Lipopolysaccharides Possessing Twol-Glycero-d-manno-heptopyranosyl-α-(1→5)-3-deoxy-d-manno-oct-2-ulopyranosonic Acid Moieties in the Core Region

The carbohydrate backbone of the core-lipid A region was characterized from the lipopolysaccharides (LPSs) of the plant-pathogenic bacterium Burkholderia caryophylli. For the first time, the presence of two moieties ofl-glycero-d-manno-heptopyranosyl-α-(1→5)-3-deoxy-d-manno-oct-2-ulopyranosonic acid was identified in a core region, which is of particular interest with regard to the biosynthesis of this and of LPSs in general. The LPSs of B. caryophylli were degraded by mild hydrazinolysis (de-O-acylation), treatment with 48% aqueous HF at 4 °C (cleavage of phosphate groups and destruction of the O-specific polysaccharides), reduction with NaBH4, and de-N-acylation utilizing hot KOH. The major oligosaccharide representing the carbohydrate backbone of the core region and lipid A was isolated by high-performance anion-exchange chromatography. Its analysis employing compositional and methylation analyses, matrix-assisted laser desorption/ionization mass spectrometry, and1H and 13C NMR spectroscopy applying various one-dimensional and two-dimensional experiments identified the following structure. STRUCTURE   I All sugars are pyranoses and α-linked, if not stated otherwise. Hep isl-glycero-d-manno-heptose, Kdo is 3-deoxy-d-manno-oct-2-ulosonic acid.


manno-oct-2-ulopyranosonic acid was identified in a core region, which is of particular interest with regard to the biosynthesis of this and of LPSs in general. The LPSs of B. caryophylli were degraded by mild hydrazinolysis (de-O-acylation), treatment with 48% aqueous HF at 4°C (cleavage of phosphate groups and destruction of the O-specific polysaccharides), reduction with
NaBH4, and de-N-acylation utilizing hot KOH. The major oligosaccharide representing the carbohydrate backbone of the core region and lipid A was isolated by high-performance anion-exchange chromatography. Its analysis employing compositional and methylation analyses, matrix-assisted laser desorption/ionization mass spectrometry, and 1 H and 13 C NMR spectroscopy applying various one-dimensional and two-dimensional experiments identified the following structure. Burkholderia caryophylli is a phytopathogenic Gram-negative bacterium that had earlier been included in the genus Pseudomonas (1). However, application of ribosomal RNA (rRNA) similarity studies showed that this original genus Pseudomonas is diverse and contains five distantly related groups. Of these, RNA group I contains the members of the true genus Pseudomonas. The new genus Burkholderia (RNA group II) contains species that are either plant or animal pathogens. B. caryophylli is responsible for the wilting of carnation (2), and it shares with other Gram-negative species the presence of lipopolysaccharides (LPSs) 1 in its cell wall. One characteristic feature of LPSs from the genus Burkholderia is the occurrence of two different O-specific polysaccharides. In the case of LPSs from B. caryophylli, two linear homo-polysaccharides were identified as O-specific polysaccharides, one of which is furnished from 3,6,10-trideoxy-4-C-(D-glycero-1-hydroxyethyl)-D-erythro-Dgulo-decose (caryophyllose, ␣-137-linked, caryophyllan) and the other from 4,8-cyclo-3,9-dideoxy-L-erythro-D-ido-nonose (caryose, ␤-137-linked, caryan) (3)(4)(5)(6). The caryan is acetylated in nonstoichiometric amounts, leading to a block pattern and, thus, to the establishment of repeating units in a homopolymer (7), while only the side chain the caryophyllan is randomly acetylated, and no chemical repeating unit was possible to define (8).
In LPSs, the O-specific polysaccharide is linked to the core region, which in turn is bound to the lipid A (9). All core regions identified so far (10) possess at least one residue of 3-deoxy-Dmanno-oct-2-ulosonic acid (Kdo) that links this region to the lipid A (Kdo I). A second characteristic molecule of the core region is L-glycero-D-manno-heptose (Hep); however, there are heptose-free LPSs (e.g. of the genera Chlamydia and Acinetobacter). In those cases Hep is present, Kdo I is usually substituted by a Hep residue at O-5, regardless to the number of Kdo residues in the structure, and elongation of the core occurs from this Hep residue. Thus, the presence of one Hep-␣-(135)-Kdo moiety is a characteristic feature of heptose-containing core regions of LPSs. Kdo I may further be substituted at O-4 by a second Kdo residue (Kdo II, e.g. in Salmonella enterica and * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This article is dedicated to Professor Lorenzo Mangoni on the occasion of his 70th birthday.
Escherichia coli). In a few cases, Kdo II is further substituted by neutral sugar residues, e.g. L-rhamnose or D-galactose, in particular E. coli strains. As elucidated best for LPSs of E. coli (11)(12)(13), biosynthesis of the initial parts of the core region begins with the attachment of the Kdo-␣-(234)-Kdo disaccharide to tetraacyl-lipid A (precursor IV A ). After completion of the acylation of lipid A, the first Hep is attached to O-5 of Kdo I, followed by further elongation steps.
LPSs of plant-pathogenic bacteria have been shown to play a role in phytopathogenicity (14). Since there is only little information on the structure-function relationship of LPSs from B. caryophylli, we have begun with the characterization of its LPSs. Here, we report the structure of the core region which possesses as a novel feature two Hep-␣-(135)-Kdo moieties.

EXPERIMENTAL PROCEDURES
Bacteria and Bacterial LPSs-B. caryophylli strain NCPP 2151 was cultivated as described previously (3). The LPSs were obtained from lyophilized bacteria by the phenol/water extraction method as described previously (3) (yield: 6% of the bacterial dry mass).
Isolation of Oligosaccharides-The LPSs (80 mg) were hydrolyzed in 1% acetic acid (100°C, 2 h) and the precipitate (lipid A) was removed by centrifugation (8000 ϫ g, 30 min). The supernatant was separated by gel-permeation chromatography on a column (50 ϫ 3 cm) of Sephadex G-50 (Amersham Biosciences, Inc.). Three fractions were obtained, the first of which eluted in the void volume and contained the caryan attached to the core region. A second fraction consisted of a mixture of caryophyllose oligosaccharides of molecular masses higher than trisaccharides. The third fraction (27.1 mg, 34% of the LPSs) was further purified using high-performance anion-exchange chromatography (HPAEC) on a column (4 ϫ 250 mm) of CarboPac PA100 (Dionex) that was eluted at 1 ml min Ϫ1 with a linear gradient of 1-5% 1 M sodium acetate in 0.1 M NaOH over 50 min. Several fractions were obtained containing caryophyllose oligosaccharides, and one fraction that yielded L-glycero-D-manno-heptopyranosyl-␣-(135)-3-deoxy-D-manno-oct-2ulopyranosonic acid (4.1 mg, 5% of the LPSs). For dephosphorylation and deacylation, the LPSs (400 mg) were dissolved in anhydrous hydrazine (15 ml), stirred at 37°C for 30 min, cooled, poured into ice-cold acetone (100 ml), and allowed to precipitate. The precipitate was then centrifuged (3000 ϫ g, 30 min), washed twice with ice-cold acetone, dried, and then dissolved in water and lyophilized (320 mg, 80% of LPSs). The sample was subsequently treated with 48% aqueous HF (4°C, 48 h) to remove phosphate groups and to degrade the O-specific polysaccharide, followed by extensive dialysis against water and lyophilization (53 mg, 13% of the LPSs). This material was reduced with NaBH 4 (20 -22°C, 18 h), then dialyzed and lyophilized (50 mg, 12.5% of the LPSs), and de-N-acylated with 4 M KOH as described previously (15). After desalting using a column (50 ϫ 1.5 cm) of Sephadex G-10 (Amersham Biosciences, Inc.), the resulting oligosaccharide fraction (46 mg, 11.5% of the LPS) was further separated utilizing gel-permeation chromatography with a column (50 ϫ 3 cm) of TSK-40 (Merck) in pyridine:acetic acid:water (8:20:1000, by volume). The main fraction was then subjected to analytical HPAEC eluted with a linear gradient of 13-16% 1 M sodium acetate in 0.1 M NaOH over 50 min, from which oligosaccharide 2, representing the complete carbohydrate backbone of the lipid A-core region (1 mg, 0.25% of the LPSs) was obtained.
General and Analytical Methods-Determination of Kdo, neutral sugars including the determination of the absolute configuration of the heptose residues, organic bound phosphate, absolute configuration of the hexoses, GLC, and GLC-MS were all carried out as described elsewhere (16 -19). For methylation analysis of the Kdo region, oligosaccharide 2 was first N-acetylated with acetic anhydride (15 l) in 0.5 M NaOH (150 l) for 10 min, then carboxymethylated with methanolic HCl (0.1 M, 5 min) and consecutively with diazomethane to improve its solubility in Me 2 SO. After methylation (20), 2 was hydrolyzed with 2 M trifluoroacetic acid (100°C, 1 h), carbonyl-reduced with NaB 2 H 4 , carboxy-methylated as before, carboxyl-reduced with NaB 2 H 4 (4°C, 18 h), acetylated (21), and analyzed by GLC-MS.
Methylation of the complete core region was carried out as described previously (22), and the sample was hydrolyzed with 4 M trifluoroacetic acid (100°C, 4 h), carbonyl-reduced with NaB 2 H 4 , carboxymethylated, carboxyl-reduced, acetylated, and analyzed by GLC-MS. In a third methylation analysis of the core region, the LPSs (10 mg) were methylated, and the sample was methanolyzed in 2 M methanolic HCl (85°C, 45 min) and examined by GLC-MS.
NMR Spectroscopy-For structural assignments of disaccharide 1, one-dimensional and two-dimensional 1 H NMR spectra were recorded of a solution of 4 mg in 0.5 ml of 2 H 2 O with a Bruker DRX 600 spectrometer (operating frequency: 600 MHz). 13 C NMR Spectra were recorded with a Bruker AMX-360 (operating frequency: 90 MHz). Measurements were achieved at 32°C, relative to internal acetone (␦ 1 H 2.225) and dioxane (␦ 13 C 67.4). 31 P NMR spectra were recorded using a solution of 1 mg in 0.5 ml of 2 H 2 O with a Bruker AMX 400 spectrometer (operating frequency: 162 MHz), equipped with a reverse probe, in the Fourier transform mode at 30°C. 85% Phosphoric acid was used as external standard. The correlation (COSY) and the total correlation spectroscopy (TOCSY) were recorded using standard Bruker software. The heteronuclear multiple quantum coherence (HMQC) spectrum was measured in the 1 H-detected mode via multiple quantum coherence with proton decoupling in the 13 C domain, using data sets of 2048 ϫ 512 points, and 64 scans were acquired for each t 1 value. Nucleaur Overhauser enhancement spectroscopy (NOESY) was measured using data sets (t 1 ϫ t 2 ) of 4096 ϫ 1024 points, and 16 scans were acquired. A mixing time of 200 ms was employed. For structural assignment of oligosaccharide 2, NMR spectra were recorded of a solution of 0.65 mg in 0.5 ml of 2 H 2 O with a Bruker DRX 600 spectrometer equipped with a microprobe head (Bruker PHTXI 600SB H-C/N-D-02.5). Resonances were measured relative to internal acetone (␦ 1 H 2.225 and ␦ 13 C 31.07). Coupling constants were determined on a first order basis from twodimensional phase-sensitive double quantum-filtered correlation spectroscopy (DQF-COSY) (23,24), which was measured using standard Bruker software. TOCSY and NOESY were carried out in the phasesensitive mode according to the method of States et al. (25) A decoupling in the presence of scalar interactions (26) was used in the TOCSY experiment with a mixing time of 125 ms and a spin lock power of 9400 Hz. The NOESY experiment was recorded with a mixing time of 200 ms, and the intensities were classified as strong, medium, and weak using cross-peaks from intra-ring proton-proton contacts for calibration. The 1 H, 13 C correlations were measured in the inverse mode, as HMQC and heteronuclear multiple bond correlation (HMBC) experiments (27,28). The experiments were carried out in the phase-sensitive mode according to the method of States et al. (25). A 60-ms delay was used for the evolution of long range connectivities in the HMBC experiment.

RESULTS
Compositional Analyses-The determination of the GlcN and organic bound phosphate contents of the LPSs from B. caryophylli gave a molecular ratio of about 2:1.3, respectively, suggesting that only the lipid A moiety is substituted by phosphate groups in nonstoichiometric amounts. The determination of the organic bound phosphate content of the supernatant obtained from acetic acid hydrolysis of the LPSs (containing the core region and the O-specific polysaccharides) gave negative results. Thus, the core region is free of phosphate.
Isolation and Characterization of Oligosaccharide 1-Oligosaccharide 1 (Fig. 1) was isolated from the supernatant obtained by gel-permeation chromatography and HPAEC after hydrolysis of the LPSs in 1% aqueous acetic acid. Its compositional analysis revealed the presence of Kdo and heptose in a molecular ratio of ϳ1:1, and methylation analysis yielded the derivatives of one residue each of terminally linked heptose and 5-substituted Kdo. Methanolysis of the sample and acetylation followed by analysis by GLC-MS identified the disaccharide Hep-Kdo. The structure of oligosaccharide 1 was established by 1 H and 13 C NMR spectroscopy. Chemical shifts were assigned utilizing COSY, TOCSY, NOESY, and HMQC experiments (Table I). Because of its free reducing end, the Kdo residue was present as ␣as well as ␤-pyranose, thus, two sets of signals were visible in the one-dimensional NMR spectra. Despite the similarity of the 13 C chemical shifts to published data (29), the linkage of the heptose residue to O-5 of Kdo was proven by the downfield shifts of the signal for its C-5 (␣-Kdo, 75.5 ppm; ␤-Kdo, 74.2 ppm) and the NOE connectivity between H-1 of the heptose and H-5 and H-7 of Kdo.
To confirm the presence of the disaccharide in the LPSs, the LPSs were methylated and then mildly methanolysed. Analysis of the sample by GLC-MS gave ions at m/z 263, 291, and 351 (J1 fragment), which were diagnostic for Hep-Kdo.
Isolation and Characterization of Oligosaccharide 2-After dephosphorylation, reduction and deacylation of the LPSs from B. caryophylli, which destroyed the O-specific polysaccharides, oligosaccharide 2 ( Fig. 1) was isolated by HPAEC. Its low yield was due to the fact that after treatment of the de-O-acylated LPSs with HF, followed by reduction and de-N-acylation, a mixture composed of variuos higher oligo-and smaller polysaccharides containing sugar residues from the O-specific polysaccharides was present, which could be further separated by gel-permeation chromatography. From this, only one fraction contained oligosaccharide 2, which was finally purified by HPAEC. NMR Spectroscopy of Oligosaccharide 2-The structure of oligosaccharide 2 was established by 1 H and 13 C NMR spectroscopy. Chemical shifts were assigned utilizing COSY, TOCSY, NOESY, ROESY, HMQC, and HMQC-TOCSY exper-iments. Anomeric configurations were assigned on the basis of the chemical shifts observed, and J 1,2 values, which were determined from the DQF-COSY experiment. The data are presented in Table II. The anomeric region of the 1 H NMR spectrum (Fig. 2) contained 10 signals, representing four heptose, five hexose, and one hexosamine residues. Their identification was possible by the complete assignment of all signals and the determination of the 3 J H,H vicinal coupling constants. One hexose (residue L, see Fig. 1) and the hexosamine (B) residue possessed the ␤-gluco configuration, which was supported by a NOESY experiment that yielded for both sugars intra-residual NOE connectivities from H-1 to H-3 and to H-5. Three hexoses (H, I, and M) possessed the ␣-gluco, one hexose (K) the ␣-galacto, and the heptoses (E, F, G) the ␣-manno configuration. The characteristic signals of H-3 of two Kdo residues were present at 1.994 ppm (H-3ax) and 2.081 ppm (H-3eq) (residue C) and 1.800 ppm (H-3ax) and 2.230 ppm (H-3eq) (residue D). Their ␣-configuration was established on the basis of the chemical shift of their 3eq proton and by measurement of the 3 J H7,H8a and 3 J H7,H8b coupling constants (30,31). All these sugars were pyranoses. Finally, one residue of glucosaminitol was identified, originating from dephosphorylated, reduced, and deacylated GlcN A.
The 13 C NMR chemical shifts could be assigned by an HMQC experiment, using the interpreted 1 H NMR spectrum. Ten anomeric signals were identified (Table I)   The sequence of the monosaccharide residues was determined using NOE data (Fig. 3). NOE contacts between anomeric and trans-glycosidic protons were observed for all hexose and heptose residues, and for GlcN B. An interresidual NOE contact was observed between H-1 of GlcN B to H-6a of GlcNol  Table I). The deoxy protons H-3ax and H-3eq of Kdo C gave an NOE contact to H-6 of Kdo D (32), and H-3ax of C gave one to H-5 of heptose E. These interresidual NOE  protons of H and Gal K showed NOE contacts to H-2 of K and H, respectively. Furthermore, a NOE contact was found between these two anomeric protons, definitely proving the (132)-linkage between K and H. Accordingly, proton H-4 of residue F possessed a strong NOE connectivity to H-1 of Gal K, indicating a close proximity of these residues which owes to the (132)-linkage of K and H. Finally, Glc I was linked to O-6 of H, as indicated by an NOE contact between H-1 of I and H-6a, b of H.
The HMBC spectrum confirmed the major portion of the structure assigned for oligosaccharide 2, since it contained most of all the required long range correlations to demonstrate the proximity of the residues. Together with intraresidual connectivities, the interresidual ones between H-1/C-1 of heptose N and C-5/H-5 of Kdo D, H-1/C-1 of E and C-5/H-5 of C, H-1/C-1 of L and C-4/H-4 of E, H-1/C-1 of F and C-3/H-3 of E, and between H-1/C-1 of residue H and C-3/H-3 of F were significant.
A MALDI-TOF mass spectrum of oligosaccharide 2 gave a molecular ion at m/z 2359.6 [(M ϩ H) ϩ ], which characterized a molecule consisting of five hexose, four heptose, two Kdo, one hexosamine, and one hexosaminitol residues.
In summary, we have established the structure of the carbohydrate backbone of the core-lipid A region of the LPSs from B. caryophylli as depicted in Fig. 1.   DISCUSSION B. caryophylli had been named Pseudomonas caryophylli before 1973 and, thus, was taxonomically included in the genus Pseudomonas, which, because of the diversity of functions found in its members, harbored a large number of species (1). Many attempts to develop systems of classification of Pseudo-monas species had failed during the first half of the 20 th century, and it was then the research on rRNA sequence similarities among Pseudomonas species that resulted in an internal subdivision of the genus into five RNA homology groups. This subdivision was largely confirmed by investigations on e.g. fatty acid compositions, the appearance of the outer membrane protein OprP, and genome structure and organization. The first of the RNA homology groups (RNA group 1) contains authentic Pseudomonas species, and RNA group 2 consists of species of a new genus named Burkholderia. Quite a number of structures of LPSs from Pseudomonas and some from Burkholderia species have been investigated so far. With regard to the core region of LPSs, which is structurally more conserved than the O-specific polysaccharide, published structures indicate that the core regions of Pseudomonas LPSs differ from those of Burkholderia LPSs (10,33), which is confirmed by the data presented in this paper. In particular, one residue of D-glycero-␣-D-talo-oct-2-ulopyranosonic acid (Ko) was identified in the LPSs of Burkholderia cepacia and Burkholderia pseudomallei, replacing the branching Kdo (Kdo II). This has not been identified in any LPS from Pseudomonas and, thus, might be of chemotaxonomical importance for the differentiation of both genera.
The linkages of the sugars in the O-specific polymers of the LPSs of B. caryophylli are acid-labile, and in preliminary experiments it could be shown that treatment of the LPSs with 48% aqueous HF (4°C, 48 h) not only removed the phosphate groups but also cleaved the O-specific polysaccharides. Thus, we applied this method and succeeded, after additional reduction and deacylation of the LPS, in the isolation of the complete carbohydrate backbone of the core-lipid A region. Its structure (Fig. 1) could be established from chemical and methylation analyses and from NMR spectroscopic and mass spectrometric investigations. The core region contains a structural element that commonly occurs in the Salmonella type core regions of enterobacterial LPS, e.g. from S. enterica or E. coli, namely  (10). The Glcp residue of this moiety is substituted by two hexoses, i.e. Glcp and Galp, which is similar to several enterobacterial core structures. In dissimilarity to the Salmonella type core regions, the core region from LPSs of B. caryophylli is free of phosphate and contains a Glcp residue that is ␤-(134)-linked to Hep E. The last structural element represents a characteristic feature of phosphate-deficient (e.g. Yersinia enterocolitica, Proteus mirabilis) or phosphate-free (e.g. Klebsiella pneumoniae) core regions. In the core region of LPSs from B. caryophylli it is substituted at O-6 by another ␣-Glcp residue. The same disaccharidic substituent occurs also in the core region of LPS from P. mirabilis strain R110/1959 (34,35). Most strikingly and identified for the first time, the core region of B. caryophylli Monosaccharides are as shown in Fig. 1. higher bearing. Biosynthesis of the core region is best established for the LPSs of E. coli (11)(12)(13). It begins with the attachment of two Kdo residues to precursor IV A , the tetraacylated and bisphosphorylated GlcN-disaccharide, which is performed by one Kdo-transferase WaaA (KdtA). This step occurs differently in LPS biosynthesis of Pseudomonas aeruginosa, where both Kdo residues are transferred to the completed (fully acylated) lipid A (41,42). However, here and in E. coli, after completion of lipid A employing two additional acylation steps, the Kdo that is attached to lipid A (Kdo I, residue C in Fig. 3) is substituted at O-5 by Hepp, a step that is brought about by heptosyltransferase I, which is encoded by the gene waaC. In a next step of E. coli LPS biosynthesis, this Hepp residue is then substituted at O-3 by another Hepp through the action of heptosyltransferase II, which is encoded by the gene waaF. Then further steps of core biosynthesis follow, i.e. attachment of the first Glcp (by waaG) and the third Hepp to Hep II (by waaQ), and completion of the outer core region. Possibly, decorations of the core, like the attachment of branching sugars or outer core heptose residues (e.g. Hepp in E. coli K-12 (38), DD-Hepp in K. pneumoniae (19) or P. mirabilis R110/1959 (34,35)), occur at later stages of the biosynthesis. With regard to the introduction of outer core heptose residues in LPS core biosynthesis, it is unknown whether the same heptosyltransferases that furnish the inner core region are utilized again or whether other, specific heptosyltransferases are activated. This is also true for the biosynthesis of the two L-␣-D-Hepp- However, in these cases Kdo I is substituted at O-4 by D-glycero-␣-D-talo-oct-2-ulopyranosonic acid (Ko) rather than Kdo. In the LPSs of B. caryophylli, only small amounts of Ko could be detected, and no core oligosaccharide possessing this sugar could be isolated. 2 It is thus unclear whether the same ␣-Ko-(234)-␣-Kdo disaccharide is present in this core region. Whereas in the core region of B. pseudomallei Hep II is substituted at O-3 by ␣-D-Glcp, this particular Hep carries in the core region of LPS from B. cepacia an L-Rhap residue at O-2, which represents another unusual structural feature.