Genetics and regulation of Biological Nitrogen Fixation

DNA, the genetic information of all organisms, is encoded in its deoxyribose nucleic acid (DNA). In prokaryotes, there is only one chromosome and it is present in the cytoplasm. In eukaryotes, all other organisms except viruses, there are several chromosomes and they are present in the nucleus. Plasmid DNA also carries genetic information but multiplies and moves independently of the chromosomal DNA. All cells of eukaryotic organisms carry DNA in their mitochondria. Plant cells, in addition to nuclear and mitochondrial DNA, also carry DNA in their chloroplasts.

Genetic information in DNA is encoded in a linear fashion in the order of the four bases (A, adenine; C, cytosine; G, guanine; and T, thymine). Each triplet of adjacent bases codes for a particular amino acid. A gene is a stretch of a DNA molecule that codes for one protein molecule or, in a few cases, one RNA molecule. When a gene is active, one of its DNA strands is used as a template and is transcribed into an RNA strand. Most genes encode proteins, and the transcription product is a messenger RNA (mRNA). Different genes code for different proteins, some of which are part of the structure of cell membranes, but most act as enzymes.

In many cases of host-pathogen interaction, genes in one organism are triggered to be expressed by a substance produced by the other organism. The presence of one or more genes for pathogenicity, specificity, and virulence against the particular host makes possible the development of disease in a host. All plants also have preformed and induced defenses that provide resistance against most pathogens.

However, a few pathogens can attack many kinds of host plants, often due to their diverse genes for virulence or less plant specificity than commonly more specialized pathogens. Despite the many pathogens that can infect them, sometimes countless numbers of individuals of a single plant species survive in huge land expanses year after year, either free of disease or with only minor symptoms, even though most other plants have been killed. DNA, the genetic information of all organisms, is encoded in its deoxyribose nucleic acid (DNA). In prokaryotes, there is only one chromosome and it is present in the cytoplasm. In eukaryotes, all other organisms except viruses, there are several chromosomes and they are present in the nucleus. Plasmid DNA also carries genetic information but multiplies and moves independently of the chromosomal DNA. All cells of eukaryotic organisms carry DNA in their mitochondria. Plant cells, in addition to nuclear and mitochondrial DNA, also carry DNA in their chloroplasts.

Genetic information in DNA is encoded in a linear fashion in the order of the four bases (A, adenine; C, cytosine; G, guanine; and T, thymine). Each triplet of adjacent bases codes for a particular amino acid. A gene is a stretch of a DNA molecule that codes for one protein molecule or, in a few cases, one RNA molecule.

1. Genetics and Regulation of Biological Nitrogen Fixation Presented To: Dr. Zarrin Fatima Rizvi Presented By: Mashal Nadeem Roll No. : 001

2. Content Introduction to BNF Nod Operon Structure of Nod Operon Regulation of Nod Operon Nif Operon Structure of Nif Operon Regulation of Nif Operon Hup Genes Regulation of Hup genes References

3. Introduction to BNF Biological nitrogen fixation (BNF) is a biochemical process in which atmospheric nitrogen (N 2 ) is converted into accessible form of nitrogen (NH 3 , No x ) by certain bacteria (diazotroph) possessing nitrogenase enzyme. It is an energy consuming process. It consumes 16 ATP molecules for the conversion of single N 2 into 2NH 3 . Moreover, 1 2 ATP molecules are consumed for NH 4 + assimilation and transport (Buscot, 2005). Noticeably, Nitrogenase, a multisubunit enzyme, is the key enzyme for the conduction of BNF. Genetically, the activity of this enzyme is controlled by various nif genes.

4. Genetics of BNF Mainly, the process of BNF is controlled by 3 genes: Nif genes (Nitrogen Fixation Genes) Nod genes (Nodulation Genes) Hup genes (Hydrogen Uptake Genes)

5. Nif Genes Nitrogen fixing genes (also known as fixers). Present in both symbiotic and non symbiotic bacteria. Activate in anaerobic condition. Firstly studied in Klebsiella pneumoniae (Dean, 1992) where it is found in the form of : Group of seven operons 24kb length Comprised of 15 to 20 genes. Of which 17 genes are used in nitrogen fixation. These genes slightly vary in different organisms. Some are structural genes responsible for nitrogenase enzyme formation while others are regulator genes to control the remaining nif genes

6. Structure of Nif Operon

7. Subunits of Nitrogenase Enzyme

8. Nif Genes Nif Genes Functions Nif Genes Functions Nif H α subunit of dinitrogenase reductase Nif x Involved in FeMo-cosynthesis Nif D α 2 β 2 subunit of nitrogenase (65kd) Nif A Involved in positive regulation Nif K β subunit of nitrogenase (60kd) Nif L Involved in negative regulation Nif J Pyruvate flavodoxin (120kd) Nif M + S Activates nitrogen reductase (16kd) Nif N Helps in formation of Fe-Mo co factor (46kd) and protein for Nif B Nif U Involved in mobilization of Fe-S cluster synthesis Nif E Helps in formation of Fe-Mo cofactor α 2 β 2 (50kd) Nif Q + V + W + Y Unknown Nif F Helps in electron transport factors Nif T Unknown Nif B Required for FeMo-cosynthesis and repair

9. Nod Genes Nodule Forming genes Firstly, Nod factors were identified in Rhizobium meliloti (Lerouge et al., 1990). Located near nif genes Present on megaplasmid in case of symbiotic bacteria Control the activity of 2 phytoactivators i.e., IAA and Cytokinin. 8.5kb length (10 genes) Of which 4 are responsible for nodulation remaining ones are host specific.

10. Structure of Nod Operon

11. Nod Genes Categories of Genes Nod Genes Functions 1. Early Nodulation Genes Nod A,B,C,D Nod A,B,C are responsible for the production of core structure of Nod factor. Nod D regulates the expression of other genes (A,B,C). 2. Nodulation Regulation Genes Nod D,E,L Nod D Respond to flavonoid signals. Nod E and L control Nod factor production rate. 3. Nodulation Signalling Genes Nod L,N,O They help to ensure proper communication and recognition between host and bacteria. 4. Nodulation Export Genes Nod I,J They are responsible for exporting Nod factor from the bacterial cell to the external environment. 5. Nodulation Enzymes Nod M,W Modify the Nod factor by the addition of various elements in core structure. 6. Nodulation Specificity Genes Nod X,Y Helps in recognition of specific opegume by the rhizobia.

12. Hup Genes Hydrogen uptake genes. Helps in the maturation and formation of hydrogenase enzyme. Recycle the hydrogen which is formed during nitrogen fixation. 30 to 50 % hydrogen loss recoverd.

13. Uptake of Hydrogen by hydrogenase

14. Hup Genes Hup Genes Functions Hup S + L Encodes the structural components of hydrogenase enzyme. Hup S encodes small subunit. Hup L encodes large subunit. Hup D + F Involved in the maturation of hydrogenase enzyme by the insertion of metallic cofactor (Ni + Fe). Hup C Encodes a chaperon protein for correct folding and assemblage of hydrogenase enzyme. Hup H + G + E Biosynthesis of hydrogenase cofactor ( Fe – S cluster). Regulatory genes It controls the expression of hup genes in response to various environmental factors such as, hydrogen gas availability and other metallic cofactor.

15. Regulatory Genes Type of genes Role

16. Regulation of BNF

17. References Buscot, F. (2005). Microorganisms in soils: roles in genesis and functions. Klipp, W., Masepohl, B., Gallon, J. R., & Newton, W. E. (Eds.). (2004).  Genetics and regulation of nitrogen fixation in free-living bacteria  (Vol. 2). Springer Science & Business Media. Dean, D. (1992). Biochemical genetics of nitrogenase.  Biological nitrogen fixation . Lerouge, P., Roche, P., Faucher, C., Maillet, F., Truchet, G., Promé, J. C., & Dénarié, J. (1990). Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal.  Nature ,  344 (6268), 781-784.