16S rRNA Sequencing for Species Identification in Mixed Cultures for New Bio Preparations in

Based on the research findings that both synergistic interactions among bacterial species and the composition of the bacterial community are important in determining the level of ecosystem functioning [1], we proposed to use bacterial communities with different plant growth-stimulating and biological diseases control mechanisms to develop new bio preparations for agriculture. In this work there was a problem of isolation of pure bacterial culture from the mixed cultures (or natural bacterial communities) with closely related bacteria. In addition to that, the separation of natural microbial associations may leads to lose its activity. In this regard, is necessary to monitor the qualitative composition of bacterial associations to develop, to store and to use of biological preparations. We used the method of 16 S-rRNA sequencing for species identification in mixed bacterial cultures with closely related bacteria.


Introduction
Based on the research findings that both synergistic interactions among bacterial species and the composition of the bacterial community are important in determining the level of ecosystem functioning [1], we proposed to use bacterial communities with different plant growth-stimulating and biological diseases control mechanisms to develop new bio preparations for agriculture. In this work there was a problem of isolation of pure bacterial culture from the mixed cultures (or natural bacterial communities) with closely related bacteria. In addition to that, the separation of natural microbial associations may leads to lose its activity. In this regard, is necessary to monitor the qualitative composition of bacterial associations to develop, to store and to use of biological preparations. We used the method of 16 S-rRNA sequencing for species identification in mixed bacterial cultures with closely related bacteria.

Material and methods
Bacterial mixed cultures. We used 4 samples of bacterial mixed cultures. The samples 1 and 3 were mixed cultures; the samples 2,4 were natural microbial communities. All bacteria were isolated from plant's rhizosphere and Galega and Pisum plants nodules. Bacterial ribosomal RNA sequences by direct sequencing. Extraction of bacterial DNA was carried out in triplicate in three independently grown cultures. Extraction of DNA was carried out by the chloroform-saline standard method based on the lysis of cells and denaturation of cellular proteins with a solution that contains guanidine thiocyanate, and followed by ethanol precipitation of nucleic acids [2].

Advances in Biotechnology & Microbiology
Electrophoresis was carried out in 8% polyacrylamide gel with a gradient of 45 to 80% (100% gel contains 7 M urea solution, and 40% deionized formamide solution). The gel was stained for 30min in Tris-acetate-EDTA buffer containing ethidium bromide, washed with deionized water, visualized under ultraviolet light. Fragments were excised from the gel with amplification products and homogenized in 1.5ml tubes. 25ul buffer was added to the DNA elute and incubated for 15min at 37 °C. Then the mixture was frozen and thawed several times. Incubating was carried out overnight at 37 °C and centrifuged at 13k rpm for 10min. Purified fragments were used for sequencing reactions formulation using a set Genome Lab DTCS-Quick Start Kit (Beckman Coulter Inc., USA).
To analyze the reaction product was sequenced purified precipitate and dissolved in SLS-buffer containing formamide and placed in a genetic analyzer Genome Lab GeXP. The obtained sequences were compared with those available in the Gen Bank database. The primary analysis of the obtained sequences was performed using the NCBI. In each case, a comparison of sequences carried out on the three most identical sample nucleotide sequences with a match of at least 99%. Alignment of the nucleotide sequences was performed in the program MEGA version 5, using an algorithm CLUSTAL V [4].

Results and Discussion
The presence of two closely related bacteria Rhizobium galegae and Rhizobium leguminusarum was shown for the sample №1. The natural bacterial community (the sample№2) consisted of closely related bacteria Enterobacter sp. 638 and Enterobacter cloacae. For the sample №3 the presence of three bacterial cultures, which sequences exhibited high 99% identity to the sequences of the following bacteria Paenibacillus polymyxa, Paenibacillus peoriae and Microbacterium testaceum was shown. The bacteria Mycobacterium gilvum and Microbacterium testaceum were identified in natural bacterial community of the sample № 4. Take into accounts the data of 16S rRNA sequencing; we excluded Enterobacter cloacae strain (as a potential pathogenic bacterium) from the further work. The strain which exhibited high levels (99%) of DNA sequence identity with the Enterobacter sp. 638 needs further identification. It was concluded that 16S rRNA sequencing might be used to monitor the qualitative composition of bacterial associations to develop, to store and to use of biological preparations.