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 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; 3 Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, Russia; 4 Belarussian Research Institute for Potato Growing, Samokhvalovitchi, Minsk Region, Belarus; 5 DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany Structures of cell wall anionic polymers of the strain Strep- tomyces sp. 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 repeating unit fi6)-a- D -Glcp-(1fi4)-b- D -ManpNAc3NAcA-(1fi,ab-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 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- D -galacto-non-2-ulopyranosonic acid (Kdn), along with small amount of glycerol teichoic acid, has been found in the cell wall of the plant pathogen Streptomyces sp. 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 25497 T based on the analysis of 16S rRNA gene sequence. MATERIALS AND METHODS The strain VKM Ac-2124 was isolated from common scab lesions of potatoes, Solanum tuberosum, cultivar ÔIzoraÕ (Leningrad region, Russia) on ISP2 agar [6] as reported by Loria & Davis [7]. For studying phenotypical characteris- tics, the methods and media described by Schirling and Gottlieb [6] were used. Extraction and purification of DNA was carried out as reported [8]. The 16S rRNA gene was amplified by PCR using prokaryotic 16S rDNA universal primers 27f (5¢-AGAGTTTGATCCTGGCTCAG-3¢)and 1522r (5¢-AAGGAGGTGATCCARCCGCA-3¢) and puri- fied as described [8]. 16S rDNA was sequenced using a Big Dye Terminator Kit (Perkin Elmer) with an a model ABI- 310 automatic DNA Sequencer (Perkin Elmer) according to 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: 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 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 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 22–24 °C in the darkness. To obtain cell wall, the culture was grown on a peptone/ yeast medium [15] on a shaker at 28 °C and harvested by centrifugation in the middle of the exponential growth phase (24–30 h). The cells were washed with 0.95% (v/v) NaClandstoredfrozenat)20 °C before use. The native cell walls were obtained from crude mycelium by fractional centrifugation after preliminary disruption by sonication, and purified using 2% (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 2–4 °Cfor24h each time; with constant stirring. The extracts were separ- ated from cell debris, combined, dialyzed against distilled water and freeze-dried. Descending chromatography 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) 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 4 as the external standard for 31 PNMR.Pre- saturation of the HDO signal (1 s) was used in the accumu- lation of the 1 H NMR spectra. Two-dimensional spectra were obtained using standard pulse sequences from the Bruker software. Mixing times 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 in 1 H, 13 CHMBCand 1 H, 31 PHMQCexperiments. RESULTS AND DISCUSSION 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 25497 T (D63872) and S. caviscabies ATCC 51928 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- 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 containing glucose 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 13 C 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 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 7.9 Hz) and 4.66 (J 1,2 7.9 Hz) (Table 2). Two signals at d1.93 and 2.07 were observed in the region of resonances of the CH 3 CO– groups. The presence of 3-deoxynonulosonic acid with the b-configuration of the glycosidic bond followed from two doublets of doublets at d2.20 ( 2 J 3,3¢ 13.0 Hz; 3 J 3,4 4.9 Hz) and 1.78 (J 3¢,4 12.4 Hz). The 1D NMR spectra could be interpreted from the analysis of 2D homonuclear 1 H, 1 HCOSY,TOCSYand ROESY spectra and 2D heteronuclear 1 H, 13 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- D -Glcp-(1fi (A) 100.2 72.5 73.9 70.05 72.3 69.4 fi4)-b- D -ManpNAc3NAcA-(1fi (B) 101.2 52.45 54.05 73.2 78.8 175.1 Polymer II (C) 176.0 97.6 40.4 70.35 71.6 72.6 68.0 79.4 61.9 (D) 102.8 74.6 77.0 71.0 77.1 61.9 Polymer III (E) 68.0 71.8 72.5 80.4 65.8 (F) 103.6 74.6 77.0 70.9 77.1 62.0 * 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 Stre ptomyces sp. VKM Ac-2124. The signal at d97.6 is marked with an arrow. 6022 A. S. Shashkov et al. (Eur. J. Biochem. 269) Ó FEBS 2002 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 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)/ 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)at4.90/ 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 repeating unit was identified with that found earlier in the Streptomyces sp. VKM Ac-2090 cell wall [3] based on the coincidence of the 1 Hand 13 C 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)andH-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)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 C, and 31 P 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), and the ribitol teichoic acid (polymer III) from cell wall of Streptomyces sp. VKM Ac-2124. Carbon Residue H-1 H-1¢ H-2 H-3 H-3 ax H-3 eq H-4 H-5 H-5¢ H-6 H-6¢ H-7 H-8 H-9 H-9¢ Polymer I fi6)-a- D -Glcp-(1fi (A) 5.07 3.36 3.58 3.40 3.69 3.98 3.88 fi4)-b- D -ManpNAc3NAcA-(1fi (B) 4.90 4.40 4.33 3.93 4.03 Polymer II (C) 1.78 2.20 3.95 3.55 4.01 4.08 3.96 3.93 3.83 (D) 4.55 3.31 3.50 3.38 3.39 3.80 3.66 Polymer III (E) 4.09 3.97 3.96 3.81 4.18 4.17 4.09 (F) 4.66 3.32 3.50 3.40 3.43 3.90 3.72 * or 2); 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 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, along with teichuronic and teichoic acids is a constituent of the cell wall of plant pathogenic strain Streptomyces sp. VKM Ac-2124, which is phylogenetically the closest to S. setonii and S. caviscabies. As mentioned above, the Kdn-containing polymer 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-containing structures 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 causing scab diseases of potatoes and root crops. 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