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Selection of Effective Nitrogen Fixing Microorganisms


Selection of Effective Nitrogen Fixing Microorganisms Isolated from Different Soil-Climatic Zones of the Caucasus for Bio-Fertilizers

Tech Area / Field

  • BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
  • BIO-CHM/Biochemistry/Biotechnology
  • BIO-MIB/Microbiology/Biotechnology
  • AGR-PPR/Plant Protection/Agriculture

3 Approved without Funding

Registration date

Leading Institute
Durmishidze Institute of Biochemistry and Biotechnology, Georgia, Tbilisi


  • CNRS / Institut National de la Recherche Agronomique / Centre de Microbiologie du Sol et de l’Environnement, France, Dijon\nJohn Innes Centre / Department of Molecular Microbiology, UK, Norwich\nSwedish University of Agricultural Sciences, Sweden, Uppsala\nUniversidad de Salamanca, Spain, Salamanca\nInstituto de Recursos Naturales y Agrobiologia - Centro Superior de Investigaciones Cientificas, Spain, Salamanca

Project summary

Selection of Effective Nitrogen Fixing Microorganisms Isolated From Different Soil-Climatic Zones of The Caucasus for Bio-fertilizers.

Biological nitrogen fixation (BNF) is a prioritary area in biology, it has made original contributions both for the specific area and for the advancement of science in general, and it has the potential to improve sustainable agriculture. An, interdisciplinary, research project at Durmischidze Institute of Biochemistry and Biotechnology aimed isolation and selection of highly active, industrially valuable nitrogen-fixing microorganisms (creation of a collection) from different soil-climatic regions of the caucasus for creation of new biological fertilizers and also at understanding the molecular mechanism of action of bacterial genes that participate in N fixation (nif genes) for future goals for the manipulation of the genome of both bacteria and plants to improve existent symbiosis with legumes, the extension of symbiotic nitrogen fixation to other crops and, eventually, the production of plants able to fix nitrogen.

Nowadays the research in biological N fixation has advanced enormously in the last years due to the effort of different laboratories around the world. Some of the important issues that are now known in this area are: the structure of the enzyme responsible for the reduction of N to ammonia: nitrogenase; The nature of chemical signals involved in the bacteria-plant communication; bacterial genes that participate in N fixation (nif genes) and in the symbiotic interaction with plants; regulatory circuits which sense environmental conditions (particularly oxygen concentration and fixed N) that trigger N fixation; specific plant genes and their proteins (nodulins) which are expressed precisely during nodule development and function. Nevertheless, the advances in the understanding of the molecular and biochemical components regulating symbiotic nitrogen fixation have yet to be translated into applied improvements.

A new point of the suggested project is a complex approach to resolve this issue. Based on knowledge molecular biology and biotechnology the project will result in creation of a collection of nitrogen-fixing highly active microorganisms' characterized with different genetic peculiarities, isolated from different soil-climatic zones of the Caucasus.

Scientific significance of the project An, interdisciplinary, research project aimed at understanding the molecular mechanism of biological nitrogen fixation. In addition to the direct interest in BNF, research in this area has contributed to the general advancement of science, with major achievements such as: the discovery of a specific system - the two component system - for the regulation of bacterial gene expression in response to environmental stimuli; the definition of the chemical nature of communication signals between a bacteria and a plant; the definition of the structure of a complex enzymatic system - the nitrogenase; the definition of the complete DNA sequence of a bacterial megaplasmid.

Practice significance of project. The project will result in creation of a collection of nitrogen-fixing microorganisms’ characterized with different genetic peculiarities, isolated from different soil-climatic zones of the Caucasus. On the basis of application of highly active strains will be produced biological fertilizers, for raising of crop yields and receipt of ecologically pure products.

The role of foreign collaborators will be expressed as follows: information exchange in the course of project implementation; consultations on the methods used in the study and help with analysis and presentation of results; provide comments to the technical reports (quarterly, annual final, etc.), submitted by project participants to the ISTC; provide necessary consultations and help on this project and help with preparation of this proposal; joint discussion of obtained results at working seminars;joint attendance of international meetings.

To achieve goals of the project it is necessary to solve the following tasks:

1. Biopersity and taxonomy of nitrogen fixing bacteria.

1.1. Isolation of nitrogen-fixing microorganisms (freely populated, associative and symbiotic) from different soil-climatic zones of the Caucasus (creation of a collection).

1.2. Study of morphological, physiological and biochemical characteristics of microorganisms of the collection.

1.3. Selection of highly active strains and comparison of the current data, with the characteristics of microorganisms isolated from the analogous soil-climatic zones of the planet.

2. Genome Dynamics.

2.1. Study of the amplicon structure of different Rhizobium etli and Azospirillum brasilense (as a model system) genomes.

2.2. Study of the symbiotic capacity of Rhizobium and associative capacity of Azospirillum strains.

2.3. Manipulation of the Rhizobium and Azospirillum genome by inducing and selecting strains harboring natural and artificial DNA amplifications in order to improve symbiotic properties.

3. Identification and characterization of saprophytic and symbiotic functions of plasmids in selected microorganisms.

3.1. Identification of genetic determinants participating in plasmid transfer.

3.2. Studying the regulation of new genes involved in nodulation and nitrogen fixation.

4. Characterization of an Rhizobium and Azospirillum mutants with enhanced N2 fixation.

4.1. DNA manipulation, cloning and sequence analysis.

4.2. b-Glucuronidase and nitrogenase assays.

Methodology. For identification of bacteria will be used the Guide of Bergy and Taranda and other descriptions; including the phase-contrast microscopy. For selection of highly active microorganisms either gas-chromatographic and mass-spectrophotometric analyses will be applied. The method of acetylene for determination of nitrogen-fixing activity of microbes. Biomass of active strains will be obtained by the method of microorganisms’ cultivation in flasks and fermenters, regarding the preliminary selection of cultivation conditions. Nucleotide sequence accession number. The nucleotide sequence of the DNA fragment containing R.. etli rpoN and associated genes will be deposited with DDBJ-EMBL-GenBank under accession no. U23471. Construction of mutants. To mutagenize the R.. etli rpoN gene, the 1.8-kb EcoRI fragment of pFAJ1150 will be blunt-end ligated into the SmaI site of pJQ200-UC1. The resulting plasmid will be digested with PstI. Two plasmids carrying the gusA-Kmr cassette in opposite orientations will be obtained. These insertional mutations will be finally recombined into the R.. etli CNPAF512 chromosome. Insertion of these mutations will be verified by Southern blot hybridization with the appropriate probes. For the inactivation of ptsN, a 1.7-kb fragment containing ptsN will be amplified by PCR. PCR conditions. Amplification of DNA fragments by PCR will be performed in a TRIO-Thermoblock (Biometra). Twenty-five-microliter reaction mixes, containing 0.65 U of Taq DNA polymerase (Boehringer), each of the deoxynucleoside triphosphates at 200 µM, and each of the primers at 1 µM, will be subjected to 30 cycles of incubation at 94 °C for 60 s, 60 °C for 60 s, and 72 °C for 210 s. DNA methods. General DNA manipulations will be performed as described previously (12), (13) DNA fragments will be recovered from agarose gels by using the Nucleotrap kit (Macherey-Nagel). Southern blotting and hybridizations will be carried out as described previously (12, 13, 14). DNA probes will be labeled with a digoxigenin labeling and detection kit (Boehringer). To generate blunt ends to incompatible DNA fragments, DNA will be incubated with Klenow or T4 DNA polymerase in the presence of the four deoxynucleoside triphosphates. Automated DNA sequencing will be performed on a Pharmacia A.L.F. sequencer with fluorescein-labeled universal and synthetic oligonucleotide primers. Both strands of overlapping pUC18 subclones covering the 5,600-bp DNA fragment will be read with multiple sequencing. Growth tests. Tests of growth of strains in liquid medium will be carried out in acid minimal salts medium (15) containing CaCl2. Cells will be first grown overnight in TY, will be washed in MgSO4, brought to an optical density at 600 nm of 0.02 in a Perkin-Elmer lambda 2 spectrometer, and diluted 100 times in AMS medium. Carbon and nitrogen sources will be added to the appropriate concentrations with a sterile concentrated stock solution. Cell growth will be monitored in terms of turbidity at 600 nm in a microtiterplate reader. Carbon sources will be D-(-)-mannitol, D-(+)-glucose, sucrose, L-(+)-arabinose, D-(+)-galactose, D-sorbitol, D-(-)-fructose, glycerol, trisodium citrate, cis-aconic acid, DL-isocitric acid, -ketoglutaric acid, succinic acid, fumaric acid, DL-malic acid, oxaloacetic acid, and pyruvic acid. Nitrogen sources will be NH4Cl, L-alanine, L-glutamine, and KNO3. For growth tests at various pH values, 3 (N-morpholino)propanesulfonic acid (pH 7.0) and 2(N-morpholino)ethanesulfonic acid (pH 6.5, 6.0, and 5.5) will be used at a concentration of 30 mM.

For growth tests on plates, appropriate combinations of mannitol, ammonium, or amino acids will be added to AMS agar. Plates will be supplied with (i) only the amino acid, (ii) the amino acid and ammonium, (iii) the amino acid and mannitol, or (iv) the amino acid, ammonium, and mannitol. Plates will be incubated at 30 °C, and colony size will be monitored over a period of 3 to 7 days.

The project will give a possibility to the Georgian scientists previously involved in military research to redirect towards civilian activities.The technology of obtaining new biocomposite materials for agricultural applications and increase the level of the preparations they have create to meet the international requirements. All this will create sound background for the preparations to be introduced to the international markets.Realization of the project will promote the application of elaborated methods of not only in Georgia, but in other countries too by governmental ecological organizations, as well as by private companies.


The International Science and Technology Center (ISTC) is an intergovernmental organization connecting scientists from Kazakhstan, Armenia, Tajikistan, Kyrgyzstan, and Georgia with their peers and research organizations in the EU, Japan, Republic of Korea, Norway and the United States.


ISTC facilitates international science projects and assists the global scientific and business community to source and engage with CIS and Georgian institutes that develop or possess an excellence of scientific know-how.

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