porri NCPPB 3365 and NCPPB 3364T possess multiple oligosaccharide O repeats, some of which are linear and composed ofL-rhamnose L-Rha, whereas the major O repeats are branched withL-rham
Trang 1Structure of the O polysaccharides and serological classification
Evelina L Zdorovenko1, George V Zatonskii1, Nina A Kocharova1, Aleksander S Shashkov1,
Yuriy A Knirel1and Vladimir V Ovod2
1
N D Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia;2Institute 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 3364T
possess multiple oligosaccharide O repeats, some of which
are linear and composed ofL-rhamnose (L-Rha), whereas
the major O repeats are branched withL-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 3364T
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
syringaeare 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-gaeand 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)
Trang 2[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-ienshave 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 22C for
24 h LPS were isolated by extraction with Tris/EDTA
buffer as described [23] The LPS of P syringae pv porri
NCPPB 3364T (GSPB 2654) and NCPPB 3365 (GSPB
2655) were degraded by hydrolysis with 2% (v/v) HOAc
for 1.5 h at 100C 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 2M
CF3CO2H (120C, 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 160C (1 min) to
250C at 3 C min)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 CH3I in dimethyl 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% D2O 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
Geographical origin
Year of
Trang 3in 99.96% D2O The 1H and 13C NMR spectra were
recorded on Bruker DRX-500 and DRX-600 spectrometers
(Germany) at 60C Chemical shifts were determined with
acetone as internal standard (dH2.225, dC31.45) Spectra
were run using standard Bruker software, and theXWINNMR
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 Ps4a1 (IgM) and Ps4e2
(IgG3) were generated against P syringae pv garcae ICMP
8047, MAbs Ps9c (IgG2a) and Ps9 c1 (IgG2a) against
P syringae pv porri NCPPB 3364T, and MAb Ps4a2
(IgM) was produced against P syringae pv delphinii
NCPPB 1879T 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]
Crude and proteinase K-digested LPS and isolated OPS
were used as antigens to coat Nunc-Immuno MaxiSorp
Surface ELISA plates (Nunc, Roskilde, Denmark)
Results
Serological characterization and classification
of strains ofP syringae pv porri in serogroup O9
Two MAbs, Ps9c and Ps9c1, were produced against type
strain of P syringae pv porri, NCPPB 3364T 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 Ps9c1recognized only strains NCPPB
3364T 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 Ps9c1, strains
of P syringae pv porri were classified in a new serogroup
O9as two serotypes designated correspondingly as O9(9c)
and O9(9c,9c1) 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 9c1 (Table 1) The
inability of MAbs Ps9c and Ps9c1to 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
3364Tand NCPPB 3545 cross-reacted in ELISA with MAb
Ps4a1, which is specific to LPS of strains from P syringae
serogroup O4 [17,29] However, the reaction was only weak,
epitope 4a1was 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 Pscor1reacted with the LPS P syr-ingaepv 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 syringaeLPS 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 syringaepv porri strains too (data not shown)
Structural studies of the OPS ofP 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 ofL-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 1H and 13C NMR spectra of the OPS (Fig 1A) showed signals of different intensities, thus indicating a structural heterogeneity The13C NMR spectrum contained
Fig 1.13C 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-dimensional1H,13C HSQC spectra).
Trang 4signals for anomeric carbons at d 101.8–103.9, CH3-C
groups (C6 of Rha residues) at d 17.9, one HOCH2-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 (CH3at d 23.6, CO at
d 175.4)
The assignment of the1H and13C NMR spectra of the
OPS was performed using two-dimensional1H,1H COSY,
TOCSY and1H,13C 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 J1,2
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- and b-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: RhaI H1/RhaIV H3, RhaII H1/RhaI H3, RhaIII H1/RhaII H3, RhaIV H1/RhaIII H2 and GlcNAcI H1/ RhaII 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 RhaII is the site of attachment of the GlcNAc side chain The NOESY data were in agreement with the methylation analysis data, and the glycosylation pattern was further confirmed by the13C NMR chemical shift data (Table 3) Particularly, the positions of substi-tution of the rhamnose residues followed from downfield displacements of the signals for C3 of RhaI and RhaIV, C2 of RhaIII, C2 and C3 of RhaIIto d 77.3–79.1, i.e by 6–8 p.p.m as compared with their positions in the
Table 2.1H 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
P syringae pv porri NCPPB 3365
O repeat 1
fi3)-a- L -Rhap I
fi3)-a- L -Rhap IV
P syringae pv porri NCPPB 3364 T
O repeat 2
fi2,3)-a- L -Rhap I
fi2)-a- L -Rhap IV
Table 3 13 C NMR data of O polysaccharides of P syringae pv porri (d, p.p.m) Assignment of the signals for N˜5 e` N˜6 of rhamnose residues could
be interchanged.
Monosaccharide residue
Chemical shift for
P syringae pv porri NCPPB 3365
O repeat 1
fi3)-a- L -Rhap I
fi3)-a- L -Rhap IV
P syringae pv porri NCPPB 3364T
O repeat 2
fi2)-a- L -Rhap III
fi2)-a- L -Rhap IV
Trang 5spectrum 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
3364T (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 ofP syringae
pv porri NCPPB 3364T Sugar analysis of the OPS isolated by mild acid degradation
of the LPS from P syringae pv porri NCPPB 3364T showed the presence ofL-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 having a main chain of L -rhamnose tetrasaccharide O repeats and side chains of single
D -GlcNAc residues.
Pathovar and
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
› 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
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-RhapIV-(1fi2)-a-L-RhapIII-(1fi3)-a-L-RhapII-(1fi3)-a-L-RhapI-(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)
Trang 6revealed 2- and 3-substituted, and 3,4-disubstituted Rha in
the ratios 10 : 1 : 3 as well as terminal GlcNAc
The 1H and 13C NMR spectra of the OPS (Fig 1B)
showed signals of different intensities, thus indicating a
structural heterogeneity The13C NMR spectrum contained
signals for anomeric carbons at d 101.7–103.7, CH3-C
groups (C6 of Rha residues) at d 17.9–18.1, one HOCH2
-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 d 70.2–79.1 and one N-acetyl group (CH3 at
d 23.9, CO at d 175.6)
The assignment of the1H and13C 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 J1,2coupling 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: RhaIH1/RhaIV H2, RhaIIH1/RhaIH3,
RhaIII H1/RhaIIH3, RhaIV H1/RhaIIIH2 and GlcNAcI
H1/RhaIH2 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 RhaII, C2 of RhaIIIand
RhaIV, and C2 and C3 of RhaI 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 3364Tdemonstrated
that, in addition to the major O repeat 2, there are two
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 (RhaIV) in the main chain and the site
of attachment of the GlcNAc side chain (at RhaII or RhaI) 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 3364T(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 allL-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 Ps9c1 produced against
P syringae pv porri NCPPB 3364T 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,9c1) (Table 1) From the two correspond-ing epitopes on the LPS, only epitope 9c, which is common for all strains, was stable, whereas epitope 9c1, 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-gaepv porri NCPPB 3364Tand NCPPB 3545 was observed
in ELISA with MAb Ps4a1 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 ofL-rhamnan main chain in OPS of P syringae pv porri (O repeat 1) and
P syringaepv garcae ICMP 8047 [18] (O repeat 5)
fi2)-a-L-RhapIV-(1fi2)-a-L-RhapIII-(1fi3)-a-L-RhapII-(1fi3)-a-L-RhapI-(1fi (2)
2
› 1 b-D-GlcpNAc
fi2)-a-L-RhapIV-(1fi2)-a-L-RhapIII-(1fi3)-a-L-RhapII-(1fi3)-a-L-RhapI-(1fi (5)
4
› 1 a-D-Fucp3NAc
Trang 7LPS of smooth strains of P syringae pv porri did not
react with MAb Pscor1, which is specific to the core
oligosaccharide and recognizes LPS of most other
P syringaestrains 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
3364T, 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(4a1,4e) (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 3683T
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 3364T: 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 syringaepv atrofaciens IMV 948 (chemotype 3C) nor
P syringaepv 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
Genetic and antigenic (chemical and serological)
similarities suggest the same ancestral origin of the
strains from pathovars delineated in genomospecies 4,
and their antigenic diversity may result from a divergent
evolution of the bacteria during a relatively short period
of time
Acknowledgment This work was supported by the Russian Foundation for Basic Research (grant 02-04-48721) and INTAS (grant YS 00–12). References
1 Young, J.M., Saddler, G.S., Takikawa, Y., DeBoer, S.H., Vau-terin, L., Gardan, L., Gvozdyak, R.I & Stead, D.E (1996) Names
of plant pathogenic bacteria 1864–1995 ISPP Subcommittee on Taxonomy of Plant Pathogenic Bacteria Rev Plant Pathol 75, 721–763.
2 Hirano, S.S & Upper, C.D (1990) Population biology and epi-demiology of Pseudomonas syringae Annu Rev Phytopathol 28, 155–177.
3 Lattore, B.A & Jones, A.L (1979) Evaluation of weeds and plant refuse as potential sources of inoculum of Pseudomonas syringae in bacterial canker of cherry Phytopathology 69, 1122–1125.
4 Gardan, L., Shafik, H., Belouin, S., Brosch, R., Grimont, F & Grimont, P.A.D (1999) DNA relatedness among the pathovars of
P syringae and description of Pseudomonas tremae sp nov & Pseudomonas cannabina sp nov (ex Sutic and Dowson 19 59 ) Int.
J Syst Bacteriol 49, 469–478.
5 Schaad, N.W., Vidaver, A.K., Lacy, G.L., Rudolph, K & Jones, J.B (2000) Evaluation of proposed amended names of several Pseudomonads and Xanthomonads and recommendations Phyto-pathology 90, 208–213.
6 Pecknold, P.C & Grogan, R.C (1973) Deoxyribonucleic acid homology groups among phytopathogenic Pseudomonas species Int J Syst Bacteriol 23, 111–121.
7 Palleroni, N.J (1984) Genus I Pseudomonas Migula 1894 237AL.
In Bergey’s Manual of Systematic Bacteriology, 1 (Krieg, N.R & Holt, G.D., eds), pp 141–199 William & Wilkins, Baltimore, London.
8 Denny, T.P., Gilmour, M.N & Selander, R.K (1988) Genetic diversity and relationships of two pathovars of Pseudomonas syringae J Gen Microbiol 134, 1949–1960.
9 Janse, J.D., Rossi, P., Angelucci, L., Scortichini, M., Derks, J.H., Akkermans, A.D.L., De Vrijer, R & Psallidas, P.G (1996) Reclassification of Pseudomonas syringae pv avellanae as Pseu-domonas avellanae (spec nov.), the bacterium causing cancer of hazelnut (Corylus avellana L.) System Appl Microbiol 19, 59 8– 595.
10 Manceau, C & Horvais, A (1997) Assessment of genetic diversity among strains of Pseudomonas syringae by PCR-restriction frag-ment length polymorphism analysis of rRNA operons with special emphasis on P syringae pv tomato Appl Environ Microbiol 63, 498–505.
11 Clerc, A., Manceau, C & Nesme, X (1998) Comparison of ran-domly amplified polymorphic DNA with amplified fragment length polymorphism to assess genetic diversity and genetic relatedness within genospecies III of Pseudomonas syringae Appl Environ Microbiol 64, 1180–1187.
12 Young, J.M., Takikawa, Y., Gardan, L & Stead, D.E (1992) Changing concepts in the taxonomy of plant pathogenic bacteria Annu Rev Phytopathol 30, 67–105.
13 Young, J.M & Triggs, C.M (1994) Evaluation of determinative tests for pathovars of Pseudomonas syringae van Hall 1902 J Appl Bacteriol 77, 195–207.
14 Ovod, V., Knirel, Y.A., Samson, R & Krohn, K (19 9 9 ) Immunochemical characterization and taxonomic evaluation of the O polysaccharides of the lipopolysaccharides of Pseudomonas syringae serogroup O1 strains J Bacteriol 181, 6937–6947.
15 Knirel, Y.A & Zdorovenko, G.M (1997) Structures of O-poly-saccharide chains of lipopolyO-poly-saccharides as the basis for classifi-cation of Pseudomonas syringae and related strains In
Trang 8Pseudomonas Syringae Pathovars and Related Pathogens
(Rudolph, K., Burr, T.J., Mansfield, J.W., Stead, D., Vivian, A &
von Kietzell, J., eds), pp 475–480 Kluwer Academic Publishers,
Dordrecht, Boston, London.
16 Knirel, Y.A., Ovod, V.V., Paramonov, N.A & Krohn, K (1998)
Heterogeneity in the O polysaccharide structure of Pseudomonas
syringae pv coriandricola GSPB 2028 (W-43) Eur J Biochem.
258, 716–721.
17 Knirel, Y.A., Ovod, V.V., Zdorovenko, G.M., Gvozdyak, R.I &
Krohn, K (1998) Structure of the O polysaccharide and
immunochemical relationships between the lipopolysaccharides of
Pseudomonas syringae pathovar tomato and pathovar maculicola.
Eur J Biochem 258, 657–661.
18 Zdorovenko, E.L., Ovod, V., Shashkov, A.S., Kocharova, N.A.,
Knirel, Y.A & Krohn, K (1999) Structure of the
O-poly-saccharide of the lipopolyO-poly-saccharide of Pseudomonas syringae pv.
garcae ICMP 8047 Biochemistry (Moscow) 64, 765–773.
19 Ovod, V., Zdorovenko, E.L., Shashkov, A.S., Kocharova, N.A &
Knirel, Y.A (2000) Structure of the O polysaccharide and
ser-ological classification of Pseudomonas syringae pv ribicola
NCPPB 1010 Eur J Biochem 267, 2372–2379.
20 Zdorovenko, G.M., Shashkov, A.S., Zdorovenko, E.L.,
Kochar-ova, N.A., Yakovleva, L.M., Knirel, Y.A & Rudolph, K (2001)
Characterization of the lipopolysaccharide and structure of the
O-specific polysaccharide of the bacterium Pseudomonas syringae
pv atrofaciens IMV 948 Biochemistry (Moscow) 66, 369–377.
21 Zdorovenko, E.L., Zatonsky, G.V., Zdorovenko, G.M.,
Pasich-nik, L.A., Shashkov, A.S & Knirel, Y.A (2001) Structural
het-erogeneity in the lipopolysaccharides of Pseudomonas syringae
with O-polysaccharide chains having different repeating units.
Carbohydr Res 336, 329–336.
22 Zdorovenko, E.L., Ovod, V., Zatonsky, G.V., Shashkov, A.S.,
Kocharova, N.A & Knirel, Y.A (2002) Structure of the O
polysaccharide of Pseudomonas syringae pv delphinii NCPPB
1879T having side chains of multiple
3-acetamido-3,6-dideoxy-D -galactose residues Biochemistry (Moscow) 67, 558–565.
23 Ovod, V., Rudolph, K., Knirel, Y.A & Krohn, K (1996)
Immunochemical characterization of O polysaccharides
compos-ing the a- D -rhamnose backbone of lipopolysaccharide of
Pseu-domonas syringae and classification of bacteria into serogroups O1
and O2 with monoclonal antibodies J Bacteriol 178, 6459–6465.
24 Koike, S.T., Barak, J.D., Henderson, D.M & Gilbertson, R.L.
(1999) Bacterial blight of leek: a new disease in California caused
by Pseudomonas syringae Plant Dis 83, 165–170.
25 Samson, R., Shafik, H., Benjama, A & Gardan, L (19 9 8)
Description of the bacterium causing blight of leek as
Pseudo-monas syringae pathovar porri (pathovar nov.) Phytopathology
88, 844–850.
26 Sawardeker, J.S., Sloneker, J.H & Jeanes, A (1965) Quantitative determination of monosaccharides as their alditol acetates by gas liquid chromatography Anal Chem 37, 1602–1603.
27 Leontein, K., Lindberg, B & Lo¨nngren, J (19 78) Assignment of absolute configuration of sugars by g.1.c of their acetylated glycosides formed from chiral alcohols Carbohydr Res 62, 359–362.
28 Ciucanu, I & Kerek, F (1984) A simple and rapid method for the permethylation of carbohydrates Carbohydr Res 131, 209–217.
29 Ovod, V., Rudolph, K & Krohn, K (1997) Serological classifi-cation of Pseudomonas syringae pathovars based on monoclonal antibodies towards the lipopolysaccharide O-chains In Pseudo-monas Syringae Pathovars and Related Pathogens (Rudolph, K., Burr, T.J., Mansfield, J.W., Stead, D., Vivian, A & von Kietzell, J., eds), pp 526–531 Kluwer Academic Publishers, Dordrecht, Boston, London.
30 Ovod, V., Knirel, Y & Krohn, K (1997) Demonstration of the immunochemical diversity of O-chains of lipopolysaccharide of Pseudomonas syringae and inferring of the serogroup- and sero-type-specific epitopes with monoclonal antibodies In Pseudo-monas Syringae Pathovars and Related Pathogens (Rudolph, K., Burr, T.J., Mansfield, J.W., Stead, D., Vivian, A & von Kietzell, J., eds), pp 532–537 Kluwer Academic Publishers, Dordrecht, Boston, London.
31 Ovod, V., Ashorn, P., Yakovleva, L & Krohn, K (1995) Classi-fication of Pseudomonas syringae with monoclonal antibodies against the core and O-side chains of the lipopolysaccharide Phytopathology 85, 226–232.
32 Jansson, P.-E., Kenne, L & Widmalm, G (1989) Computer-assisted structural analysis of polysaccharides with an extended version of CASPER using1H- and13C-n.m.r data Carbohydr Res 188, 169–191.
33 Zdorovenko, E.L., Knirel, Y.A & Ovod, V.V (1999) Structures
of O-polysaccharide chains of Pseudomonas syringae pv garcae LPS In Abstract 13th International Congress of the Hungarian Society for Microbiology, Budapest, Hungary, 29August )1 Sep-tember 19 9 9 , p 112.
34 Knirel, Y.A., Zdorovenko, G.M., Shashkov, A.S., Mamyan, S.S., Gubanova, N.Y., Yakovleva, L.M & Solyanik, L.P (1988) Antigenic polysaccharides of bacteria 30 Structure of the polysaccharide chain of the Pseudomonas syringae pv syringae
281 (serogroup I) lipopolysaccharide Bioorg Khim 14, 180–186.
35 Knirel, Y.A., Zdorovenko, G.M., Shashkov, A.S., Yakovleva, L.M., Gubanova, N.Y & Gvozdyak, R.I (1988) Antigenic polysaccharides of bacteria 29 Structure of the O-specific poly-saccharide chain of the Pseudomonas holci 8300 (serogroup I) lipopolysaccharide Bioorg Khim 14, 172–179.