Báo cáo khoa học: Structure of the O polysaccharides and serological classification of Pseudomonas syringae pv. porri from genomospecies 4 doc

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:
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.
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.SuticandDowson1959).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. 237

AL
.
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. 19,598–
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.(1999)
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 lipopolysaccharides as the basis for classifi-
cation of Pseudomonas syringae and related strains. In
26 E. L. Zdorovenko et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Pseudomonas 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 lipopolysaccharide 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
1879
T
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.(1998)
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.(1978)Assignmentof
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 using
1
H- and
13
C-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, 29 August)1Sep-
tember1999,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.
Ó FEBS 2003 O polysaccharides of Pseudomonas syringae pv. porri (Eur. J. Biochem. 270)27