Báo cáo Y học: A polymer with a backbone of 3-deoxy-D-glycero -D-galacto -non-2ulopyranosonic acid, a teichuronic acid, and a b-glucosylated ribitol teichoic acid in the cell wall of plant pathogenic Streptomyces sp. VKM Ac-2124 pdf

The strain contains three anionic glycopolymers, viz., a teichuronic acid with a disaccharide repeatingunitfi6-a-D -Glcp-1fi4-b-D-ManpNAc3NAcA-1fi, a b-glucosylated polymer of 3-deoxy-D-gly

A polymer with a backbone of 3-deoxy- D - glycero - D - galacto -non-2-ulopyranosonic acid, a teichuronic acid, and a b-glucosylated ribitol

VKM Ac-2124

Alexander S Shashkov1, Larisa N Kosmachevskaya2, Galina M Streshinskaya2, Lyudmila I Evtushenko3, Olga V Bueva3, Viktor A Denisenko4, Irina B Naumova2and Erko Stackebrandt5

1 N.D Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia; 2 School of Biology,

M.V Lomonosov Moscow State University, Moscow, Russia;3Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, Russia;4Belarussian Research Institute for Potato Growing,

Samokhvalovitchi, Minsk Region, Belarus;5DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,

Braunschweig, Germany

Structures of cell wall anionic polymers of the strain

Strep-tomycessp VKM Ac-2124, a causative agent of potato scab,

which is phylogenetically the closest to plant pathogenic

species S setonii and S caviscabies, were studied The strain

contains three anionic glycopolymers, viz., a teichuronic acid

with a disaccharide repeatingunitfi6)-a-D

-Glcp-(1fi4)-b-D-ManpNAc3NAcA-(1fi, a b-glucosylated polymer of

3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid (Kdn),

and a b-glucosylated 1,5-poly(ribitol phosphate) The strain

studied is the second representative of plant pathogenic streptomycetes inducingpotato 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

repeatingunit fi4)-b-D-ManpNAc3NAcA-(1fi3)-a-D

-GalpNAc-(1fi was identified in the cell wall of

Streptom-yces lavendulocolorVKM Ac-215T[2] Recently, a polymer

of 3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid

(Kdn), alongwith small amount of glycerol teichoic acid,

has been found in the cell wall of the plant pathogen

Streptomycessp VKM Ac-2090 [3] This nine-carbon sugar,

which may be regarded as a modification of sialic acid, is

abundant in animal tissues [4] and, presumably, plays a role

in intercell interactions [5]

In the present work, we investigated cell wall polymers

of yet another representative of streptomycetes, viz., of the strain VKM Ac-2124, a causative agent of potato scab, which is the closest to Streptomyces setonii ATCC

25497T based on the analysis of 16S rRNA gene sequence

M A T E R I A L S A N D M E T H O D S

The strain VKM Ac-2124 was isolated from common scab lesions of potatoes, Solanum tuberosum, cultivar
Izora (Leningrad region, Russia) on ISP2 agar [6] as reported by Loria & Davis [7] For studyingphenotypical characteris-tics, the methods and media described by Schirlingand Gottlieb [6] were used Extraction and purification of DNA was carried out as reported [8] The 16S rRNA gene was amplified by PCR usingprokaryotic 16S rDNA universal primers 27f (5¢-AGAGTTTGATCCTGGCTCAG-3¢) and 1522r (5¢-AAGGAGGTGATCCARCCGCA-3¢) and puri-fied as described [8] 16S rDNA was sequenced usinga Big Dye Terminator Kit (Perkin Elmer) with an a model

ABI-310 automatic DNA Sequencer (Perkin Elmer) accordingto the manufacturer’s protocol The sequences of the highest scores were chosen from NCIB database usingBLASTsearch [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 linensDSM 20425T(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: naumova@microbiol.bio.msu.su

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

accordingto the earlier nomenclature [9].

(Received 5 July 2002, revised 11 September 2002,

accepted 20 September 2002)

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

the same software

To evaluate the pathogenic activity of the strain, the

aseptically cultured potato microtubers in vitro were used as

described by Lawrence et al [14] The microtubers were

immersed for 5–10 min in a suspension of 14-day-old agar

culture (mainly, spore mass) grown on Czapek’s agar [6]

followed by incubation at 100% relative humidity for

5 days at 22–24C in the darkness

To obtain cell wall, the culture was grown on a peptone/

yeast medium [15] on a shaker at 28C and harvested by

centrifugation in the middle of the exponential growth

phase (24–30 h) The cells were washed with 0.95% (v/v)

NaCl and stored frozen at)20 C before use The native cell

walls were obtained from crude mycelium by fractional

centrifugation after preliminary disruption by sonication,

and purified using2% (w/v) SDS to avoid possible

contamination with membrane compounds, including

lipoteichoic acids, washed several times with water, and

freeze-dried To isolate polymers, cell walls were extracted

twice with 10% (v/v) trichloroacetic acid at 2–4C for 24 h

each time; with constant stirring The extracts were

separ-ated from cell debris, combined, dialyzed against distilled

water and freeze-dried

Descendingchromatography and electrophoresis were

performed on Filtrak FN-13 paper Electrophoresis was

performed in pyridinium acetate buffer (pH 5.6) to separate

phosphate esters and to purify ribitol teichoic acid

(20 VÆcm)1, 5 h) Paper chromatography was performed in

a pyridine-benzene-butanol-water (3 : 1 : 5 : 3, v/v/v/v)

sol-vent system to separate ribitol and glucose Phosphoric esters

were detected with the molybdate reagent, reducing sugars,

with aniline hydrogenphthalate; and ribitol and

monosac-charides, with 5% (w/v) AgNO3in aqueous ammonia

Acid hydrolysis was carried out with 2MHCl for 3 h at

100C; alkaline hydrolysis was performed with 1MNaOH

for 3 h at 100C; 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 D2O at 30C

with acetone (dH2.225 dC231.45) as the internal standard,

and 80% H3PO4as the external standard for31P NMR

Pre-saturation of the HDO signal (1 s) was used in the

accumu-lation of the1H NMR spectra Two-dimensional spectra

were obtained usingstandard pulse sequences from the

Bruker software Mixingtimes of 100 and 200 ms were used

in TOCSY and ROESY experiments, respectively A 60-ms

delay was used for the evolution of long-range connectivities

in1H,13C HMBC and1H,31P HMQC experiments

R E S U L T S A N D D I S C U S S I O N

To identify the strain VKM Ac-2124 isolated from common

potato scab, an almost complete 16S rRNA gene sequence

(1470 nucleotides) was determined Phylogenetic analysis

indicated it to be the closest (99.6% 16S rDNA binary

sequence similarity) to S setonii ACTT 25497T (D63872) and S caviscabies ATCC 51928T (AF112160), which are also causative agents of potato scab These three strains and

S griseusISP 5236T(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 Testingof plant pathogenicity of the strain VKM Ac-2124 showed that it induced rough, corky lesions such as those resultingfrom 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-positive compound was also detected, which migrated to the cathode in the electrophoresis, but was absent from phosphates produced upon alkaline hydrolysis

Ribitol mono- and bisphosphates were subjected to the action of phosphomonoesterase; these were identified based

on the ribitol/phosphate ratio Glucosylribitol phosphate was identified based on its electrophoretic mobility (pyridi-nium acetate buffer) in comparison with an analogous ester obtained upon alkaline hydrolysis of glucosylated ribitol teichoic acid from the cell wall of S azureus RIA 1009 [24] and based on the analysis of the products formed upon acid and enzymatic hydrolysis Acid hydrolysis afforded glucose and ribitol monophosphate, while a glycoside containingglucose and ribitol (1 : 1 molar ratio) was produced under the action of phosphomonoesterase Low content of teichoic acid-linked phosphorus (0.8%) in the cell wall as well as high percentage of glucose and the presence

of an unidentified ninhydrin-positive component suggest that the cell wall contains other polymer(s) in addition to ribitol teichoic acid

The polymers present in the cell wall were investigated using NMR spectroscopy The 13C NMR spectrum of the preparation revealed the presence, in the region typical of anomeric carbon atoms

of carbohydrates, of five signals of unequal intensities at d103.6, 102.8, 101.2, 100.2, and 97.6 (Table 1) As followed from the APT spectrum (Fig 1), four signals

at d100.2–103.6 belonged to the protonated anomeric 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

CH2-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

(CH3CON), and three signals for CO groups at d174.5–

176.0 The resonances for the CH2O 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

1H NMR spectrum (Fig 1) contained two abundant signals

at d4.90 (J1,2< 2 Hz) and 5.07 (J1,23.6 Hz) and two signals

of lower intensities at d4.55 (J1,2 7.9 Hz) and 4.66 (J1,2

7.9 Hz) (Table 2) Two signals at d1.93 and 2.07 were

observed in the region of resonances of the CH3CO– groups

The presence of 3-deoxynonulosonic acid with the

b-configuration of the glycosidic bond followed from two

doublets of doublets at d2.20 (2J3,3¢13.0 Hz;3J3,44.9 Hz) and 1.78 (J3¢,412.4 Hz)

The 1D NMR spectra could be interpreted from the analysis of 2D homonuclear1H,1H COSY, TOCSY and ROESY spectra and 2D heteronuclear 1H, 13C HSQC (Fig 1) and HMBC and 1H, 31P 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 repeatingunit 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 C NMR chemical shifts (d, p.p.m) for the teichuronic acid(polymer I), the Kdn-containing polymer (polymer II), andthe ribitol teichoic acid (polymer III) from cell wall of Streptomyces sp VKM Ac-2124.

Carbon

Polymer I

Polymer II

Polymer III

* or 2);

CH 3 CON, d 23.3; CH 3 CON, d 174.8 and 174.

Fig 1 Part of the HSQC spectrum of anionic cell wall polymers from Streptomyces sp VKM Ac-2124 The signal at d97.6 is marked with an arrow.

chromatography [25] The absolute configuration of

ManpNAc3NAcA(D-) in the polymer I was inferred from

the glycosylation effect on C-3 of this

manno-monosacchar-ide The small absolute magnitude of the b-effect

(< 0.5 p.p.m) suggests identical absolute configurations

of the glycosylating sugar (glucose) and the 4-substituted

ManpNAc3NAcA residue [26,27] The signals for a-D-Glcp

and b-D-ManpNAc3NAcA were identified in the1H COSY

and TOCSY spectra The anomeric configuration of the

Glcp residue was a, followed from the couplingconstant

value (3JH-1,H-2¼ 3 Hz) The b-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 correspondingto the carboxy group

The interresidue cross-peak H-1(B)/H-6(A) and H-1(B)/

H-6¢(A) in the ROESY spectrum at 4.90/3.98 and

3.88 p.p.m and the correlation H-1(B)/C-6(A) at 4.90/

69.40 p.p.m in the HMBC spectrum suggest that the

b-D-ManNAc3NAcA residue is 1fi6-linked to the a-D

-Glcp residue In turn, that the b-D-ManNAc3NAcA

residue is substituted at position 4 with the a-D-Glcp

residue, followed from the presence of the correlation

peaks H-1(A)/H-4(B) at 5.07/3.93 p.p.m in the ROESY

spectrum and H-4(B)/C-1(A) at 3.93/100.20 p.p.m in the

HMBC spectrum

Two other polymers were present in nearly equal

amounts One of them was shown to be a Kdn-containing

polymer (polymer II) The structure of its repeatingunit was

identified with that found earlier in the Streptomyces sp

VKM Ac-2090 cell wall [3] based on the coincidence of the

1H and 13C chemical shifts in the NMR spectra of both these polymers This was confirmed additionally by the observation of the correlation peaks H-1(D)/H-8(C) and H-1(D)/H-9(C) and H-1(D)/H-9¢(C) at 4.55/3.96 p.p.m 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) at 4.55/ 79.40 p.p.m in the HMBC spectrum The downfied shift

of the C-4 resonance of the b-Kdn residue in the13C NMR 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, which was concluded in particular from the couplingconstant value (3JH-1, H-2¼ 8 Hz)

Thus, the polymer II has the followingrepeatingunit:

The signals of the terminal monosaccharide residues were not detected This fact allows one to suggest that the polymer contains no less than 20 repeatingunits

The third cell wall polymer was identified as 1,5-poly(ribitol phosphate) partially substituted with b-glucose (3JH-1, H-2¼ 8 Hz) at position 4(2) (polymer III) based on

1H,13C, and31P NMR spectroscopic data The structure of this polymer followed from the coincidence of the chemical shifts in the respective NMR spectra with those in the spectra of glucosylated ribitol teichoic acid from

Table 2 1 H NMR chemical shifts (d, p.p.m) for the teichuronic acid(polymer I), the Kdn-containing polymer (polymer II), andthe ribitol teichoic acid (polymer III) from cell wall of Streptomyces sp VKM Ac-2124.

Carbon

Polymer I

Polymer II

Polymer III

* or 2);

CH 3 CON, d1.93 and 2.07.

Streptomyces azureusRIA 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 repeatingunit fi6)-a-D-Glcp-(1fi4)-b-D

-ManpNAc3NAcA-(1fi, (I) the b-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 takinginto 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 the1H 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

10 % of the mass of the cell wall, respectively The three

polymers altogether constitute 50 % of the mass of dry

cell wall

Thus, the present study shows that the Kdn-containing

polymer, alongwith teichuronic and teichoic acids is a

constituent of the cell wall of plant pathogenic strain

Streptomycessp VKM Ac-2124, which is phylogenetically

the closest to S setonii and S caviscabies As mentioned

above, the Kdn-containingpolymer was also revealed in the

cell wall of a streptomycete strain isolated from common

scab lesions of the potatoes [3], which induced scab disease

in potato tubers, while such polymers have been never

reported in other numerous Streptomyces spp [29]

It is known, that the virulence of Gram-negative

bac-teria is often correlated with the structures of surface

polysaccharides [30] An acidic polysaccharide containing

3-deoxy-D-manno-octulosonic acid (formerly,

2-keto-3-deoxy-octonic acid, Kdo), belonging to the same family of

higher 3-deoxyulosonic acids to which Kdn belongs too,

from the plant pathogen Agrobacterium tumefaciens has

been shown to be involved in the attachment of the

microorganism to carrot (host) cells, this being an early step

in crown gall tumor formation [31] A lipopolysaccharide

from Pseudomonas corrugata, a plant pathogenic bacterium,

contains 5,7-diamino-5,7,9-trideoxynon-2-ulosonic acid

[32], yet another derivative of sialic acid Probably, the

localization of Kdn-containingstructures in the

near-surface regions of actinomycete hyphae is essential for their

growth taxis and their attachment to potato tuber The

presence of Kdn might be characteristic of plant pathogenic

streptomycete strains causingscab 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

A C K N O W L E D G E M E N T S

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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