Structure of the O polysaccharides and serological classification of Pseudomonas syringae pv. porri from genomospecies 4 Evelina L. Zdorovenko 1 , George V. Zatonskii 1 , Nina A. Kocharova 1 , Aleksander S. Shashkov 1 , Yuriy A. Knirel 1 and Vladimir V. Ovod 2 1 N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia; 2 Institute of Medical Technology, University of Tampere, Tampere, Finland Strains of Pseudomonas syringae pv. porri are characterized by a number of pathovar-specific phenotypic and genomic characters and constitute a highly homogeneous group. Using monoclonal antibodies, they all were classified in a novel P. syringae serogroup O9. The O polysaccharides (OPS) isolated from the lipopolysaccharides (LPS) of P. syringae pv. porri NCPPB 3365 and NCPPB 3364 T possess multiple oligosaccharide O repeats, some of which are linear and composed of L -rhamnose ( L -Rha), whereas the major O repeats are branched with L -rhamnose in the main chain and GlcNAc in side chains (structures 1 and 2). Both branched O repeats, which differ in the position of substitution of one of the Rha residues and in the site of attachment of GlcNAc, were found in the two strains stud- ied, O repeat 1 being major in strain NCPPB 3365 and 2 in strain NCPPB 3364 T . The relationship between OPS chemotype and serotype on one hand and the genomic characters of P. syringae pv. porri and other pathovars delineated in genomospecies 4 on the other hand is discussed. Keywords: lipopolysaccharide; O polysaccharide structure; serological classification; monoclonal antibody; Pseudo- monas syringae. Strains of the phytopathogenic bacterium Pseudomonas syringae are characterized by a high degree of heterogeneity in respect to phenotypic and genotypic characters. More than 50 infraspecific taxa, so-called pathovars, of P. syrin- gae and related species have been described based on the distinctive pathogenicity of strains to one or more host plants [1]. However, P. syringae is known to be an opportunistic pathogen that includes both nonpathogenic (epiphytes) and pathogenic strains, all of which are able to induce the hypersensitive reaction to tobacco [2,3]. There- fore, pathovars have no taxonomic impact [2,4,5]. P. syringae strains of different pathovars also reveal heterogeneity of their genomic characters [6–11]. Recently, pathotype strains of 48 pathovars of P. syringae and eight related phytopathogenic pseudomonads have been delinea- ted in nine genomospecies [4]. However, the genomospecies cannot be properly discriminated based on the nutritional characters of strains [2,4,7,12,13]. Therefore, new pheno- typic characters are necessary for discrimination between pathovars/genomospecies and identification of the bacteria. Recently, it has been suggested that chemotype of the lipopolysaccharide (LPS) and the corresponding O serotype of P. syringae are conserved phenotypic characters, which may correlate with pathovars and genomospecies [14]. Previously, we have elucidated the structures of the O polysaccharide chains (OPS) of LPS of a number of P. syringae strains belonging to different pathovars fi3)-a- L -Rhap-(1fi2)-a- L -Rhap-(1fi3)-a- L -Rhap-(1fi3)-a- L -Rhap-(1fi (1) 2 › 1 b- D -GlcpNAc fi2)-a- L -Rhap-(1fi2)-a- L -Rhap-(1fi3)-a- L -Rhap-(1fi3)-a- L -Rhap-(1fi (2) 2 › 1 b- D -GlcpNAc Correspondence to Yuriy A. Knirel, N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 47, 119991 Moscow, GSP-1, Russia. Fax: +7 095 1355328, Tel.: +7 095 9383613, E-mail: knirel@ioc.ac.ru Abbreviations: HSQC, heteronuclear single-quantum coherence; LPS, lipopolysaccharide; OPS, O polysaccharide; Rha, rhamnose. (Received 30 August 2002, revised 24 October 2002, accepted 7 November 2002) Eur. J. Biochem. 270, 20–27 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03354.x [14–23]. Here we report on structural and serological studies of LPS of strains of P. syringae pv. porri, the causative agent of the bacterial blight of leek (Allium porrum) [24,25], which together with P. syringae pvs. garcae, atropurpurea, oryzae, striafaciens, zizaniae, and Pseudomonas coronafac- iens have been delineated in genomospecies 4 [4]. Materials and methods Cultivation of bacteria, isolation of lipopolysaccharides and polysaccharides Bacterial strains of pathovars delineated in genomospecies 4 (Table 1) were cultivated on potato agar at 22 °Cfor 24 h. LPS were isolated by extraction with Tris/EDTA buffer as described [23]. The LPS of P. syringae pv. porri NCPPB 3364 T (GSPB 2654) and NCPPB 3365 (GSPB 2655) were degraded by hydrolysis with 2% (v/v) HOAc for 1.5 h at 100 °C. The OPS were isolated by gel- permeation chromatography on a column (70 · 2.6 cm) of Sephadex G-50 using pyridinium acetate buffer pH 4.5 (4 mL pyridine and 10 mL HOAc in 1 L water) and monitoring of elution with a differential refractometer (Knauer, Germany). Chemical analyses For sugar analysis, the OPS was hydrolyzed with 2 M CF 3 CO 2 H (120 °C, 2 h), monosaccharides were identified by GLC as the alditol acetates [26] on a Hewlett-Packard 5880 chromatograph (USA) equipped with a DB-5 capillary column using a temperature gradient of 160 °C(1min)to 250 °Cat3°Cmin )1 . The absolute configurations of the monosaccharides were determined by GLC of the acetyl- ated glycosides with (S)-octan-2-ol [27]. Methylation was carried out with CH 3 Iindimethyl sulfoxide in the presence of solid NaOH [28]. Hydrolysis of the methylated polysaccharides was performed as in sugar analysis, partially methylated monosaccharides were con- verted into the alditol acetates and analyzed by GLC/MS on a Hewlett Packard 5890 chromatograph (USA) equipped with a DB-5 capillary column and a NERMAG R10–10 L mass spectrometer (France) in the same chromatographic conditions as above. NMR spectroscopy For NMR spectroscopy, samples were deuterium- exchanged by freeze-drying from 99.9% D 2 O and dissolved Table 1. LPS-based serological and chemical classification of strains of Pseudomonas syringae pathovars and Pseudomonas coronafaciens from genomospecies 4 [4]. CFBP, French Collection of Phytopathogenic Bacteria (INRA, Angers, France); ICMP, International Collection of Micro- organisms from Plants (Auckland, New Zealand); NCPPB, National Collection of Plant Pathogenic Bacteria (Harpenden, UK). P. syringae pathovar or Pseudomonas species Strain Host plant Geographical origin Year of isolation Serotype Chemotype atropurpurea NCPPB 2397 T Lolium multiflorum Japan 1967 O3(3c,3c 1 )3C NCPPB 2396 Lotium sp. Japan 1967 O3(3c,3c 1 )3C NCPPB 1768 Agrostis sp. UK 1965 O4(4a,4e) 4E0 P. coronafaciens NCPPB 600 T Avena sativa UK 1958 O3(3c) 3C NCPPB 1253 Avena sativa UK 1962 O3(3c) 3C NCPPB 1356 Avena sativa Canada 1954 Rough NCPPB 1357 Avena sativa Canada 1962 O4(4a 1 ,4e) 4E1-I NCPPB 1327 Avena sativa Canada 1962 O4(4a 1 ,4e) 4E1-I NCPPB 874 Avena sativa Germany 1959 O4(4a 1 ,4e) 4E1-I NCPPB 2481 Avena sativa Kenya 1970 O4(4a 1 ,4e) 4E1-I NCPPB 2680 Avena sativa New Zealand 1969 O4(4a 1 ,4e) 4E1-I NCPPB 2816 Avena sativa Canada 1933 O4(4a 1 ,4e) 4E1-I garcae NCPPB 588 T Coffea arabica Brazil 1956 O4(4a 1 ,4e) 4E1-I NCPPB 512 Coffea arabica Brazil 1956 O4(4a 1 ,4e) 4E1-I ICMP 5802 Coffea arabica Brazil 1976 O4(4a 1 ,4e) 4E1-I NCPPB 2708 Coffea arabica Kenya 1972 O4(4a,4a 1 ,4a 2 )4A NCPPB 2710 Coffea arabica Kenya 1973 O4(4a,4a 1 ,4a 2 )4A ICMP 8047 Coffea arabica Kenya 1974 O4(4a 1 ,4e 2 ) 4E2 oryzae NCPPB 3683 T Oryza sativa Japan 1983 O8(8c) 8C CFBP 4363 Oryza sativa Japan 1983 O4(4a 1 ,4e) 4E1-I porri NCPPB 3364 T Allium porrum France 1978 O9(9c,9c 1 )9C NCPPB 3365 Allium porrum France 1964 O9(9c) 9C NCPPB 3366 Allium porrum France 1975 O9(9c) 9C NCPPB 3367 Allium porrum France 1979 Rough NCPPB 3545 Allium porrum Netherlands 1984 O9(9c,9c 1 )9C striafaciens NCPPB 1898 T Avena sativa unknown 1966 Rough NCPPB 2480 Avena sativa Zimbabwe 1971 O3(3c) 3C NCPPB 2713 Secale & Triticum sp. Mexico 1973 O1[(1–2)a,(1–2)a 1 ,1a,1b] 1B ICMP 4483 Avena sativa New Zealand Unknown O4(4a,4e) 4E0 ICMP 8815 Avena sativa Mexico 1973 O1[(1–2)a,(1–2)a 1 ,1a,1b] 1B zizaniae NCPPB 3690 T Zizania aquatica USA 1983 O4(4a 1 ,4e) 4E1-I Ó FEBS 2003 O polysaccharides of Pseudomonas syringae pv. porri (Eur. J. Biochem. 270)21 in 99.96% D 2 O. The 1 Hand 13 C NMR spectra were recorded on Bruker DRX-500 and DRX-600 spectrometers (Germany)at60°C. Chemical shifts were determined with acetone as internal standard (d H 2.225, d C 31.45). Spectra were run using standard Bruker software, and the XWINNMR 2.1 program was used to acquire and process the data. A mixing time of 100 and 200 ms was used in TOCSY and NOESY experiments, respectively. Production of monoclonal antibodies and serological tests Murine MAbs Ps3c, Ps4a, Ps4e, and Ps8c have been produced and characterized previously [17,19,29–31]. New O polysaccharide-specific MAbs Ps4a 1 (IgM) and Ps4e 2 (IgG 3 ) were generated against P. syringae pv. garcae ICMP 8047, MAbs Ps9c (IgG 2a )andPs9c 1 (IgG 2a ) against P. syringae pv. porri NCPPB 3364 T ,andMAbPs4a 2 (IgM) was produced against P. syringae pv. delphinii NCPPB 1879 T . Immunization protocol, hybridomas gen- eration, selection of specific clones and determination of MAb isotypes were performed as described earlier [14,23,29,31]. ELISA, SDS/PAGE and Western immuno- blotting were performed essentially as described [23,29,31]. CrudeandproteinaseK-digestedLPSandisolatedOPS were used as antigens to coat Nunc-Immuno MaxiSorp Surface ELISA plates (Nunc, Roskilde, Denmark). Results Serological characterization and classification of strains of P. syringae pv. porri in serogroup O9 Two MAbs, Ps9c and Ps9c 1 , were produced against type strain of P. syringae pv. porri, NCPPB 3364 T . In ELISA, both MAbs strongly reacted with the homologous LPS whether it was crude or digested with proteinase K. In Western immunoblotting, only MAb Ps9c was reactive (data not shown). MAb Ps9c cross-reacted with all strains of P. syringae pv. porri (Table 1), except for strain NCPPB 3367, whereas MAb Ps9c 1 recognized only strains NCPPB 3364 T and NCPPB 3545. Strains of none of the other pathovars delineated in genomospecies 4 (Table 1) reacted with these MAbs. Based on the reactivity with MAbs Ps9c and Ps9c 1 ,strains of P. syringae pv. porri were classified in a new serogroup O9 as two serotypes designated correspondingly as O9(9c) and O9(9c,9c 1 ). A stable epitope 9c is present in all strains of pathovar porri studied that have an S-form LPS, whereas only a few strains coexpose epitope 9c 1 (Table 1). The inability of MAbs Ps9c and Ps9c 1 to recognize P. syringae pv. porri NCPPB 3367 was accounted for by the R-form of LPS of this strain revealed by SDS/PAGE (data not shown). The crude LPS from strains P. syringae pv. porri NCPPB 3364 T and NCPPB 3545 cross-reacted in ELISA with MAb Ps4a 1 , which is specific to LPS of strains from P. syringae serogroup O4 [17,29]. However, the reaction was only weak, epitope 4a 1 was not stably expressed by OPS of this strain and was absent from LPS of P. syringae pv. porri NCPPB 3365. Therefore, the observed cross-reactivity is not suffi- cient for classification of strains of P. syringae pv. porri in serogroup O4 rather than in a new serogroup O9. Remarkably, MAb Pscor 1 reactedwiththeLPSP. syr- ingae pv. porri rough strain NCPPB 3367 but with none of the other, smooth strains of P. syringae pv. porri. This MAb is known to be specific to the outer core region of P. syringae LPS and reactive in Western immunoblotting with R- and SR (semirough)-form LPS, which are coex- pressed with S-form LPS in smooth strains of most P. syringae pathovars [23,31]. Other epitopes related to the LPS core, which are common for all P. syringae strains, were recognized by the corresponding core-specific MAbs in P. syringae pv. porri strains too (data not shown). Structural studies of the OPS of P. syringae pv. porri NCPPB 3365 A high-molecular-mass OPS was isolated by mild acid degradation of the LPS from P. syringae pv. porri NCPPB 3365 followed by gel-permeation chromatography on Sephadex G-50. Sugar analysis of the OPS, including determination of the absolute configurations of monosac- charides, demonstrated the presence of L -rhamnose ( L -Rha) and 2-amino-2-deoxy- D -glucose ( D -GlcN). Methylation analysis of the OPS revealed 2-substituted, 3-substituted, and 3,4-disubstituted Rha in the ratios 3 : 3 : 2 as well as terminal GlcNAc. The 1 Hand 13 C NMR spectra of the OPS (Fig. 1A) showed signals of different intensities, thus indicating a structural heterogeneity. The 13 C NMR spectrum contained Fig. 1. 13 C NMR spectra of the O polysaccharides of P. syringae pv. porri NCPPB 3365 (A) and NCPPB 3364 T (B). Signals for anomeric carbons of the major O repeats are designated in the expansions as follows:G,GlcNAc;RI,Rha I ;R2,Rha II ; RIII, Rha III ;RIV,Rha IV ; other major anomeric signals are superpositions of signals from minor O repeats (data of the two-dimensional 1 H, 13 CHSQCspectra). 22 E. L. Zdorovenko et al. (Eur. J. Biochem. 270) Ó FEBS 2003 signals for anomeric carbons at d 101.8–103.9, CH 3 -C groups (C6 of Rha residues) at d 17.9, one HOCH 2 -C group (C6 of GlcN) at d 61.9, one nitrogen-bearing carbon (C2 of GlcN) at d 57.1, sugar ring carbons linked to oxygen at d 70.5–79.1 and one N-acetyl group (CH 3 at d23.6, CO at d 175.4). The assignment of the 1 Hand 13 C NMR spectra of the OPS was performed using two-dimensional 1 H, 1 HCOSY, TOCSY and 1 H, 13 C HSQC experiments, and spin systems for four major residues of Rha and one residue of GlcNAc were identified (Tables 2 and 3). A relatively large J 1,2 coupling constant value of 8 Hz showed that the GlcNAc residue is b-linked. The a configuration of all rhamnosidic linkage followed from the comparison of the H5 and C5 NMR chemical shifts (Tables 2 and 3) with published data for a-andb-rhamnopyranose [32]. Therefore, the major O repeat of the OPS is a pentasaccharide consisting of four residues of a- L -Rha and one residue of b- D -GlcNAc. The linkage and sequence analyses of the OPS were performed using a NOESY experiment. The NOESY spectrum contained the following correlations between the anomeric protons and the protons at the linkage carbons: Rha I H1/Rha IV H3, Rha II H1/Rha I H3, Rha III H1/Rha II H3, Rha IV H1/Rha III H2 and GlcNAc I H1/ Rha II H2 at d 5.05/3.85, 5.25/3.91, 5.25/3.99, 4.96/4.04 and 4.63/4.15, respectively. These data defined the sequence of rhamnose residues in the main chain and showed that Rha II isthesiteofattachmentofthe GlcNAc side chain. The NOESY data were in agreement with the methylation analysis data, and the glycosylation pattern was further confirmed by the 13 CNMRchemical shift data (Table 3). Particularly, the positions of substi- tution of the rhamnose residues followed from downfield displacements of the signals for C3 of Rha I and Rha IV , C2 of Rha III , C2 and C3 of Rha II to d 77.3–79.1, i.e. by 6–8 p.p.m. as compared with their positions in the Table 2. 1 H NMR data of O polysaccharides of P. syringae pv. porri (d, p.p.m.). Assignment of the signals for H6 of rhamnose residues could be interchanged. Monosaccharide residue Chemical shift for H1 H2 H3 H4 H5 H6a H6b CH 3 CON P. syringae pv. porri NCPPB 3365 O repeat 1 b- D -GlcpNAc-(1fi 4.63 3.71 3.62 3.49 3.43 3.77 3.89 2.09 fi3)-a- L -Rhap I -(1fi 5.05 4.15 3.91 3.59 3.87 1.32 fi2,3)-a- L -Rhap II -(1fi 5.25 4.15 3.99 3.51 3.72 1.34 fi2)-a- L -Rhap III -(1fi 5.25 4.04 3.86 3.54 3.77 1.34 fi3)-a- L -Rhap IV -(1fi 4.96 4.16 3.85 3.58 3.75 1.27 P. syringae pv. porri NCPPB 3364 T O repeat 2 b- D -GlcpNAc-(1fi 4.61 3.72 3.59 3.47 4.42 3.76 3.89 2.09 fi2,3)-a- L -Rhap I -(1fi 5.18 4.19 3.89 3.48 3.71 1.27 fi3)-a- L -Rhap II -(1fi 5.00 4.12 3.76 3.62 3.78 1.34 fi2)-a- L -Rhap III -(1fi 5.17 4.08 3.96 3.49 3.83 1.30 fi2)-a- L -Rhap IV -(1fi 5.12 4.09 3.91 3.47 3.70 1.29 Table 3. 13 C NMR data of O polysaccharides of P. syringae pv. porri (d, p.p.m). Assignment of the signals for N ˜ 5e ` N ˜ 6 of rhamnose residues could be interchanged. Monosaccharide residue Chemical shift for C1 C2 C3 C4 C5 C6 CH 3 CON CH 3 CON P. syringae pv. porri NCPPB 3365 O repeat 1 b- D -GlcpNAc-(1fi 103.9 57.1 74.4 71.3 76.8 61.9 23.6 175.4 fi3)-a- L -Rhap I -(1fi 103.4 71.2 79.1 72.6 70.5 17.9 fi2,3)-a- L -Rhap II -(1fi 102.5 79.1 77.3 73.5 70.6 17.9 fi2)-a- L -Rhap III -(1fi 101.8 79.1 71.4 73.5 70.6 17.9 fi3)-a- L -Rhap IV -(1fi 103.1 71.1 79.1 72.7 70.6 17.9 P. syringae pv. porri NCPPB 3364 T O repeat 2 b- D -GlcpNAc-(1fi 103.2 56.9 74.4 71.1 76.8 61.8 23.9 175.6 fi2,3)-a- L -Rhap I -(1fi 101.9 78.6 78.9 72.9 70.4 17.9 fi3)-a- L -Rhap II -(1fi 103.7 71.0 78.8 72.8 70.2 17.9 fi2)-a- L -Rhap III -(1fi 101.9 78.5 71.3 73.4 70.2 18.1 fi2)-a- L -Rhap IV -(1fi 101.7 79.1 71.0 73.4 71.0 17.9 Ó FEBS 2003 O polysaccharides of Pseudomonas syringae pv. porri (Eur. J. Biochem. 270)23 spectrum of nonsubstituted a-rhamnopyranose [32]. The C2–C6 chemical shifts of the GlcNAc residue were close to those of nonsubstituted b-GlcNAc [32]. These data together showed that the major O repeat of the OPS of P. syringae pv. porri NCPPB 3365 has structure 1. Studies of minor series in the NMR spectra of this OPS, including tracing connectivities in the two-dimensional spectra, showed that, in addition to the major O repeat 1, there is another branched O repeat, which is identical to the major O repeat in the OPS of P. syringae pv. porri NCPPB 3364 T (structure 2, see below), and a linear O repeat 3 having the following structure: The O repeat 3 has been previously found as one of two linear O repeats in the OPS of P. syringae pv. garcae NCPPB 2708 [33]. Similar NMR spectroscopic studies of the OPS of P. syringae pv. atrofaciens IMV 948 showed that, in addition to the branched O repeat 4, whose structure was determined by us earlier [20] (Table 4), it also contains the minor O repeat 3. Structural studies of the OPS of P. syringae pv. porri NCPPB 3364 T Sugar analysis of the OPS isolated by mild acid degradation of the LPS from P. syringae pv. porri NCPPB 3364 T showed the presence of L -rhamnose ( L -Rha) and 2-amino-2- deoxy- D -glucose ( D -GlcN). Methylation analysis of the OPS Table 4. Structures of the O polysaccharides of P. syringae havingamainchainof L -rhamnose tetrasaccharide O repeats and side chains of single D -GlcNAc residues. Pathovar and strain O repeat structure Chemotype Serotype Reference Porri NCPPB 3365, fi3)-a- L -Rhap-(1fi2)-a- L -Rhap-(1fi3)-a- L -Rhap-(1fi3)-a- L -Rhap-(1fi 1 a 9C O9(9c) This work porri NCPPB 3364 T 2 O9(9c,9c 1 ) › 1 b- D -GlcpNAc fi2)-a- L -Rhap-(1fi2)-a- L -Rhap-(1fi3)-a- L -Rhap-(1fi3)-a- L -Rhap-(1fi 2 a 2 › 1 b- D -GlcpNAc Atrofaciens IMV 948 fi2)-a- L -Rhap-(1fi2)-a- L -Rhap-(1fi3)-a- L -Rhap-(1fi3)-a- L -Rhap-(1fi 4 3C O3(3c) [20] 2 › 1 b- D -GlcpNAc Ribicola NCPPB 1010 fi2)-a- L -Rhap-(1fi2)-a- L -Rhap-(1fi3)-a- L -Rhap-(1fi3)-a- L -Rhap-(1fi 6 8C O8(8c) [19] 3 › 1 b- D -GlcpNAc fi3)-a- L -Rhap-(1fi2)-a- L -Rhap-(1fi3)-a- L -Rhap-(1fi3)-a- L -Rhap-(1fi 7 3 › 1 b- D -GlcpNAc a The O repeat 1 is major in strain NCPPB 3365 and minor in strain NCPPB 3364 T , and the O repeat 2 is major in strain NCPPB 3364 T and minor in strain NCPPB 3365. fi3)-a- L -Rhap IV -(1fi2)-a- L -Rhap III -(1fi3)-a- L -Rhap II -(1fi3)-a- L -Rhap I -(1fi (1) 2 › 1 b- D -GlcpNAc fi2)-a- L -Rhap-(1fi2)-a- L -Rhap-(1fi3)-a- L -Rhap-(1fi3)-a- L -Rhap-(1fi (3) 24 E. L. Zdorovenko et al. (Eur. J. Biochem. 270) Ó FEBS 2003 revealed 2- and 3-substituted, and 3,4-disubstituted Rha in the ratios 10 : 1 : 3 as well as terminal GlcNAc. The 1 Hand 13 C NMR spectra of the OPS (Fig. 1B) showed signals of different intensities, thus indicating a structural heterogeneity. The 13 C NMR spectrum contained signals for anomeric carbons at d 101.7–103.7, CH 3 -C groups (C6 of Rha residues) at d 17.9–18.1, one HOCH 2 - C group (C6 of GlcN) at d 61.8, one nitrogen-bearing carbon (C2 of GlcN) at d 56.9, sugar ring carbons linked to oxygen at d70.2–79.1 and one N-acetyl group (CH 3 at d 23.9, CO at d 175.6). The assignment of the 1 Hand 13 C NMR spectra of the OPS was performed as described above and the results are given in Tables 2 and 3. Again, the major pentasac- charide O repeat of the OPS was identified, which consists of four residues of L -Rha and one residue of D -GlcNAc. A relatively large J 1,2 coupling constant value of 8 Hz for the H1 signal of the GlcNAc residue and the NMR chemical shifts of H5 and C5 of the rhamnose residues showed that the former is b-linked and the latter are a-linked. The NOESY experiment revealed the following correla- tions between the anomeric protons and the protons at the linkage carbons: Rha I H1/Rha IV H2, Rha II H1/Rha I H3, Rha III H1/Rha II H3, Rha IV H1/Rha III H2 and GlcNAc I H1/Rha I H2 at d 5.18/4.09, 5.00/3.89, 5.17/3.76, 5.12/4.08 and 4.61/4.19, respectively. The glycosylation pattern was confirmed by downfield displacements of the signals for the linkage carbons, namely C3 of Rha II ,C2ofRha III and Rha IV , and C2 and C3 of Rha I to d 78.5–79.1 (by 6–8 p.p.m.), and the similarity of the C2-C6 chemical shifts of the GlcNAc residue to those of nonsubstituted b-GlcNAc [32]. These data showed that the major O repeat of the OPS has structure 2: Analysis of minor series in the NMR spectra of the OPS of P. syringae pv. porri strain NCPPB 3364 T demonstrated that, in addition to the major O repeat 2,therearetwo minor O repeats: the branched O repeat 1 and the linear O repeat 3. Discussion Two major branched O repeats 1 and 2 present in the OPS of P. syringae pv. porri have the same monosaccha- rides composition and similar structures differing from each other in the position of substitution of one of the rhamnose residues (Rha IV )inthemainchainandthesite of attachment of the GlcNAc side chain (at Rha II or Rha I ). Remarkably, both O repeats are present in each strain of P. syringae pv. porri studied, the O repeat 1 being major in strain NCPPB 3365 and 2 in strain NCPPB 3364 T (Table 4). In previous studies of structurally heterogeneous OPS of P. syringae having an L -rhamnan backbone, it has been demonstrated that both major and minor O repeats enter into the same polysaccharide chain, where they form blocks of structurally identical oligosaccharides [19,21,34,35]. This could be determined making use of a different behavior of the O repeats towards Smith degradation, from which only one was oxidized, whereas the other was stable. In the OPS of P. syringae pv. porri both major and minor O repeats are oxidizable by periodate, and therefore this approach could not be used to solve the problem. Assuming that biosyn- thesis of all L -rhamnan-based OPS of P. syringae proceeds by the same mechanism, it can be concluded that the O repeats of both types occur in the same polysaccharide chain in P. syringae pv. porri strains too. The structural data of the OPS revealed the molecular basis for strong serological cross-reactivity of these strains and their classification in the same serogroup O9. Serolog- ical studies using MAbs Ps9c and Ps9c 1 produced against P. syringae pv. porri NCPPB 3364 T showed that all and only smooth strains of P. syringae pv. porri fell in the novel serogroup O9, which can be divided into two serotypes, O9(9c) or O9(9c,9c 1 ) (Table 1). From the two correspond- ing epitopes on the LPS, only epitope 9c, which is common for all strains, was stable, whereas epitope 9c 1 , present only in a few strains, could be revealed only in ELISA and therefore can be considered as a conformational epitope. Epitope Ps9c, which is restricted to strains of P. syringae pv. porri, is evidently associated with the lateral b-GlcNAc residue but it remains unknown which O repeat, 1, 2 or both, carries this epitope. A weak cross-reactivity of the crude LPS from P. syrin- gae pv. porri NCPPB 3364 T and NCPPB 3545 was observed in ELISA with MAb Ps4a 1 . This MAb has been produced against P. syringae pv. garcae ICMP 8047 and is specific to the L -rhamnan backbone. The cross-reactivity could be accounted for by the presence of the same of L -rhamnan main chain in OPS of P. syringae pv. porri (O repeat 1)and P. syringae pv. garcae ICMP 8047 [18] (O repeat 5). fi2)-a- L -Rhap IV -(1fi2)-a- L -Rhap III -(1fi3)-a- L -Rhap II -(1fi3)-a- L -Rhap I -(1fi (2) 2 › 1 b- D -GlcpNAc fi2)-a- L -Rhap IV -(1fi2)-a- L -Rhap III -(1fi3)-a- L -Rhap II -(1fi3)-a- L -Rhap I -(1fi (5) 4 › 1 a- D -Fucp3NAc Ó FEBS 2003 O polysaccharides of Pseudomonas syringae pv. porri (Eur. J. Biochem. 270)25 LPS of smooth strains of P. syringae pv. porri did not react with MAb Pscor 1 , which is specific to the core oligosaccharide and recognizes LPS of most other P. syringae strains studied [23,31]. This suggests a difference in either the LPS core structure or/and in the mode of the attachment of the OPS to the core. Therefore, strains of pathovar porri are clearly distinct from other P. syringae pathovars in serology of both OPS moiety and LPS core. Strains of this pathovar are also distinguished in a number of other phenotypic and genotypic characters [4,24,25]. These data together suggest that P. syringae pv. porri is a separate ancestral line that can be identified on the basis of distinctive chemical characters. The pathotype strain of P. syringae pv. porri, NCPPB 3364 T , was delineated in genomospecies 4 [4]. It showed as much as 78–95% DNA–DNA homology with the patho- type strains of the other pathovars delineated in genomo- species 4, namely P. syringae pvs. garcae, atropurpurea, oryzae, porri, striafaciens, zizaniae, and Pseudomonas coronafaciens, which altogether constitute a distinct ribo- group F [4]. Studies of the representative strains of these pathovars using ELISA and Western immunoblotting with MAbs specific to the OPS and LPS core showed their serological heterogeneity (Table 1). Most strains from genomospecies 4 belong to three serotypes: O3(3c) [29], O4(4a 1 ,4e) (authors’ unpublished data), and O9(9c) (this work), which correspond to OPS chemotypes 3C, 4E1-I, and 9C, respectively. The less common serotype O8(8c) and the corresponding chemotype 8C, which has been described earlier for P. syringae pv. ribicola NCPPB 1010 [17], is characteristic of only one strain from genomospecies 4, namely the pathotype strain of P. syringae pv. oryzae, NCPPB 3683 T . OPS of strains from genomospecies 4 have marked compositional and structural similarities. Particularly, they all have a backbone of a-(1fi2)- and a-(1fi3)- linked L -rhamnose residues and lack a strict regularity owing to the occurrence of several types of O repeats in the main chain. The OPS are either linear (chemotype 4A) or branched with side chains of single a- D -Fuc3NAc residues (chemotypes 4E0, 4E1-I, and 4E2) or b- D - GlcNAc residues (chemotypes 3C, 8C, and 9C) (Table 1). The OPS of chemotypes 3C, 8C and 9C differ from each other in the site of the attachment of b- D -GlcNAc residues to the main L -rhamnan chain (Table 4). The OPS of P. syringae pv. atrofaciens IMV 948 [20] resembles most closely that of P. syringae pv. porri NCPPB 3364 T : both OPS have similar major branched O repeats 4 and 2, respectively (Table 4), and the same minor linear O repeat 3. In spite of this similarity, neither P. syringae pv. atrofaciens IMV 948 (chemotype 3C) nor P. syringae pv. ribicola NCPPB 1010 [19] (chemotype 8C, O repeats 6 and 7, Table 4) is serologically related to P. syringae pv. porri (chemotype 9C), and, accordingly, they were classified into different serogroups O3 and O8, respectively. 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