Structual Peculiarities of Exopolysaccharides of Phototrophic Bacteria Rhodobacter sphaeroides

For the first time, properties and structural peculiarities of exopolysaccharides produced by phototrophic bacteria Rhodobacter sphaeroides isolated from hydrocarbonate mineral waters in Armenia with a degree of mineralization in limits of 3.5-5.0g/l, pH 7.2 have been studied. It is noteworthy that despite membership of the isolated strains in one and the same genus, these strains are significantly different by the nature of the produced exooligosaccharide. The studies implemented show that phototrophic bacteria are able to synthesize exopolysaccharides with different compositions and structures: cyclic and linear, depending on the cultivation conditions.

Phototrophic microorganisms represent considerable interest for productions various biologically active compounds -vitamins, carotenoids, organic acids, enzymes, etc. However, the biosynthesis and properties of polysaccharides produced by phototrophic bacteria are not entirely studied and represent prospective areas of study.
The aim of these researches is to study the properties and structural peculiarities of exopolysaccharides produced by the strains of phototrophic bacteria Rhodobacter sphaeroides.

Materials and Methods
Strains and media. Phototrophic bacteria Rhodobacter sphaeroides, designated as A-1 and D-1 have been isolated from hydrocarbonate water sources of Armenia (Arzni and Jermuk) with a degree of mineralization of 3.5-5.0 g/l, pH 7.2. The strain of Rh. sphaeroides A1 was cultivated in the under stationary conditions with illumination intensity of 2500-3000 lux, at 30˚C, during 8 days in slightly modified Ormerod's nutrition medium [20]: (g/l) KH 2 PO 4 -0. 6 The total quantity of sugars was determined by anthrone method [21].
Antimicrobial activity of polysaccharides was identified by the agar diffusion methods [22].
NMR spectra were registered on spectrometer MERCURY 300Vx (Varian) with resonance frequency of 300.077 and 75.465 MHz, on 1 H and 13 C nuclei, respectively. Heavy water was used as solvent. In the NMR spectrum, 13 C signal of methanol group (δ (CH 3 ) 49 ppm) acted as the internal standard.
Isolation of exopolysaccharids was carried out according to the following method developed by as (diagram). The mentioned cultures grew well in the cultivation medium, containing the salts of organic acids -acetate, piruvate, lactate, malate, fumarate, succinate as well as glucose, fructose, maltose, saccharose and other sugars. Microscopic studies have shown that bacterial cells form a mucous capsule.
Studies of synthesis of exopolysaccharides have demonstrated that their content reaches maximum in the 6-7 days of cultivation, which coincides with the maximum of culture growth. Under further cultivation the level of sugars is not changed, as the case with the biomass (Figure 2). Microbial polysaccharides are known to be biologically active [ 2,11,18,22].. Our studies of the effect of the isolated exopolysaccharides on microorganisms-E. coli, B. subtilis, Candida albicans -have not indicate any antimicrobial activity.
Structural peculiarities of the noted exopolysaccharide have been studied by means of nuclear-magnetic resonance (NMR) spectroscopy of hydrogen and carbon atom nuclei. The 13 C NMR spectrum of the exopolysaccharide from Rh. sphaeroides strain A-1 contains six absorption signals from six non-equivalent carbon atoms (Figure 3). The comparison of the abovementioned spectrum with 13 C NMR spectrum of α-glucose has revealed good coincidence of signals chemical shifts for carbon atoms in positions 2 -5, whereas carbon atom signals in positions 1 and 6 were significantly different. This indicates that α-glucose is the base of the oligosaccharide, and glycosidic residues are connected in positions C1 and C6 (Figure 4). Analysis of the proton spectra of the oligosaccharide in question agrees well with the abovementioned ( Figure 5 and Table 1).
The doublet signal of anomeric hydrogen atom with the spin-spin interaction constant J = 3.6 Hz indicate the evidences of α-configuration of the monosaccharide. Secondly, detailed analysis of multiplet structure and determination of the spin-spin interaction constant between nuclei 1 and 2, 2 and 3, 3 and 4, 4 and 5 indicates that α-glucose is the monosaccharide residue. and 7 molecules are connected in positions C1 and C6, forming a cyclic structure with certain elements of symmetry and molecular mass 1135 ( Figure 6).   It is important to note that spectra of oligosaccharide with linear structure and, therefore, without symmetry elements, would have appeared to be much more complex than the observed spectra. In particular, the 13 C NMR spectrum of such oligosaccharide should have consisted of several tens of signals.
The influence of different sources of carbon on the peculiarity of exopolysaccharide produced by Rh. sphaeroides D-1 has been carried out. Investigations of 13 C NMR spectrum of exopolysaccharide of phototrophic bacteria Rh. sphaeroides D-1 grown in acetate containing medium as the only source of carbon reveled the presence of numerous different intensity signals (Figure 7). The results obtained indicate that the investigating polysaccharide consists of two different monomers of glucose and galactose. The same results have been obtained by proton NMR spectrum. It should also be mentioned that the exopolysaccharide of Rh.sphaeroides D-1 is a linear heteropolysaccharide.
In literature, it is assumed that the monomeric composition and structure of synthesized polysaccharide by microorganisms depend on the cultivation conditions, particularly from source of carbon [7,12,14].
In order to study the possibility of changing the monomer composition and structure of exopolysaccharide of phototrophic bacteria of Rh. sphaeroides D-1, the cultivation has been carried out on Ormerud medium with succinate as the only source of carbon, instead of acetate. The studies of structural properties of isolated exopolysaccharide by 13 C NMR spectrum have shown the presence of six identical carbon signals which have proved that the polysaccharide is composed of identical monomers (Figure 8). In the proton NMR spectrum of investigated polysaccharide is observed 5.06mm doublet swallowing with constant spin-spin interacts 3.8 Hz, indicating that the polysaccharide is a part of the monomer α-glucopyranoside ( Figure 9).

Conclusion
Strains of phototrophic bacteria have been isolated from mineral hydro carbonic water (Armenia). The studies of morphological, physiological and biological properties allowed identifying them as strains of Rhodobacter sphaeroides. The isolated bacteria are gram positive and can utilize organic substrates: acetate, piruvate, lactate, fumarate, succinate as well as glucose, fructose, maltose, saccharose and other sugars as in anaerobic condition in the light and in aerobic conditions in the dark.
For the first time it has been shown that the phototrophic bacteria (Rh. spaeroides A1, D1) produce exopolysaccharides and it is noteworthy that in despite that isolated bacteria belonging the same genera can produced exopolysaccharides significantly differ by the structure and composition: in our study thr cyclic and linear forms have been described.