Structural and serological relatedness of the O-antigens of Proteus penneri 1 and 4 from a novel Proteus serogroup O72 Zygmunt Sidorczyk 1 , Filip V. Toukach 2 , Krystyna Zych 1 , Dominika Drzewiecka 1 , Nikolay P. Arbatsky 2 , Alexander S. Shashkov 2 and Yuriy A. Knirel 2 1 Department of General Microbiology, Institute of Microbiology and Immunology, University of èodz  , Poland; 2 N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation O-speci®c polysaccharides (O-antigens) of the lipopolysac- charides (LPS) of Proteus penneri strains 1 and 4 were studied using sugar analysis, 1 Hand 13 C NMR spectroscopy, including 2D COSY, H-detected 1 H, 13 CHMQC,and rotating-frame N OE s pectroscop y ( ROESY). The following structures of the t etrasaccharide (strain 1 ) and pentasac- charide (strain 4) repeating units of the polysaccharides were established: In the polysaccharide of P. penneri strain 4, glycosylation with the lateral Glc residue ( 75%) a nd O- acetylation of the lateral GalNAc residue (55%) are nonstoichiometric. This polysaccharide contains also other, minor O-acetyl groups, whose positions were not determined. The structural similarity of the O-speci®c polysaccha- rides was consistent with the close serological relatedness of the LPS, which was demonstrated b y immunochemical studies with O-an tisera against P. penneri 1and4.Basedon these data, it was proposed to classify P. penneri strains 1 and 4 into a new Proteus serogroup, O72, as two sub- groups, O72a and O72a,b, respectively. Serological cross- reactivity of P. penneri 1 O-antiserum with the LPS of P. penneri 40 and 41 was substantiated by the presence of an epitope(s) on the LPS core region shared by all P. penneri strains studied. Keywords: Proteus penneri; O-antigen; O-speci®c poly- saccharide; O-serogroup; lipopolysaccharide. 1 Gram-negative bacteria of the genus Proteus are common in human and animal intestines but under favourable con di- tions they cause infections of wou nds, burns, skin, eyes, ears, nose and throat, as well a s intestinal and urinary tract infections. Strains of two species, Proteus mirabilis and Proteus vulgaris, have been classi®ed into 60 O -serogroups [1,2]. Proteus p enneri is a new species proposed for strains formerly described as Prot eus v ulgaris biogroup I [3,4]. In our immunochemical studies of the outer-membrane lipopolysaccharides (LPS) aiming at creation of a classi®- cation scheme for P. penneri, we have found that their O-speci®c polysaccharide chains are acidic or, less c ommon, neutral polymers composed of tri- to hexa-saccharide repeating units [5,6]. As a result of the chemical and serological studies of LPS, a number of new Proteus serogroups was proposed for P. penneri strains [6,7]. Here, we report on the structures of two neutral structurally related O-speci®c polysaccharides from P. penneri strains 1 and 4 and p ropose to classify these strains into a new Proteus serogroup, O72, as two subgroups. MATERIALS AND METHODS Bacterial strains P. penneri strains 1 (3960±66) and 4 (3266±68) were kindly provided by D. J. Brenner (Center for Diseases Control, Atlanta, GA, USA). They were isolated from the urine of patients with bacteriuria and a urinary tract infection in Michigan and Porto Rico (USA), respectively. Further, 66 P. penneri strains came from the Collection of the Department of General Microbiology, University of èo  dz (Poland). Thirty-seven strains of P. mirabilis and 28 strains of P. vulgaris were from the Czech National Collection of Type Cultures (CNCTC, National Institute of Public Health, Prague, Czech Republic). Dry bacterial cells of P. penneri 1 and 4 were obtained from aerated cultures as d escribed previously [8]. 6)- - D -Glcp-(1 3)- - D -GalpNAc-(1 4)- - D -Galp-(1 3 1 - D -GalpNAc 6)- - D -Glcp-(1 3)- - D -GalpNAc-(1 4)- - D -Galp-(1 6 3 1 1 - D -Glcp - D -GalpNAc 6 OAc Correspondence to Z. Sidorczyk, Department of General Microbiology, Institute of Microbiology and Immunology, University of Lodz, Banacha 12/16, 90-237 èodz , Poland. Fax: + 48 42 784932, E-mail: zsidor@biol.uni.lodz.pl Abbreviations: LPS, lipopolysaccharide; ROESY, rotating-frame NOE spectroscopy. (Received 31 July 2001, revised 17 October 2001, accepted 7 November 2001) Eur. J. Biochem. 269, 358±363 (2002) Ó FEBS 2002 Isolation of the LPS and O-speci®c polysaccharides Lipopolysaccharides of P. penneri 1and4wereisolatedin yields of 4.1 and 9.3% by extraction of bacterial mass with a hot phenol/water mixture [9] followed by treatment with cold aqueous 50% CCl 3 CO 2 H a s d escribed previously [10]. Degradation o f the LPS was performed with aqueous 1% HOAc at 100 °C for 2 h, a lipid precipitate was removed by centrifugation (13 000 g, 20 min), and the carbohydrate portion was fractionated by gel-permeation chromatogra- phy on a column (3 ´ 65 cm) of Sephadex G-50 using 0.05 M pyridinium acetate buffer pH 4.5 as eluent to give the corresponding O-speci®c polysaccharides in a yield of 20% of the LPS mass. Rabbit antisera and serological assays Polyclonal O-antisera were obtained by immunization of rabbits with heat-inactivated bacteria of P. penneri 1and4 according to a published p rocedure [11]. Agglutination and precipitation tests, SDS/PAGE, elec- trotransfer of L PS from gels to nitrocellulose sheets, immu- nostaining, and absorption experiments were carried out as described in detail previously [12]. LPS±BSA complexes were used as solid-phase antigen in enzyme immunosorbent assay [13], and passive immunohemolysis was performed with increasing amounts (2±200 lg) of alkali-treated LPS [12]. Chemical methods The polysaccharides were hydrolysed with 2 M CF 3 CO 2 H (100 °C, 4 h). Amino sugars were identi®ed with a Biotronik LC-2000 amino-acid analyser on a Ostion LG AN B cation-exchange resin in the standard 0.35 M sodium citrate buffer pH 5.28 at 80 °C. Neutral sugars were analysed with a Biotronik LC-2000 sugar analyser on a c olumn of a Dionex Ax8-11 anion-exchange resin in 0.5 M sodium borate buffer pH 8.0 at 65 °C. The absolute con®gurations of monosaccharides were determined by GLC of a cetylated (S)-2-butyl glycosides [14±16] on an Ultra 2 column using a Hewlett-Packard 5890 chromato- graph and a temperature program 150±290 °Cat 10 °Cámin )1 . O-deacetylation of the strain 4 polysaccharide was performed with aqueous 12% ammonia at 60 °Cfor2h, the modi®ed polysaccharide was isolated by gel-permeation chromatography as described above. NMR spectroscopy 1 Hand 13 C NMR spectra were recorded with Bruker AM-300 and Bruker DRX-500 spectrometers in D 2 Oat 60 °C using internal acetone (d H 2.225, d C 31.45) as 2D spectra were obtained u sing standard Bruker s oftware, and XWINNMR 2.1 program (Bruker) was used to acquire and process NMR data. A mixing time of 230 ms was used in a ROESY experiment. RESULTS AND DISCUSSION Structure of the O-speci®c polysaccharide from P. penneri strain 1 Sugar analysis of the polysaccharide f rom P. penneri 1 revealed glucose and galactose in almost equal amounts as well as 2-amino-2-deoxygalactose. All sugars were assigned to the D series using GLC of the acetylated (S)-2-butyl glycosides [14±16]. The 13 C NMR spectrum of the polysaccharide (Fig. 1) contained signals for four anomeric carbons at d 99.7±105.6, two carbons bearing nitrogen at d 52.5 and 54.1, three nonsubstituted CH 2 OH groups at d 61.9±62.5 and one O-substituted group at d 66.8(C6ofhexoses;dataofthe attached-proton test [17]), other sugar ring carbons in the region d 68.8±81.6, and two N-acetyl groups at d 23.7 (CH 3 ) and 176.0 (CO). Accordingly, the 1 H NMR spectrum contained, inter alia, signals for four anomeric protons at d 4.59±4.96 and two N-acetyl groups at d 2.05 and 2.06. Therefore, the polysaccharide has a tetrasaccharide repeating unit containing one residue each of D -Glc and D -Gal and two D -residues of GalNAc. The 1 H- and 13 C NMR spectra of the polysaccharide were assigned using 2D COSY, H,H-relayed COSY, and H-detected 1 H, 13 CHMQCexperiments(Tables1and2). Signals for H1±H4 of each monosaccharide were assigned directly from the 2D spectra. Signals for H5 of b-linked sugars (Glcp,GalpNAc I and GalpNAc II , J 1,2  8Hz)and that of a-Galp (J 1,2 4 Hz) were recognized by H1/H5 and H4/H5 correlations, respectively, which were shown by a 2D ROESY experiment. Signals for H6a,6b of Glc were assigned on the basis of the H6/C6 correlations, which were observed in the H-detected 1 H, 13 C HMQC spectrum. The spin system of Glc was distinguished from those of Gal and GalNAc on the basis of the 3 J H,H coupling constants values [18], and the spin systems of two GalNAc residues were Fig. 1. 125-MHz 13 C NMR spectrum of the O-speci®c polysaccharide of P. penneri 1. The region of CO resonances is not shown. Ó FEBS 2002 O-antigens of Proteus penneri 1 and 4 (Eur. J. Biochem. 269) 359 distinguished from t hat of Gal by lower-®eld positions of the signals for H2 (d 4.07 and 4.02, as compared to d 3.76, respectively) and by their correlation to the corresponding nitrogen-bearing carbons at d 52.5 and 54.1. The values 1 J C1,H1 162.5±165.4 Hz determined from a nondecoupled 1 H, 13 C HMQC spectrum con®rmed the b con®gu ration of Glc and both GalNAc residues, whereas 1 J C1,H1 171.1 con®rmed the a con®guration of the Gal residue [19]. Signi®cant low-®eld displacements of the signals for C6 of Glc, C3 and C4 of Gal, and C3 of GalNAc I to d 66.8, 81.3, 77.1, and 81.6 in the 13 C NMR spectrum of the polysac- charide, compared with their positions in the spectra of the corresponding u nsubstituted monosaccharides at d 61.9, 70.4, 70.6, and 72.4 [20], respectively, were due to the effects of glycosylation and showed that the polysaccharide is branched, Glc is 6-substituted, Gal 3,4-disubstituted, and GalNA c I 3-substituted. No signi®cant displacements were observed for C2-C6 of GalNAc II and, hence, it occupies the terminal position in the side chain. A ROESY experiment revealed the following correla- tions between the anomeric and linkage protons: Gal H1/Gl c H6b at d 4.95/3.75, Glc H1/GalNAc I H3 at d 4.59/3.89, GalNAc I H1/Gal H4 at d 4.96/4.39, and GalNA c II H1/Gal H3 at d 4.61/3.96. These data ®tted well with the substitution pattern of the sugar residues determined by the 13 C NMR chemical shift data and de®ned the full sequence of the monosaccharide residues in the repeating unit. Therefore, on the basis of the data obtained, it was concluded that the O-speci®c polysaccharide of P. penneri 1 has structure 1. 6)- - D -Glcp-(1 3)- - D -GalpNAc I -(1 4)- - D -Galp-(1 1 3 1 - D -GalpNAc II Structure of the O-speci®c polysaccharide from P. penneri strain 4 Sugar analysis of the polysaccharide from P. penneri 4 showed the presence o f the same monosaccharides as in the polysaccharide from P. penneri 1 but the r elative content of D -glucose was twice as high. Table 2 . 13 C NMR data (d, p.p.m.). Chemical shifts for NAc groups are d 23.7 (CH 3 ) and 176.0 (CO). Sugar residue C1 C2 C3 C4 C5 C6 O-speci®c polysaccharide of P. penneri 1 ® 6)-b- D -Glcp-(1 ® 105.6 74.3 76.9 70.4 75.4 66.8 ® 3)-b- D -GalpNAc I -(1 ® 102.6 52.5 81.6 69.2 75.7 61.9 ® 4)-a- D -Galp-(1 ® 99.7 68.8 81.3 77.1 71.5 62.2 3  b- D -GalpNAc II -(1 ® 105.1 54.1 72.5 69.1 76.2 62.5 O-deacetylated polysaccharide of P. penneri 4 ® 6)-b- D -Glcp I -(1 ® 105.6 74.3 77.0 70.3 75.5 66.6 ® 3,6)-b- D -GalpNAc I -(1 ® 102.6 52.4 81.4 69.4 73.7 68.1 ® 4)-a- D -Galp-(1 ® 99.8 68.9 81.1 77.6 71.7 62.5 3  b- D -GalpNAc II -(1 ® 105.0 54.1 72.5 69.1 76.2 62.6 a- D -Glcp II -(1 ® 99.8 72.6 74.5 71.0 73.3 62.0 Table 1 . 1 HNMRdata(d, p.p.m.). Chemical shifts for NAc groups are d 2.05 and 2.06. Sugar residue H1 H2 H3 H4 H5 H6a, H6b O-speci®c polysaccharide of P. penneri 1 ® 6)-b- D -Glcp-(1 ® 4.59 3.31 3.51 3.55 3.59 4.05, 3.75 ® 3)-b- D -GalpNAc I -(1 ® 4.96 4.07 3.89 4.12 3.66 a ® 4)-a- D -Galp-(1 ® 4.95 3.76 3.96 4.39 3.96 a 3  b- D -GalpNAc II -(1 ® 4.61 4.02 3.74 3.99 3.69 a O-deacetylated polysaccharide of P. penneri 4 ® 6)-b- D -Glcp I -(1 ® 4.62 3.32 3.51 3.62 3.60 4.05, 3.75 ® 3,6)-b- D -GalpNAc I -(1 ® 4.99 4.09 3.92 4.16 3.87 3.95, 3.73 ® 4)-a- D -Galp-(1 ® 4.97 3.76 3.95 4.36 3.97 a 3  b-D-GalpNAc II -(1 ® 4.61 4.03 3.74 4.00 3.69 a a- D -Glcp II -(1 ® 4.96 3.58 3.70 3.44 3.66 3.87, 3.77 a Signals for H6a and H6b are in the region d 3.65±3.85. 360 Z. Sidorczyk et al. (Eur. J. Biochem. 269) Ó FEBS 2002 The 13 C (Fig. 2) and 1 H NMR spectra of the polysac- charide demonstrated a structural heterogeneity, most likely, owing to nonstoichiometric O-acetylation [there were signals for CH 3 of O-acetyl groups at d H 2.14 and 2.15, d C 21.7 (major), 21.4 and 21.5 (both minor)]. After O-deacetylation with aqueous ammonia, the spectra showed a higher degree of regularity bu t a number of minor signals were still present. Assignment of the major series in the 1 H and 13 C NMR spectra of the O-deacetylated polysaccharid e using 2D COSY and 1 H, 13 CHMQCexperiments(Tables1 and 2) revealed the same linkage pattern and sugar sequence as in the polysaccharide of P. penneri 1 (structure 1) and one additional sugar residue (a-Glcp II ) attached a t position 6 of GalNAc I .Thea-linkage of Glc II followed from the chemical shifts for C2-C5, w hich were close t o those for a-glucopyr- anose [20]. The s ite of attachment o f this monosaccharide was established by displacements of the signals for C5 and C6 of GalNAc I to d 73.7 and 68.1, compared with their positions at d 75.7 and 61.9, respectively, in the spectrum of the P. penneri 1 polysaccharide, which are typical of glycosylation by an a-linked monosaccharide at position 6 [20]. Accordingly, in the 1 HNMRspectrummarked displacements were observed for the s ignals of GalNAc I (in particular, the signal for H5 shifted from d 3.67 to d 3.87), whereas the positions of signals for the other sugar residues were essentially the same. Therefore, the m ajor repeating unit of the O-deacetylated polysaccharide from P. penneri 4 is a pentasaccharide having structure 2. 6)- - D -Glcp I -(1 3)- - D -GalpNAc I -(1 4)- - D -Galp-(1 2 6 3 1 1 - D -Glcp II - D -GalpNAc II A minor series of signals in the NMR spectra of the O-deacetylated polysaccharide from P. penneri 4 resembled the spectra of the P. penneri 1 polysaccharide and belonged to a tetrasaccharide repeating unit lac king Glc II and, hence, having the structure 1. In particular, the signal for C5 of GalNAc I in the minor series had the same chemical shift, d 75.7, as in the spectrum of the P. penneri 1 polysaccharide (the signal for C6 of this residue could not be clearly observed o wing to a coincidence with the signal for C6 of Glc II at d 62.0). Therefore, in the P. penneri 4 polysacchar- ide the Glc II residue is present in a nonstoichiometric amount. As judged by the ratio of the integral intensities of the signals in the major and minor series, the average degree of glycosylation with Glc II is  75%. Comparison of the 13 C NMR spectra of the initial and O-deacetylated polysaccharides from P. penneri 4 showed that in the former a part of the signals for C5 and C6 of GalNAc II are shifted from d 76.2 and 62.6 to d 73.6 and 65.0, respectively. Such displacements are characteristic for the effects o f O-acetylation of this monosaccharide a t position 6 [21]. This conclusion was con®rmed by higher-®eld positions of the signals for H6a and H6b of GalNAc II at d 3.70±3.85 in the 1 H NMR spectrum of the O-deacetylated polysaccharide compared to those at d 4.70 and 4.62 in the spectrum of the initial polysaccharide (a deshielding effect of the O-acetyl group). The average degree of O-acetylation at this position was estimated as  55%. The sites of attachment of other, minor O-acetyl groups were not determined. Judging from the ratio of in tegral intensities of the signals for the O-acetyl and N-acetyl groups in the 1 H NMR spectrum, the total content o f the O-acetyl groups in the repeating unit is 0.75. In conclusion, the repeating unit of the O-speci®c polysaccharide of P. penneri 4hasastructuresimilarto that of P. penneri 1 and differs in the presence of the second side chain of an a- D -glucopyranose residue and O-acetyl groups. The additional substituents occur in nonstoichio- metric amounts, thus indicating that in biosynthesis of the P. penneri 4 O-antigen glucosylation a nd O-acetylation are postpolymerization modi®cations [22]. Serological studies Lipopolysaccharides from 38 strains of P. penneri and 65 strains from 49 O-serogroups of P. mirabilis and P. vulgaris were tested in agglutination t est with rabbit polyclonal O-antisera against P. penneri 1 and 4. Only three strains, P. penneri 4, 40, and 41, cross- reacted with P. penneri 1 O-antiserum and only one strain, P. penneri 1, with P. penneri 4 O-antiserum. LPS, alkali-treated LPS, and LPS±BSA complexes were obtained from the cross-reactive strains and tested in passive immunohemolysis and enzyme im munosorbent ass ay (Table 3). In all tests, the reactivity of P. penneri 1 O-antiserum with the LPS of P. penn eri 1and4wasalmost identical, whereas P. penneri 4 O-antiserum reacted with the LPS of P. penneri 1 more weakly than with the homologous LPS. The LPS of P. penneri 40 and 41 showed a weak but remarkably similar cross-reactivity with P. penneri 1 O-antiserum and no cross-reactivity with P. penneri 4 Fig. 2. 125-MHz 13 C NMR spectrum of the O-speci®c polysaccharide of P. penneri 4. The region of CO resonances is not show n. Ó FEBS 2002 O-antigens of Proteus penneri 1 and 4 (Eur. J. Biochem. 269) 361 O-antiserum. The speci®city of the cross-reactions was con®rmed by p recipitation test and in hibition of passive immunohemolysis using various LPS as inhibitors in both homologous alkali-treated LPS/O-antiserum test systems (Table 3). O-Antisera against P. penneri 1 and 4 were absorbed with the alkali-treated LPS from various strains and tested in passive immunohemolysis again (Table 4). The reactivity of P. penneri 1 O-antiserum with all tested antigens was completely abolished when it was abso rbed with either the homologous alkali-treated LPS or that of P. penneri 4. In contrast, absorption of P. pe nneri 4 O-antiserum with the alkali-treated LPS of P. penneri 1 removed all antibodies to P. penneri 1 but only a part o f antibo dies to P. penneri 4. Absorption of P. penneri 1 O-antiserum with the alkali- treated LPS of P. penneri 40 and 41 abolished binding of these antigens and signi®cantly decreased binding of the antigens of P. penneri 1and4. In Western b lot analysis (Fig. 3), P. penneri 1O-antise- rum reacted with both the slow and fast migrating bands of the P. penneri 1 and 4 LPS, which correspond to high- and low-molecular-mass LPS species consisting of the core-lipid A moiety with and without a long-chain O-speci®c polysac- charide attached, respectively. Antibodies to P. penneri 1 bound also to low-molecular-mass LPS s pecies of P. penneri 40 and 41. Absorpion of P. penneri 1 O-antiserum with the P. penneri 40 LPS abolished binding to low-molec ular-mass LPS species of all strains but remained binding to high- molecula r-mass LPS spec ies of P. penneri 1and4(datanot shown). P. penneri 4 O -antiserum clearly r ecognized high- molecular-mass LPS species of P. penneri 1 and 4 but only weakly bound to low-molecular-mass LPS species. These data ®tted well with a marked similarity of the structures 1 and 2 of the O-speci®c polysaccharides of the P. penneri 1 and 4 LPS, respectively. The two O-antigens share the major epitope, wh ich was recognized by both O-antisera in all serological tests used, and that of P. penneri 4 exposes also a minor epitope, which was bound by P. penneri 4 O-antiserum only and, most likely, is associated with the lateral glucose residue (structure 2). The structures 1 and 2 are unique among Proteus O-antigens, and, accordingly, O-antisera against these strains showed no signi®cant cross-reactivity with O-antigens of any strain from the other known Proteus serogroups. Based on these data, we propose to classify P. penneri 1 and 4 into a new Proteus serogroup, O72, as subroups O72a and O72a,72b, respectively, where a is the major, common epitope and b is a particular epitope of P. penneri 4. Western blot data showed also that, as opposite to P. penneri 4 O-antiserum, P. penneri 1 O -antiserum contained a signi®cant amount o f antibodies to an LPS core epitope(s), which is shared by all strains studied. Such Table 3. Reactivit y of O -antis era against P. penneri 1and4withtheLPSofP. pe nneri. LPS and alkali-treated LPS were used as antigen in enzyme immunosorbent assay and passive immunohemolysis test, respectively. The d ata of the homologous LPS are shown in bold type. LPS from P. penneri strain Reciprocal titre for the LPS in Minimal dose of the LPS in Enzyme immunosorbent assay Passive immunohemolysis Precipitation test (lg) Inhibition of passive immunohemolysis (ng) P. penneri 1 O-antiserum 1 512000 25600 31.7 0.5 4 256000 25600 15.80.5 40 16000 12800 250.07.8 41 16000 12800 250.07.8 P. penneri 4 O-antiserum 1 256000 6400 125.08 4 1024000 51200 7.9 1 40 1000 100 > 1000 > 1000 41 1000 100 > 1000 > 1000 Table 4. Passive immunohemolysis of the alkali-treated LPS with absorbed O-antisera against P. penneri 1 and 4. Sheep red blood cells were used as control. NT, not tested. O-antisera absorbed with the alkali-treated LPS from P. penneri strain Reciprocal titre with absorbed O-antisera for the alkali-treated LPS from P. penneri strain 144041 P. penneri 1 O-antiserum Control 25600 25600 12800 12800 1 < 100 < 100 < 100 < 100 4 < 100 < 100 < 100 < 100 40 3200 3200 < 100 < 100 41 3200 3200 < 100 < 100 P. penneri 4 O-antiserum Control 6400 51200 < 100 < 100 1 < 100 6400 NT NT 4 < 100 < 100 NT NT 362 Z. Sidorczyk et al. (Eur. J. Biochem. 269) Ó FEBS 2002 antibodies are present also in P. penneri 40 O-antiserum, whose reactivity with the alkali-treated LPS of P. penneri 1, 4, 40 and 41 in passive immunohemolysis (titres 1 : 25 600, 1 : 12 800, 1 : 25 600 and 1 : 25 600, respectively) was completely abolished by any of the four antigens. This ®nding was consistent with the absence of a long-chain O-speci®c polysaccharide from the LP S of P. penneri 40 (data not shown) and demonstrated the i dentity of t he LPS core epitope(s) in P. penneri 1, 4, 40 and 41. ACKNOWLEDGEMENTS This work was supported by grant 6 P04 A 074 20 from the Sciences Research Committee (KBN, Polan d) and grant 99 -04-4827 9 from the Russian Foundation for Basic Research. REFERENCES 1. Larsson, P. (1984) Serology of Proteus mirabilis and Proteus vulgaris. Methods Microbiol. 14, 187±214. 2. Penner, J.L. & Hennessy, J.N. (1980) Separate O-grouping schemes for serotyping clinical isolates of Proteus vulgaris and Proteus mirabilis. J. Clin. Microbiol. 12, 304±309. 3. Hickman, F.W., Steigerwalt, A.G., Farmer, J.J. III & Brenner, D.J. (1982) Identi®cation o f Proteus penneri sp. novum form erly known as Proteus vulgaris indole negative or as Prote us vulgaris biogroup 1. J. Clin. Microbiol. 15, 1097±1102. 4. List 11 (1983) Validation of the publication of new names and new combinations previously eectively published outside the IJSB. Int. J. Syst. Bacteriol. 33, 672±674. 3 5. K nirel, Y.A., Vinogradov, E.V., Shashkov, A.S., S idorczyk, Z., Rozalski, A., Radziejewska-Lebrecht, I. & Kaca, W. (1993) Structural study of O-speci®c polysaccharides of Proteus. J. Car- bohydr. Chem. 12, 379±414. 6. Knirel, Y .A., Kaca, W., Ro   zalski, A. & Sidorczyk, Z. (1999) Structure of the O-antigenic polysaccharides of Proteus bacteria. Polish J. Chem. 73, 895±907. 7. Zych, K., Ko walczyk, M., Knirel, Y.A. & Sidorczyk, Z. (2000) New serog roups of genus Prote us consisting of Proteus penneri strains only. In Genes and Proteins Underlying Microbial Urinary Tract Virulence. Basic Aspects and Applications (Hacker, J., B lum- Ochtev, G., Pal, T. & Emody, L., eds), p p. 339±344. Kluwer Academic/Plenum Publishers, New York. 8.Kotelko,K.,Gromska,W.,Papierz,M.,Sidorczyk,Z.,Kra- jewska-Pietrasik, D. & Szer, K. (1977) Core region in Proteus mirabilis lipopolysaccharide. J. Hyg. Epidemiol. Microbiol. Immunol. 21, 271±284. 9. Westphal,O.&Jann,K.(1965)Bacteriallipopolysaccharides. Extraction with p henol-water and further applications of the procedure. Methods Carbohydr. Chem. 5, 83±91. 10. Z ych, K., Toukach, F.V., Arbatsky, N.P., Kolodziejska, K., Senchenkova, S.N., Shashkov, A.S., Knirel, Y .A. & Sidorcz yk, Z. (2001) Structure of the O-speci®c polysaccharide of Proteus m ir- abilis D52 and typing this strain to Proteus serogroup O33. Eur. J. Biochem. 268, 4346±4351. 11. Zych, K., S  wierzko, A. & Sidorczyk, Z. (1992) S erological char- acterization of Proteus penneri species novum. Arch. Immunol. Ther. Exp. 40, 89±92. 12. Sidorczyk, Z., S  wierzko, A., Knirel, Y.A., Vinogradov, E.V., Chernyak, A.Y., Kononov, L.O., Cedzyn Ä ski, M., Ro   zalski, A., Kaca, W., Shashkov, A.S. & Kochetkov, N.K. (1995) Structure and epitope speci®city of the O-speci®c polysaccharide of Proteus penneri 12 (ATCC 33519) containing amide of D -galacturonic acid with threonine. Eur. J. Biochem. 230, 713±721. 13. Fu, Y., Baumann, M., Kosma, P., Brade, L. & Brade, H. (1992) A synthetic glycoconjugate representing t he genus-spec i®c epitope of Chlamydial lipopolysaccharide exhibits the same speci®city as its neutral counterpart. Infect. Immun. 60, 1314±1321. 14. Ge rwig, G.J., Kamerling, J.P. & Vliegenthart, J.F.G. (1978) Deter- mination of the D and L con®guration of neutral monosaccharides by high-resolution capillary g.l.c. Carbohydr. Res. 62, 349±357. 15. L eontein , K., Lindberg, B. & L o È nngren, J. (1978) Assignment of absolute con®guration of sugars by g.l.c. o f t heir acetylated gly- cosides formed f rom chiral alcohols. Carbohydr. Res. 62 , 359±362. 16. Shashkov, A.S., Senchenkova, S.N., Nazarenko, E.L., Zubkov, V.A., Gorshkova, N.M., Knirel, Y.A. & Gorshkova, R.P. (1997) Structure of a phosphorylated polysaccharide from Shewanella putrefaciens strain S29. Carbohydr. Res. 303, 333±338. 17. Patt, S.L. & Shoolery, J .N. ( 1982) A ttached p roton test for car- bon-13 NMR. J. Magn. Reson. 46, 535±539. 18. Altona, C. & Haasnoot, C.A.G. (1980) Prediction of anti and gauche vicinal proton-proton coupling constants in carbohydrates: a simp le additivity rule for pyranose rings. Org. Magn. Reson. 13 , 417±429. 19. Bock, K. & Pedersen, C. (1974) A study of 13 CH coupling con- stants in hexopyranoses. J. Chem. Soc. P erkin Trans. 2, 293±297. 20. L ipkind, G.M ., Shashkov, A.S., Knirel, Y.A., Vinogradov, E.V. & Kochetkov, N.K. (1988) A computer-assisted structural analysis of regular p olysaccharides on the b asis of 13 C-n.m.r. data. Car- bohydr. Res. 175, 59±75. 21. Jansson,P E.,Kenne,L.&Schweda,E.(1987)Nuclearmagnetic resonance and con formational stud ies on m onoacetylate d methy l D -gluco- and D -galacto-pyranosides. J. Chem. Soc., Perkin Trans. 1, 377±383. 22. J ann, K. & Jann, B. (1984) Struc ture and biosynthesis of O-anti- gens. In Handbook of Endotoxin, Vol. 1 Chemistry of Endotoxin (Rietschel, E.T., ed.), pp. 138±186. Elsevier, Amsterdam. Fig. 3. Western blots of the LPS of P. penneri strains 1, 4, 40, and 41 with O-antisera ag ainst P. penneri 1 (A) and 4 (B). Ó FEBS 2002 O-antigens of Proteus penneri 1 and 4 (Eur. J. Biochem. 269) 363 . Structural and serological relatedness of the O-antigens of Proteus penneri 1 and 4 from a novel Proteus serogroup O72 Zygmunt Sidorczyk 1 , Filip. penneri 1and4 .Basedon these data, it was proposed to classify P. penneri strains 1 and 4 into a new Proteus serogroup, O72, as two sub- groups, O7 2a and O7 2a, b,