Nitrogen

• Nitrogen fixation


  The chemical conversion of N2 to NH3 which cannot be used directly by plants or animals to synthesize amino acids and nucleotides
• Nitrogen fixation


  1. Requires large amount of energy because of triple bond of N2 (N≡N) which is very stable

  2. Energy obtained from hydrolysis of ATP

  3. Chemical conversion of N2 to NH3 requires high temp and pressure
• 100 million tons of fixed N2 required annually to sustain global food production
• Sources of fixed nitrogen

  • Chemical fertilizers (half)
  • Diazotrophic (nitrogen fixing) bacteria (remainder)
• Examples of diazotrophic bacteria

  • Rhizobium
  • Frankia
  • Azospirillum
  • Azotobacter
  • Cyanobacteria
• Prohibitive cost of chemical fertilizers acts as an incentive for the development of biofertilizers
• Most important organisms used in agriculture

  • Rhizobium
  • Bradyrhizobium
• Life cycle of rhizobial bacteria

  Rhizobial bacteria invade plant root cells and initiate developmental changes that lead to the formation of root nodules
• Protection of nitrogenase from O2 in root nodules

  1. O2 does not readily diffuse into a nodule

  2. Nitrogenase content is regulated by leghemoglobin (heme moiety synthesized by bacterium, globin portion encoded by plant gene)
• Inside the nodule


  • The plant provides the bacterium with photosynthetically fixed carbon (needed for bacterial growth)
  • The plant receives fixed nitrogen from the bacterium
• Nitrogenase


  All known nitrogenases have two oxygen sensitive components
• Component I of nitrogenase

  • Two β- protein subunits (60,000 daltons each)
  • Two α- protein subunits (50,000 daltons each)
  • 22 molecules of Fe
  • 2 molecules of molybdenum
  • Iron-molybdenum cofactor called FeMoCo
• Component II of nitrogenase


  • Two α – protein subunits (32,000 daltons each)
  • Associated iron-molybdenum molecules
• Catalysis of nitrogen to ammonia by nitrogenase


  Requires the combination of components I and II, a complex of magnesium and ATP, and a source of reducing equivalents
• Nitrogenase can reduce the gas acetylene to ethylene, which can be used as a measure of nitrogenase activity
• Functional nitrogenase is dependent on 15 to 20 additional accessory proteins
• Genetic complementation to isolate nif genes


  Identify and characterize clones of a wild-type library that restore nitrogen fixation to various mutants of the original organism
• The first nif genes identified by complementation were isolated from clone banks of Klebsiella pneumoniae
• Isolation protocol for nif genes

  1. Treat K. pneumonia cells with a mutagenic agent to obtain Nif- mutants

  2. Construct a clone bank of chromosomal DNA from wild type (Nif+) K. pneumoniae into a plasmid vector in E. coli

  3. Conjugate the Nif- K. pneumoniae cells with the E. coli cells carrying the clone bank

  4. Select for the Nif+ phenotype in the transformed K. pneumoniae cells
• The entire set of nif genes in K. pneumoniae has been isolated and characterized, occupying a single 24 kb cluster containing 7 operons encoding 20 distinct proteins
• NifA protein


  A positive regulatory factor that turns on the transcription of all other nif genes except its own, by binding to a specific DNA sequence
• NifL protein


  A negative regulatory factor that acts as an antagonist to NifA in the presence of O2 or fixed N, turning off the transcription of all other nif genes
• To genetically engineer nitrogen fixation in important plant growing bacteria, nif genes from K. pneumoniae have been used to isolate nif genes from other diazotrophic microorganisms
• It may be possible to increase the amount of nitrogen fixed by diazotrophic microorganisms by manipulating nifA and nifL genes
• The addition of nif genes to a non-diazotrophic microorganism may not confer the ability to fix nitrogen due to transcriptional, translational and regulatory problems, and the lack of plant promoters that respond to the nifA protein
• The introduction of functional nitrogen fixation capabilities in plants is extremely unlikely
• Rhizobial respiration plays an important role in nitrogen fixation by providing the energy needed and removing O2 to prevent inactivation of nitrogenase
• A strain of Rhyzobium tropici with a deletion mutant for glycogen synthase was able to increase both the nodule number and dry weight of treated plants, but the inability to synthesize glycogen can have deleterious effects and require reinoculation in the field
• Oxygen is inhibitory to nitrogenase and is a negative regulator of nif gene expression
• Plant cells do not process multigene transcripts
• Nitrogen fixation


  Plays an important role in N fixation
• Nitrogen fixation


  1. Oxidative phosphorylation provides the energy needed for N fixation

  2. Respiration removes O2 to prevent inactivation of nitrogenase
• It would be more advantageous if the C reserves were driven towards ATP production instead of glycogen synthesis
• Glycogen synthase mutants


  • A strain of Rhyzobium tropici was constructed with a deletion mutant for glycogen synthase
  • This strain was unable to produce glycogen and was able to increase both the nodule number and the dry weight of treated plants
  • Deletion mutants cannot revert to the wild type
  • Good for agriculture
  • Inability to synthesize glycogen can have deleterious effects hence these microbes will not persist for a long time
  • Reinnoculation field necessary
• Engineering oxygen levels


  1. Introduction of leghemoglobin that binds free oxygen tightly

  2. This will ensure that the nif genes and functioning of nitrogenase are not impaired
• It is possible to engineer rhizobial strains with genes encoding the bacterial equivalent of the leghemoglobin
• The hemoglobin containing strain can grow to a greater extent under conditions where oxygen is limited
• The expression of bacterial hemoglobin genes maybe advantagous to Rhyzobium both when they are free living and when they are bacteriods
• Hydrogenase


  1. Undesirable side effect of nitrogen fixation is the conversion of H+ to H2 gas by nitrogenase

  2. This utilizes a lot of energy and H2 is lost to the atmosphere

  3. As a result only 40-60% of the electrons flux through the nitrogenase system is transferred to N2

  4. Efficiency of nitrogen fixation is reduced
• Theoretically if H2 could be recycled back to H+ the energy loss would be less and the nitrogen fixing process more efficient