Разнообразие гликополимеров клеточных стенок актинобактерий (стр. 4 )

So the cell wall of S. albus Ac-35T contained three glycopolymers. Two of them (minor polymers) were the TA: unsubstituted 1,5-poly(ribitol phosphate) and 1,3-poly(glycerol phosphate) with the β-glucopyranose (β-D-Glcp) residues at O-2 of the most of glycerol residues. The third glycopolymer was the Kdn-TULA of following structure: β-D-Glcp-(1→8)-α-Kdn-(2[(→6)-β-D-Glcp-(1→8)-α-Kdn-(2→]n 6)-β-D-Glcp-(1→8)-β-Kdn-(2-OH, were n ≥ 3. Taking into account the lability of the Kdn-TULA it can be assumed that the length of the native polymer can be greater.

Streptomyces albus J1074 and R1-100

The cell walls of S. albus J1074 and R1-100 contained 0.8 – 0.9% of phosphate-containing polymers phosphorus. The yield of preparation 1 (of both organisms) were ca. 2% of the cell walls dry mass.

The compositions of acid hydrolysates (2 M HCl, 100 °С, 3 h) of the both preparations 1 and the cell walls themselfs were studied by electrophoresis and chromatography on paper, they were found to be qualitatively identical. Hydrolysis afforded the following products: inorganic phosphate, glycerol, its mono - and bisphosphates, and galactose.

The electrophoretic study of native preparations 1 led to the formation of two zones having different mobilities: mGroP 1.3 and mGroP 0.65, that suggested the presence of several polymers in the cell wall of streptomycetes under study.

The same data were obtained under the chemical and electrophoretic studies of native preparations 2 (from the both organisms, the yield was ca 9.5% of the cell walls dry mass).

The preparations 1 and 2 have been studied separately by NMR spectroscopy. The results showed qualitative identity of the polymers from investigated strains (Fig. 4a, b).

The 13С and 1H NMR spectra of the preparation 1 showed signals corresponding to the carbon atoms of the unsubstituted 1,3- and 2,3-poly(glycerol phosphates) - at δC 67.8 and 70.9 and δC 62.2, 76.6 and 66.0, accordingly (Table 2, Fig. 5). The 31P NMR spectrum (not shown) of the preparation 1 contained the minor signal at δP +0.5 and +0.8 (Table 2). So, the unsubstituted 1,3- and 2,3-poly(glycerol phosphates) were found in the cell wall of investigated actinomycetes.

We did not find the Kdn-TULA structure in preparation 1. So the structure of Kdn-TULA was established by NMR investigation of preparation 2.

The 13С and 1H NMR spectra (Table 2) of the preparations 2 contained the signals of different integral intensity in the anomeric carbon resonance region at δC 96.4 – 104.6, including signals for quaternary carbons at δC 96.4 and 99.8 characteristic for C-2 of nonulosonic acid (Fig. 6, Table 2). The 13С NMR spectrum also contained the signals of CH–CH2–C group at δC 40.0 – 40.4, characteristic for C-3 of nonulosonic acid (Fig. 4a, b, top left).

The 1H NMR spectra of the preparations 2 contained signals for anomeric protons at δH 4.50 (Table 2), signals for a CH–CH2–C group at δH 2.71 and 1.78 (H-3eq and H-3ax of nonulosonic acid, accordingly, (Fig. 3, top left).

The signals in the 1D 13С and 1H NMR spectra of the preparations were assigned using 2D homonuclear 1H,1H COSY, TOCSY, ROESY, heteronuclear 1H,13C HSQC, HSQC-TOCSY, HMBC, and 1H,31P HMBC experiments, and spin-systems for β-Galp, α- and β-Kdn were identified (Table 2).

The 1H,1H ROESY spectra (not shown) of preparations 2 revealed the contacts of anomeric proton Н-1 β-Galp (Table 2) with the protons H-9, 9′ of α-Kdn and the proton H-3ax of α-Kdn and H-3 β-Galp besides the trivial contacts of the protons of the same residue. Based on the data obtained on the NMR spectra, the following structure of the repeating unit of Kdn-TULA was concluded: →3)-β-D-Galp-(1→9)-α-Kdn-(2→.

The 1H,13C HSQC spectrum (Fig. 4a, b) showed, that preparations contained the residues of α-Kdn and β-Kdn. This fact indicates that the above-mentioned polymer from preparation 2 was partially cleaved.

The bond -α-Kdn-(2→3)-β-D-Galp- for the deep hydrolysis product of the preparations under study were independently confirmed due to the presence of correlation peak 3G/2Kα in fairly well-resolved spectrum 1H/13C HMBC (Fig. 6). The spectra also confirms the bond β-D-Galp-(1→9)-α, β-Kdn (Table 2).

So the cell walls of S. albus R1-100 and J1074 contained three glycopolymers: two TA - 1,3- and 2,3-poly(glycerol phosphates) and the Kdn-TULA of the following structure:

β-D-Galp-(1→9)-α-Kdn-(2[(→3)-β-D-Galp-(1→9)-α-Kdn-(2→]n 3)-β-D-Galp-(1→9)-β-Kdn-(2-OH, where n~7-8.

S. albus sbsp. pathocidicus VKM Ac-598 T

The cell wall of this organism contained 2.4% of phosphate-containing polymers phosphorus. The yield of the preparation 1 was ca. 17.4 % of the cell wall 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, glycerol, its mono - and bisphosphates, glucosamine, and a small amount of glucose. In addition, lysine was detected in the processing of the cell wall and preparation 1 with aqueous ammonia.

The electrophoretic study of native preparations 1 led to the formation of two zones having different mobilities: mGroP 0.6; mGroP 0.36 that suggested the possibility of the presence of two polymers in the cell wall of streptomycete under study.

The preparation 1 has been studied by spectroscopy, and the 13С, 31P and 1H NMR spectra were recorded. 31P NMR spectrum (not shown) contained two signals of the phosphate groups, the most intense of them were at δР +0.4 and minor δР -1.3 (Table 3). These data indicated the possible presence of two different phosphate containing polymers. The 13С and 1H NMR spectra showed that one of the polymers had signals, characteristic for a TA of following structure: 1,3-poly(glycerol phosphate) partially glycosylated with 2-acetamido-2-deoxy-α-D-glucopyranose and/or O-acylated with L-lysine at O-2 of glycerol (Fig. 7, Table 3). The spectra of a similar polymer are given in the works (Shashkov et al. 2006; Tul’skaya et al. 2007 b). In addition minor signals belonging to the other phosphate containing polymer were found.

Analysis of the 2D spectrum 1H/ 13C HSQC (Fig. 7, Table 3) demonstrated that the second polymer is built of disaccharide residues α-D-Glcp-(1→6)-α-D-GlcpNAc combined in the polymeric chain by phosphodiester bonds between О-1 α-D-GlcpNAc and О-6 α-D-Glcp. Based on these data, the structure of the repeating unit of disaccharide 1-phosphate polymer can be presented as follows: -6)-α-D-Glcp-(1→6)-α-D-GlcpNAc-(1-P-.

This polymer was destroyed partially in the process of extraction, and during a long-term recording of the NMR spectra with the cleavage of the С-1-O-Р bond. So, the 2D spectrum 1H/ 13C HSQC (Fig. 7 bottom left) contained signals of terminal units at the reducing end of the chain -6)-α-D-GlcpNAc-ОН and -6)-β-D-GlcpNAc-ОН. This disaccharide 1-phosphate polymer was described for the first time in Gram-positive bacteria.

So the cell wall of S. albus sbsp. pathocidicus VKM Ac-598 T contained two phosphate-containing polymers: 1,3-poly(glycerol phosphate) partially glycosylated with α-D-GlcpNAc and/or O-acylated with L-lysine at O-2 of glycerol and a GPof following repeating unit -6)-α-D-Glcp-(1→6)-α-D-GlcpNAc-(1-P-.

Discussion

In this paper the cell wall glycopolymers structures and composition were identified by chemical, NMR spectroscopic and ESI-MS methods. The combination of techniques made it possible to establish the structure of major polymers, as well as to reveal the presence of some different minor polymers in the cell walls of the strains under study.

The cell wall of the type strain S. albus VKM Ас-35Т contained two TA, viz. unsubstituted 1,5-poly(ribitol phosphate) and 1,3-poly(glycerol phosphate) with b-D-Glcp at O-2 of glycerol. The former TA previously detected in the cell walls of S. chrysomalus (Streshinskaya et al. 1995), and the second one – quite spread in the cell walls of Gram-positive bacteria (Potekhina et al. 2003; Streshinskaya et al. 2011). The major glycopolymer was Kdn-TULA with the repeating unit: →6)-β-D-Glcp-(1→8)-α-Kdn-(2→. The glycopolymers of the same structure were recently found in the cell walls of a number of actinobacteria, viz. B. aurantiacum VKM Ac-2111Т, Arthrobacter protophormiae VKM Ac-2104T, S. coelicolor VKM Ac-738T (Streshinskaya et al. 2015).

Two other strains S. albus J1074 and R1-100 differed significantly from the above S. albus VKM Ас-35Т on composition and structure of cell wall glycopolymers. The cell walls of S. albus J1074 and R1-100 were absolutely identical on the composition and structure of glycopolymers. Among them there were unsubstituted 1,3- and 2,3-poly(glycerol phosphates) commonly occurs in the streptomycetes cell walls (Potekhina et al. 1996; Tul’skaya et al. 1997; 2007b; 2011). The major glycopolymer was Kdn-TULA with the repeating unit: →3)-β-D-Galp-(1→9)-α-Kdn-(2→. This polymer was found for the first time in Gram-positive bacteria. The topology of ketosidic bond (-α-Kdn-(2®3)-b-D-Galp) was the main difference between the last Kdn-TULA from that found early in S. coelicolor M145 (Shashkov et al. 2012).

The cell wall of S. albus sbsp. pathocidicus VKM Ac-598 T contained two phosphate-containing glycopolymers. The maior polymer was 1,3-poly(glycerol phosphate) partially O-glycosylated with α-D-GlcpNAc and/or O-acylated with L-lysine at O-2 of glycerol. TA of this structure was found early in the cell walls of a number of streptomycetes (Shashkov et al. 2006; Tul’skaya et al. 2007 b). The minor polymer comprised the GP of new structure with the following repeating unit: -6)-α-D-Glcp-(1→6)-α-D-GlcpNAc-(1-P-.