Разнообразие гликополимеров клеточных стенок актинобактерий (стр. 3 )

S. albus J1074 is an auxotrophic mutant derived from S. albus G defective in SalIG1 restriction-modification system and has a valin-isoleucine auxotrofic phenotype (Chater and Wilde, 1976). This strain is characterized by a very fast and dispersed growth, absence of restriction barriers, simplicity of genetic manipulations and high sensitivity to moenomicin A. The genome of S. albus J1074 has been completely sequenced in 2014 (Zaburannyi et. al. 2014). S. albus R1-100 is a spontaneous moenomicin A resistant variant of S. albus J1074. Both strains are maintained in the collection of microorganisms of Ivan Franko National University of Lviv (http://lv-microbcollect. ).

The biomass of the mention above streptomycetes were accumulated by growing the cultures aerobically in a liquid peptone-yeast medium to the middle of the exponential phase in shaking flasks at 28 °C as described early (Potekhina et al. 2011). The mycelium was harvested by centrifugation, washed with 0.95% NaCl, stored at -18 °C and used for preparation the cell wall.

Cell walls preparation and the glycopolymers extraction

The native cell walls were obtained from crude mycelium by fractional centrifugation after preliminary disruption by sonication in ice water (UP 100H, Hielscher, Germany, 30 kHz) and purified using 2% sodium dodecyl sulfate to avoid possible contamination with membrane components, including lipoteichoic acids, washed several times with water, and freeze-dried.

Glycopolymer preparations were isolated from the cell walls by different extraction methods to obtain preparations enriched in one or another polymer: i) the glycopolymer preparation (Preparation 1) were isolated from cell walls with 10% trichloroacetic acid at 2–4 °C by three successive extractions for 24, 48, and 72 h; the extracts were separated from cell debris, combined, dialyzed against distilled water, and freeze dried; ii) the glycopolymer preparation (Preparation 2) were isolated from cell walls with 0.05 M NaOH-glycine buffer (pH 8.2 – 8.8) at 2–4 °C by two successive extractions for 24 h; the extracts were separated from cell debris, combined, dialyzed against distilled water, and freeze dried.

Primary structural determination and analytical procedures

Acid hydrolysis of the cell walls and the preparation of glycopolymers, dephosphorylation of the preparation 1, determination of the glycopolymers phosphorus, primary structural determination and other analytical procedures have been described previously (Potekhina et al. 2011). Ammonolysis of TA was carried out as described earlier (Streshinskaya et al. 1981).

Chromatography and electrophoresis

Descending paper chromatography and electrophoresis were carried out on Filtrak FN-3 paper (Germany) with using different solvent systems. The molybdate reagent was used for detection of phosphate-containing compounds and zones of native glycopolymers; ninhydrin - for amino sugar, lysine and its amide; 5% AgNO3 in aqueous ammonia - for polyols, monosaccharides and glycoside, and aniline hydrogen phthalate reagent - for reducing sugars. All procedures were carried out as described in (Potekhina et al. 2011).

Determination of the absolute configuration

The absolute configuration of six-carbon sugars were determined by GLC following their conversion into acetylated (S)-octan-2-yl (α- and β-Galp) or (S)-butan-2-yl (α-GlcpNAc) and comparison with referent samples (Gerwig et al, 1979); and of lysine as described (Shashkov et al. 2006).

NMR spectroscopy

Spectra were recorded with a Bruker AV-600 spectrometer in D2O at 30 oC. Chemical shifts are reported related to TSP (δH 0.0, δC -1.6) and external 85% H3PO4 (δP 0.0). Standard pulse sequences were 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 the TOCSY experiments. A spin-lock time of 150 ms was used in the ROESY experiments. Both 1H,13C and 1H,31P 2D HMBC experiments were optimized for the coupling constants 8 Hz.

ESI-MS

High resolution mass spectra (HR MS) were measured on a Bruker microTOF II instrument using electrospray ionization. The measurements were done in a positive ion mode (interface capillary voltage was 4500 V) or in a negative ion mode (3200 V); mass range from m/z 50 to m/z 3000 Da; internal and external calibration was done with Electrospray Calibrant Solution (Fluka). M / z values of the most intense experimental peaks of the isotopic clusters are shown. A syringe injection was used for solutions MeCN-H2O (1:1) (flow rate 3 µL/min). Nitrogen was applied as a dry gas; interface temperature was set at 180 ºC.

Results

The structures of glycopolymers were established by using a combination of chemical, NMR spectroscopic and ESI-MS methods. The Kdn-TULA were found in the cell walls of three streptomycetes under study. Stepwise extraction of the freeze-dried cell walls with 10% trichloroacetic acid (4 °С, 3 ´ 24 h each) resulted in TCA-preparation (preparation 1) which was used in structural studies of the anionic glycopolymers. However, Kdn-TULA were highly unstable in acidic medium furthermore the polymers are destroyed to the repeating units thereof in the process of separation, during chromatography and even for a long-term recording of the NMR spectra. Extraction of the polymers under alkaline conditions yielded a preparation enriched of Kdn-TULA (preparation 2).

The absolute configuration of six carbon sugars were D, and of lysine was L.

Streptomyces albus VKM Ac-35T

The cell wall of S. albus Ac-35T contained 1.2% of phosphate-containing polymers phosphorus. The yield of preparation 1 was ca. 7.7% of the cell walls dry mass.

The compositions of acid hydrolysates (2 M HCl, 100 °С, 3 h) of the obtained preparation 1 and the cell wall itself were found to be qualitatively identical. Hydrolysis afforded the following products: inorganic phosphate, minor amount of glycerol, its mono- and bisphosphates, ribitol, and its mono - and bisphosphates, anhydroribitol phosphate, and glucose.

Dephosphorylation (HF, 4 °С, 24 h) of the preparation 1 yielded glycerol, ribitol, and a glycoside with mobility RGlc 1.07 (chromatography on paper). The last stained with AgNO3 and not stained with aniline phthalate, and under hydrolysis the equimolar proportion of glucose and glycerol (Glc:Gro ~ 1:1) were found. So, the glycoside represented glucosyl-(1®2)-glycerol (Tul’skaya et al. 1993).

The electrophoretic study of native preparation 1 led to the formation of two zones, stained in different ways with the molybdate reagent and of different mobilities: mGroP 1.2 – blue; mGroP 0.76 – gray, that suggested the presence of several polymers in the cell wall of the streptomycete under study, among which were presumably teichoic and teichulosonic acids (Tulskaya et al. 2007b). All chemical studies of the preparation 2 (the yield was ca 8.8% of the cell walls dry mass) led to the similar results.

The preparations 1 and 2 have been studied by NMR spectroscopy. 1H, 13C and 31P NMR spectra of both preparations were recorded. Signals in one-dimensional NMR spectra were assigned using two-dimensional techniques 1H/1H COSY, TOCSY, ROESY, 1H/13C HSQC, HMBC and 1H/31P HMBC.

Analysis of the spectra revealed the presence in the preparations 1 two TA. The 31P NMR spectrum contained several broad signals of the phosphate groups, the most intense of them were at δР +1.1 and -0.3 (Fig. 1, Table 1). Thus, the preparation 1 contained 1,5-poly(ribitol phosphate) and 1,3-poly(glycerol phosphate) with the β-glucopyranose (β-Glcp) residues at O-2 of the most of glycerol residues. All characteristic signals of similar polymers which have been described in our earlier work (Streshinskaya et al., 1995; Tul’skaya et al., 1993; Potekhina et al., 2003) were found in the NMR spectra of preparations 1. Intense signals of the terminal residues indicated short chains for both polymers.

As noted above the Kdn-TULA became labile during extraction from the cell wall (preparation 1), chromatographic purification and even during recording of the NMR spectra. In all cases the disaccharide β-Glcp-(1→8)-β-Kdn was found as the final degradation product (Fig. 2, Formula 1) with a molecular mass of M-H 411.1145 (calculated mass 411.1144 for ESI-Mass Spectra data).

Since the Kdn-TULA in the preparation 1 was degraded, we present the NMR spectroscopic data on the preparation 2, containing oligosaccharide fractions of Kdn-TULA where dominated α-Kdn. The preparation 2 contained also some amount of TA (Fig. 3).

The structure of the initial Kdn-polymer was deducted from the analysis of sub-spectra relating to the oligomer containing the α-Kdn residues. High field region of 1H and 13C NMR spectra (Fig. 3, top; Table 1) showed signals that are characteristic for H-3 and C-3 of Kdn with α- (δС 41.0; δН 1.67 and 2.62) and β- (δС 40.4; δН 1.79 and 2.18) glycoside center configuration. The ratio of H-3 α- and β-Kdn signals was approximately 4:1. Several signals of anomeric carbons of sugar residues (Fig. 3, bottom) and weak signals of quaternary carbon atoms of Kdn residues were observed at the downfield region (Table 1).

Analysis of NMR spectra allowed us to identify signals of disaccharide moiety →6)-β-D-Glcp-(1→8)-α-Kdn-(2→, as well as in the repeating unit of the polymer from Brevibacterium aurantiacum VKM Ac-2111 (Shashkov et al., 2015). However, these signals were minor compared to those belonging residues of α-Kdn with strongly downfield shifted signals of H-9 and H-9' (Fig. 3, Table 1). This shift is characteristic of O-acylated molecular fragments. We supposed that the cause of this effect is the formation of intramolecular 1-9 macrocyclic Kdn-lactons during isolation of the polymer (Fig. 2, Formula 2).

Highfield shifting of the signal C-8 of residues with 1-9 lactone compared with C-8 in fragments →6)-β-D-Glcp-(1→8)-α-Kdn-(2→ (Fig. 3) confirmed our assumption. Attempts to uncover lactone macrocycle led to the formation of the disaccharide β-Glcp-(1→8)-β-Kdn. These observations suggested that the native polymer was identical to that isolated from the cell wall of B. aurantiacum VKM Ac-2111 (Shashkov et al., 2015).