Structures of two O-chain polysaccharides of Citrobacter gillenii O9a,9b lipopolysaccharide A new homopolymer of 4-amino-4,6-dideoxy- D -mannose (perosamine) Tomasz Lipin  ski 1 , George V. Zatonsky 2 , Nina A. Kocharova 2 , Michel Jaquinod 3 , Eric Forest 3 , 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 ø aw, Poland; 2 N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation; 3 CNRS and CEA, Institut de Biologie Structurale, LSMP, Grenoble, France Mild acid degradation of the lipopolysaccharide of Citro- bacter gillenii O9 a,9b released a polysaccharide (PS), which was found to consist of a single monosaccharide, 4- acetamido-4,6-dideoxy- D -mannose ( D -Rha4NAc, N-acetyl- D -perosamine). P S was studied by methylation a nalysis and 1 H-NMR and 13 C-NMR spectroscopy, u sing two-dimen- sional 1 H, 1 H COSY, TOCSY, NOESY, and H-detected 1 H, 13 C h eteronuclear corr elation experiments. It was found that PS in cludes two structurally dierent polysaccharides: an a1 ® 2-linked homopolymer of N-acetyl- D -perosamine [ ® 2)-a- D -Rhap4NAc-(1 ® , PS2] and a polysaccharide composed of tetrasaccharide repeating units (PS1) with the following structure: ® 3)-a- D -Rhap4NAc-(1 ® 2)-a- D -Rhap4NA c-(1 ® 2) -a- D -Rhap4NA c-(1 ® 3) -a- D -Rhap4 N Ac2Ac-(1 ® where the degree of O-acetylation of a 3-substituted Rha4NAc residue at position 2 is  70%. PS could be fractionated into PS1 and PS2 by gel-perme- ation c hromatography on TSK H W-50S. Matrix-assisted laser desorption ionization MS data indicate sequential chain elongation o f both PS1 and PS2 by a single sugar unit, with O-acetylation in PS1 beginning at a certain chain length. Anti-(C. gillenii O9a,9b) serum re acted with PS1 in double immunodiffusion and immunoblotting, whereas neither PS2 nor the lipopolysaccharide of Vibrio cholerae O1 with a struc turally related O -chain polysac charide were reactive. Keywords: 4-acetamido-4,6-dideoxy- D -mannose; Citro- bacter gillenii; lipopolysaccharide; O-antigen; polysaccharide structure. Strains of g enus Citrobacter are inhabitants of t he intestinal tract and, accordingly, are present in sewage, surface waters, and food contaminated with faecal material. Outbreaks of febrile gastroenteritis associated with Citrobacter have been described. Citrobacter strains may cause opportunistic infections, including urinary and respiratory tract infections, especially in the immunocompromised host, and are also associated with meningitis, brain abscesses, and neonatal sepsis [1,2]. Currently, strains o f the ge nus Citrobacter are classi®ed into 11 species [3] and 43 O-serogroups [1,4]. Serological heterogeneity of Citrobacter strainsisde®nedby the diversity in structures of the cell-surface lipopolysac- charide (LPS) [1,5]. With the aim of creating a molecular basis for classi®cation of strains and substantiating their serological cross-reactivity, structures of the O-chain poly- saccharides of LPS ( O-antigens) of more t han 20 serologi- cally different Citrobacter strains have been established [6±8]. Now we r eport s tructural studies of LPS from C. gillenii O9a,9b, w hich is distinguished by the presence of two structurally different polysaccharide chains. Strains of this serogroup are often isolated from patients [1]. MATERIALS AND METHODS Bacterial strain, isolation and degradation of LPS Citrobacter gillenii O9a,9b:48 (strain PCM 1537) came originally from the Cze ch National Collection of Type Cultures, Prague (IHE Be 65/57, Bonn 16824 [1,5,9]) and was obtained from the collection of the Institute of Immunology and Experimental Therapy. Bacteria were cultivated in Davis broth supplemented with casein hydro- lysate and yeast ex tract (Difco) with aeration at 37 °Cfor 24 h; they were then harvested and freeze-dried. LPS was isolated by phenol/water extraction and puri®ed by ultra- centrifugation [10]. The yield of LPS was 3.2% of d ry bacterial mass. A portion of LPS (200 mg) was heated with 1% acetic acid (20 mL) for 3 h at 100 °C, and the carbohydrate- containing supernatant was fractionated on a column (1.6 ´ 100cm)ofBio-GelP4()400 mesh) in 0.05 M aqueous pyridinium acetate buffer, pH 5.6, at a ¯ow rate of 4 mLáh )1 . The yield of polysaccharide material was 34 mg. Alternatively, carbohydrate material from another Correspondence to A. Gamian, L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wrocøaw, Poland. Fax: + 48 71 3732587, Tel.: + 48 71 3732316, E-mail: gamian@immuno.iitd.pan.wroc.pl Abbreviations: HSQC, heteronuclear single-quantum coherence; MALDI, matrix-assisted laser desorption ionization; LPS, lipopoly- saccharide; PS, O-chain polysaccharide; Rha4NAc, 4-acetamido-4,6- dideoxymannose. (Received 22 June 2001, revised 22 October 2001, accepted 23 October 2001) Eur. J. Biochem. 269, 93±99 (2002) Ó FEBS 2002 portion of LPS ( 200 m g), degraded as above to obtain the carbohydrate-containing supernatant, was fractionated on a column (1.6 ´ 100 cm) of TSK H W-50S in the same pyridinium acetate buffer at a ¯ow rate of 8 mLáh )1 .The yields of fractions 1, 2 (PS1), 3 (PS2), and 4 were 2.6, 11.4, 18.0 and 16.8%, r espectively. Chemical methods O-deacetylation of PS (30 mg) was carried out with aqueous 12% ammonia a t r oom temp erature overnight followed by gel-permeation chromatography on a column (1.6 ´ 80 cm) of TSK HW-40S in water. For sugar analysis, PS (0.4 mg) was hydrolysed with 10 M HCl for 30 min at 80 °C, and the alditol acetates derived were analysed by GLC-MS using a Hewlett±Packard 5971A system with an HP-1 glass capillary column (0.2 mm ´ 12 m) and temperature program of 8 °ámin )1 from 150 to 270 °C. For determination of the a bsolute con®guration [11,12], LPS (0.8 mg) was subjected to 2-butanolysis [300 lL(R)-2-butanol and 20 lL acetyl chloride, 100 °C, 3 h ]; the products were acetylated and analysed by GLC-MS as above. Methylation of PS (0.4 mg) was performed by the Hakomori procedure [ 13]; products were recovered by extraction with chloroform/water (1 : 1, v/v), hydrolysed with 10 M HCl for 30 min at 8 0 °C, and the p artially methylated alditol acetates d erived were analysed by GLC- MS as above. NMR spectroscopy Samples were freeze-dried twice from a 2 H 2 O solution and dissolved in 99.96% 2 H 2 O. 1 H-NMR and 13 C-NMR spectra were recorded with a Bruker DRX-500 spectrometer at 60 °C; chemical shifts are reported with internal acetone (d H 2.225, d C 31.45) as reference. Two-dimensional exper- iments were per formed using standard Bruker software. A mixing t ime of 200 ms was used i n TOCSY and HMQC- TOCSY experiments and 300 ms in a NOESY ex periment. Matrix-assisted laser desorption ionization (MALDI) MS MALDI mass spectra were recorded on a RETOF (time- of-¯ight) i nstrument from Perseptive Biosystems ( Framing- ham, MA, U SA) e quipped w ith a pulsed delay source extractor [14]. Spectra were recorded from 256 laser shots (nitrogen laser, 337 nm) with a n accelerating voltage of 20 kV in linear mode. For a matrix, 2,5-dihydroxybenzoic acid was dissolved in aqueous 70% acetonitrile containing 0.1% tri¯uoroacetic acid . Then 1 lL matrix was mixed with 1 lL sample, placed on top o f the matrix surface, and allowed to dry by itself. The spectra were calibrated using insulin (1 pmolálL )1 ; m/z 5736) in the same conditions. Mass numbers were rounded to the nearest integer. Rabbit antiserum, antigens and serological techniques Rabbit antiserum against whole cells of C. gillenii O9a,9b was prepared as described previously [15]. LPS of Hafnia alvei PCM 1186 wa s from p revious st udies [1 5], LPS o f V. cholerae O1 was a gift from O. Holst (Forschungszen - trum Borstel, Germany), and that of Escherichia c oli O157 was a gift from B. MaÎczyn  ska a nd A. Przondo-Mordarska (Medical Academy, Wrocøaw, Poland). S DS/PAGE and immunoblotting with LPS and double immunodiffusion with LPS and polys accharides were performed as described previously [15±17]. RESULTS AND DISCUSSION A high-molecular-mass PS was isolated by mild acid degradation of LPS of C. gillenii O9a,9b followed by gel- permeation chromato graphy of the carbohydrate portion on Bio-Gel P-4. Sugar analysis of PS revealed a 4-amino- 4,6-dideoxyhexose as the single monosaccharide c onstitu- ent. This was identi®ed as 4-amino-4,6-dideoxy- D -mannose ( D -Rha4N, D -perosamine) by comparison with the corres- ponding authentic s amples from LPS of V. cholerae O1 [18] using G LC-MS o f the alditol acetates and acetylated (R)-2- butyl glycosides. Methylation analysis of PS revealed 4 ,6-dideoxy-3-O- methyl-4-(N-methyl)acetamidomannose and 4,6-dideoxy- 2-O-methyl-4-(N-methyl)acetamidomannose in the ratio  2 : 1, which were identi®ed by GLC-MS of partially methylated alditol acetates (retention times 8.98 and 9.03 min, respectively). The former compound was char- acterized by the presence in the mass spectrum of intense ion peaks for the C1±C3, C1±C4, and C4±C6 primary fragments at m/z 190, 275, a nd 172, respectively. The mass spectrum of the latter compound showed intense i on peaks for the fragments C1 ±C2, C1±C4, and C4±C6 at m/z 118, 275, and 172, respectively. Hence, PS is linear and contains 2-substituted and 3-substituted p erosamine residues. Further studies showed that PS includes two polysaccharides with the same sugar composition but different structures. The 13 C-NMR s pectrum of PS (Fig. 1, top) contained signals with different inte gral intensities that c ould be due to nonstoichiometric O-acetylation (there was a signal for CH 3 COO at d 21.5). Some minor signals could belong to the LPS core c onstituents as they were still present after O-deacetylation of PS with aqueous ammonia. The 13 C-NMR spectrum of the O-deacetylated polysaccharide (PS NH 4 OH , F ig. 1 , bottom) was less c omplex than the spectrum of the initial PS and contained signals for several different Rha4NAc residues including signals for anomeric carbons (C1) at d 101.6±102.9, carbons bearing nitrogen (C4) at d 52.9±54.3, CH 3 -C groups (C6) at d 18.0±18.3, and N-acetyl groups at d 23.3±23.5 (CH 3 ) and 175.0±175.7 (CO). In each carbon group, some signals were two to ®ve times as intense as the single signal. Accordin gly, the 1 H-NMR spectrum of PS NH 4 OH (Table 1) contained, among other things, signals for ano- meric protons (H1) at d 4.96±5.13, CH 3 -C groups (H6) at d 1.17±1.22, and N-acetyl groups at d 2.04. The t wo-dimen- sional COSY and TOCSY spectra of PS NH 4 OH revealed spin systems for ®ve different Rha4NAc residues, all signals for one of them (Rha4NAc I ) being about twice as intense as signals for each of four other residues (Rha4NAc II ± Rha4NAc V ). At the H1 co-ordinate, the TOCSY spectrum showed cross-peak s with H 2±H6 for Rha4NAc II ±Rha4- NAc V but o nly two c ross-peaks, w ith H2 and H3, for Rha4NAc I . At the H6 co-ordinate, th e spectrum showed cross-peaks f or the whole spin system o f each m onosaccha- ride residue. The COSY spectrum a llowed differentiation 94 T. Lipin  ski et al.(Eur. J. Biochem. 269) Ó FEBS 2002 between protons within each spin system. Dif®culties associated with coincidence o f signals for s ome neighbour- ing p rotons (H3 and H4 of Rha4NAc I and Rha4NAc II ) were overcome using an H-detected 1 H, 13 C heteronuclear single-quantum coherence (HSQC) experiment. This also con®rmed the assignment for H4 by t heir correlation to C4 located i n the r esonance region o f carbons bearin g nitrogen (d 52.9±54.3). The 13 C-NMR s pectrum of PS NH 4 OH (Table 2) was assigned using a 1 H, 13 C HSQC experiment. The assignment for C2 was additionally con®rmed by a combined 1 H, 13 C HMQC-TOCSY experiment (Fig. 2), which r evealed clear correlation between H1 and C2. Chemical shifts for C 5 (d 69.3±69.6) in the 13 C-NMR spectra of PS NH 4 OH and an a1 ® 2-linked D -Rha4NAc homopolymer from V. chole- rae bio-serogroup Hakata [19] (serogroup O140 [20]) were close and, hence, all Rha4NAc residues are a-linked (C5 of b-pyranosides is known t o resonate in a lower ®eld than C5 of a-pyranosides [21]). The relatively low-®eld position at d 78.0±79.3 of the signals for C3 of Rha4NAc II and Rha4NAc V and C 2 o f three other R ha4NAc demonstrated the mode of substitution of the monosaccharides (compare the position a t d 69.0±70.6 of the signals for nonlinked C2 and C3 of Rha4NAc; Table 2). A NOESY experiment (Fig. 3 ) r evealed strong intrares- idue H1/H2 c orrelations for R ha4NAc I and Rha4NAc II at d 5.13/4.12 and 4.97/3.85 and weaker H1/H2 correlations for R ha4NAc III ±Rha4NAc V (the latter are below the level shown in Fig. 3). M ost importantly, t he spectrum contained interresidue cross-peaks between the following transglycos- idic protons: Rha4NAc II H1/Rha4NAc V H3 at d 4.97/3.98, Rha4NAc V H1/Rha4NAc IV H2 at d 5.03/4.13, Rha4NAc IV H1/Rha4NAc III H2 at d 5.10/3.79, and Rha4NAc III H1/ Rha4NAc II H3 at d 4.96/3.91. These d ata a re in agreement with the 13 C-NMR chemical-shift data and show a Rha4NAc homopolysaccharide with a tetrasaccharide repeating unit ( PS1 NH 4 OH ; Fig. 4). No interresidue cross- peak was observed f or Rha4NAc I but a strong intraresidue H1/H2 cross-peak at d 5.13 /4.12 and a weak H1/H5 cross- peak at d 5.13/3.82 typical of a1 ® 2-linked sugars with the manno con®guration. Hence, Rha4NAc I residues are a1 ® 2-linked a nd bu ild a nother polysaccharide chain (PS2; Fig. 4 ). Comparison of the 1 H-NMR, 13 C-NMR, and 1 H, 13 C HMQC spectra of PS NH 4 OH and PS enabled the determi- nation of the site of attachment of t he O-acetyl group. In the 1 H, 13 C HMQC s pectrum, the intensity of the H2/C2 Fig. 1. 125-MHz 13 C-NMR spectra of the initial (PS, top) and O-deacetylated (PS NH 4 OH , bottom) po lysaccharides from C. gillenii O9a,9b. Table 1. 1 H-NMR data. Additional chemical shift for the N-acetyl groups is d 2.04. Sugar residue Chemical shift (p.p.m.) H1 H2 H3 H4 H5 H6 O-Deacetylated PS1 ® 3)-a- D -Rhap4NAc II -(1 ® 4.97 3.85 3.91 3.91 3.84 1.21 ® 2)-a- D -Rhap4Nac III -(1 ® 4.96 3.79 3.99 3.86 3.89 1.20 ® 2)-a- D -Rhap4NAc IV -(1 ® 5.10 4.13 4.05 3.91 3.80 1.22 ® 3)-a- D -Rhap4NAc V -(1 ® 5.03 4.17 3.98 3.99 3.87 1.18 PS2 ® 2)-a- D -Rhap4NAc I -(1 ® 5.13 4.12 4.03 3.89 3.82 1.17 Ó FEBS 2002 Polysaccharides of C. gillenii 9a,9b (Eur. J. Biochem. 269)95 cross-peak of Rha4NAc II at d 3.85/70.6 markedly decreased and a new cross-peak appeared at d 5.00/72.1. The 13 C-NMR spectrum displayed displacements of parts of the signals for C1 and C3 of Rha4NAc II from d 102.9 and 78.0 t o d 101.6 a nd 76.6, respectively, which a re typical of b-effe cts of acetylation at O2 [22]. Therefore, part of the Rha4NAc II residues is O-acetylated at position 2, and PS1 thus has the structure shown in Fig. 4 . As judged by the ratio of t he integral intensities of the signals f or the O-acetylated and non-O-acetylated residues, the average degree of O-acetylation of Rha4NAc II in PS1 is  70%. PS2 contains no O-acetyl group. To con®rm the existence of two polysaccharides, the carbohydrate portion obtained after mild acid degradation of C. gillenii O9a,9b LPS was fractionated by gel-perme- ation chromatography on TSK HW-50S to give six fractions (Fig. 5). The MALDI mass spectrum of fraction 1 revealed a series of hexose increments with m/z 1 62, and this fraction was considered to be a glucan-type contami- nant. Fraction 4 represented a core oligosaccharide, and fractions 5 and 6 contained low-molecular-mass compounds released from LPS. 1 H-NMR and 13 C-NMR sp ectroscopic analysis showed that the perosamine-containing polysaccharides PS1 and PS2 were present in fractions 2 and 3, respectively. Therefore, the two polysaccharides could b e separated and thus belonged to separate LPS molecules. The MALDI mass spectrum of PS1 showed a series of ion p eaks with differences between ions of 187 or 229 Da, which corresponded to non-O-acetylated and O-acetylated Rha4NAc, respectively (Fig. 6). The low-molecular-mass polysaccharide species (below 4258 Da) were devoid of O-acetyl groups. The difference between the ions at m/z 4258 and 4487 corresponded to the O-acetylated Rha4NAc residue (Rha4NAc2Ac II ), and the next three peaks in this series at m/z 4674, 4861 and 5048 re¯ected further chain elongation by non-O-acetylated residues (Rha4NAc III ± Rha4NAc V ) to complete the tetrasaccharide r epeating unit of PS1. Then, starting from the ion peak at m/z 5048, the pattern iterated. The next ion peaks with a difference of 790 D a for the mono-O-acetylated tetrasaccharide ( indicat- ed by arrows), as well as the intermediate i on peaks (shown by asterisks), were clearly observed up to m/z 7418. Some of the minor peaks may be due to heterogeneity of the core oligosaccharide. The 18 D a difference between ions (at 2949 and 2967 m/z and the next peaks in this series) may result from the dehydrated a nd hydrated forms of 3-deoxy- octulosonic acid ( Kdo) residue, respectively, at the reducing end of the polysaccharide. The O-acetylation of PS1 begins at a certain polysac- charide chain length (about three t etrasaccharide repeating units). These data are in agreement with the NMR spectroscopic data (see above), w hich showed that only  70% tetrasaccharide repeating units in PS1 are O-acet- ylated. Table 2. 13 C-NMR data. Additional c he mical shifts for t he N-acetyl groups a re: d 23.3±23.5 (CH 3 ) and 175.0±175.7 (CO). Sugar residue Chemical shift (p.p.m.) C1 C2 C3 C4 C5 C6 O-Deacetylated PS1 ® 3)-a- D -Rhap4NAc II -(1 ® 102.9 70.6 78.0 53.1 69.6 a 18.0 b ® 2)-a- D -Rhap4NAc III -(1 ® 101.9 79.3 69.0 54.3 69.3 a 18.0 b ® 2)-a- D -Rhap4NAc IV -(1 ® 101.9 78.2 69.0 54.3 69.4 a 18.3 b ® 3)-a- D -Rhap4NAc V -(1 ® 102.7 70.1 78.2 52.9 69.6 a 18.0 b PS2 c ® 2)-a- D -Rhap4NAc I -(1 ® 101.6 78.2 69.0 54.3 69.6 18.0 (101.33) (77.86) (68.67) (53.91) (69.33) (17.64) a,b Assignment could be interchanged. c Data from [19] for the O-speci®c polysaccharide of V. cholerae bio-serogroup Hakata (serogroup O140 [20]) are given in parentheses. The dierences in the chemical shifts are due to the use of dierent references for calibration (dioxane in the published work [19] and acetone in this work). Fig. 2. Part of a 1 H, 13 C HMQC-TOCSY spectrum of the O-deacetyl- ated polysaccharide (PS1 NH 4 OH )fromC. gillenii O9a,9b. The corres- ponding parts of 13 C-NMR and 1 H-NMR spectra are displayed along the vertical a nd horizontal axes, r espectively. 96 T. Lipin  ski et al.(Eur. J. Biochem. 269) Ó FEBS 2002 The MALDI mass spectrum of PS2 (not shown) displayed a series of ion peaks with a difference between ions of 187 Da, which corresponded to sequential chain elongation by one non-O-acetylated Rha4NAc residue. The intensities of the ®rst peaks for the short-chain polysac- charide species were h igh and those o f the following peaks decreased, but the series could be traced up to 20 and more Rha4NAc I residues. The data obtained suggested that growth of both PS1 and PS2 in C. gillenii O9a,9b proceeds by sequential t ransfers of single sugar units. A biosynthetic model involving sequential single sugar transfers to the nonreducing end of the growing chain has been suggested for the A-band polysaccharide ( D -rhamnan) in Pseudomonas aeruginosa LPS [ 23] as w ell as for linear homopolysaccharide O-antigens of Escherichia coli O8 and O9 ( D -mannans) and D -galactan I from Klebsiella pneumoniae (reviewed in [24]). This model requires participation of several distinct transferases for the same monosaccharide, as demonstr ated for biosynthesis of the A-band polysaccharide [23]. A polysaccharide with the same structure as PS2 has been previously reported to be the O-chain of the LPS of V. chole rae bio-serogroup Hakata [19] (serogroup O140 [20]), whereas PS1 is new. Inte restingly, a polysaccharide of a1 ® 2-linked and a1 ® 3-linked 4-formamido-4,6- dideoxy- D -mannose (N-formyl- D -perosamine) having a pentasaccharide repeating unit has been found in Brucella melitensis LPS [25]. Published structural data [25] do not exclude the occurrence of t wo separate polysaccharide chains in the LPS of B. melitensis. The O-chain homo- polymer from Escherichia hermannii LPS composed of a1 ® 2-linked and a1 ® 3-linked D -Rhap4NAc residues has been reported t o h ave a pentasaccharide repeating unit containing the tetrasaccharide sequence present in PS1 [26]. Fig. 3. Part of a NOESY spe ctrum of the O-deacetylated polysaccharide (PS NH 4 OH ) from C. gillenii O9a,9b. The corresponding parts o f the 1 H-NMR spectrum a re displayed along t he axes. Arabic numerals refer t o pro- tons in sugar residues denoted by roman numerals as shown in F ig. 4 . Fig. 4. Structures of the pol ysaccharides (PS1 and PS2) and the O -deacetylated poly sacchar- ide (PS1 NH 4 OH )fromC. gillenii O9a,9b. Fig. 5. Fractionation on TSK HW-50S of the carbohydrate material obtained b y mild acid hydrolysis o f C. gillenii O9a,9b L PS. For e xpla- nation of fractions, s ee the t ext. Ó FEBS 2002 Polysaccharides of C. gillenii 9a,9b (Eur. J. Biochem. 269)97 LPS of C. gillenii O9a,9b reacted w ith homologous anti- O serum in double immunodiffusion (data not shown). In SDS/PAGE and immunoblotting (Fig. 7), anti-(C. gille- nii O9a,9b) s erum reacted mainly with slowly moving, hig h- molecular-mass LPS species. O-Deacylation of C. gillenii O9a,9b LPS had no effect on its serological reactivity. From the separated O-chain polysaccharides, only P S1 reacted i n double immunodiffusion with anti-(C. gillenii O9a,9b) serum, whereas PS2 was inactive, p robably, because of a lower molecular mass. No signi®cant cross-reactivity was observed between anti- (C. gillenii O9a,9b) s erum and V. cholerae O1 LPS in double immunodiffusion (not shown) and immunoblotting (Fig. 7 ). This can be accounted for b y different N-acyl substituents at D -Rha4N: N-acetyl or N-[(S)-2,4-dihydroxy- butyryl] g roup in the O-antigens of C. gillenii and V. chole- rae [18], respective ly. The LPS from E. coli O157, which also contains D -Rha4N [24], a lso d id not react with anti- (C. gillenii O9a,9b) s erum in double i mmunodiffusion (data not shown). ACKNOWLEDGEMENTS We thank Professor O. Holst (Forschungszentrum Borstel, Germany) for the gift of V. cholerae O1 LPS and Dr B. MaÎczyn  ska and Professor A. Przondo-Mordarska (Medical Academy, Wrocøaw, Poland) for t he gift of E. coli O157 LPS. This work was supported b y grant 99-04- 48279 from the Russian Foundation for Basic Research and grant 500- 1-15 from the Polish Academy of Sciences. REFERENCES 1. La  nyi, B. (1984) B iochemical a nd serologic al characteriz ation o f Citrobacter. Methods Microbiol. 15, 144±171. 2. Badger, J .L., Stins, M.F. & Kim, K.S. (1999) Citrobacter freundii invades and replicates in human brain microvascular endothelial cells. Infect. I mmun. 67, 4 208±4215. 3. 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