A polymer with a backbone of 3-deoxy-
D
-
glycero
-
D
-
galacto
-non-2-
ulopyranosonic acid, a teichuronic acid, and a b-glucosylated ribitol
teichoic acid in the cell wall of plant pathogenic
Streptomyces
sp.
VKM Ac-2124
Alexander S. Shashkov
1
, Larisa N. Kosmachevskaya
2
, Galina M. Streshinskaya
2
, Lyudmila I. Evtushenko
3
,
Olga V. Bueva
3
, Viktor A. Denisenko
4
, Irina B. Naumova
2
and Erko Stackebrandt
5

studied is the second representative of plant pathogenic
streptomycetes inducing potato scab disease, the cell wall
anionic polymers of which were shown to contain a Kdn-
polymer. Presumably, the presence of Kdn-containing
structures in the surface regions of pathogens is essential for
their efficient attachment to host plant cells.
Keywords: NMR spectroscopy; teichuronic acid; teichoic
acid; Kdn; Streptomyces.
Cell walls of the majority of Gram-positive bacteria
belonging to the genus Streptomyces (the order Actino-
mycetales) contain teichoic acids, the anionic glycopolymers
which are covalently bound to peptidoglycan and are
situated between other cell wall layers and at the cell surface.
They impart a negative charge to the cell surface, which is
essential for the physiological functioning of the cells and
cell coaggregation [1]. In addition to teichoic acids, other
anionic polymers have been found in the cell wall of
streptomycetes. A teichuronic acid with a disaccharide
repeating unit fi4)-b-
D
-ManpNAc3NAcA-(1fi3)-a-
D
-
GalpNAc-(1fi was identified in the cell wall of Streptom-
yces lavendulocolor VKM Ac-215
T
[2]. Recently, a polymer
of 3-deoxy-
D
-glycero-

the manufacturer’s protocol. The sequences of the highest
scores were chosen from NCIB database using
BLAST
search
[10]. Other 16S rDNA sequences of the plant pathogenic
streptomycetes and related strains used in the analysis
were selected from NCIB database. The sequence of
Brevibacterium linens DSM 20425
T
(X77451) was used as
an outgroup. Nucleotide substitution rates were calculated
as described by Kimura & Ohta [11] and the phylogenetic
Correspondence to I. B. Naumova, School of Biology,
M.V. Lomonosov Moscow State University, Moscow 119899, Russia.
E-mail:
Abbreviations: PME, phosphomonoesterase; Kdn, 2-keto-3-deoxy-
nononic acid.
Enzyme: phosphomonoesterase (EC 3.1.3.1).
Note: Kdn is the abbreviation of 2-keto-3-deoxy-nononic acid, named
according to the earlier nomenclature [9].
(Received 5 July 2002, revised 11 September 2002,
accepted 20 September 2002)
Eur. J. Biochem. 269, 6020–6025 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03274.x
tree was constructed by the neighbour-joining method [12]
with
CLUSTAL W
software [13]. Three topologies were
evaluated by bootstrap analysis of the sequence data with
thesamesoftware.
To evaluate the pathogenic activity of the strain, the

were detected with the molybdate reagent, reducing sugars,
with aniline hydrogenphthalate; and ribitol and monosac-
charides, with 5% (w/v) AgNO
3
in aqueous ammonia.
Acid hydrolysis was carried out with 2
M
HCl for 3 h at
100 °C; alkaline hydrolysis was performed with 1
M
NaOH
for 3 h at 100 °C; enzymatic hydrolysis with phospho-
monoesterase (PME) from calf intestine (EC 3.1.3.1; Sigma)
was conducted in ammonium acetate buffer, pH 9.8 at 37°
for 18–20 h.
Analytical methods used and the scheme of identification
of a glucosylribitol were the same as described previously
[16,17].
NMR spectra were recorded with a DRX-500 (Bruker,
Germany) spectrometer for 2–3% solutions in D
2
Oat30 °C
with acetone (d
H
2.225 d
C
231.45) as the internal standard,
and 80% H
3
PO

T
(AF112160), which are
also causative agents of potato scab. These three strains and
S. griseus ISP 5236
T
(AY094371) formed a tight cluster with
a 100% bootstrap replication value (not presented), which is
significantly distant from other validly described plant
pathogenic streptomycete species [18–21]. At the pheno-
typical level, the strain was most similar to S. setonii in
accordance to characteristics of S. setonii described previ-
ously [18,22,23]. Spore mass of VKM Ac-2124 was usually
grey or yellowish grey on glycerol-asparagine agar [6], the
spores were smooth, and borne in mature fexuous chains.
Substrate mycelium was yellow or brownish-yellow on most
tested media. Melanoid pigment was not produced on
tyrosine or peptone iron agar while pale or greyish to light
yellowish brown diffusible pigment was formed on some
media. Testing of plant pathogenicity of the strain VKM
Ac-2124 showed that it induced rough, corky lesions such as
those resulting from natural infections, and the lesions
covered about 70% of the tuber surface.
The anionic polymers were isolated from the cell wall and
investigated. Glucosylribitol monophosphate and small
amounts of ribitol mono- and bisphosphates were identified
as alkaline hydrolysis products. Acid hydrolysis afforded
ribitol monophosphates and bisphosphates, anhydroribitol
phosphate, anhydroribitol, ribitol, inorganic phosphate and
glucose. The amount of the latter exceeded considerably
that bound to ribitol phosphate. An unidentified ninhydrin-

carbon atoms, while the fifth signal of low intensity at
d97.6 belonged to the nonprotonated carbon atom,
presumably, to the anomeric atom C(2) of an ulosonic
acid. The presence of 3-deoxyulosonic acid was also
suggested based on the identification of a signal for a
Ó FEBS 2002 Kdn-polymer of plant pathogenic streptomycete (Eur. J. Biochem. 269) 6021
CH
2
-group at d40.4. The spectrum contained also two
signals in the region of resonances of carbon atoms bound
to nitrogen at d52.45 and 54.05, a signal at d23.3
(CH
3
CON), and three signals for CO groups at d174.5–
176.0. The resonances for the CH
2
O groups were found at
d61.9, 62.0, 65.8, 68,0, and 69.4. Other signals of the
spectrum were found at d67.9–80.4, i.e. in the region of
resonances of CH groups bound to one oxygen atom.
The region of resonances of the anomeric protons in the
1
H NMR spectrum (Fig. 1) contained two abundant signals
at d4.90 (J
1,2
<2Hz)and5.07(J
1,2
3.6 Hz) and two signals
of lower intensities at d4.55 (J
1,2

CHSQC
(Fig. 1) and HMBC and
1
H,
31
P HMQC spectra. The
spectroscopic data obtained suggested the presence of three
different types of anionic glycopolymers (Tables 1,2).
Teichuronic acid (polymer I) with the repeating unit fi6)-
a-
D
-Glcp-(1fi4)-b-
D
-ManpNAc3NAcA-(1fi was the
major component of the cell wall preparation. The absolute
configuration of glucose (
D
-) isolated after hydrolysis of the
total cell wall preparation was determined by its transfor-
mation in 2-octyl glycoside and by comparison of the
derivative obtained with standard samples of (S+)-
and (R-)-2-octyl glucopyranosides using gas-liquid
Table 1.
13
CNMRchemicalshifts(d, p.p.m) for the teichuronic acid (polymer I), the Kdn-containing polymer (polymer II), and the ribitol teichoic acid
(polymer III) from cell wall of Streptomyces sp. VKM Ac-2124.
Carbon
Residue C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9
Polymer I
fi6)-a-

ManpNAc3NAcA residue [26,27]. The signals for a-
D
-Glcp
and b-
D
-ManpNAc3NAcA were identified in the
1
HCOSY
and TOCSY spectra. The anomeric configuration of the
Glcp residue was a, followed from the coupling constant
value (
3
J
H-1,H-2
¼ 3Hz).Theb-anomeric configuration of
the
D
-Manp NAc3NAcA unit was established from both
the presence of the intraresidue correlation peak (H-1/H-5)
in the ROESY spectrum and the low-field chemical shift of
C-5 of this residue (HSQC spectrum). The C-2 and C-3
atoms resonated in the region typical of carbon atoms
bound to nitrogen (HSQC spectroscopic data, see Table 1),
which proves the position of the acetamido groups at C-2
and C-3 of this sugar. The signal of the H-5 of this sugar
appeared as a doublet, which suggests the absence of
protons at H-6. In addition, the HMBC spectrum has
shown a correlation of H-4 and H-5 with a low-field signal
at d175.1 corresponding to the carboxy group.
The interresidue cross-peak H-1(B)/H-6(A)andH-1(B)/

and at 4.55/3.93 and 3.83 p.p.m. in the ROESY spectrum
and the correlation peak H-1(D)/C-8(C)at4.55/
79.40 p.p.m. in the HMBC spectrum. The downfied shift
of the C-4 resonance of the b-Kdn residue in the
13
CNMR
spectrum of this polymer equal to 2 p.p.m. as compared to
that of nonsubstituted b-Kdn [28] revealed the 2fi4 linkage
between the Kdn units in the polysaccharide. The anomeric
configuration of the glucose residue was b,whichwas
concluded in particular from the coupling constant value
(
3
J
H-1, H-2
¼ 8Hz).
Thus, the polymer II has the following repeating unit:
The signals of the terminal monosaccharide residues were
not detected. This fact allows one to suggest that the
polymer contains no less than 20 repeating units.
The third cell wall polymer was identified as 1,5-
poly(ribitol phosphate) partially substituted with b-glucose
(
3
J
H-1, H-2
¼ 8 Hz) at position 4(2) (polymer III)basedon
1
H,
13

CH
3
CON, d1.93 and 2.07.
Ó FEBS 2002 Kdn-polymer of plant pathogenic streptomycete (Eur. J. Biochem. 269) 6023
Streptomyces azureus RIA 1009 [24] and from the presence
of the correlation peaks H-1(F)/H-4(E) at 4.66/4.18 p.p.m.
in the ROESY spectrum and H-1(F)/C-4(E) at 4.66/
80.40 p.p.m. in the HMBC spectrum.
Thus, the cell wall of Streptomyces sp. VKM Ac-2124
contains three anionic glycopolymers, viz., the teichuronic
acid with the repeating unit fi6)-a-
D
-Glcp-(1fi4)-b-
D
-
ManpNAc3NAcA-(1fi,(I)theb-glucosylated Kdn-based
polymer (II), and b-glucosylated ribitol teichoic acid (III).
The percentage of the teichoic acid ( 10 % mass of the cell
wall) was calculated from the content of the teichoic acid-
linked phosphorus (0.8%) and taking into account the
structure of the polymer (the phosphate:glucose molar ratio
in the poly(ribitol phosphate) purified by electrophoresis
was equal to 1 : 0.9).
The ratio of the cell wall glycopolymers I : II : III was
calculated as 1 : 0.33 : 0.33 based on the integral intensities
of the signals in the
1
H NMR spectrum. It is likely that the
percentages of the teichuronic acid, the Kdn-containing
polymer, and the ribitol teichoic acid are 30 %, 10 %, and

growth taxis and their attachment to potato tuber. The
presence of Kdn might be characteristic of plant pathogenic
streptomycete strains causing scab diseases of potatoes and
root crops. Further studies of the cell wall anionic polymers
in the plant pathogenic streptomycetes, including S. scabies,
S. acidiscabies, S. caviscabies, S. setonii, S. turingiscabies,
S. europaeiscabiei and S. reticuliscabiei, where cell wall
anionic polymers have not been analysed yet, will testify
to or against our suppositions.
ACKNOWLEDGEMENTS
This work was supported in part by INTAS no. 01–2040 (Brussels,
Belgium) and the Russian Foundation for Basic Research (Project no.
01-04-48769).
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