Carbohydrate Research 359 (2012) 70–75

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Carbohydrate Research

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Novel teichulosonic acid from cell wall of Streptomyces coelicolor M145

Alexander S. Shashkov a, Bohdan E. Ostash b, Victor A. Fedorenko b, Galina M. Streshinskaya c,⇑,Elena M. Tul’skaya c, Sof’ya N. Senchenkova a, Lidiya M. Baryshnikova d, Ludmila I. Evtushenko d

a N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospect, 47, Moscow 119991, Russian Federationb Ivan Franko National University of Lviv, Department of Genetics and Biotechnology, Lviv 79005, Ukrainec School of Biology, M. V. Lomonosov Moscow State University, Moscow 119991, Russian Federationd All-Russian Collection of Microorganisms (VKM), G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms,Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russian Federation

a r t i c l e i n f o

Article history:Received 19 April 2012Received in revised form 14 May 2012Accepted 15 May 2012Available online 23 May 2012

Keywords:StreptomycesCell wallTeichulosonic acidGlycosyl 1-phosphate polymerNMR spectroscopy

0008-6215/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.carres.2012.05.018

⇑ Corresponding author. Tel.: +7 495 9395601; fax:E-mail address: streshinskaya@mail.ru (G.M. Stres

a b s t r a c t

The cell wall of Streptomyces coelicolor M145, a prototrophic plasmidless (SCP1� SCP2�) variant of strainS. coelicolor A3(2) contains the main glycopolymer represented by Kdn-containing teichulosonic acid withunusual structure which has not been described so far:

The minor polymer was found to be a poly(diglycosyl 1-phosphate) with the following repeating unit:-6)-a-Galp-(1?6)-a-GlcpNAc-(1-P-. The structures of both glycopolymers were established by using acombination of chemical and NMR spectroscopic methods.

� 2012 Elsevier Ltd. All rights reserved.

Members of the genus Streptomyces (streptomycetes) are the microbial biology,2,10 such as cell division, hyphal growth11, and

Gram-positive bacteria with high mol % G + C of the DNA, whichproduce branching vegetative and aerial hyphae bearing longchains of reproductive spores.1 Streptomycetes are the most abun-dant natural source of various bioactive secondary metabolites andare therefore of great interest for medicine and industry. Represen-tatives of the genus Streptomyces are widely used in genetic re-search related to antibiotic production, bacterial physiology, andcell differentiation.2

Our previous studies showed a large diversity of anionic poly-mers in the cell wall of Streptomycetes species, and numerous novelstructures of teichoic and teichuronic acids were revealed.3–8 Inaddition, a natural Kdn-polymer (teichulosonic acid) was discov-ered for the first time.9 In the present work, we report an unusualheteropolymer that is a Kdn-containing teichulosonic acid, alongwith a poly(diglycosyl 1-phosphate), found in the cell wall ofS. coelicolor M145, a prototrophic plasmidless (SCP1� SCP2�) deriv-ative of strain A3(2). M145 is genetically the best known actinomy-cete strain and considered a model organism for various aspects of

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+7 495 9394309.hinskaya).

antibiotic resistance.12 In this respect, our results provide new de-tails on the chemical composition of M145 cell wall and thusbroaden an understanding of this model microbe.

Stepwise extraction of the freeze-dried cell wall with 10% tri-chloroacetic acid (4 �C, 3 � 24 h each) resulted in preparations thatwere used in structural studies of the anionic glycopolymers. Theyield was ca 15% of the cell wall dry mass. The freeze-dried prep-arations were severally used in composition and NMR analyses.Their similarity allowed us to combine them for the further chem-ical and NMR spectroscopic analyses.

The compositions of acid hydrolysates (2 M HCl, 100 �C, 3 h) ofthe preparations obtained and the cell wall itself were studied bypaper electrophoresis and paper chromatography, they were foundto be qualitatively identical.

Hydrolysis afforded the following products: inorganic phos-phate, two phosphorous esters with the mobility mGrop 0.21 and0.45 (electrophoresis) and monosaccharides: galactose and aminosugar (chromatography).

The results suggested the presence of phosphate-containingpolymers. Judging by the number of identified phosphate esterstheir content in the cell wall of S. coelicolor M145 was small.

Figure 1. 13C NMR spectrum of preparation from cell wall of Streptomyces coelicolor M145. Arabic numerals refer to the atoms in the sugar residues as designated in Table 1.

Table 113C and 1H NMR data of the cell wall polymers of Streptomyces coelicolor M145

Polymer C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9Residue H-1 H-2 H-3 (H-3e,3a) H-4 (H-4a,4b) H-5 H-6 (H-6a,6b) H-7 H-8 H-9

Polymer I?6)-b-D-Galp-(1? (GI) 104.7 72.1 73.8 69.9 74.6 64.7

4.45 3.56 3.65 3.93 3.77 3.93, 3.53?9)-a-Kdn-(2? (Ka) 174.8 101.6 40.8 71.5 70.9 74.7 68.9 70.1 72.1

2.68, 1.68 3.57 3.57 3.69 3.98 4.05 4.25, 3.85?9)-a-Kdn-(2? (Ka,GN) 174.5 101.5 37.8 76.3 69.1 74.7 68.9 70.1 72.14) 2.72, 1.63 3.70 3.69 3.69 3.98 4.05 4.25, 3.85"a-D-GlcpNAc-(1 (GNI) 95.5 54.8a 72.1 71.1 73.0 61.6

5.03 3.93 3.80 3.53 3.93 3.81, 3.81?9)-a-Kdn-4-OMe-(2? (Ka,Me) 174.6 101.6 37.6 80.9b 69.8 74.7 68.9 70.1 72.1

2.86, 1.55 3.33 3.63 3.69 3.98 4.05 4.25, 3.85

Oligomers from polymer Ib-D-Galp-(1? (Gt) 104.6 72.1 73.9 69.8 76.3 62.2

4.43 3.56 3.66 3.92 3.69 3.78, 3.75?9)-b-Kdn-(2-OH (Kb) 174.2 97.2 40.0 70.0 71.4 72.9 68.8 70.5 72.9

2.23, 1.80 4.00 3.59 3.99 4.02 3.91 4.15, 3.84?9)-b-Kdn-(2-OH 176.1 97.2 36.9 75.7 69.3 72.8 68.8 70.5 72.94) (Kb,GN) 2.27, 1.75 3.98 3.72 3.99 4.02 3.91 4.15, 3.84"a-D-GlcpNAc-(1 (GNI) 95.6 54.8a 72.1 71.0 73.0 61.6

5.03 3.92 3.77 3.53 3.95 3.83, 3.83?9)-b-Kdn-4-OMe-(2-OH (Kb,Me) 176.1 97.4 36.8 79.6b 69.9 73.0 68.8 70.5 72.9

2.43, 1.68 3.71 3.66 3.99 4.02 3.91 4.15, 3.84

Polymer II-6)-a-Galp-(1? (GII) 99.4 69.6 70.6 69.9 70.8 65.9

5.00 3.83 3.92 4.11 4.12 4.02, 3.97?6)-a-GlcpNAcb-(1-P- (GNII) 95.3 54.8 72.2 70.4 72.8 66.4

5.47 3.98 3.75 3.71 4.03 4.12, 3.70

a CH3CON at dC 23.4 and dC 175.8–176.1.b OMe at dC 57.8 and dH 3.45.

A. S. Shashkov et al. / Carbohydrate Research 359 (2012) 70–75 71

The structures of the cell wall polymers of S. coelicolor M145were established by NMR spectroscopic methods.

The 13C NMR spectrum of the preparation (Fig. 1, Table 1) wastypical of a non-regular polymer or a mixture of polymers. It con-

tains inter alia signals of different integral intensities in the ano-meric carbon resonance region at d 92.2–104.7, including signalsfor quaternary carbons at d 97.2–97.4 and 101.5–101.6 (data ofthe APT spectrum, not shown).

Figure 3. 1H/31P HMBC spectrum of preparation from cell wall of Streptomyce coelicolor M145. Arabic numerals refer to the protons in the sugar residues as designated inTable 1.

Figure 2. DOSY spectrum of preparation from cell wall of Streptomyces coelicolor M145.

72 A. S. Shashkov et al. / Carbohydrate Research 359 (2012) 70–75

The spectrum also showed signals for C–CH2–C groups at d37.6–40.8, nitrogen-bearing carbons at d 54.8, HO2C–C groups at

d 174.1–174.6, and N-acetyl groups (CH3 at dC 23.4 and CO at dC

175.8–176.1).

Figure 4. Parts of 1H/13C HMBC spectrum of preparation from cell wall of Streptomyces coelicolor M145. Arabic numerals before slash refer to the protons and after slash referto carbon in the sugar residues as designated in Table 1.

A. S. Shashkov et al. / Carbohydrate Research 359 (2012) 70–75 73

The 1H NMR spectrum of the preparation (Fig. 2, up) containedsignals for anomeric protons at d 5.47 (broadened multiplet), 5.03and 5.00 (both doublets, J1,2 �3 Hz), d 4.45 and 4.43 (both doublets,J1,2 �8 Hz), signals for a CH–CH2–CH group at d 2.23–2.86 (dd) and1.80–1.55 (t), and N-acetyl groups at d 2.02–2.06. The 1H DOSYspectrum (Fig. 2) revealed at list three compounds with differentdiffusion coefficients for their structural units: 1.4e�10, 1.9e�10,and 2.4e�10.

The 31P NMR spectrum of the preparation contained an intensesignal at d �1.4 and a minor signal at d 0.7 (Fig. 3, left).

The 1D NMR spectra of the preparation were assigned using 2D1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC, HSQC-TOCSY, HMBC, and1H,31P HMBC experiments, and spin-systems for a- and b-Galp, a-GlcpNAc, a- and b-Kdn were identified (Table 1). The absolute D-configuration of six-carbon sugars was determined by GLC follow-ing their conversion into acetylated (S)-2-octyl (a- and b-Galp) or(S)-2-butyl (a-GlcpNAc) and comparison with referent samples.13

The signals for C-6 of a-Galp and some residues of b-Galp, C-9 ofboth a- and b-Kdn, C-4 of some residues of a- and b-Kdn, and C-6 of some residues of GlcNAc were shifted downfield, as comparedwith their positions in the corresponding non-substituted mono-saccharides. Accordingly, the C-2–C-5 chemical shifts of theremaining parts of b-Gal and a-GlcpNAc were close to those ofthe corresponding non-substituted sugars, thus indicating theirterminal positions. The C-3 and C-4 chemical shifts of minor partsof a- and b-Kdn were characteristic of the residues devoid of anysubstituent at position 4. These data revealed the substitution pat-tern in the polymer.

The 1H,31P HMBC spectrum (Fig. 3) revealed the location of thephosphodiester group at position 1 of a-GlcpNAc and position 6 ofa-Galp. The 1H,13C HMBC spectrum (Fig. 4) indicated the (1?6)-gly-cosidic linkage between the a-Galp and a-GlcpNAc residues (corre-lation peaks dH-1/dC-6 5.00/66.4 and dH-6/dC-1 4.12 and 3.70/99.4).Therefore, one of the components in the preparation is a polymerwith the following disaccharide-phosphate repeating unit:

-6)-a-D-Galp-(1?6)-a-D-GlcpNAc-(1-P-

The polymer showed the smallest diffusion coefficient (1.4e�10)in the 1H DOSY spectrum and is designated on Figure 2 aspolymer II.

The two other components of the preparation with the diffusioncoefficients 1.9e�10 and 2.4e�10 contain both a- and b-Kdn. Analysisof the 1H,13C HMBC spectrum indicated that all a- and b-Kdn resi-dues are substituted at position 9 by b-Galp residues (correlationpeaks at dH-1/dC-9 4.43/72.9, 4.45/72.1, 4.15/104.6, 3.84/104.6,4.25, and 3.85/104.7). The chemical shift of C-2 of all b-Kdn residues(d 97.2) is characteristic of the monosaccharide at the reducing endof an oligosaccharide or a polysaccharide. In contrast, the C-2 chem-ical shifts of all a-Kdn residues (d 101.5 or 101.6) are typical oflinked pyranoses. The HMBC spectrum demonstrated the glycosidiclinkage between a-Kdn and O-6 of b-Galp (a correlation peak atdH-6/dC-2 3.93/101.6). In addition, most of Kdn residues aresubstituted at position 4 by either a-GlcpNAc (correlation peaksat dH-1/dC-4 5.03/76.3, 5.03/75.7 and dH-4/dC-1 3.70/95.6) or OMe(correlation peaks dH/dC-4 3.45/80.9 and 3.45/79.6). Based on the

74 A. S. Shashkov et al. / Carbohydrate Research 359 (2012) 70–75

data obtained, it was concluded that the Kdn-containing compo-nent of the preparation has the following structure:

Analysis of the DOSY spectrum (Fig. 2) and NMR data (Table 1)showed that the largest diffusion coefficient of 2.4e�10 refers to therepeating unit b-D-Galp-(1?9)[RO?4)]-b-Kdn-(2-OH and shortoligomers with n = 0, whereas the coefficient of 1.9e�10 is charac-teristic of more long oligomers or a polymer.

Thus, the cell wall of S. coelicolor M145 contains poly(diglycosyl1-phosphate) with following repeating unit: -6)-a-Galp-(1?6)-a-GlcpNAc-(1-P- as a minor component, whereas the main polymeris represented by Kdn containing teichulosonic acid with unusualstructure which has not been described so far.

The Kdn-containing glycoconjugates are widespread in animals(vertebrates and higher invertebrates) and in Gram-negative bac-teria.14,15 The Kdn-residues typically occupy the distal end of gly-can chains. This ‘outermost’ location makes them suitable forinteraction with other cells and with environmental agents.15

Kdn-containing teichulosonic acids having the chains consistingof 20 or more residues of this monosaccharide3 are characteristic ofrepresentatives of soil microflora belonging to some genera of theorder Actinomycetales (Streptomyces, Brevibacterium, Arthrobac-ter).16 Common structural feature of the polymers consists of hom-ogenic backbone built up the b-(2?4)-linked Kdn residues. TheKdn residues of the polymers usually substituted at position 8and/or 9 by sugar residues, which are different for the various spe-cies. Glucopyranose, galactopyranose, 3-O-methyl-galactopyra-nose, and 2-acetamido-2-deoxy-glucopyranose in b-anomericconfiguration were found as the branching sugars.16 The Kdn con-taining polymer of S. coelicolor M145 is fundamentally distin-guished from the earlier studied polymers by two structuralfeatures: (i) the backbone of S. coelicolor M145 polymer is hetero-genic and includes residue of neutral sugar b-D-Galp together withKdn; (ii) anomeric configuration of the Kdn residues is a-, but notb- in the backbone.

The cell wall of the studied S. coelicolor M-145 has a smallamount of poly(glycosyl 1-phosphate) polymer with the repeatingunit structure: -6)-a-Galp-(1?6)-a-GlcpNAc-(1-P-. The poly(gly-cosyl 1-phosphate) polymer of the same structure was found inthe thermophilic streptomycete.4

It should also be noted that the cell walls of actinomycetes stud-ied early comprise, in addition to teichulosonic acids, other glyco-polymers of different nature, viz. teichoic and teichuronic acids aswell as neutral polysaccharides.16

1. Experimental

1.1. Materials

S. coelicolor M145 (=ATCC BAA-471) is a prototrophic plasmid-less (SCP1� SCP2�) variant of strain S. coelicolor A3(2). The genomeof M145 has been completely sequenced in 2002.10 The M145strain was provided by Prof. M. Bibb from the Streptomyces strainscollection of John Innes Centre (UK).

The biomass of S. coelicolor M145 was accumulated by growingthe culture aerobically in a liquid peptone–yeast medium to themiddle of the exponential phase in shaking flasks at 28 �C as de-scribed by Naumova et al.17

The mycelium was harvested by centrifugation, washed with0.95% NaCl, and used for cell-wall preparation.

A native cell wall was obtained from crude mycelium by frac-tional centrifugation after preliminary disruption by sonication(UP100H, Hielscher, Germany, 30 kHz, (5–10) � 2 min in icewater), and purified using 2% sodium dodecyl sulfate to avoid pos-sible contamination with membrane components.

The glycopolymer preparations were isolated from cell wallswith 10% trichloroacetic acid at 2–4 �C by three successive extrac-tions for 24, 48, and 72 h. The extracts were separated from celldebris, combined, dialyzed against distilled water, and freeze-dried.6

1.2. Acid hydrolysis

Acid hydrolyses of the cell wall and the preparation of glyco-polymers were carried out with 2 M HCl at 100 �C for 3 h. Identifi-cation of neutral monosaccharides has been described elsewhere.6

Descending paper chromatography and electrophoresis were car-ried out on a Filtrak FN-3 paper (Germany) in a pyridine-ben-zene-butan-1-ol-water (3:1:5:3 v/v) solvent system and apyridinium acetate buffer, pH 5.6, respectively.6

The molybdate reagent was used for detection of phosphate-containing compounds; amino sugars were detected with ninhy-drin; monosaccharides were detected with 5% AgNO3 in aqueousammonia or aniline hydrogenphthalate reagent.6

1.3. Determination of the absolute configuration

The absolute configuration of six-carbon sugars was determinedby GLC following their conversion into acetylated (S)-2-octyl(a- and b-Galp) or (S)-2-butyl (a-GlcpNAc) and comparison withreferent samples.13

1.4. NMR spectroscopy

Spectra were recorded with a Bruker AV-600 spectrometer inD2O at 30 �C. Chemical shifts are reported related to TSP (dH 0.0,dC �1.6) and external 85% H3PO4 (dP 0.0). Standard pulse sequenceswere used for 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC, HMBC,and 1H,31P HMBC spectra. A mixing time was set to 100 ms in theTOCSY experiments. A spin-lock time of 150 ms was used in theROESY experiments. Both 1H,13C and 1H,31P 2D HMBC experimentswere optimized for the coupling constants 8 Hz.

DOSY experiment was performed using stebpgp pulse sequencefrom the Bruker library. The following parameters were appliedduring acquisition of the spectra: Z-gradient from 2% to 95% at512 steps, D = 0.1s, d = 0.01s.

Acknowledgments

This work was supported by the programs of Presidium of Rus-sian Academy of Sciences ‘Molecular and Cell Biology’, the Ministryof Education and Science of the Russian Federation (contract no.16.518.11.7035), and by grant Bg-98f of the Ministry of Educationand Science, Youth and Sports of Ukraine.

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