Tài liệu Báo cáo khoa học: A novel type of highly negatively charged lipooligosaccharide from Pseudomonas stutzeri OX1 possessing two 4,6-O-(1-carboxy)-ethylidene residues in the outer core region ppt

Eur J Biochem 271, 2691–2704 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04197.x A novel type of highly negatively charged lipooligosaccharide from Pseudomonas stutzeri OX1 possessing two 4,6-O-(1-carboxy)-ethylidene residues in the outer core region Serena Leone1, Viviana Izzo2, Alba Silipo1, Luisa Sturiale3, Domenico Garozzo3, Rosa Lanzetta1, Michelangelo Parrilli1, Antonio Molinaro1 and Alberto Di Donato2 Dipartimento di Chimica Organica e Biochimica and 2Dipartimento di Chimica Biologica, Universita` degli Studi di Napoli Federico II, Napoli, Italy; 3Istituto per la Chimica e la Tecnologia dei Materiali Polimerici – ICTMP – CNR, Catania, Italy Pseudomonas stutzeri OXI is a Gram-negative microorganism able to grow in media containing aromatic hydrocarbons A novel lipo-oligosaccharide from P stutzeri OX1 was isolated and characterized For the first time, the presence of two moieties of 4,6-O-(1-carboxy)-ethylidene residues (pyruvic acid) was identified in a core region; these two residues were found to possess different absolute configuration The structure of the oligosaccharide backbone was determined using either alkaline or acid hydrolysis Alkaline treatment, aimed at recovering the complete carbohydrate backbone, was carried out by mild hydrazinolysis (de-Oacylation) followed by de-N-acylation using hot KOH The lipo-oligosaccharide was also analyzed after acid treatment, attained by mild hydrolysis with acetic acid, to obtain information on the nature of the phosphate and acyl groups The two resulting oligosaccharides were isolated by gel permeation chromatography, and investigated by compositional and methylation analyses, by MALDI mass spectrometry, and by 1H-, 31P- and 13C-NMR spectroscopy These experiments led to the identification of the major oligosaccharide structure representative of core region-lipid A All sugars are D-pyranoses and a-linked, if not stated otherwise Based on the structure found, the hypothesis can be advanced that pyruvate residues are used to block elongation of the oligosaccharide chain This would lead to a less hydrophilic cellular surface, indicating an adaptive response of P sutzeri OX1 to a hydrocarbon-containing environment Environmental pollution is recognized worldwide as an emergency for its negative effects on the biosphere and on human health Bioremediation strategies have recently been devised, based on microbial biotransformations, given the metabolic potential of selected microorganisms, in particular by Gram-negative bacteria, and their adaptability to many different pollutants [1] Pseudomonas stutzeri OX1 is a Gram-negative bacterium isolated from the activated sludge of a wastewater treatment plant, and endowed with unusual metabolic capabilities for the degradation of aromatic hydrocarbons [2] In fact, in contrast with other Pseudomonas strains, this microrganism is able to grow on a large spectrum of aromatic compounds including phenol, cresol and dimethylphenol, and on nonhydroxylated molecules such as toluene and o-xylene, the most recalcitrant isomer of xylene Moreover, it is able to metabolize tetrachloroethylene (PCE), one of the groundwater pollutants commonly resistant to degradation [3] Degradation of aromatic hydrocarbons by aerobic bacteria comprises an upper pathway, which produces dihydroxylated aromatic intermediates by the action of monooxygenases, and a lower pathway, which processes these intermediates to molecules that enter the citric acid cycle [4] We have recently cloned, expressed and characterized three different enzymatic systems from P.stutzeri OX1: (a) toluene-o-xylene monooxygenase (ToMO) [5], endowed with a broad substrate specificity [6] and (b) phenol hydroxylase (PH) [7], both belonging to the upper pathway; and (c) catechol 2,3 dioxygenase (C2,3O) (A di Donato, unpublished observations), the ÔgatewayÕ enzyme to the lower pathway However, chemical toxicity of wastes can hamper the use of this and other microorganisms in bioremediation strategies, especially when organic solvents are present at high concentrations Several mechanisms have been described that contribute to solvent resistance in Gram-negative bacteria, all based on structural changes in outer and inner membranes [8] Different short- and long-term responses have been observed including modifications of the fatty acid and phospholipid composition of the membrane, extrusion mechanisms using vesicles, and energy-dependent active efflux pumps that export toxic organic solvents outside the cytoplasm [9] Correspondence to A Molinaro, Dipartimento di Chimica Organica e ` Biochimica, Universita di Napoli Federico II, Complesso Universitario Monte S Angelo, via Cintia 4, 80126 Napoli, Italy Fax: + 39 081 674393, Tel.: + 39 081 674123, E-mail: molinaro@unina.it Abbreviations: DEPT, distorsionless enhancement by polarization transfer; GlcN, 2-amino-2-deoxy-glucose; Hep, L-glycero-Dmanno-heptose; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; LOS, lipooligosaccharide; LPS, lipopolysaccharide (Received March 2004, revised 23 April 2004, accepted 30 April 2004) Keywords: Pseudomonas stutzeri OXI; lipopolysaccharide; NMR spectroscopy; mass spectrometry; pyruvic acid Ó FEBS 2004 2692 S Leone et al (Eur J Biochem 271) Even though lipopolysaccharides (LPSs) are major components of the outer membrane of Gram-negative bacteria, little is known about their role and their chemical modifications under environmental stress [1,9] It is certain, however, that LPSs are unique and vital components of these microorganisms and that they play an important role in their survival and their interaction with the environment [10,11] Smooth-form lipopolysaccharides (S-LPSs) include three regions, the O-specific polysaccharide (or O-antigen), the oligosaccharide region (core region) and the lipid part (lipid A) Conversely, rough (R) form LPSs not possess an O-specific polysaccharide and are frequently named lipooligosaccharides (LOSs) LOSs have been found either in wild-type strains and in mutant strains harboring mutations in the genes encoding enzymes of the biosynthesis and/or the transfer of the O-specific polysaccharide [12,13] The core region from both smooth and rough forms of enteric bacteria generally includes oligosaccharides built of up to 11 units [12,13], and consists of two distinct domains: an inner core, characterized by the presence of 3-deoxy-Dmanno-oct-2-ulosonic acid (Kdo) and L-glycero-D-mannoheptose (Hep), and an outer core, which contains common sugars It is worth noting that the core oligosaccharide of LOSs has been reported to play a role in the interaction of the microorganism with the environment [12,13] In this paper, the structural characterization of the carbohydrate backbone of the rough form LPS of P stutzeri OX1 is reported, obtained by chemical analyses, MALDITOF mass spectrometry and two-dimensional NMR spectroscopy This novel oligosaccharide chain was found to possess unusual structural features, which might be biologically relevant Among these is a GalN residue substituted by two gluco-configured residues, which are blocked at position O-4 and O-6 by a pyruvate ketal linkage, a structure peculiar and new to lipopolysaccharide core regions Based on this finding, the hypothesis can be advanced that the insertion of pyruvate residues at the end of the oligosaccharide chain blocks its elongation, thereby leading to a shorter LOS and hence to a less hydrophilic cellular surface Moreover, as it has already been proposed [1,9], these residues may also contribute to the rigidity and stability of the Gram-negative cell wall by binding cations Experimental procedures Bacterial growth and LPS extraction Cells were routinely grown on M9-agar plates supplemented with 10 mM malic acid as the sole carbon source, at 27 °C For growth in liquid medium, mL was inoculated with a single colony from a fresh plate, and grown for 18 h at 27 °C with constant shaking This saturated culture was used to inoculate 100 mL of the same medium and grown at 27 °C until D600  Final growth was started by inoculating the appropriate volume of the latter culture into L of fresh medium, to D600 ¼ 0.02 Cells were grown at 27 °C, until D600 ¼ was reached and then recovered by centrifugation at 3000 g for 15 at °C, washed with an isotonic buffer and lyophilized Growth was carried out in M9 salt medium supplemented with mM phenol as the sole carbon and energy source Dried cell yield was 0.13 gỈL)1 Dried cells were extracted three times with a mixture of aqueous 90% phenol/chloroform/petroleum ether (50 mL, : : v/v/v) as described previously [14] After removal of the organic solvents under vacuum, the LOS fraction was precipitated from phenol with water, washed first with aqueous 80% (v/v) phenol, and then three times with cold acetone, each time centrifuged as above, and lyophilized (the yield was 90 mg of LOS, about 4.3% of the dry mass) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) was performed as described previously [15] For detection of LPS and LOS, gels were stained with silver nitrate [15] Isolation of oligosaccharides An aliquot of LOS (40 mg) was dissolved in anhydrous hydrazine (2 mL), stirred at 37 °C for 90 min, cooled, poured into ice-cold acetone (20 mL), and allowed to precipitate The precipitate was then centrifuged (3000 g, 30 min, °C), washed twice with ice-cold acetone, dried, dissolved in water and lyophilized (32 mg, 80% of the LOS) This material was de-N-acylated with M KOH as described [16] Salts were removed using a Sephadex G-10 (Pharmacia) column (50 · 1.5 cm) The resulting oligosaccharide constitutes the complete carbohydrate backbone of the lipid A-core region (16 mg, 40% of the LOS) Another aliquot of LOS (40 mg) was hydrolyzed in 1% (v/v) acetic acid (100 °C, h) and the precipitate (lipid A) was removed by centrifugation (8000 g, 30 min) The supernatant was separated by gel-permeation chromatography on a P-2 column (85 · 1.5 cm) Two fractions were obtained, the first contained oligosaccharide (28 mg, 70% of the LOS), whereas the second fraction contained a mixture of reducing pyranose, furanose, anhydro and lactone forms of 3-deoxy-D-manno-oct-2-ulosonic acid (3 mg, 7.5% of the LPSs) General and analytical methods Determination of Kdo, neutral sugars, carbamoyl analysis, including the determination of the absolute configuration of the heptose residues, organic bound phosphate, absolute configuration of the hexoses, fatty acids and their absolute configuration, GLC and GLC-MS were all carried out as described previously [17–21] For methylation analysis of Kdo region, LOS was carboxy-methylated with methanolic HCl (0.1 M, min) and then with diazomethane to improve its solubility in dimethyl sulfoxide Methylation was carried out as described [22,23] LOS was hydrolyzed with M trifluoroacetic acid (100 °C, h), carbonyl-reduced with NaBD4, carboxy-methylated as described above, carboxylreduced with NaBD4 (4 °C, 18 h), acetylated and analyzed by GLC-MS Methylation of the complete core region was carried out as described previously [22–24] The sample was hydrolyzed with M trifluoroacetic acid (100 °C, h), carbonyl-reduced with NaBD4, acetylated and analyzed by GLC-MS NMR spectroscopy For structural assignments of oligosaccharides and 2, 1D and 2D 1H-NMR spectra were recorded on a solution of Ó FEBS 2004 LPS from Pseudomonas stutzeri OX1 (Eur J Biochem 271) 2693 mg in 0.6 mL of D2O, at 55 °C or at 30 °C, at pD 14 and (uncorrected values), respectively 1H- and 13C-NMR experiments were carried out using a Varian Inova 500 or a Varian Inova 600 instrument, whereas for 31P-NMR spectra a Bruker DRX-400 spectrometer was used Spectra were calibrated with internal acetone [dH 2.225, dC 31.45] Aqueous 85% phosphoric acid was used as external reference (0.00 p.p.m.) for 31P-NMR spectroscopy Nuclear Overhauser enhancement spectroscopy (NOESY) and rotating frame Overhauser enhancement spectroscopy (ROESY) were measured using data sets (t1 · t2) of 4096 · 1024 points, and 16 scans were acquired A mixing time of 200 ms was used Double quantum-filtered phase-sensitive COSY experiments were performed with 0.258 s acquisition time, using data sets of 4096 · 1024 points, and 64 scans were acquired Total correlation spectroscopy experiments (TOCSY) were performed with a spinlock time of 80 ms, using data sets (t1 · t2) of 4096 · 1024 points, and 16 scans were acquired In all homonuclear experiments the data matrix was zero-filled in the F1 dimension to give a matrix of 4096 · 2048 points and was resolution enhanced in both dimensions by a shifted sine-bell function before Fourier transformation Coupling constants were determined on a first-order basis from 2D phase sensitive double quantum filtered correlation spectroscopy (DQF-COSY) [25,26] Intensities of NOE signals were classified as strong, medium and weak using cross-peaks from intraring proton-proton contacts for calibration Heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) experiments were measured in the 1H-detected mode via single quantum coherence with proton decoupling in the 13C domain, using data sets of 2048 · 512 points, and 64 scans were acquired for each t1 value Experiments were carried out in the phase-sensitive mode according to the method of States et al [27] A 60 ms delay was used for the evolution of long-range connectivities in the HMBC experiment In all heteronuclear experiments the data matrix was extended to 2048 · 1024 points using forward linear prediction extrapolation [28,29] MALDI-TOF analysis MALDI mass spectra were carried out in the negative polarity in linear or in reflector mode on a Voyager STR instrument (Applied Biosystems, Framingham, MA, USA) equipped with a nitrogen laser (k ¼ 337 nm) and provided with delayed extraction technology Ions formed by the pulsed laser beam were accelerated through 24 kV Each spectrum is the result of approximately 200 laser shots A saturated solution of 2,4,6-trihydroxyacetophenone was used as the matrix Results Isolation and characterization of the LOS fraction The LOS fraction was isolated from dried cells by extraction with phenol/chloroform/petroleum ether, and further purified with gel permeation chromatography SDS/PAGE showed, after silver nitrate gel staining, the presence of fast migrating species in agreement with their oligosaccharide nature Compositional monosaccharide analysis of the LOS fraction led to the identification of L,D-Hep, D-GalN, D-GlcN, D-Glc, Kdo (2 : 1.0 : 3.2 : 1.1 : 1.8) and trace amounts of L-Rha 7-O-Carbamoyl-L,D-Hep was present in a stoichiometric ratio with L,D-Hep Methylation analysis showed the presence of terminal Kdo, 6-substituted-HexN, 3-substituted-Hep, 4,5-disubstituted-Kdo, 3,4-disubstitutedHexN, 4,6-disubstituted-Glc, 4,6-disubstituted-HexN and, in small amounts, terminal-Rha and 6-substituted-Glc In addition, the disaccharide 7-O-carbamoyl-Hep-(1fi3)Hep was found Fatty acid analysis revealed the presence of typical fatty acids of pseudomonads LPS [30], i.e (R)-3-hydroxydodecanoic acid [C12:0 (3-OH)], present exclusively in amide linkage and (R)-3-hydroxydecanoic [C10:0 (3-OH)] (S)-2hydroxydecanoic [C12:0 (2-OH)] and dodecanoic acid (C12:0), present in ester linkage Moreover, phosphate colorimetric assays gave positive results The LOS fraction was then subjected to both alkaline and acid degradations and complete structural characterization NMR spectroscopy and MALDI-TOF MS spectrometry of oligosaccharide Oligosaccharide was isolated by gel permeation chromatography after complete deacylation of the LOS of P stutzeri OX1 The complete structure of fully deacylated oligosaccharide (Fig 1) was determined by 1H-, 31P- and 13 C-NMR spectroscopy Chemical shifts were assigned using DQF-COSY, TOCSY, NOESY, ROESY, 1H,13CDEPT-HSQC, 1H,31P-HSQC, 1H,13C-HMBC and 1H,13CHSQC-TOCSY experiments Anomeric configurations were assigned on the basis of 1H and 13C chemical shifts, of 3JH1,H2 values determined from the DQF-COSY experiment (Table 1), and of 1JC1,H1 values derived by 1H,13CHSQC spectrum recorded without decoupling during acquisition All sugars were present as pyranose rings, as indicated by H- and 13C-NMR chemical shifts and by the HMBC spectrum that showed for all residues intraresidual scalar connectivity between H-1/C-1 and C-5/H-5 atoms (for Kdo units, between C-2 and H-6) The anomeric region of the H-NMR spectrum (Fig 2) showed seven major signals in the region between 5.46 and 4.47 p.p.m relative to seven different spin systems (A–G, in order of decreasing chemical shift), and in addition two AB methylene resonances at high fields, typical of Kdo residues (I–L) Each spin system was completely assigned by COSY and TOCSY starting from anomeric resonances For Kdo residues I and L the starting point was the H-3 diastereotopic methylene resonance Both spin systems A and D (5.46 and 5.27 p.p.m.) were characterized by low 3JH1, H2 and 3JH2, H3 values, indicative of two a-manno-configured residues Moreover, all other cross peaks within each spin system were assigned in the TOCSY spectrum from H-2 proton signals, leading to their identification as two heptoses Residue B was identified as a-gluco-configured hexosamine on the basis of chemical shifts and 3JH,H values Moreover, based on its anomeric signal at 5.42 p.p.m present as a double doublet (3JH1,H2 ¼ 2.9 Hz and 3JH1,P ¼ 8.3 Hz), with one of the couplings due to a phosphate signal as shown below, it was identified as GlcN I of the lipid A skeleton The spin system at Ó FEBS 2004 2694 S Leone et al (Eur J Biochem 271) Fig The structure of oligosaccharide obtained by alkaline hydrolysis of the core region of the LPS of P stutzeri OX1 Table 1H, 13C and 31P NMR chemical shifts (p.p.m) of deacylated core-lipid A backbone (oligosaccharide 1) of LOS from P stutzeri OX1 Chemical shifts are relative to acetone and external aq 85% (v/v) phosphoric acid (1H, 2.225 p.p.m.; 13C, 31.45 p.p.m.; 31P, 0.00 p.p.m at 55 °C) Residue Nucleus A Hep B GlcN C GalN D Hep E Glc F GlcN G GlcN I Kdo L Kdo S-Pyr R-Pyr H C 31 P H 13 C 31 P H 13 C H 13 C 31 P H 13 C H 13 C H 13 C 31 P H 13 C H 13 C H 13 C H 13 C 13 3ax/ 4.09 76.7 4.20 69.8 3.86/4.08 63.9 3.64 73.7 4.46 72.0 4.0 3.45 70.8 4.39 73.7 5.42 95.1 3.0 5.32 101.3 5.27 102.7 4.39 73.8 1.9 2.73 55.9 4.09 72.0 4.30/3.74 70.1 3.22 51.5 4.43 69.9 4.06 78.3 4.14 78.2 4.27 77.5 4.05 67.4 4.02 71.5 4.16 71.7 4.73 105.8 4.67 104.7 4.47 103.5 3.44 75.3 2.70 58.2 2.67 56.9 3.71 72.9 3.54 76.6 3.66 73.5 3.43 75.8 3.71 77.7 3.82 73.4 3.9 4.27 65.9 4.12 71.8 3.66 67.7 3.36 67.2 3.47 76.9 3.71 61.7 4.45 74.2 4.3 3.93/3.65 64.6 4.04/3.93 64.7 3.71/3.45 63.7 4.08 67.7 4.24 69.5 3.69 73.0 3.67 73.6 5.46 97.8 175.0 101.7 175.0 100.9 175.5 101.9 175.8 99.5 1.81/2.07 36.1 2.00/2.34 35.0 1.48 25.2 1.62 17.2 3.75/4.09 63.7 4.03 70.1 3.93 70.6 3.86/3.70 63.7 3.86/3.69 63.9 Ó FEBS 2004 LPS from Pseudomonas stutzeri OX1 (Eur J Biochem 271) 2695 Fig 1H-NMR spectrum of oligosaccharide The spectrum was recorded under the following conditions: mg of oligosaccharide in 0.6 mL D2O, pD 14 at 30 °C 5.32 p.p.m (C; 3JH1, H2 ¼ 3.6 Hz) was identified as a-GalN by its JH,H values for H-3/H-4 and H-4/H-5, diagnostic of a galacto configuration (3.4 Hz and less than Hz, respectively) Three spin systems E, F and G (doublets; 3JH1, H2 ¼ 8.6, 7.8 and 7.7 Hz, respectively) were identified as b-glucoconfigured monosaccharides given their large 3JH,H-values A further indication of their b configuration was the observation of NOE contacts in the ROESY spectrum among H-1, H-3 and H-5, for all E, F, G residues The H-3 methylene signals of two a-Kdo residues were present at 1.82 p.p.m (H-3ax) and 2.07 p.p.m (H-3eq) (residue I), and 2.00 p.p.m (H-3ax) and 2.34 p.p.m (H-3eq) (residue L), respectively Their a configuration was established on the basis of the chemical shift of their H-3eq protons and by measurement of the 3JH7,H8a and JH7,H8b coupling constants [31,32] Two methyl singlet signals were present at higher fields, at 1.48 and 1.62 p.p.m., respectively Each methyl signal was in a : ratio with the anomeric signals, i.e in a stoichiometric ratio The 13C-NMR chemical shifts could be assigned by a DEPT-HSQC experiment, using the assigned 1H-NMR spectrum Seven anomeric carbon resonances were identified (Table 1), numerous carbon ring signals and four nitrogen-bearing carbon signals assigned to C-2 of B, C, F and G spin systems Considering the 13C chemical shifts of nonsubstituted residues [33], several low-field shifted signals indicated substitutions at O-3 of residue A, O-6 of residue B, O-3 and O-4 of residue C, O-3 of residue D, O-4 and O-6 of residues E and F, O-6 of residue G, O-5 and O-4 of residue L, whereas I was a terminal residue In the high field region of the spectrum two cross peaks at 1.48/25.2 and 1.62/ 17.2 p.p.m were present Phosphate substitution was established on the basis of 31 P-NMR spectroscopy The 31P-NMR spectrum showed the presence of five monophosphate monoester signals (Table 1) The site of substitution was inferred by a H,31P-HSQC spectrum that showed correlations of 31P signals with H-1 B (GlcN), H-4 A and H-2 A (Hep I), H-4 G (GlcN) and H-6 D (Hep II) The sequence of the monosaccharide residues was determined using NOE effects of the ROESY (Fig 3) and NOESY spectra, and by 1H,13C-HMBC correlations The typical lipid A carbohydrate backbone was eventually assigned on the basis of the NOE signal between H-1 G and H-6a,b B In the case of Kdo units, which lack the anomeric proton, the sequence was inferred by NOE contacts between the methylene-proton H-3eq of Kdo L and H-6 of Kdo I, whereas Kdo L was substituted by heptose A as indicated by the NOE effect found between H-1 A and H-5 L, and, in addition, between H-5 A and H-3ax L All of these NOE contacts were characteristic of the sequence a-L-glycero-D-manno-heptose-(1fi5)-[a-DKdo-(2fi4)]-a-D-Kdo [34,35] Heptose A was, in turn, substituted at the O-3 position by heptose D, as demonstrated by the NOE cross peak between H-1 D and H-3 A A disaccharide 7-O-carbamoyl- 2696 S Leone et al (Eur J Biochem 271) Ó FEBS 2004 Fig Section of the ROESY spectrum of oligosaccharide Monosaccharide labels are as indicated in Fig NOE cross-peaks are in black, in antiphase with diagonal (grey lines) Spectrum was recorded at pD 14, 55 °C Hep-(1fi3)-Hep was also identified by methylation analysis of the intact LOS, thus, the carbamoyl group should be attached to O-7 of the heptose moiety D The GalN C was attached to the O-3 position of this last heptose as shown by the NOE effect between the anomeric proton of GalN and H-3 of D GalN is the branching point of the chain and, consequently, it should carry two sugar residues at O-3 and O-4 Indeed, the anomeric proton of b-glucose E gave a NOE effect with H-3 of GalN, whereas the anomeric proton of b-glucosamine F gave a NOE effect with H-4 of GalN In determining the L Kdo location, its linkage to unit G was deduced by exclusion In particular, the linkage to O-6 of G was inferred by taking into account the downfield shift of the carbon signal C-6 (63.7 p.p.m., Table 1) indicating its involvement in a glycosydic linkage The HMBC spectrum confirmed the sequence proposed for oligosaccharide 1, as it contained the significant longrange correlations required for the determination of the sequence and of the attachment points In fact, together with intraresidual long-range cross-peaks, interresidual long-range connectivity was found between H-5/C-5 L and C-1/H-1 A, H-3/C-3 A and C-1/H-1 D, H3/C-3 D and C-1/H-1 C, H-1/C-1 E and C-3/H-3 C, H-1/C-1 F and C-4/ H-4 C, H-1/C-1 G and C-6/H-6 B The HMBC experiment was also crucial for the identification and localization of the two methyl groups belonging to noncarbohydrate constituents Plain long-range correlations (Fig 4A) were found in the spectrum for each methyl signal The signal at 1.48 p.p.m correlated to two different carbon signals at 101.9 and 175.5 p.p.m., whereas the signal at 1.62 p.p.m correlated to two other signals at 99.5 and 175.8 p.p.m None of these four carbon signals was present in the HSQC spectrum These data pointed to two cyclic ketals of pyruvic acid present on two distinct residues, namely E and F, whose C-4 and C-6 signals experienced a downfield displacement This was confirmed by the HMBC spectrum where each ketal carbon signal of pyruvate residues correlated to H-4 and H-6 of E and F residues (b-D-Glc and b-D-GlcN, respectively) It should be noted that the signal discrepancy in the 1H and 13C chemical shifts of the two pyruvate moieties is due to the different absolute configuration at C-2 In fact, the methyl signal occurring at 1.48 and 25.2 p.p.m is assigned to the S-pyruvate group, whereas the one occurring at 1.62 and 17.2 p.p.m is assigned to an R-pyruvate group, as already described [36] Moreover, the ROESY spectrum (Fig 4B) was in complete agreement with the assignment above In fact, the methyl signal of the R-pyruvate residue at 1.62 p.p.m gave a strong NOE effect with H-4 and H-6a of residue F This is in agreement with an axial orientation of the methyl group on a 1,3-dioxane ring in a chair-like conformation in which H-4 and H-6a are sin diaxial with respect to it The methyl signal of S-pyruvate, being in equatorial orientation, only gave NOE effect with the Ó FEBS 2004 LPS from Pseudomonas stutzeri OX1 (Eur J Biochem 271) 2697 Fig Sections of the high field region of the (A) ROESY and (B) HMBC spectra Correlations of pyruvate methyl groups are shown (A) The 4,6 Pyr-GlcN residue is drawn in the middle of the figure with arrows indicating the relevant NOE contacts between methyl protons of the R-pyruvate group and H-4 and H-6 of GlcN residue Spectra were recorded at pD 14, 55 °C adjacent H-6 of residue E Thus, all main spin systems were assigned in the NMR spectra, and all chemical data found a rational explanation The presence of a minor spin system (10%) belonging to rhamnose (anomeric signal at 4.89 p.p.m) and 6-substituted glucose (overlapped with terminal glucose) might be explained by the presence of a second outer core glycoform in which rhamnose is attached at O-6 of the glucose residue, which obviously lacks the pyruvate group The MALDI mass spectrum confirmed the proposed structure In fact, an ion peak at m/z 2188.4 (Fig 5A) was present, corresponding to the complete carbohydrate backbone bearing five phosphate goups and two pyruvic acid acetal residues Moreover, at higher laser intensity (Fig 5B) various ion peaks related to fragments were found, all fitting with the structure shown in Fig In conclusion, the data above allowed the identification of the carbohydrate backbone from alkaline degradation of the rough form LPS from P stutzeri OX1 Isolation, NMR and MS analyses of oligosaccharide from acetic acid hydrolysis Further information on alkaline labile groups that could be present in the core region (i.e acyl groups) was obtained by treating the LOS with acetic acid to split the Kdo linkage An oligosaccharide mixture was isolated after gel permeation chromatography, which was purified further and the resulting oligosaccharide (Fig 6) analyzed by compositional/methylation analyses, 2D NMR and mass spectrometry Compositional and methylation analyses led to the identification of 3-substituted-L,D-Hep, 7-O-carbamoyl3-substituted-L,D-Hep 3,4-disubstituted-D-GalN, terminal D-glucose and terminal D-GlcN Traces of 5-substituted Kdo, 6-substituted-D-Glc and terminal L-rhamnose were also found The 1H-NMR spectrum revealed the absence of anomeric signals from GlcN I and GlcN II of Lipid A, the lack of pyruvate methyl groups, as a consequence of the cleavage of the ketal group under acid treatment, and the presence of singlet signals at 2.00 p.p.m Methylene signals of Kdo were spread because of its presence as reducing end unit, i.e pyranose, furanose, anhydro and lactone forms present at same time The anomeric region of the spectrum consisted of six main signals (Fig 6), five of which belonging to the main oligosaccharide backbone, named U–Z All resonances of the monosaccharides (Table 2) were obtained from 2D NMR spectroscopy (DQF-COSY, TOCSY, NOESY, ROESY, 1H,13C-DEPT-HSQC 1H,31P-HSQC, 1H,13CHMBC and 1H,13C-HSQC-TOCSY) Evaluation of chemical shifts and of 3JH,H coupling constants led to identification of residues Hep (U), 7-O-carbamoyl-Hep (V), GalN (X), GlcN (W), Glc (Z) 2698 S Leone et al (Eur J Biochem 271) Ó FEBS 2004 Fig Negative ion MALDI-TOF mass spectra of oligosaccharide obtained in linear mode at normal (A) and higher laser intensity (B) Assignments of main ion peaks are shown P, phosphate; Pyr, pyruvic acid Low-field shifted signals were present in the HSQC spectrum indicating substitutions at O-3 (U, V and X) and O-4 (X), whereas residues W and Z were not substituted Cross-peaks were also detected only for two nitrogen atoms bearing carbon signals, at 4.26/49.8 p.p.m (H-2/C-2 X) and 3.67/56.1 p.p.m (H-2/C-2 W), in agreement with the absence of Lipid A disaccharide and with the presence of a a-galacto and a b-gluco configured 2-amino-2-deoxy hexoses Moreover, given the downfield H-2 chemical shifts of the X and W residues, the amino groups should have been present as acylamido The high field proton region of the HMBC gave clues for the identification of the nature of acyl groups Three different singlet signals were present in this region (Fig 6), two with a smaller area that probably account for the same methyl group that experienced oligosaccharide heterogeneity All signals in the region of 2.0 p.p.m showed longrange correlations with a carbonyl signal at 174.6 p.p.m which in turn correlated to protons at 3.67 p.p.m (H-2 W) and 4.26 p.p.m (H-2 X) These correlations indicated the presence of two acetamido groups at the C-2 of GlcN W and GalN X Thus, the two smaller methyl signals are both due to GalN X and are consequences of oligosaccharide heterogeneity, possibly due either to the adjacent heptose V bearing heterogeneous phosphate substitution (see below) or to a reducing Kdo residue In addition, all diagnostic interresidue NOE effects were found in the ROESY spectrum This confirmed the oligosaccharide sequence as determined in the previous paragraph Other information on noncarbohydrate substituents (phosphate and carbamoyl groups) was gained by the observation of the downfield displaced heptose signals, namely H-2/C-2 and H-4/C-4 of heptose U and H-6/C-6 and H-7a,b of heptose V The H-7a,b downfield shift was clearly due to the presence of a carbamoyl group that did not undergo hydrolysis in mild acid conditions, and that has already been located at position O-7 of the second heptose residue on the basis of methylation analysis In agreement with this assignment, a Ó FEBS 2004 LPS from Pseudomonas stutzeri OX1 (Eur J Biochem 271) 2699 Fig 1H-NMR spectrum of oligosaccharide obtained by acetic acid hydrolysis The spectrum was recorded under the following conditions: mg of oligosaccharide in 0.6 mL D2O, pD Monosaccharides are as shown; rhamnose residue anomeric signal is not labeled as it belongs to the minor oligosaccharide Dotted bonds indicate a nonstoichiometric linkage Chemical shifts are shown in Table signal at 160.0 p.p.m in the HMBC spectrum correlated with both protons H-7 of V The degree of phosphorylation and localization of phosphate substituents was established by 1D and 2D 31 P-NMR spectroscopy (Fig 7, Table 2) Several signals were found in the 31P-NMR spectrum, whose chemical shift clearly indicated that they derived from phosphate groups present in different magnetic/chemical environments In fact, in addition to a number of phosphate monoester signals in the region of 1.4–3.2 p.p.m., two peaks of lower intensity were present at )5.5 and )9.7 p.p.m These last two signals derived from a diphosphate monoester bond, i.e a pyrophosphate group In particular, the signal at )5.5 p.p.m could be identified as the distal phosphate group, while the phosphate at )9.7 p.p.m was identified as the proximal phosphate group The 1H,31P-HSQC spectrum showed correlation between H-2 and H-4 of heptose U, with typical signals of a phosphate monoester group The H-6 V resonance, present as two different signals, showed two different cross-peaks, one with a phosphate monoester group at 4.46/3.2 p.p.m and the other at 4.63/)9.7 p.p.m., with the proximal phosphate of a diphosphate monoester residue Thus, heptose U is substituted at O-2 and O-4 by a phosphate group, whereas heptose V carries at O-6 a phosphate group or alternatively, a pyrophosphate group The MALDI-TOF mass spectrum (Fig 8) of oligosaccharide confirmed all the assignments, as all ion peaks corresponding to the structures above were present In fact, ion peaks characteristic of an oligosaccharide were found, composed of two HexNAc, one Hex, two Hep, one Kdo, one carbamoyl group and two, three, four and five phosphate groups Moreover, additional peaks at Dm/z 146 accounted for the presence of a second core glycoform, which differs from the most abundant one by an additional rhamnose residue that must be linked at O-6 of glucose Ion peaks derived from the loss of water from molecular ions, probably Kdo lactone or anhydro-Kdo forms, were also present Furthermore, the MALDI-TOF mass spectrum also accounted for the presence of a very small amount of pentaphosphorylated species, which was not detected by NMR Because no different phosphate substitution was visible in 1D and 2D 31P-NMR, we propose that the fifth phosphate group is present as pyrophosphate on heptose U In conclusion, information derived from both acid and alkaline hydrolysis leads to the proposal of the following structure of the major oligosaccharide from the LOS of P stutzeri OX1 Ó FEBS 2004 2700 S Leone et al (Eur J Biochem 271) Table 1H, 13C and 31P NMR chemical shifts (p.p.m.) of the oligosaccharide product deriving from acetic acid treatment of the LOS from P stutzeri OX1 O-6 V resonances are given in parentheses when this position is monophosphorylated P/P refers to both resonances of pyrophosphate Because Kdo signals are spread due to its multiple forms, resonances are not given Resonances of the minor fragment Rha-C1fi6)-Glc are also shown at the bottom Chemical shifts are relative to acetone and external aq 85% (v/v) phosphoric acid (1H, 2.225 p.p.m.; 13C, 31.45 p.p.m.; 31 P, 0.00 p.p.m at 30 °C) Residue 13 H/ C/ P U Hep 5.23 99.2 V Hep 5.14 102.7 X GalN W GlcN Z Glc Ac 5.14 99.8 4.91 101.2 4.53 104.4 Cm Glc Rha 4.56 73.8 1.4 4.39 69.17 4.21 76.8 4.23 74.5 4.07 70.9 3.77 64.1 4.02 78.1 4.68 73.7 1.8 4.11 67.9 4.15 71.6 4.59/3.96 62.4 4.26 49.8 3.67 56.1 3.29 73.5 2.0–2.1 22.3 4.12 77.3 3.67 72.6 3.49 75.6 4.39 75.0 3.61 73.6 3.48 70.1 4.12 73.5 3.41 70.0 3.40 76.9 4.63 (4.46) 71.2 (70.2) )9.7/)5.5 (3.2) 3.72 60.1 3.612/3.88 61.2 3.74/3.89 60.7 3.30 73.5 3.96 70.5 3.52 76.5 3.73 70.2 3.79 70.9 3.42 72.9 3.80 75.1 4.01 69.2 3.93 69.7 1.26 17.0 31 174.6–175.0 160.0 4.53 104.4 4.86 101.7 Fig Section of the 1H,31P-HSQC spectrum of oligosaccharide The spectrum shows cross peaks relevant for the localization of the phosphate groups Ó FEBS 2004 LPS from Pseudomonas stutzeri OX1 (Eur J Biochem 271) 2701 Discussion Several core oligosaccharides from Pseudomonas strains have already been isolated and characterized [12,13], mainly from Pseudomonas aeruginosa strains [21,37–43] The carbohydrate backbone of the so-called inner core region of Pseudomonas LPS determined so far has always been found to be identical in different strains [12,13,21,37–43], which indicates a strict biosynthetic control It contains two residues of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), two residues of L-glycero-D-manno-heptose (Hep), one O-carbamoyl group (Cm), which links the O-7 of a Hep residue, and a 2-amino-2-deoxy galactose (GalN), which is the branching point of the oligosaccharide The amino function of the GalN residue is frequently acylated with alanine, but in a few cases an acetyl group has been found [38] The inner core region is always characterized by the presence of a large number of negative charges, usually carried by phosphate groups, which are linked to heptose residues, in addition to the key phosphate residues attached to the lipid A backbone The outer core is more variable than the inner part, usually resulting in two outer core glycoforms However, the architecture of the outer core is common, with a GalN residue linked by two glucose moieties, one of which could carry a rhamnose residue, the key residue substituent for the O-polysaccharide transfer [40–42] The structure of the inner core region of the LOS from P stutzeri OX1, as determined in this study, was found to agree closely with the general structure described above It includes the characteristic monosaccharide residues, a large number of phosphate groups at the proper location, a carbamoyl moiety, and an acetyl group It is worth noting that the nature and the localization of phosphate substituents on heptose residues in core oligosaccharides of Pseudomonas have recently been resolved, mainly by MS techniques following alkaline and acid degradations [37–43] Also in this case, the comparative analysis by NMR and MS of products obtained by either Fig Negative ion MALDI-TOF-MS spectrum of oligosaccharide recorded in reflector mode Assignments of main ion peaks are shown Dm/z 18 is due to Kdo present in reducing or lactone form P, phosphate; Cm, 7-O-carbamoyl; Ac, acetyl Ó FEBS 2004 2702 S Leone et al (Eur J Biochem 271) alkaline and acid degradation allowed the complete identification and localization of the labile groups, i.e pyrophosphate groups, which are commonly lost in alkaline treatment As for the outer core structure of the LOS from P stutzeri OX1, the structure we have determined was found to be of special interest, both for its novelty with respect to the structures already determined [12,13,21,37–43], and for its biosynthetic implications A GalNAc residue substituted by two gluco-configured residues, at O-3 by glucose and at O-4 by GlcNAc, was found To our knowledge, a GlcNAc residue directly linked to GalN has never been found in the core region structure of Pseudomonas Moreover, both of the two gluco-configured residues are blocked at position O-4 and O-6 by a pyruvate ketal linkage The presence of pyruvate residues in the core region of lipopolysaccharides is also new Although pyruvate residues are frequently found in bacterial exopolysaccharides [44,45], and in O-polysaccharides [46] as a postpolymerization decoration, they have never been found as core constituents In our opinion, this finding could be relevant to the understanding of adaptive chemical alterations of the outer membrane of P stutzeri OX1 as a consequence of its exposure to solvents, which has already been documented in other cases [1,8,9] Most of the modifications described essentially involve phospholipid head groups and fatty acid composition, whereas little is known about LPS alterations On the basis of this novel structure, we can advance the hypothesis that the presence of pyruvate residues in lipopolysaccharides is a new, structurally mediated, biochemical response to a harsh and odd environment, such as that in which P stutzeri OX1 was selected Our hypothesis is based on the consideration that insertion of two bulky pyruvate groups at the end of the oligosaccharide chain blocks its elongation, by creating a structural hindrance to the action of glycosyl transferases This leads to a shorter LOS, and hence to a less hydrophilic cellular surface Thus, the presence of pyruvate moieties might represent a chemical camouflage of the glycosyl transferase substrate, obtained by linking a key molecule from the primary metabolism in a simple and mild chemical bond This chemical protection is very similar to the isopropylidene group, one of the most widespread protecting groups in coupling reactions of oligosaccharide syntheses It should be added that this hypothesis is also supported by the finding that an alternative minor glycoform of the LOS has been found, with the typical oligosaccharide structure of a potential substrate for chain elongation and O-polysaccharide attachment [40–42] In this latter chain a rhamnose residue is present at O-6 of the glucose residue substituting the pyruvate moiety This finding would confirm that pyruvate residues are used to block chain elongation in the LOS of P stutzeri OX1 Thus, the structure of the R-type LPS (LOS) that we have found in Pseudomonas stutzeri OX1 would represent an adaptive response of the microorganism to a hydrocarboncontaining environment, because the presence of a long hydrophilic O-polysaccharide chain could have hindered its suitability to an external medium characterized by the presence of aromatic hydrocarbons Moreover, the peculiar presence of bulky pyruvate residues as blocking groups might offer an additional advantage to P stutzeri OX1, as these residues could help prevent the massive entrance of external organic compounds, which could be detrimental to its catabolism In fact, their presence increases the total negative charges of the LOS, thus altering the physical properties of the external membrane It is already known [1,8,9,47,48] that polyanionic LOS molecules electrostatically bind divalent cations; thus, an increased capability to bind cations might favor better packing of membrane molecules and constitute a selective barrier to the entrance of organic molecules Acknowledgements This work was supported by grants (to A.D.D and M.P.) from the Ministry of University and Research (PRIN/2000 and PRIN/2002) A.M thanks Hermann Moll (Research Center Borstel) for carbamoyl and methylation analyses References Ramos, J.L., Duque, E., Gallegos, M.T., Godoy, P., Ramos´ Gonzalez, 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Several core oligosaccharides from Pseudomonas strains have already been isolated and characterized [12,13], mainly from Pseudomonas aeruginosa strains [21,37–43] The carbohydrate backbone of the. .. Elucidation of the structure of the ă lipopolysaccharide core and the linkage between the core and the O-antigen in Pseudomonas aeruginosa immunotype using strong alkaline degradation of the lipopolysaccharide... carbohydrate backbone from alkaline degradation of the rough form LPS from P stutzeri OX1 Isolation, NMR and MS analyses of oligosaccharide from acetic acid hydrolysis Further information on alkaline labile