. In searching for a candidate glucosaminyltransferase the wabH gene product was characterized (12). The WabH protein catalyzes the incorporation of GlcNAc from UDP-GlcNAc into outer core LPS. This transferase activity is dependent on the presence of the outer core D-galacturonic acid (GalA) residue in the acceptor core LPS (12). But because GlcN instead of GlcNAc is found in the K. pneumoniae outer core either an unknown glucosaminyltransferase remains to be identified or a mechanism to deacetylate the WabH-transferred GlcNAc residue should be involved. In this work we report the identification and characterization of such a core LPS-GlcNAc deacetylase activity in K. pneumoniae and Serratia marcescens sharing the outer core disaccharide ␣GlcN-(1,4)-␣GalA.

MATERIALS AND METHODS
Bacterial Strains, Plasmids, and Growth Conditions-Bacterial strains and plasmids used in this study are shown in TABLE ONE. Bacterial strains were grown in LB broth and LB agar (13). LB medium was supplemented with kanamycin (50 g/ml), ampicillin (100 g/ml), chloramphenicol (20 g/ml), and tetracycline (25 g/ml) when needed.
General DNA Methods-Standard DNA manipulations were done essentially as described previously (14). DNA restriction endonucleases, T4 DNA ligase, E. coli DNA polymerase (Klenow fragment), and alkaline phosphatase were used as recommended by the suppliers.
Mutant Construction-K. pneumoniae 52145 and C3 individual genes were mutated by creating in vitro in-frame deletions of each gene (15). Each mutated gene was transferred to the chromosome by homologous recombination using the temperature-sensitive suicide plasmid pKO3 containing the counterselectable marker sacB (15). Mutations were made in wabH and wabN. The plasmids containing the engineered in-frame deletions (pKO3⌬wabH C3 , pKO3⌬wabH 52145 , pKO3⌬waNH C3 , and pKO3⌬wabN 52145 ) were transformed into K. pneumoniae 52145, C3, 52145⌬waaL, and C3⌬waaL by electroporation. Mutants were selected based on growth in LB agar containing 10% sucrose and loss of the chloramphenicol resistance marker of vector pKO3. The mutations were confirmed by sequencing of the whole constructs in amplified PCR products.
Plasmid Constructions for Gene Overexpression and Mutant Complementation Studies-For gene overexpression studies the wabN genes from K. pneumoniae strains 52145 and C3 and S. marcescens N28b were PCR-amplified by using specific primers pairs and transferred into plasmid pABD18-Cm. Each gene was expressed from the arabinose-inducible and glucose-repressible pABD18-Cm promoter (16). Repression from the araC promoter was achieved by growth in medium containing 0.4% (w/v) glucose, and induction was obtained by adding L-arabinose to a final concentration of 0.02% (w/v). Briefly a culture was grown for 18 h at 37°C in LB medium supplemented with chloramphenicol and 0.4% glucose. These cultures were diluted 1:100 in fresh medium (without glucose) and grown until they reached an A 600 nm of about 0.2. L-Arabinose was then added, and the cultures were grown for another 2 h. Repressed controls were maintained in glucose-containing medium.
For pBAD-18-wabN 52145 construction a 2,313-bp DNA fragment containing the wabN gene was PCR-amplified from K. pneumoniae 52145 chromosomal DNA with primers NA 52145 and ND 52145 . This fragment was used as template to amplify an internal 1,649 bp with primers NFw 52145 (5Ј-TTCGATTCTAGAGGGATCAG-3Ј) and ND 52145 . This fragment was digested with XbaI and HindIII to obtain a 1,204-bp fragment that was ligated to the same sites in pBAD18-Cm.
Plasmid pBAD18-Cm-wabN C3 was constructed by PCR amplification of a 2,308-bp DNA fragment from strain C3 chromosomal DNA using primers and NA C3 and ND C3. This fragment was used as template to amplify an internal 1,644 bp with primers FN 52145 and ND C3 . This fragment was digested with BglII, blunt-ended, and then digested with XbaI to obtain a 1,638-bp fragment. This fragment was ligated to pBAD18-Cm, HindIII-digested, blunt-ended, and XbaI-digested.
LPS Isolation and Electrophoresis-LPS was extracted from dry cells of K. pneumoniae grown in LB. The phenol/chloroform/light petroleum ether method (18) was used for strains producing rough LPS, whereas the phenol/water procedure (19) was used for the strains producing the O-antigen domain (smooth LPS). For screening purposes LPS was obtained after proteinase K digestion of whole cells (20). LPS samples were separated by SDS-PAGE or Tricine-SDS-PAGE and visualized by silver staining as described previously (21,22).
Preparation of Oligosaccharides-The LPS (20 mg) was hydrolyzed in 1% acetic acid (100°C for 120 min), and the precipitate was removed by centrifugation (8,000 ϫ g for 30 min) and lyophilized to give lipid A (10 mg, 50% of LPS). The supernatant was evaporated to dryness, dissolved in water, and lyophilized (6 mg, 30% of LPS).
Glycosyl and Lipid Analysis-LPS (1 mg) was dried over P 2 O 5 overnight and treated with 1 M HCl/CH 3 OH (1 ml) at 80°C for 20 h to analyze both glycosyl and fatty acid composition. The crude reaction was extracted twice with hexane; the two extracts were pooled, dried under a stream of air, and treated with acetic anhydride (100 l) at 100°C for 15 min. The methanol layer was neutralized with Ag 2 CO 3 , dried, and acetylated. Both samples were injected into the gas chromatography-MS system, and acetylated fatty acid methyl esters were recovered in the hexane phase, whereas the methylglycoside derivatives were in the methanolic phase.
NMR Spectroscopy-1 H NMR spectra were recorded on a solution (0.5 ml) of K. pneumoniae 52145⌬waaL (8 mg) and 52145⌬waaL wabN (2 mg) core oligosaccharides, respectively, in D 2 O at 400 MHz with a Bruker DRX 400 Avance spectrometer equipped with a reverse probe in the Fourier transform mode at 303 K.
Mass Spectrometry Studies-Positive and negative ions reflectron time-of-flight mass spectra (MALDI-TOF) were acquired on a Voyager DE-PRO instrument (Applied Biosystems) equipped with a delayed extraction ion source. Ion acceleration voltage was 25 kV, grid voltage was 17 kV, mirror voltage ratio was 1.12, and delay time was 150 ns. Samples were irradiated at a frequency of 5 Hz by 337 nm photons from a pulsed nitrogen laser. Postsource decay was performed using an acceleration voltage of 20 kV. The reflectron voltage was decreased in 10 successive 25% steps. Mass calibration was obtained with a maltooligosaccharide mixture from corn syrup (Sigma). A solution of 2,5-dihydroxybenoic acid in 20% CH 3 CN in water at a concentration of 25 mg/ml was used as the MALDI matrix. One microliter of matrix solution was deposited on the target followed by loading of 1 l of the sample. The droplets were allowed to dry at room temperature. Spectra were calibrated and processed under computer control using the Applied Biosystems Data Explorer software.
Methylation Analysis-A core oligosaccharide sample (1 mg) obtained from 1% AcOH hydrolysis was first reduced with NaBH 4 and then methylated (23). Linkage analysis was performed as follows. The methylated sample was carboxymethyl-reduced with lithium triethylborohydride (Super-Hydride, Aldrich), hydrolyzed by mild acid to cleave ketosidic linkage, reduced by means of NaBD 4 , then totally hydrolyzed, reduced with NaBD 4 , and finally acetylated as described previously (24).
Preparation of Cell-free Extracts Containing Core LPS Biosynthetic Enzymes-The K. pneumoniae strains 521453⌬wabH (pBAD18-Cm-wabH 52145 ) and 52145⌬wabN (pBAB18Cm-wabN 52145 ) were used to overexpress the WabH and WabN proteins, respectively. Cultures of these strains and controls (52145⌬wabH and 52145⌬wabN harboring pBAD18-Cm) were grown and arabinose-induced as described above. The cells were harvested, washed once with 50 mM Tris-HEPES (pH 7.5), and then frozen until needed. To prepare lysates cell pellets were resuspended in 50 mM HEPES (pH 7.5) and sonicated on ice (for a total of 2 min using 10-s bursts followed by 10-s cooling periods). Unbroken cells, cell debris, and the membrane fraction were removed by ultracentrifugation at 100,000 ϫ g for 60 min. Protein expression was monitored by SDS-PAGE, and protein contents of cell-free extracts were determined using the Bio-Rad Bradford assay as directed by the manufacturer.
Assay reactions using UDP-GlcNAc as the substrate were carried out in 0.02 ml at a final concentration of 50 mM Tris-HCl (pH 8.0) containing 10 mM MgCl 2 , 1 mM dithiothreitol, 1 mM UDP-GlcNAc, and 0.003 mg of 52145⌬wabH mutant LPS as the acceptor. The reaction was started by addition of 0.04 mg each of the cell-free extracts from 52145⌬wabH (pBAD18Cm-wabH 52145 ) and 52145⌬wabN (pBAD18-Cm-wabN 52145 ). As controls reactions containing cell-free extracts (0.04 mg) of 52145⌬wabH (pBAD18-Cm-wabH 52145 ), 52145⌬wabH (pBAD18-Cm) and/or 52145⌬wabN (pBAD18-Cm-wabN 52145 ) were used. The mixtures were incubated at 37°C for 2 h, and the reactions were stopped by adding 0.08 ml of SDS-PAGE sample buffer and boiling for 10 min. Proteinase K diluted in SDS-PAGE sample buffer was then added to a final concentration of 0.

RESULTS
Identification of a New Core LPS Gene-In the Enterobacteriaceae the genes coding for enzymes involved in core LPS biosynthesis are usually found clustered in a region (wa gene cluster) with the exception of Yersinia sp. where two different clusters exist. Inspection of the known wa gene clusters from Enterobacteriaceae shows a high level of gene density with very short spacing between the different genes. In addition, overlapping start and stop codons between adjacent genes is quite frequent. By contrast, in the K. pneumoniae C3 (type 1 core) and 52145 (type 2 core) wa gene clusters, regions of about 1,000 bp of unknown coding potential, are located upstream of the waaQ gene. The analysis of this region in strain 52145 revealed a potential 328-amino acid residueencoding open reading frame (ORF) suggesting that a previously missed gene could play a role in type 2 core OS biosynthesis; this putative gene was named wabN. The 1,066-bp region was PCR-amplified using oligonucleotides A and B and K. pneumoniae 52145 chromosomal DNA, and the amplicon was cloned in plasmid pGEM-T and transformed into E. coli DH5␣. Plasmid pGEM-T-wabN 52145 was used in an in vitro transcriptional/translation reaction with [ 35 S]methionine. As a control the vector pGEM-T was used. The radiolabeled proteins were resolved by SDS-PAGE, showing a specific radiolabeled polypeptide with a molecular mass of about 38 kDa (data not shown).
The 984-nucleotide wabN gene stop codon is adjacent to the wabM stop codon, and the 5Ј-end of wabN is located 79 nucleotides upstream from waaQ. In a similar position wabN homologues were found in K. pneumoniae C3 (type 1 core) and in S. marcescens (Fig. 1). The strain C3 wabN homologue was located 53 and 32 nucleotides upstream of genes wabI and waaQ, respectively. The S. marcescens homologue was found between ORF7 and waaQ. Additional putative protein homologues were found in Proteus mirabilis HI 4320 and Photorhabdus luminescens laumondii TT01. All these WabN proteins shared similar length and high levels of amino acid identity (58.43%) and similarity (76.25%) (Fig.  2). An InterPro scan search (www.ebi.ac.uk/InterProScan/index.html) identified a domain shared by the glycoside hydrolase/deacetylase superfamily (25).
Isolation of Non-polar wabN Mutants-To determine the role of the wabN gene in core LPS biosynthesis non-polar wabN mutations were isolated by an internal in-frame deletion approach (15). The engineered in vitro wabN mutations were introduced by double recombination (see "Materials and Methods") to wild type K. pneumoniae strains C3 and 52145 and to mutant strains C3⌬waaL and 52145⌬waaL. Candidate mutants were checked by PCR amplification of the mutated chromosomal region, using oligonucleotides C and D, and nucleotide sequence determination.
An initial characterization of the effect of the mutation on core LPS biosynthesis was carried out by LPS isolation and analysis by both SDS-PAGE and Tricine-SDS-PAGE. The core-lipid A region of LPS preparations from strains C3⌬wabN and 52145⌬wabN and their derived dou-ble waaL wabN mutants migrated faster than LPS from wild type strains C3 and 52145 (Fig. 3). This suggests that the wabN mutation causes the loss of one or more sugar residues from the core. In addition LPS preparations from strains C3⌬wabN and 52145⌬wabN lacked O-antigen polysaccharide (Fig. 3). The K. pneumoniae O-antigen polysaccharide is linked to outer core Kdo residue in type core 1 (26) and probably to the last Glc residue in type core 2 (7), suggesting that the residues lost in the wabN mutants reside in the main core LPS branch. The cloned wabN from strain C3 (pGEM-T-wabN C3 ) was introduced into C3⌬wabN and restored wild type LPS migration and O-antigen polysaccharide production (Fig. 3). Both K. pneumoniae wabN homologues (wabN KpC3 and wabN Kp52145 ) complemented the wabN mutations in either of the two genetic backgrounds, C3 or 52145 (Fig. 3). The S. marcescens wabN homologue in pGEM-T-wabN Sm also complemented the K. pneumoniae wabN mutants (Fig. 3). These results demonstrate the presence of a new gene shared by K. pneumoniae and S. marcescens encoding an enzymatic activity necessary for core LPS synthesis.
Characterization of the K. pneumoniae wabN Mutant Core Polysaccharide-To determine the core LPS changes produced by the wabN mutation, LPS was obtained from strain 52145⌬wabN waaL by the phenol, chloroform, and petroleum ether method (18). Composition analysis by gas chromatography-MS of acetylated methyl glyco-sides revealed the presence of LD-Hep, GalA, D-Glc, D-GlcN, and Kdo. Acetic acid hydrolysis gave one major compound as shown by the negative ion reflectron MALDI-TOF spectrum (TABLE TWO). In this spectrum the signals at m/z 1,531.13 and 1,513.16 correspond to pseudomolecular ions (M Ϫ H) Ϫ and (M Ϫ H Ϫ 18) Ϫ , respectively, confirming the presence of one major core OS structure. In particular, the signal at m/z 1,531.13 was in agreement with the calculated average molecular mass (1,532.30 Da) of a structure with one hexose, three heptoses, two hexuronic acids, one N-acetylhexosamine, and one Kdo unit. The signals at m/z 1,531.13 and 1,513.16 were accompanied by the corresponding adduct signals with sodium ions at m/z 1,553.13 and 1,535.15, respectively. From these data it can be assumed that this oligosaccharide is truncated in respect to the complete core structure (7) at the level of D-GlcN, which is N-acetylated. To test this hypothesis a positive ion postsource decay MALDI-TOF experiment (TABLE  THREE) was performed. This spectrum is dominated by fragment ions of B and C series (27) in addition to some double fragmentation (TABLE  THREE). Starting from the pseudomolecular ion at m/z 1,554.5 the signal at m/z 1,316.6 was attributed to a B 4 ion ([M ϩ Na] ϩ Ϫ 220 Ϫ 18) followed by a B 3 ion at m/z 962.6 and B 2 ion at m/z 402.1. From these signals it was possible to identify some ions derived from them by multiple Y-type cleavages. Starting from the C 3 signal at m/z 980.5 there are FIGURE 1. K. pneumoniae oligosaccharide core OS structures and genetic organization of the core OS biosynthetic clusters. A shows the K. pneumoniae types 1 (5, 6) and 2 (7) core OS structures. Depending on the K. pneumoniae strain, residues J and K could be hydrogen or GalA, and residue P could be hydrogen or Hep. Broken lines denote the truncation level for the different core biosynthetic gene mutations (7-12): 3-deoxy-D-manno-octulopyranosonic acid (Kdop), LD-Hepp, D-glucopyranose (Glcp), glucosamine (GlcNp), N-acetylglucosamine (GlcpNAc), and galacturonic acid (GalAp). B shows a diagram of the K. pneumoniae core types 1 (strain C3, O8:K66) (8) and 2 (strain 52145, O1:K2) (7) and S. marcescens N28b (17) wa gene clusters. Inner core genes (black arrows), outer core genes (gray arrows), wabN homologues (striped arrows), and waaL (O-antigen-lipid A-core ligase) are shown. two peaks at m/z 804.6 and 600.8, which were derived from the loss of one GalA and one GlcNAc, respectively. Another fragment sequence was generated from the B 4 ion, which has lost a GalA unit (m/z 1,140.5; 1,316.6 Ϫ 176) and a GalA and a GlcNAc unit (m/z 938.0; 1,316.6 Ϫ 176 -203). These results are consistent with the core oligosaccharide structure in Structure 1.
To confirm this hypothesis a methylation analysis on the core oligosaccharide alditol mixture was performed; the results were in agreement with the proposed structure. In particular, the 4-linked GalA and the terminal GlcNAc residues confirmed the presence of the disaccharide GlcNAc-(134)-GalA. Moreover terminal Glc, terminal GalA, 7-linked Hep, 3,4-linked Hep, and 3,7-linked Hep units were found.
The core OS structure of the 52145⌬wabN waaL mutant was definitively confirmed by the comparison of its 1 H NMR spectrum with that of the core structure of 52145⌬waaL mutant (Fig. 4) (7). In particular the presence of an intense singlet signal at 2.11 ppm (Fig. 4), attributable to the methyl of an acetyl group, supported the N-acetylation of the GlcN residue. In addition the comparison of the anomeric region confirmed the lack of the 6-␣-Glc and the terminal ␤-Glc residues, while the form and the chemical shifts of the other signals indicated the anomeric configuration to be the same as those of the corresponding region of 52145⌬waaL mutant (Fig. 4).
Isolation and Characterization of Non-polar wabH Mutants-Previously a K. pneumoniae wabH mutant was constructed by insertion of a non-polar kanamycin resistance cassette in a strain producing type 1 core LPS (12). This construction results in the insertion of an 850-bp DNA fragment in the targeted gene. Although this mutagenesis approach is reported to be devoid of polar effects on downstream genes, alterations in the stability of the mRNA cannot be ruled out. Furthermore this approach results in a kanamycin-resistant mutant precluding the use of this marker to introduce additional mutations. Thus, we decided to use the internal in-frame deletion approach (see "Materials and Methods") to construct wabH mutations in both core types 1 (C3) and 2 (52145) strains. Analysis of LPS isolated from 52145⌬wabH and C3⌬wabH confirmed that core OSs from 52145⌬wabH and C3⌬wabH were devoid of O-antigen (data not shown).
The core OS fraction obtained from 52145⌬waaL wabH by mild acid hydrolysis was analyzed by transformed negative ion electrospray ionization-MS (TABLE TWO). The major pseudomolecular ion signal (M Ϫ H) Ϫ at m/z 1,327.8 (TABLE TWO) was in agreement with the calculated average molecular mass (1,329.11 Da) of the expected molecular structure (KdoHep 3 GlcGalA 2 ). Thus, the wabH mutation precludes the extension of the core beyond the outer core GalA residue (depicted in Fig. 1).
Core LPS Deacetylase Assays-The core OS structure determined from strain 52145⌬waaL wabN lacks the last two outer core residues and presents GlcNAc instead of GlcN. Interestingly the same core OS STRUCTURE 1  structure is predicted as the result of the WabH protein activity (12). The WabH protein catalyzes the transfer of GlcNAc to outer core OS from UDP-GlcNAc (12). The presence of a putative domain corresponding to the superfamily glycoside hydrolase/deacetylase in the WabN protein suggests that it could be responsible for the deacetylation of the GlcNAc-containing core LPS intermediate. To test this hypothesis two in vitro assays were developed. The first assay takes advantage of the differences in Tricine-SDS-PAGE migrations between LPS samples from 52145⌬waaL wabN and 52145⌬waaL wabK. The 52145⌬waaL wabK mutant LPS lacks the last two outer core Glc residues, thus containing core residues up to GlcN (7). The difference in migration between these two LPS preparations is attributable to the acetate group present in LPS from 52145⌬waaL wabN but absent from 52145⌬waaL wabK LPS (TABLE TWO). This assay is based in the one previously described for WabH (12) using UDP-GlcNAc as substrate and LPS from mutant 52145⌬waaL wabH as acceptor and using as the enzyme sources cell-free extracts from K. pneumoniae strains 52145⌬waaL wabH (pBAD18-Cm-wabH 52145 ) and 52145⌬waaL wabN (pBAD18-Cm-wabN 52145 ) overexpressing the WabH and WabN proteins, respectively. As controls cell-free extracts from strains 52145⌬waaL wabH (pBAD18-Cm) and 52145⌬waaL wabN (pBAD18-Cm) were used. Tricine-SDS-PAGE analysis of reaction products formed by extract from 52145⌬waaL wabH (pBAD-wabH 52145 ) showed reduced lipid A core migration in comparison to the acceptor LPS (obtained from 52145⌬waaL wabH) as described previously (12) (Fig.  5). Product from the reaction containing both 52145⌬waaL wabH (pBAD18-Cm-wabH 52145 ) and 52145⌬waaL wabN (pBAD18-Cm-wabN 52145 ) extracts migrated to the same extent as LPS from 52145⌬waaL wabK (Fig. 5). Reactions containing 52145⌬waaL wabH (pBAD18-Cm) extract alone migrated as 52145⌬waaL wabN LPS, and reactions containing 52145⌬waaL wabN (pBAD18-Cm) extract migrated as the acceptor LPS.
These results indicate that the GlcNAc residue transferred to core LPS by the WabH protein is deacetylated by the WabN enzyme. The deacetylation step is necessary for core completion by addition of the last two outer core residues.

DISCUSSION
We identified a novel gene involved in K. pneumoniae core OS biosynthesis. Analysis of LPS extracted from non-polar wabN mutants, constructed in K. pneumoniae strains producing either type 1 or type 2 core LPS, revealed that these OSs are truncated and that the non-reducing end GlcNAc replaces the wild type GlcN residue. In addition nonpolar wabH mutants were constructed in strains producing type 1 (C3) and type 2 (52145) core LPS by internal in-frame deletion. Chemical characterization by mass spectrometry of LPS from these wabH mutants showed that they produce truncated core OS extending up to the GalA residue as shown preciously for a strain producing type 1 core LPS (12). It has been shown that the WabH enzyme catalyzes the in vitro incorporation of GlcNAc from UDP-GlcNAc into core LPS truncated at the level of the GalA residue (12). These results did not explain how the GlcN residue is incorporated into outer core LPS, although the functions of the reported genes of the K. pneumoniae wa gene clusters were known (7, 8, 10 -12).
In both the lipid A and nodulation factor biosynthesis a GlcNAc residue is first incorporated from UDP-GlcNAc and later deacetylated to GlcN (for reviews, see Refs. 28 and 29). Because the core LPS structure generated in vitro by the WabH enzyme is also found in wabN mutants we hypothesized a LPS-GlcNAc deacetylase function for WabN. This hypothesis was confirmed by in vitro reactions using LPS from mutant wabH, UDP-GlcNAc, and cell extracts containing WabH, WabN, or both enzymes.
Our work clearly resolves for the first time the complete biosynthesis of the K. pneumoniae core LPS and strongly suggests that the three genes wabG (galacturonosyltransferase), wabH (N-acetylglucosaminyltransferase), and wabN (LPS-GlcNAc deacetylase) could be used to identify bacterial species containing the disaccharide ␣GlcN-(1,4)-␣GalA linked to inner core HepII. Furthermore the lack of reported UDP-GlcN in bacterial cells (32), the results obtained in K. pneumoniae, and the full complementation achieved by the S. marcescens wabN led us to conclude that this could be a general mechanism in the biosynthesis of GlcN-containing enterobacterial core OS (a GlcNAc residue is first incorporated and later deacetylated to GlcN). The limited similarities shown by the WabN homologues to members of the hydrolase/ deacetylase superfamily (25) and thus to known deacetylase enzymes of this superfamily probably reflect the specificity of these enzymes for their natural substrates (chitin, lipid A, and core LPS). The established function for K. pneumoniae WabN, LPS-GlcNAc deacetylase, would be extremely useful to identify similar deacetylases in other core OSs containing GlcN in different bacteria.