Báo cáo khoa học: Structural and serological studies on a new 4-deoxy-D-arabino-hexosecontaining O-specific polysaccharide from the lipopolysaccharide of Citrobacter braakii PCM 1531 (serogroup O6) pptx

Structural and serological studies on a new 4-deoxy- D - arabino -hexose- containing O-specific polysaccharide from the lipopolysaccharide of Citrobacter braakii PCM 1531 (serogroup O6) Ewa Katzenellenbogen 1 , Nina A. Kocharova 2 , George V. Zatonsky 2 , Danuta Witkowska 1 , Maria Bogulska 1 , Alexander S. Shashkov 2 , Andrzej Gamian 1 and Yuriy A. Knirel 2 1 L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroc ł aw, Poland; 2 N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia The O-specific polysaccharide of Citrobacter braakii PCM 1531 (serogroup O6) was isolated by mild acid hydrolysis of the lipopolysaccharide (LPS) and found to contain D -fucose, L -rhamnose, 4-deoxy- D -arabino-hexose and O-acetyl groups in molar ratios 2 : 1 : 1 : 1. On the basis of methylation analysis and 1 Hand 13 C NMR spectroscopy data, the structure of the branched tetrasaccharide repeating unit of the O-specific polysaccharide was established. Using various serological assays, it was demonstrated that the LPS of strain PCM 1531 is not related serologically to other known 4-deoxy- D -arabino-hexose-containing LPS from Citrobacter PCM 1487 (serogroup O5) or C. youngae PCM 1488 (sero- group O36). Two other strains of Citrobacter, PCM 1504 and PCM 1505, which, together with strain PCM 1531, have been classified in serogroup O6, were shown to be serologi- cally distinct from strain PCM 1531 and should be reclassi- fied into another serogroup. Keywords: Citrobacter braakii; lipopolysaccharide; O-anti- gen structure; serological specificity; 4-deoxy- D -arabino- hexose. Certain strains of the genus Citrobacter often cause serious opportunistic infections. Most frequently, these bacteria are the aetiological factor of enteric diseases but they are also associated with extraintestinal disorders, among which the most significant are neonatal meningitis and brain abscesses [1,2]. On the basis of genetic studies, the genus Citrobacter has been recently classified into 11 species [3]. Based on their lipopolysaccharides (LPS), strains of Citrobacter are divided into 43 O-serogroups [4] and 20 chemotypes [5]. The structures of about 30 different Citrobacter O-antigens (polysaccharide chains of the LPS) have been established and chemical data employed to improve the serological classification of Citrobacter strains and to substantiate multiple cross-reactions between Citrobacter and other genera of the family Enterobacteriaceae,suchas Hafnia, Escherichia, Klebsiella and Salmonella [6]. Now we report on the structure of the O-specific polysaccharide (OPS) of Citrobacter braakii PCM 1531, which belongs to serogroup O6 (O6,4b,5b:72) and chemotype VI [5], and on the occurrence in this OPS of 4-deoxy- D -arabino-hexose (ara4dHex). Serological studies were undertaken to deter- mine whether there is any serological relatedness between the LPS of the strain studied and those of Citrobacter strains PCM 1487 and PCM 1488, which also contain the same rarely occurring monosaccharide, as well as of two other Citrobacter strains, PCM 1504 and 1505, which, together with strain PCM 1531, are included in serogroup O6. Materials and methods Gel chromatography and GLC-MS Gel-permeation chromatography was carried out on a column (2 · 100 cm) of Sephadex G-50 in 0.05 M pyridinium acetate buffer, pH 5.6, and monitored by the phenol–H 2 SO 4 reaction. GLC-MS was performed with a Hewlett-Packard 5971 instrument (Palo Alto, CA, USA), equipped with an HP-1 glass capillary column (12 m · 0.2 mm), using a temperature program of 150– 270 °Cat8°CÆmin )1 . NMR spectroscopy Samples were deuterium-exchanged by freeze-drying three times from D 2 O, and examined in a solution of 99.96% D 2 O. Spectra were recorded using a Bruker DRX-500 spectrometer (Karlsruhe, Germany) at 30 °C. A mixing time of 150 and 200 ms was used in two-dimensional TOCSY and NOESY experiments, respectively. Chemical shifts are reported in relation to internal acetone (d H 2.225; d C 31.45). Correspondence to E. Katzenellenbogen, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-14 Wrocław, Poland. Fax: + 48 71 3371382; Tel.: + 48 71 3371172; E-mail: katzenel@immuno.iitd.pan.wroc.pl Abbreviations: ara4dHex, 4-deoxy- D -arabino-hexose; Fuc, fucose; Hep, L -glycero- D -manno-heptose; LPS, lipopolysaccharide; OPS, O-specific polysaccharide; Rha, rhamnose; R Glc , TLC mobility related to that of glucose; R Rha , TLC mobility related to that of rhamnose; t R , GLC retention time relative to that of glucitol acetate (sugar analysis) or to 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylglucitol (methylation analysis). (Received 5 February 2003, revised 11 April 2003, accepted 28 April 2003) Eur. J. Biochem. 270, 2732–2738 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03640.x Bacterial strains, isolation and degradation of the LPS C. braakii O6,4b,5b:72 (PCM 1531, IHE Be 58/57, St U260B) [5,7] was used in the structural studies and Citrobacter strains PCM 1504 (IHE Be 15/50), PCM 1505 (IHE Be 16/50), PCM 1487 (O5), PCM 1488 (O36) and PCM 1525 (IHE Be 52/57) (O4) were derived from the collection of the L. Hirszfeld Institute of Immunology and Experimental Therapy (Wrocław,Poland).Bacteriawere cultured in a liquid medium [8]. The LPS were obtained from acetone-dried bacteria by phenol–water extraction [9]. For structural studies, the LPS from strain PCM 1531 was isolated from both water (LPS- I) and phenol (LPS-II) phases and purified as described previously [10]. For serological studies, the LPS from strains PCM 1504, PCM 1505 and PCM 1487 were obtained following treatment with proteinase K [11]. The LPS from strains PCM 1487, PCM 1488 and PCM 1525 were as isolated previously [12–14]. In order to obtain the OPS and the core oligosaccharides, LPS-I and LPS-II were hydrolysed with aqueous 1% HOAc (100 °C, 2 h), and, after removal of a lipid sediment, the carbohydrate-containing supernatant was fractionated by gel-permeation chromatography on a column of Sephadex G-50 to give OPS-I or OPS-II (fraction P 1 ), core oligosac- charides (fraction P 3 ) and a low-molecular-mass material containing 3-deoxy- D -manno-oct-2-ulosonic acid. For sero- logical studies, OPS-II was O-deacetylated by treatment with aqueous 12% ammonia at 20 °C for 18 h. Sugar and methylation analyses The OPS was hydrolysed with 2 M CF 3 CO 2 H (120 °C, 2 h), 10 M HCl (80 °C, 30 min) or 0.5 M CF 3 CO 2 H (100 °C, 1 h), and monosaccharides were converted conventionally into alditol acetates [15] and analysed by GLC-MS. The content of ara4dHex was estimated by using the Cynkin–Ashwell method [16], the content of O-acetyl groups by the Hestrin procedure [17] and the content of 6-deoxyhexose according to Dische [18]. TLC was carried out on DC-Fertigplatten Kieselgel plates in a system of EtOAc/pyridine/HOAc/water (5 : 5 : 1 : 3, v/v/v/v). An authenticsampleof D -ara4dHex was obtained from the OPS of Citrobacter PCM 1487 [12] and PCM 1525 [13] by hydrolysis with 0.1 M HCl (80 °C, 2 h) or 0.5- M CF 3 CO 2 H (100 °C, 1 h). The sugar was stained on the chromatograms using the molybdate/H 2 SO 4 reagent [10 g Ce(SO 4 ) 2 ,25g (NH 4 ) 2 MoO 4 , 100 mL of concentrated H 2 SO 4, 900 mL of H 2 O] at 100 °C for 2–5 min. The absolute configurations of the monosaccharides were established by GLC-MS of the acetylated (S)-2-octyl glycosides [19]. Methylation of the OPS was performed according to the method of Gunnarsson [20]. Polysaccharide (0.5 mg) dissolved in dimethylsulfoxide (0.5 mL) was methylated with MeI (0.25 mL) in the presence of solid NaOH. The finely powdered NaOH ( 20 mg) was added to the solution of OPS prior to methylation and mixed vigrously, using a shaker or ultrasonic bath, for 5–10 min at 20 °C. After methylation (20 °C, 20 min) the reaction mixture was neutralized with 1 M acetic acid (1–2 mL) and the methy- lated product was purified by extraction (three times) with CHCl 3 /H 2 O (1 : 1, v/v) and recovered from the chloroform phase by evaporation. The methylated product was hydro- lysed, as described above for sugar analysis, and the partially methylated monosaccharides derived were conver- ted into the alditol acetates and analysed by GLC-MS. Smith degradation OPS-I, OPS-II and the O-deacetylated OPS-II (aqueous 12% NH 4 OH, 20 °C, 18 h) were oxidized with periodate (2.5 mg of OPS in 0.25 mL of 0.1- M NaIO 4 ;4°C for 72 h). Ethylene glycol (0.01 mL) was added to destroy the excess NaIO 4 , and the product was reduced with NaBH 4 (10 mg, 20 °C, 18 h), neutralized with aqueous 50% HOAc and codistilled four times with methanol. The oxidized OPS was dialysed against distilled water (3 · 10 L), hydrolysed with aqueous 2% HOAc (100 °C, 2 h), and the modified OPS was subjected to sugar and methylation analyses. Serological methods Rabbit antisera against Citrobacter strains PCM 1531 and PCM 1487 were prepared as described previously [21]. Passive haemagglutination and inhibition of passive haemagglutination were performed, using horse erythro- cytes, according to Romanowska & Mulczyk [8]. The erythrocytes were coated with a suspension of 1 mgÆmL )1 LPS in NaCl/P i (PBS: 0.15 M NaCl, 0.01 M Na 2 HPO 4 / NaH 2 PO 4 ,pH7.3). SDS/PAGE of LPS and proteinase K-treated bacteria [11] was performed by the method of Laemmli [22]. The gels were stained using the silver reagent [23]. Immunoblotting was carried out as described previously [24]. Briefly, after separation by SDS/PAGE, the LPS were transblotted from the gel onto an Immobilon P (Millipore Corp., Bedford, MA, USA) membrane. The transblot was incubated with antiserum, washed with Tris-buffered saline (20 m M Tris/HCl, 50 m M NaCl, pH 7.5) and incubated with alkaline phosphatase-conjugated goat anti-(rabbit IgG). The immunoblot was visualized with a staining reagent (nitro-blue tetrazolium and 5-bromo-4-chloro-3- indolyl phosphate in 0.05 M Tris/HCl, pH 9.5, containing 5m M MgCl 2 ). Double immunodiffusion was performed according to Ouchterlony [25], using 1% agarose in NaCl/P i (PBS) containing 1% polyethylene glycol 6000. Results and discussion Isolation and chemical analysis of the O-specific polysaccharide On phenol–water extraction [9], the LPS of C. braakii PCM 1531 was recovered from both aqueous layer (LPS-I) and phenol layer (LPS-II) in yields of 0.34% and 0.74%, respectively. Following SDS/PAGE, both LPS-I and LPS- II showed the same ladder-like pattern characteristic of S-type LPS (Fig. 1A, lanes 1 and 2). Mild acid hydrolysis of the LPS, followed by fraction- ation of the carbohydrate material (63% of the LPS mass) on Sephadex G-50, produced the main fractions P 1 (OPS) and P 3 (core oligosaccharide). The yields of OPS-I (from Ó FEBS 2003 O-polysaccharide from Citrobacter braakii O6 (Eur. J. Biochem. 270) 2733 LPS-I) and OPS-II (from LPS-II) were 24.5% and 52.3% of the total material eluted from the column, respectively. Sugar analysis of both OPS by GLC-MS revealed fucose (Fuc) and rhamnose (Rha) in the molar ratio 1.8 : 1.0 or 2.4 : 1.0 when 0.5 M CF 3 CO 2 Hor10 M HCl was used for hydrolysis, respectively. An additional sugar component, ara4dHex, which was first detected by NMR spectroscopy (see below), was identified by a positive Cynkin–Ashwell reaction [16] (the content  10%), TLC (R Rha 1.06, R Glc 1.45) and GLC-MS (t R ¼ 0.87; ara4dHex : Rha molar ratio ¼ 0.2 : 1) using the authentic sample from the OPS of Citrobacter PCM 1487 [12] and PCM 1525 [13]. The absolute L configuration of Rha and D configuration of Fuc and ara4dHex were determined by GLC-MS of the acetylated (S)-2-octyl glycosides [19]. The content of O-acetyl groups [17] and 6-deoxyhexoses [18], determined by colorimetric methods, was 6% (1.4 lmolÆmg )1 )and67% (4.1 lmolÆmg )1 ), respectively, which yields one O-acetyl group per three 6-deoxyhexose residues. Smith degradation of OPS-I, OPS-II and O-deacetylated OPS-II resulted in the complete loss of ara4dHex, but no destruction of the other monosaccharides. Methylation analysis of OPS-I and OPS-II by GLC-MS of the partially methylated alditol acetates (Table 1) revealed terminal ara4dHex, 3-substituted Fuc and 3,4-disubstituted Rha as the main constituents, as well as a small amount of 3-substituted Rha, which could be obtained as a result of the incomplete substitution with ara4dHex or partial cleavage of terminal ara4dHex during isolation of the OPS by mild acid degradation of the LPS. The fully methylated derivative of ara4dHex had the same GLC retention time and a similar electron impact mass spectrum with the same major fragment ions (m/z 43, 71, 85, 101, 115, 117, 127 and 175) as the corresponding derivative from the OPS of C. braakii PCM 1487 [12]. Methylation analysis of the Smith-degraded OPS (Table 1) showed 3-substituted Fuc and 3-substituted Rha in the molar ratio  2:1. These data suggest that the OPS-I and OPS-II have an identical branched tetrasaccharide repeating unit. It consists of the main chain containing one 3-substituted Rha and two 3-substituted Fuc residues and a terminal residue of ara4dHex, which is 1fi4-linked to a residue of Rha at the branching point. Elucidation of the structure of the O-specific polysaccharide by NMR spectroscopy The 13 C NMR spectra of OPS-I and OPS-II were essentially identical, and therefore only the former polysaccharide was studied further. The 13 C NMR spectrum (Fig. 2) contained signals for four anomeric carbons in the region d 94.8–99.8, three CH 3 -C groups (C6 of Rha and Fuc), one C-CH 2 -C group at d 29.5, other sugar ring carbons in the region d 67.6–78.1, and one O-acetyl group at d 21.8 (Me) and 175.0 (CO). The 1 H NMR spectrum of the OPS-I (Fig. 2) contained, inter alia, signals for four anomeric protons in Fig. 1. Silver-stained SDS/PAGE gels (A) and immunoblotting with anti-(Citrobacter braakii PCM 1531) serum (B) and anti-(Citrobacter PCM 1487) serum (C) of the lipopolysaccharide (LPS)-I (lane 1) and LPS-II (lane 2) of C. braakii PCM 1531, LPS of Citrobacter PCM 1504 (lane 3), LPS of Citrobacter PCM 1505 (lane 4) and LPS of Citrobacter PCM 1487 (lane 5). Table 1. Methylation analysis data. GLC retention time (t R ) for the corresponding alditol acetate relative to that of 1,5-di-O-acetyl-2,3,4,6-tetra-O- methylglucitol (2,3,4,6-Me 4 Glc). Hydrolysis conditions: A, 2- M CF 3 CO 2 H, 120 °C,2h;B,10- M HCl, 80 °C, 0.5 h; C, 0.5- M CF 3 CO 2 H, 100 °C, 1 h. DA, O-deacetylated; OPS, O-specific polysaccharide; SD, Smith-degraded. Sugar derivative t R GLC detector response OPS-I OPS-I-SD OPS-II OPS-II-SD OPS-II-DA-SD AB ABABCABA B 2,3,6-Me 3 ara4dHex 0.84 0.4 0.5 0.7 2,4-Me 2 Rha 0.92 0.2 0.13 1 1 0.2 0.3 0.2 1 1 1 1 2,4-Me 2 Fuc 0.96 1.4 1.9 1.8 1.9 1.7 2.0 1.7 1.8 2.0 2.4 2.3 2-MeRha 1.08 1 1 1 1 1 2734 E. Katzenellenbogen et al. (Eur. J. Biochem. 270) Ó FEBS 2003 the region d 4.97–5.10, three CH 3 -C groups (H6 of Rha and Fuc) at d 1.21–1.40, one C-CH 2 -C group at d 1.76 and 1.52 (H4ax and H4eq of ara4dHex, respectively) and one O-acetyl group at d 2.19. These data were in agreement with a tetrasaccharide repeating unit containing three residues of 6-deoxy sugars, one residue of ara4dHex and one O-acetyl group. In addition to the major signals described above, the NMR spectra contained minor signals, which could originate from ara4dHex-lacking repeating units (see above) and/or from the core monosaccharides. The 1 H- and 13 C-NMR spectra of the OPS-I were assigned using two-dimensional COSY, TOCSY, NOESY and 1 H, 13 C HSQC (Fig. 2) experiments, and spin systems for one Rha, one ara4dHex and two Fuc residues (Fuc I and Fuc II ), all present in the pyranose form, were identified (Table 2). Having the D configuration, ara4dHex, Fuc I and Fuc II occur in the 4 C 1 conformation and L -Rha in the 1 C 4 conformation. In the 1 H NMR spectrum, all four anomeric protons gave poorly resolved signals (singlets for Rha and ara4dHex and poorly resolved doublets for Fuc I and Fuc II ), and therefore J 1,2 coupling constant data could not be used for reliable determination of the anomeric configurations. The 1 Hand 13 C NMR chemical shifts for H5 and C5, d 3.58 and 72.8 in Rha, d 4.26 and 68.0 in Fuc I and d 4.02 and 67.6 in Fuc II , respectively, indicated that Rha is b-linked and both Fuc Fig. 2. Part of a two-dimensional 1 H, 13 C HSQC spectrum of the O-specific polysaccharide (OPS) of Citrobacter braakii PCM 1531. One-dimen- sional 1 Hand 13 C NMR spectra are displayed along the horizontal and vertical axes, respectively. Table 2. 500-MHz 1 H and 125-MHz 13 C NMR data of the O-specific polysaccharide (d, p.p.m.). The chemical shift for the O-acetyl group is d H 2.19, d C 21.8 (Me) and 175.0 (CO). Sugar residue Chemical shift (p.p.m.) H1 H2 H3 H4 H5 H6 fi3)-a- D -Fucp I -(1fi 5.07 3.85 4.10 3.96 4.26 1.21 fi3,4)-b- L -Rhap-(1fi 4.97 5.66 4.06 3.94 3.58 1.40 fi3)-a- D -Fucp II -(1fi 4.98 3.84 3.84 3.98 4.02 1.26 a- D -ara4dHexp-(1fi 5.10 3.47 4.06 1.76 a 3.97 3.64 C1 C2 C3 C4 C5 C6 fi3)-a- D -Fucp I -(1fi 94.8 67.7 78.1 70.4 68.0 16.7 fi3,4)-b- L -Rhap-(1fi 97.3 68.7 75.2 76.5 72.8 18.5 fi3)-a- D -Fucp II -(1fi 98.3 67.1 78.0 70.1 67.6 16.5 a- D -ara4dHexp-(1fi 99.8 70.4 69.2 29.5 73.1 65.5 a H4ax, H4eq resonates at d 1.52. Ó FEBS 2003 O-polysaccharide from Citrobacter braakii O6 (Eur. J. Biochem. 270) 2735 residues are a-linked (compare published data for a-and b-rhamnopyranose, a-andb-fucopyranose [26]). This con- clusion was confirmed and the a configuration of ara4dHex established using a NOESY experiment. This showed H1,H3 and H1,H5 correlations for Rha at d 4.97/4.06 and 4.97/3.58, which are characteristic for b-linked sugars, and H1,H2 correlations for the a-linked Fuc I ,Fuc II and ara4dHex at d 5.07/3.85, 4.98/3.84 and 5.10/3.47, respectively. In the 13 C NMR spectrum, the signals for C3 of Fuc I and Fuc II were shifted downfield by 7.7–7.8 p.p.m. as compared with their respective chemical shifts in the spectra of the corresponding nonsubstituted monosaccharides [26]. These displacements were caused by the glycosylation effects on the linkage carbons and confirmed the sugar-substitution pattern determined by methylation analysis (see above). Smaller downfield displacements of the signals for C3 and C4 of Rha by 1.4 and 3.7 p.p.m., respectively, were also in agreement with 3,4-disubstitution of this sugar residue. A strong downfield displacement of the signal for H2 of Rha (> 1 p.p.m.) was caused by the deshielding effect of the O-acetyl group and defined the O-acetylation site as position 2ofRha. One b-Rha and two a-Fuc residues in the main chain (see data of chemical studies above) may form only sequence. This sequence and the site of attachment of ara4dHex were confirmed by the NOESY spectrum of OPS-I, which showed the following cross-peaks between the linkage and anomeric protons: Fuc II H1/Rha H3, Rha H1/Fuc I H3 and ara4dHex H1/Rha H4 at d 4.98/4.06, 4.97/4.10 and 5.10/ 3.94, respectively. Fuc I H1 gave a cross-peak at d 5.07/3.84, which could be assigned to a superposition of an intra- residue correlation with Fuc I H2 and an inter-residue correlation with Fuc II H3. Therefore, based on the data obtained, it was concluded that the OPS-I, as well as OPS-II, of C. braakii PCM 1531 have a branched tetrasaccharide-repeating unit with the structure 1 showninFig.3. The OPS studied is distinguished by the presence of a monosaccharide – ara4dHex – which occurs rarely in nature. This sugar has been found only in two other OPS of Citrobacter (Fig. 3). One, from Citrobacter PCM 1487 and PCM 1528 (O5), is built up of branched trisaccharide repeating units, in which D -ara4dHexisattachedasaside- chain to a GlcNAc residue in a disaccharide main chain (structure 2) [6,12]. The other OPS, shared by C. youngae PCM 1525 (O4), PCM 1488 (O36) and C. werkmanii PCM 1560 (O27), is a linear homopolymer of D -ara4dHex (structure 3) [13,14]. Chemical studies on the core oligosaccharide Fraction P 3 (core oligosaccharide) was obtained from LPS-I and LPS-II at a yield of 44% and 21% of the total material eluted from the column of Sephadex G-50, respectively. A preferable distribution to the water phase of LPS-I with more core oligosaccharide lacking the OPS substitution could be a result of the hydrophobic nature of the OPS that contains deoxy sugars only. GLC-MS analysis of the alditol acetates showed that the core oligosaccharide from both LPS contains Glc, Gal, GalN and L -glycero- D -manno-heptose (Hep) in the molar ratio 3.1 : 1.0 : 0.9 : 1.7, respectively. Methylation analysis of the core oligosaccharide revealed similar amounts of 2,3,4,6-Me 4 Glc, 3,4,6-Me 3 Glc, 2,4,6- Me 3 Glc, 4,6-Me 2 Gal, 3,4,6-Me 3 GalN and 2,4,6-Me 3 Hep, which correspond to terminal Glc, 2-substituted Glc, 3-substituted Glc, 2,3-disubstituted Gal, terminal GalN, and 3,7-disubstituted Hep, respectively. The same sugar composition and the same substitution pattern have been previously demonstrated in the LPS core of Citrobacter PCM 1487 [27]. Therefore, the LPS of C. braakii PCM 1531 (O6) and PCM 1487 (O5) may have an identical core region, a suggestion which remains to be proved unambiguously. Serological studies In order to determine whether the LPS of C. braakii PCM 1531 (serogroup O6) and the other Citrobacter LPS that contain D -ara4dHex (Fig. 3), including Citrobacter PCM 1487 (serogroup O5), are serologically related, they were studied by double immunodiffusion, passive haemaggluti- nation and inhibition of passive haemagglutination, SDS/ PAGE and immunoblotting using O-antisera against C. braakii PCM 1531 and PCM 1487. In double immunodiffusion (Fig. 4), the LPS of C. bra- akii PCM 1531 and PCM 1487 reacted with the homolog- ous antisera only. Two precipitin lines were observed in the gel between the LPS of strain PCM 1531 and the homologous antiserum, which suggest the presence of two populations of antibodies directed against different parts of the OPS or against the OPS and the core region of the LPS. In the passive haemagglutination test, anti-(C. braakii PCM 1531) serum and anti-(Citrobacter PCM 1487) serum reacted with the homologous LPS at titres of 1 : 1280 and 1 : 10 240, respectively, and again no cross-reaction was observed. The reaction in the system C. braakii PCM 1531 LPS/anti-(C. braakii PCM 1531) serum was inhibited only by homologous OPS-I and OPS-II and not with the OPS of Fig. 3. Structures of the O-specific polysaccharide (OPS) of Citrobacter containing 4-deoxy-D-arabino-hexose ( D -ara4dHex). 1, OPS from C. braakii PCM 1531 (this work); 2, OPS from Citrobacter PCM 1528 and PCM 1547 (O5) [6,12]; 3, OPS from C. youngae PCM 1525 (O4), PCM 1488 (O36) and C. werkmanii PCM 1560 (O27) [13,14]. 2736 E. Katzenellenbogen et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Citrobacter PCM 1487 or C. youngae PCM 1488 (sero- group O36). Neither the O-deacetylated OPS nor the Smith-degraded OPS (devoid of D -ara4dHex) from strain PCM 1531 showed any inhibitory activity. Therefore, both O-acetyl groups and D -ara4dHex play an important role in manifesting the serological specificity of C. braakii PCM 1531. In SDS/PAGE, all LPS tested showed a ladder-like pattern of slowly migrating high-molecular-mass LPS species with OPS chains of different length, as well as fast migrating bands of LPS species having the core with no OPS attached (Fig. 1A). In immunoblotting, anti-(C. bra- akii PCM 1531) serum (Fig. 1B) and anti-(C. braakii PCM 1487) (Fig. 1C) serum strongly reacted with slowly migra- ting species (S-form) of the homologous LPS only, but recognized fast-migrating LPS species (R-form) from both strains (lanes 1, 2 and 5). The data obtained showed that, in spite of the discovery that D -ara4dHex is the immunodominant sugar in both C. braakii PCM 1531 and PCM 1487 O-antigens [12], these O-antigens are serologically unrelated, which can be accounted for by the different anomeric configurations of D -ara4dHex (a in PCM 1531 and b in PCM 1487). This conclusion is in agreement with the classification of C. braakii PCM 1531 and PCM 1487 in different O-serogroups (O6 and O5, respectively). In contrast, the core regions of their LPS are serologically related, which is in accordance with the chemical data (see above). In immunoblotting experiments, Citrobacter PCM 1504 and PCM 1505 showed no cross-reactivity with anti- (C. braakii PCM 1531) serum (Fig. 1B, lanes 2 and 3). Therefore, these two strains, which have been classified in serogroup O6, are serologically distinct from strain PCM 1531 and should be reclassified into a different serogroup. References 1. Doran, T.I. (1999) The role of Citrobacter in clinical disease of children: review. Clin. Infect. Dis. 28, 384–394. 2. Badger, J.L., Stins, M.F. & Kim, K.S. (1999) Citrobacter freundii invades and replicates in human brain microvascular endothelial cells. Infect. Immun. 67, 4208–4215. 3. 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(1985) Citrobacter lipopoly- saccharides: structure elucidation of the O-specific polysaccharide from strain PCM 1487 by mass spectrometry, one-dimensional and two-dimensional 1 H-NMR spectroscopy and methylation analysis. Eur. J. Biochem. 146, 641–647. 13. Romanowska, E., Romanowska, A., Dabrowski, J. & Hauck, M. (1987) Structure determination of the O-specific polysaccharides from Citrobacter O4- and O27-lipopolysaccharides by methylation analysis and one- and two-dimensional 1 H-NMR spectroscopy. FEBS Lett. 211, 175–178. 14. Romanowska, E., Romanowska, A., Lugowski, C. & Katze- nellenbogen, E. (1981) Structural and serological analysis of Citrobacter-O36-specific polysaccharide, the homopolymer of (b1fi2)-linked 4-deoxy-D-arabino-hexopyranosyl units. Eur. J. Biochem. 121, 119–123. 15. Sawardeker, J.S., Sloneker, J.H. & Jeans, A. (1965) Quantitative determination of monosaccharides as their alditol acetates by gas liquid chromatography. Anal. Chem. 37, 1602–1604. Fig. 4. Double immunodiffusion of anti- (Citrobacter braakii PCM 1531) serum (A) and anti-(Citrobacter PCM 1487) serum (B) with lipopolysaccharide (LPS)-I (well 1) and LPS-II (well 2) of C. braakii PCM 1531, and LPS of Citrobacter PCM 1487 (well 3) and C. youngae PCM 1488 (well 4). Ó FEBS 2003 O-polysaccharide from Citrobacter braakii O6 (Eur. J. Biochem. 270) 2737 16. Cynkin, H.A. & Ashwell, G. (1960) Estimation of 3-deoxy sugars by means of the malonaldehyde-thiobarbituric acid reaction. Nature 186, 155–156. 17. Hestrin, S. (1949) The reaction of acetylcholine and other carb- oxylic acid derivatives with hydroxylamine and its analytical applications. J. Biol. Chem. 180, 249–253. 18. Dische, Z. (1962) Color reactions of 6-deoxy-, 3-deoxy, and 3,6- dideoxyhexoses. Methods Carbohydr. Chem. 1, 501–503. 19. Leontein, K., Lindberg, B. & Lo ¨ nngren, J. (1978) Assignment of absolute configuration of sugars by g.1.c. of their acetylated glycosides formed from chiral alcohols. Carbohydr. Res. 62, 359–362. 20. Gunnarsson, A. (1987) N- and O-Alkylation of glycoconjugates and polysaccharides by solid base in dimethyl sulphoxide/alkyl iodide. Glycoconjugate J. 4, 239–245. 21. Romanowska, A., Gamian, A., Witkowska, D., Katzenellenbo- gen, E. & Romanowska, E. (1994) Serological and structural features of Hafnia alvei lipopolysaccharides containing D-3- hydroxybutyric acid. FEMS Immunol. Med. Microbiol. 8, 83–88. 22. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. 23. Tsai, C. & Frash, C.E. (1982) A sensitive silver stain for detection of lipopolysaccharides in polyacrylamide gels. Anal. Biochem. 119, 115–119. 24. Gamian, A., Romanowska, E. & Romanowska, A. (1992) Immunochemical studies on sialic acid-containing lipopoly- saccharides from enterobacterial species. FEMS Microbiol. Immunol. 89, 323–328. 25. Ouchterlony, O. (1958) Diffusion-in-gel methods for immuno- logical analysis. Prog. Allergy 5, 1–78. 26. Jansson, P E., Kenne, L. & Widmalm, G. (1989) Computer- assisted structural analysis of polysaccharides with an extended version of CASPER using 1 H- and 13 C-n.m.r. data. Carbohydr. Res. 188, 169–191. 27.Romanowska,E.,Gamian,A.&Dabrowski,J.(1986)Core region of Citrobacter lipopolysaccharide from strain PCM 1487. Structure elucidation by two-dimensional 1 H-NMR spectroscopy and methylation analysis/mass spectrometry. Eur. J. Biochem. 161, 557–564. 2738 E. Katzenellenbogen et al. (Eur. J. Biochem. 270) Ó FEBS 2003 . using the authentic sample from the OPS of Citrobacter PCM 1487 [12] and PCM 1525 [13]. The absolute L configuration of Rha and D configuration of Fuc and ara4dHex. 6000. Results and discussion Isolation and chemical analysis of the O-specific polysaccharide On phenol–water extraction [9], the LPS of C. braakii PCM 1531 was recovered