The structure of the carbohydrate backbone of the lipopolysaccharide from Acinetobacter baumannii strain ATCC 19606 Evgeny V. Vinogradov 1, *, Jens é. Duus 1 , Helmut Brade 2 and Otto Holst 2 1 Department of Chemistry, Carlsberg Laboratory, Valby, Copenhagen, Denmark; 2 Division of Medical and Biochemical Microbiology, Research Center Borstel, Borstel, Germany The c hemical structure of the phosphorylated carbohydrate backbone of the lipopolysacchar ide ( LPS) from Acineto- bacter baumannii strain ATCC 19606 was investigated by chemical analysis and NMR spectroscopy of oligosaccha- rides obtained a fter deacylation or mild acid hydrolysis. From the c ombined i nformation the following carbohydrate backbones c an be deduced: -GalpNR 1 -GlcpN -Kdo 1 1 2 4 7 4 R 2 3)- -GlcpNAcA-(1 4)- -Kdo-(2 5)- -Kdo-(2 6)- -GlcpN4P-(1 6)- -GlcpN1P where R 1  HandR 2  a-Glcp-(1 ® 2) -b-Glcp-(1 ® 4)-b- Glcp-(1 ® 4)-b-Glcp-(1 as major and R 1  Ac and R 2  H as minor products. All monosaccharides are D -con®gured. Also, smaller oli- gosaccharide phosphates were identi®ed that are thought to represent d egradation products of the above structures. Keywords: Acinetobacter baumannii; lipopolysaccharide; core region; structural analysis; NMR spectroscopy. The Gram-negative bacterium Acinetobacter (Moraxella- ceae) i s isolated from soil and water w hich represent its natural habitats. However, the genus has gained increasing importance as a causative agent of nosocomial infections (e.g. bacteremia, secondary meningitis) in recent years [1]. As in other G ram-negative bacteria, Acinetobacter pos- sesses lipopolysaccharides (LPS) in t he outer membrane of the cell wall t hat have been shown to represent useful chemotaxonomic and a ntigenic markers for its identi®ca- tion and differentiation. The o ccurrence of S-form L PS in Acinetobacter has unequivocally b een proven recently, and the s tructures of a number of O-speci®c polysaccharides and t heir antigenic characterization have b een published [2±9]. T he core region of Acinetobacter LPS possesses particular structural features which clearly distinguish it from other core structures [10]. First, it belongs to the group of heptose-de®cient core regions. Second, it may contain D -glycero- D -talo-oct-2-ulopyranosonic acid (Ko) which can replace the 3-deoxy- D -manno-oct-2-ulopyrano- sonic a cid (Kdo) residue linking the core region to lipid A [11±15]. Another core type h as been identi®ed in A. baumannii strain NCTC 10303 [16] that is devoid of Ko. It comprises the tetrasaccharide a-Kdo-(2 ® 5)-[a-Kdo-(2 ® 4)-]-a- Kdo-( 2 ® 5)-a-Kdo-(2 ® [a-Kdo IV-(a-Kdo III)-a-Kdo II-a-Kdo I], of which Kdo IV is substituted at O-8 by a short-chain rhamnan and Kdo III at O-4 by the disaccha- ride a- D -GlcpNA c-(1 ® 4)-a-D-GlcpNA (GlcpNA, 2-ami- no-2-deoxy-glucopyranosuronic acid). Kdo I links the core region to the lipid A. This Kdo tetrasaccharide is unique in nature; however, a second Kdo tetrasaccharide of different structure has been identi®ed in LPS of Chlamydophila psittaci 6BC [17]. The latter has been shown to be assembled by one Kdo t ransferase, which is therefore multifunctional [18]. However, the biosynthesis of th e Kdo-tetrasaccharide from LPS of A. baumannii strain NCTC 10303 has not yet been elucidated. For A. baumannii strain ATCC 15303, which possesses i n its LPS c ore r egion the trisaccharide a-Kdo- (2 ® 5)-[a-Kdo-(2 ® 4)-]-a-Kdo-(2 ® , it has been shown that the Kdo t ransferase is able to transfer the ®rst two a-(2 ® 4)-linked Kdo residues [19]. The mechanism of the transfer of the third Kdo residue has not yet been identi®ed. In addition to the work on the determination of the chemical and antigenic structures of O-speci®c polysaccha- rides from LPS of Acinetobacter in order to establish an O-serotyping scheme, t here exists considerable interest in investigating the LPS core regions from this genus which obviously allows novel insights in structure, genetics and biosynthesis of LPS. Here, the structures of the carbohy- drate backbones of the LPS from A. baumannii strain ATCC 19606 are reported. Correspondence to O. Holst, Research Center Borstel, Parkallee 22, D-23845 Borstel, Germany. Fax: + 49 4537 188419, Tel.: + 49 4537 188472, E-mail: oholst@fz-borstel.de Abbreviations: LPS, lipopolysaccharide; GlcpNAcA, 2-acetamido-2- deoxy-glucopyranosyluronic acid; DGlcpNA, 2 -amino-2,4-dideoxy-b- L -threo-hex-4-enopyranosyluronic acid; HMBC, heteronuclear mul- tiple bond correlation; HMQC, heteronuclear multiple quantum co- herence; HPAEC, high-performance anion-exchange chromatography; Kdo, 3-deoxy- D -manno-oct-2-ulopyranosonic acid. Dedication: this article is dedicated to Prof Dr Joachim Thiem, Institute of Organic Chemistry, University of Hamburg, Germany on the occasion of his 60th birthday. *Present address: Institute for Biological Sciences, National Research Council Canada, Ottawa, Ontario K1A 0R6, Canada. (Received 23 July 2001, revised 29 October 2001, accepted 31 October 2001) Eur. J. Biochem. 269, 422±430 (2002) Ó FEBS 2002 MATERIALS AND METHODS Bacteria and Bacterial LPS A. ba umannii strain ATCC 19606 was cultivated and the LPS was obtained as described previously [11]. Preparation of oligosaccharides The LPS (80 mg) was hydrolysed in 5% acetic acid (100 °C, 5 h ), the precipitate was removed by ultracentrifugation (100 000 g, 4 h), and the supernatant was lyophilized (yield: 50 mg, 62.5% of the LPS). The latter sample was reduced with NaBH 4 , and, after working up, was d esalted by gel-permeation chromatography. High-performance anion- exchange chromatography (HPAEC) of this f raction yield- ed oligosaccharides 1 (4 mg, 5% of the LPS) and 2 (10 mg , 12.5% of the LP S), and two stereoisomers of 1 and 2 in minor amounts that differed b y the con®guration at C2 o f Kdo-ol. Another portion of the LPS (350 mg) was de-O-acylated as described previously [20] (yield: 236 mg, 67.4% of the LPS), and 150 mg of this de-O-acylated LPS was then de-N-acylated [20], and, after working up, desalted by gel-permeation chromatography. H PAEC of this sample yielded oligosaccharides 3 and 4 (10 and 25 mg, 2.9 and 7.1% of the LPS, respectively). General and analytical methods Gel-permeation chromatography was carried out on a column (3.5 ´ 40 cm) of TSK HW40 (S) gel (Merck) in water, and runs were monitored with a differential refractometer (Knauer). Preparative HPAEC was per- formed on a c olumn (9 ´ 250 mm) of CarboPac PA1 (Dionex Corp.) as described previously [20] usi ng linear gradient programs of 3 to 40% 1 M sodium acetate in 0.1 M NaOH over 80 min (for separation of oligosaccha- rides 1 and 2, F ig. 1) and 30 t o 70% o ver 8 0 min (for separation of oligosaccharides 3 and 4, F ig. 1). Samples Fig. 1. Structures of oligosaccharides 1±5. DGlcpNA, 2-amino-2,4-dideoxy-b- L -threo- hex-4-enopyranosyluronic acid (the product of the 4,5-b-eliminat ion of a-glucosaminuronic acid). Ó FEBS 2002 LPS from Acinetobacter baumannii (Eur. J. Biochem. 269) 423 Table 1 . 1 H NMR data o f oligosaccharides 1±4. Sp ectra were recorded o f solutions in 2 H 2 O relative to internal acetone (2.225 p.p.m.). Oligo, oligosaccharide. Chemical shift (in p.p.m.) and J H,H coupling constants (in Hz) of proton 123(ax)3eq 4 5 6(a) 6b 78a8b Residue Oligo J 1,2 J 2,3 J 3,4 ,J 3ax,4 J 3eq,4, J 3ax,3eq J 4,5 J 5,6 J 6a,6b J 5,6b A, a-GlcpN 3 5.62 a 3.38 3.85 3.62 4.10 3.72 4.24 3.5 12 1.9 4 5.63 b 3.39 3.86 3.63 4.12 3.73 4.26 3.5 10 10 9 9 12 1 B, b-GlcpN 3 4.83 3.03 3.82 3.74 3.72 3.38 3.65 4 4.85 3.03 3.83 3.75 3.73 3.39 3.68 8.5 10.2 10 C, a-Kdo 3 2.31 12.1 1.90 4 12.2 3.93 4.47 3.66 3.85 3.59 3.85 4 2.33 12.5 1.92 3.5 12 3.95 <1 4.48 3.67 9 c 3.86 3.60 3.86 D, a-Kdo 3 1.69 12.3 2.06 2.4 12.8 3.99 <1 3.95 <1 3.68 3.94 3.69 3.90 4 1.70 12.7 2.07 4.6 12.3 4.01 <1 3.96 <1 3.69 9 c 3.96 3.70 3.89 E, aKdo-ol/a-Kdo 1 4.06 4 d 1.93 6 e 2.08 6 3.99 6 f 4.10 6 3.73 4 6 c v 3.87 3.69 3.83 2 4.16 2.00 2.17 4.02 4.17 3.78 3.89 3.76 3.88 3 2.29 12 2.90 2.7 12.0 4.84 <1 4.43 <1 4.27 9 c 4.12 3.91 3.91 4 2.30 12 2.91 4 12 4.87 <1 4.43 <1 4.29 9 d 4.13 3.92 3.92 F, a-GlcpAN 1 4.99 3.92 3.86 3.71 4.12 3.5 10 10 10 2 4.97 4.20 4.29 3.94 4.25 3.5 10 9 10 3 5.45 3.54 4.45 5.88 2 4.2 4.2 4 5.470 3.762 4.658 5.982 1.6 4.1 4.1 G, a-GalpN g 1 5.36 4.09 3.82 3.93 3.90 3.63 3.70 4 10 2.5 2 5.74 3.26 3.87 3.96 3.92 3.68 3.68 3.8 9.7 2 Y, a-Glc 2 5.34 3.51 3.73 3.43 4.04 3.75 3.78 4 5.34 3.51 3.72 3.42 4.04 3.75 3.77 3.8 10 3.5 13 R, b-Glc 2 4.63 3.49 3.57 3.41 3.43 3.69 3.90 7.8 10 4 4.63 3.49 3.56 3.41 3.44 3.70 3.89 7.8 9.4 T, b-Glc 2 4.50 3.34 3.64 3.75 3.63 3.85 3.96 4 4.50 3.33 3.64 3.74 3.63 3.85 3.97 8.0 9.5 9 9 4 12 S, b-Glc 2 4.61 3.32 3.60 3.68 3.52 3.85 3.96 4 4.72 3.33 3.64 3.64 3.63 3.81 3.97 8.0 9.5 4 12 Z, b-GlcN 1 4.77 2.98 3.54 3.40 3.43 3.67 3.85 2 4.54 2.68 3.37 3.36 3.43 3.69 3.90 8.2 9.5 9.5 3 4.96 3.09 3.65 3.42 3.50 3.69 3.90 8.4 10 9 9.9 2.3 6.2 h 4 4.97 3.12 3.66 3.45 3.52 3.72 3.92 8.3 10.6 a J 1,P 6.5 Hz; b J 1,P 6.6; c J 6,7; d J 2,3; e J 2,3¢ ; f J 3¢,4 ; g additional signal in oligosaccharide 1:CH 3 CO, 2.033 p.p.m; h J 5,6a . 424 E. V. Vinogradov et al. (Eur. J. Biochem. 269) Ó FEBS 2002 were desalted using a Dowex 50 ´ 4(H + ) cation exchanger in water, and amino-group containing compounds were then eluted with 5% aqueous ammonia. GLC-MS , mono- saccharide analysis, and the determination of the absolute con®guration of monosaccharides were performed as described p reviously [14,15,21]. Conformational analysis was carried out as described previously [16]. NMR spectroscopy For structural assignments, 1D and 2D 1 Hand 13 CNMR spectra were recorded on solutions of oligosaccharides 1±4 in 2 H 2 O(500lL) with a Bruker AMX-600 spectrometer at 27 °C using standard Bruker software. Chemical shifts are given relative to internal acetone (CH 3 -, 2.225 p .p.m. for 1 H, 34.5 p.p.m. for 13 C). The assignment of the proton chemical shifts was a chieved by c orrelation spectroscopy (COSY), total correlation spectrosco py (TOCSY), and nuclear Overhauser enhancement s pectroscopy (NOESY), and the assignment of the carbon chemical shifts was performed b y heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond correlation (HMBC) experiments. Broad band 1 H-decoupled 31 P NMR spect ra w ere r ecorded at 10 1.25 MHz with a Bruker DRX 250 spectrometer at 2 7 °C, and chemical shifts are given relative to an external reference of 85% phospho- ric acid (0.0 p.p.m.). Mass spectrometry The electrospray mass spectra (ES-MS) were acquired in the negative mode on a VG Quattro triple quadropole mass spectrometer (VG Biotec h, Altrincham, Cheshire, UK) with 50% aqueous CH 3 CN as the mobile phase at a ¯ow rate of 8 lLámin )1 . Ten microlitres of a 20 l M aqueous solution of the samples were injected into the electrospray source. RESULTS AND DISCUSSION Sugar analyses of the LPS Monosaccharide a nalyses of the LPS f rom A. baumannii strain ATCC 19606 revealed the presence of D -Glc, D -GlcN, D -GalN, and Kdo. The D -con®guration of Kdo was determined on the basis of the optical rotation value [a] D  +56 ° (C 1, in water) of Kdo methyl ester a-methyl glycoside isolated after methanolysis of the LPS [14]. Table 2. 13 C NMR data of oligosaccharides 1±4. Spectra were r ecorde d of solutions in 2 H 2 O relative to internal acetone (34.5 p .p.m.). Oligo, oligosaccharide. Residue Oligo Chemical shift of carbon (in p.p.m.) 12345678 A, a-GlcpN 3 91.7 a 55.4 b 70.4 70.3 73.4 70.7 4 91.6 55.4 70.4 70.3 73.4 70.4 B, b-GlcpN 3 100.2 56.4 72.7 75.2 c 74.6 d 63.2 4 100.4 56.4 72.8 75.1 74.7 63.2 C, a-Kdo 3 175.9 100.8 35.2 73.4 70.0 73.3 70.2 64.6 4 176.1 100.8 35.1 73.4 70.0 73.3 70.3 64.6 D, a-Kdo 3 176.0 103.4 35.1 66.7 67.7 72.8 71.8 63.7 4 176.0 103.4 35.2 66.7 67.6 72.8 71.6 63.7 E, Kdo-ol/a-Kdo 1 182.4 71.2 36.6 76.8 70.5 70.0 80.5 61.9 2 182.0 70.7 37.0 76.5 70.4 69.2 80.1 61.2 3 176.0 101.9 30.0 71.8 63.1 71.0 77.3 60.6 4 176.0 101.8 30.0 71.8 63.1 71.0 77.1 60.6 F, a-GlcpAN 1 97.0 55.3 73.4 77.2 74.4 177.3 2 97.6 54.0 77.7 74.0 74.2 175.0 3 91.8 53.4 63.3 105.8 147.7 169.0 4 92.2 51.3 71.6 104.4 148.4 168.7 G, a-GalN 1 98.3 50.9 68.7 69.4 71.8 61.9 2 96.7 51.5 68.5 68.7 71.7 61.3 23.1 176.3 R, b-Glc 2 102.2 77.1 75.3 70.7 76.7 61.8 4 102.2 77.1 75.3 70.4 76.7 61.4 T, b-Glc 2 103.2 73.5 74.5 76.6 75.7 60.8 4 103.1 73.9 74.8 76.6 75.7 60.8 S, b-Glc 2 103.5 73.5 74.7 79.1 75.4 59.7 4 102.2 73.5 74.5 79.1 75.7 60.6 Y, a-Glc 2 98.2 72.1 73.5 70.1 72.4 61.1 4 98.2 72.1 73.5 70.1 72.4 61.1 Z, b-GlcN 1 100.4 57.3 74.1 71.1 77.3 61.7 2 103.3 57.4 76.4 70.7 76.7 61.4 3 98.2 56.6 72.9 71.8 76.7 61.4 4 98.1 56.6 72.9 70.7 76.7 61.5 a J 1,P 0.5 Hz; b J 2,P 7.5 Hz; c J 4,P 3.5 Hz; d J 5,P 6.5 Hz. Ó FEBS 2002 LPS from Acinetobacter baumannii (Eur. J. Biochem. 269) 425 Isolation and structural analysis of oligosaccharides 1±4 Acetic acid degradation of the LPS and reduction yielded as main products two stereoisomers each of the two oligosac- charides resulting from r eduction of the carbonyl group of the reducing Kdo residue. Oligosaccharides 1 and 2 as the major isomers were isolated by HPAEC and were examined by NMR spectroscopy (data summarized in Tables 1±3). All proton and carbon resonances could be a ssigned. Most relative con®guration s and ring sizes of the monosac- charides were determined on the basis of vicinal proton± proton coupling constants. The ano meric con®gu rations of hexoses followed from J H1,H2 coupling constants (3.5±4 Hz for a-linked and 7.8±8.4 Hz for b-linked residues). All residues were present in the pyrano se form, as deduced from the absence of low-®eld signals i n t he 13 C-NMR spectra that are characteristic for furanoses, and from the vicinal proton coupling constan ts. The NOE data (Table 3) allowed the unambiguous determination of the sequence of monosac- charide residues. In the low-®eld region of t he 1 H-NMR spectra of oligosaccharides 1 and 2 (4.4±5.4 p.p.m.), signals for t hree and seven anomeric protons were present, respectively. The two oligosaccharides differed from each other by the tetrasaccharide fragment Y-R-T-S- (see Fig. 1). In both oligosaccharides, the Kdo residue was present as an octitol derivative (H2 at 4.061 and 4.159 p.p.m., respectively). For each oligosa ccharide, all aldose residues gave NOE signals between the H1 protons and the proton on the carbon atom to which they w ere glycosidically linked (Table 3). Corre- lations between the anomeric protons and the transglycosi- dic carbon atom could also be detected in HMBC spectra, con®rming the monosaccharide sequence and attachment sites, the latter being assigned from 13 C-NMR spectroscopic data using HMQC experiments (Table 2). The structures of oligosaccharides 1 and 2 were con®rmed by e lectrospray mass spectrometry, the spectra of which gave for o ligosac- charide 1 an ion at m/z 822.4 ([M + H] + ,calculated molecular mass 821.9 Da) and for oligosaccharide 2 ions at m/z 1428.6 (calculated molecular mass 1428.4 Da) and 715.2, representing [M + H] + and [M + 2H]/2 + ,respec- tively. From the resulting mixture of deacylated LPS, the two major oligosaccharides 3 and 4,aswellasthetetrasac- charide a-Kdo- (2 ® 4)-a-Kdo-(2 ® 6)-b-GlcpN4P-( 1 ®6) -a-GlcpN1P, the trisaccharide a-Kdo- (2 ® 6)-b- GlcpN4P-(1 ® 6)-a-GlcpN1P, and several minor com- pounds were isolated by HPAEC. The tetrasaccharide and the trisaccharide were readily identi®ed on the basis of published NMR data [24,25], and the minor compounds were not further investigated. The structures of oligosac- charides 3 and 4 were fully characterized by NMR spectroscopy (Tables 1±3). They differed from each other by the tetrasaccharide fragment Y-R-T-S- (Fig. 1), as observed also for oligosaccharides 1 and 2. T he substituent at O4 of GlcpNA F, Gal pN G present i n 1 and 2,waslostby b-elimination owing to the alkaline c onditions that also converted the GlcpNA into 2-amino-2,4-dideoxy-b- L -threo- hex-4-enopyranosyluronic a cid. Oligosaccharides 3 and 4 were similar to the oligosaccharide 5,isolatedfromLPSof A. baumannii strain NCTC10303 [16], d iffering by the presence of b-GlcN Z and (in 4) b y t he tetrasaccharide sequence attached to O-3 of DGlcpNA F. Both oligosaccharides 3 and 4 contained three Kdo residues. All monosaccharide residues were present in the pyranose form, as deduced from the absence of low-®eld signals in the 13 C-NMR spectra that are characteristic for furanoses, and from the vicinal proton coupling c onstants (Tables 1 and 2). NOE contacts between H-1 of DGlcpNA FandH-3ax,H-3eq, H-4 and H-5 of one of the Kdo residues identi®ed the latter as residue E (Fig. 3), which was present as an octitol residue i n compounds 1 and 2.The Kdo residue E w as substituted by b-GlcN Z, as was apparent from the observation of an NOE between protons Z1 (b-GlcpN) and E7 ( Fig. 2, Table 3). The NOE contacts between protons C5 and E3ax,E3eq , E4, and E6 (Fig. 3) suggested t he attachment of E to C5. The sequence of the sugar residues C, D, E , and F was ®n ally proven by long- range H -C correlations that were present in t he HMBC spectrum of heptasacch aride 3, namely between proton C4 and carbon D2, C5 and E2, and F1 and E4. The signals of the anomeric carbons in Kdo residues were assigned on the basis of the intraresidual correlations between H-3eq and C-2. The a-con®guration of the Kdo residues C and D followed from the observed NOE between protons C3eq and D6 (Fig. 3), characteristic for the fragment a-Kdo- (2 ® 4)-a-Kdo [25], which were also consistent with the sequence D-(2 ® 4)-C. The a-con®guration of the Kdo residues C and D in 3 and 4 each was con®rmed by the chemical shifts of their H-3ax, signals (1.7±2.3 p .p.m.) [22,23]; however, the chemical shifts of H-3ax,ofCwere interchanged. The chemical shifts of protons H-3, H-4, H-5, and H-6 of Kdo E occurred at unusual low ®eld positions, Table 3. Interresidual NOE contacts observed in oligosaccharides 1±4. s, Stro ng NOE; m, medium NOE; w, weak NOE. Labeling i s a s f or Fig. 1. Oligosaccharides Observed NOE contact From proton To protons 3, 4 B-1 A-6a m, A-6b m 3, 4 C-3ax D-6 s, E-6 m 3, 4 C-3eq D-6 s, D-7 m 3, 4 D-3eq C-5 w, E-5 m, E-6 m 3, 4 E-3ax C-5 m, F-1 w 3, 4 E-3eq C-5 s, D-3eq m, F-1 m 3 E-4 C-5 s, F-1 s 4 E-4 C-5 s, C-7 w, D-4 w, D-7 w, F-1 s 3, 4 E-6 C-3 axw, C-5 s, C-7 s, D-3 eqm 1, 2 F-1 E-4 s, E-5 m, E-6 m 3 F-1 E-3 eqw, E-3 axw, E-4 s, E-5 s 4 F-1 E-3 eqw, E-3 axw, E-4 s, E-5 s, E-6 w, D-4 w 1, 2 G-1 F-2 w, F-3 w, F-4 s 2, 4 R-1 T-3 m, T-4 s, T-6 am, T-6 bm 2 S-1 F-3 s 4 S-1 F-2 s, F-3 s 2, 4 T-1 S-4 s, S-6 am, S-6b m 2, 4 Y-1 R-1 w, R-2 s, T-1 w, T-5 m, T-6 m 1, 2 Z-1 E-7 s 3, 4 Z-1 E-5 w, E-7 s, E-8 s 426 E. V. Vinogradov et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Fig. 2. Part of the NOESY spectrum of oligosaccharide 4. Fig. 3. Some important observed interresidual NOE connectivities in the structural element C-D-E-F of oligosaccharide 4. The depicted structure represents one minim um energy conformation. Ó FEBS 2002 LPS from Acinetobacter baumannii (Eur. J. Biochem. 269) 427 nevertheless, on the basis of conformational analysis (see below) and comparison o f the data with those of structure 5, this residue is proposed to possess th e a-con®guration. Oligosaccharide 3 possessed a structure similar t o oligo- saccharide 5 (Fig. 1), which had been isolated earlier f rom the LPS of A. baumannii strain NCTC10303 [16]. The only difference was the presence of a-GlcpNZin3. However, signi®cant differences of up to 0.4 p .p.m. w ere observed between some proton signals of 3 and 5, i.e. C3 ax,E3ax, E3eq, E4 and E5. In o rder to exclude any in¯uence o f varying experimental conditions, 1 H-NMR a nd COS Y spectra of a mixture of oligosaccharides 3 and 5 were recorded, resulting in much closer chemical shifts: the signals of C3ax,E3ax,E3eq, E4 and E5 of 5 were observed at 2.174, 2.125, 2.607, 4.632, and 4.251 p.p.m., respectively, and of 3 at 2.190, 2 .184, 2.764, 4.709 , and 4.270 p.p.m, respectively. Other signals (except those from p rotons close to the attachment point of GlcpNZ)overlappedinboth structures. The differences i n chemical shifts for 3 and 5 obtained for the isolated samples must therefore be due to small differences in sample conditions, e.g. concentration, pH or ionic strength. Also, most of the NOE contacts (Fig. 2 ) were similar. Thus, oligosaccharides 3 and 5 possess the common hexasaccharide fragment F ® E ® (D ® ) C ® B ® A, and Kdo E is proposed to be present in oligosaccharides 3 and 4 in a-pyranosidic form, a s shown earlier for the o ligosaccharide 5 isolated from the LPS of A. baumannii strain NCTC10303 [16]. The conformation o f the fragment F-C was similar in 3, 4 and 5, but not identical. An NOE contact between protons E3eq and D3eq was only observed in the oligosaccharides from strain A TCC 1960 6 (compounds 3 and 4). In contrast, an NOE contact between E3eq and F5 was only observed in the p roducts from strain NCTC 10303 (e.g. compound 5). Conformational analyses showed that a close proximity of protons E3eq to D3eq and of E3eq and E4 to C5 could not simultaneously occur in the same low energy conformation (assuming the chair confor- mation of Kdo E). Thus, the observed NOE contacts originate probably from different conformational minima or from a d istorted ring conformation of Kdo E . T he last possibility was supported by the observation of the intra- residual NOE contact between E3 eq and E6, which is n ot possible in the 5 C 2 con®guration. The existence of residue E in the equilibrium of several conformers agrees with the observation of its broadened and unresolved H-3 signals, whereas all other signals in the spectra of 3 and 4 were sharp and well resolved. 31 P NMR sp ectra o f oligosaccharides 3 and 4 showed two signals of equal intensity at 2.2 and 4.5 p.p.m., which in 1 H, 31 P-HMQC spectrum correlated with protons A1 and B4, respectively, thus proving phosphorylation at positions O-1 of A and O -4 of B. In summary, the structures of oligosaccharides 1±4 were elucidated as depicted in Fig. 1. The structures possess a common branched tetrasaccharide fragment G-F-[Z-]-E, which is substituted in compound 2 at O- 3 of GlcpNA F with the tetraglucosyl sequ ence Y-R-T-S Oligosaccharides 1 and 2 differ also in the acetylation o f the amino group of GalN G. In the octasaccharide, this amino g roup is not acetylated and H-2 of the residue G resonates at 3.264 p.p.m., whereas in the tetrasaccharide this amino- group is acetylated, thus, this signal is shifted to 4.092 p.p.m. Structures 1±4 can b e combined to a complete structure of the carbohydrate backbone of the LPS from A. baumannii strain ATCC 19606 as depicted in Fig. 4. The structures o f four core regions from LPS of d ifferent Acinetobacter strains have been characterized during this work. Two of these core regions (i.e. o f LPS from A. haemolyticus strain ATCC 17906/NCTC10305 and from Acinetobacter strain A TCC 1 7905) have i n c ommon t he presence of Ko, which replaces in part the K do residue that links the core oligosaccharide to the lipid A [14,15]. In bo th cases, this ®rst Ko or Kdo residue was substituted at O-5 by the t risaccharide a- D -Glcp-(1 ® 6)-b- D -Glcp-( 1 ® 4)-a- D - Glcp (which in the core region of strain ATCC 17906/ NCTC10305 is phosphorylated at O-6 o f the 4-substituted a-Glcp). The other two core regions were identi®ed in L PS of A. baumannii, i.e. in s trains A TCC 17904/NCTC 10303 [16] and ATCC 19606 (this w ork). Their common feature is the p rese nce o f t he trisaccharide a-Kdo-(2 ® 5)-[a-Kdo- (2 ® 4)-]-a-Kdo-(2 ® , which has also been identi®ed in the core r eg ion of LPS from A. baumannii strain ATCC 15303 [19]. Thus, it is possible t hat this t risaccharide represents a partial structure o f the core region which i s speci®c for LPS of A. b aumannii. The oligosaccharides identi®ed in the core region of LPS from A. baumannii strain ATCC 19606 suggests how a part of its b iosynthesis may occur. Approximately one-third of the core region is terminated by a D -GalpNAc residue (G in Fig. 1), as indicated b y the yield of oligosaccharide 1. Here, the D -GlcpNAcA residue F is not substituted at O -3. The other two-thirds of the core comprised an oligosaccharide in which G is a D -GalpN residue and F bears at O-3 the tetrasaccharide a-Glcp-(1 ® 2)-b-Glcp-(1 ® 4)-b-Glcp- (1 ® 4)-b-Glcp-(1 ® . Thus, it is r easonable to assume that the tetrasaccharide is introduced only after enzymatic de-N-acetylation of GalpNAc and that 1 represents a precursor o f core region biosynthesis. Additionally, the isolation of the tetrasaccharide a-Kdo-(2 ® 4)-a-Kdo- (2 ® 6)-b-GlcpN4P-(1 ® 6)-a-GlcpN1P and the trisaccha- ride a-Kdo-(2 ® 6)-b-GlcpN4P-(1 ® 6)-a-GlcpN1P from deacylated LPS suggests that the a-( 2 ® 4)-linked Kdo disaccharide is furnished ®rst in biosynthesis of t he core region, as found e.g. i n LPS biosynthesis of Escherichia coli Fig. 4. The complete chemical structure of the carbohydrate backbone of the LPS from Acinetobacter baumannii strain ATCC 19606. 428 E. V. Vinogradov et al. (Eur. J. Biochem. 269) Ó FEBS 2002 [26,27]. Then, the biosynthesis of the core region of LPS from A. baumannii strain ATCC 19606 should proceed with the introduction of a third Kdo residue to O-5 of the second Kdo. This represents a remarkable difference to the biosynthesis of LPS in E. coli (and in other bacteria), in which the second Kdo is substituted at O-5 by one residue of L -glycero- D -manno-heptopyranose [10,26,27]. The K do transf erase o f strain A TCC 1 5303 has b een cloned and characterized in vitro whereby it was shown that it is able to transfer two Kdo residues in a-(2 ® 4)-linkage to a lipid A precursor [ 19]. No evidence was obtained for a second Kdo t ransferase for which we searched by Southern blots. 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Marcel Dekker Inc., New York. 27. Gronow, S. & Brade, H. (2001) Lipopolysaccharide biosynthesis: whichstepdobacterianeedtosurvive?J. Endotoxin Res. 7, 3±23. 430 E. V. Vinogradov et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . biosynthesis of Escherichia coli Fig. 4. The complete chemical structure of the carbohydrate backbone of the LPS from Acinetobacter baumannii strain ATCC 19606. 428. The structure of the carbohydrate backbone of the lipopolysaccharide from Acinetobacter baumannii strain ATCC 19606 Evgeny V. Vinogradov 1, *,