1. Getting an N Fix Energetics N  N Haber-Bosch (100-200 atm, 400-500°C, 8,000 kcal kg -1 N) Nitrogenase (4,000 kcal kg -1 N)

2. Biological Nitrogen Fixation N2N2 NH 3 nitrogenase

3. Pea Plant R. leguminosarum nodules Pink color is leghaemoglobin a protein that carries oxygen to the bacteroids

4. Host plantBacterial symbiont AlfalfaRhizobium meliloti CloverRhizobium trifolii SoybeanBradyrhizobium japonicum BeansRhizobium phaseoli PeaRhizobium leguminosarum SesbaniaAzorhizobium caulinodans Complete listing can be found at at: http://cmgm.stanford.edu/~mbarnett/rhiz.htm Both plant and bacterial factors determine specificity Rhizobium-legume symbioses

5. Some nitrogen fixing organisms Free living aerobic bacteria –Azotobacter –Beijerinckia –Klebsiella –Cyanobacteria (lichens) Free living anaerobic bacteria –Clostridium –Desulfovibrio –Purple sulphur bacteria –Purple non-sulphur bacteria –Green sulphur bacteria Free living associative bacteria –Azospirillum Symbionts –Rhizobium (legumes) –Frankia (alden trees)

6. legume rhizobia Fixed nitrogen (ammonia) Fixed carbon (malate, sucrose)

7. MEDICAGO (alfalfa) LOTUS (birdsfoot trefoil) Obvious signs of nodulation by common rhizobial species

8. rhizosphere Flavonoids nod-gene inducers Nod-factor Very early events in the Rhizobium-legume symbiosis

9. Formation of a Root Nodule

10. The Nodulation Process Chemical recognition of roots and Rhizobium Root hair curling Formation of infection thread Invasion of roots by Rhizobia Cortical cell divisions and formation of nodule tissue Bacteria fix nitrogen which is transferred to plant cells in exchange for fixed carbon

11. The Colonization Process Signaling Rhizobia sense flavonoid compounds release by roots specific species sense particular flavonoids specific to a plant Rhizobia move by use of flagella propelling cell through soil water Rhizobia produce lipo-oligosaccharides or nod factors these initiate root hair deformation and curling and the division of cortical cells in the root at very low concentrations (< 10-9 M soil solution).

12. Role of Root Exudates General Amino sugars, sugars Specific Flavones (luteolin), isoflavones (genistein), flavanones, chalcones Inducers/repressors of nod genes Vary by plant species Responsiveness varies by rhizobia species

13. Genetics of Nodulation Legume plants secrete specific flavonoids, which are detected by interaction with bacterial NodD proteins.

15. The Biology of Symbiotic Development

16. NodD pSym nod genes activated NodD positively regulates nod genes Sinorhizobium meliloti nod-gene inducers from alfalfa roots (specificity) chromosome plasmid

17. Signals early in infection –Complex handshaking between legume root and rhizobium Incorrect signal Correct signal

19. nod Gene Expression Common nod genes Nod factor–LCO (lipo-chitin oligosaccharide)

20. Role of Root Exudates General Amino sugars, sugars Specific Flavones (luteolin), isoflavones (genistein), flavanones, chalcones Inducers/repressors of nod genes Vary by plant species Responsiveness varies by rhizobia species

21. The Colonization Process Infection Thread –Protein called recadhesin and polysaccharides from Rhizobia and lectins from plants interact to adhere the bacterium to the root hair –curling of the root hair and hydrolysis of root epidermis –Rhizobia move down centre of the root hair toward the root cortex –plant produces tube called an infection thread –in the cortex Rhizobia enter enclosed area within a plant-derived peribacteroid membrane. –membrane protect the rhizobia from plant defense responses.

22. Nod Factor: a lipooligosaccharide Nod factor biosynthesis Nod factor R-group “decorations” determine host specificity

23. LysM receptor-like kinases mediate plant recognition of symbiotic bacteria Taken from a review: Nature 425: 569-570. Taken from a review: Parniske & Downie. Locks, keys and symbioses. Nature 425: 569-570.

24. Rhizobium Formation of nodule primordia Bacteroid differentiation Nitrogen fixation Nod factor (specificity) Invasion through infection tube Attachment and infection Flavonoids (specificity)

25. Enlargement of the nodule, nitrogen fixation and exchange of nutrients Nodule development

27. (From Quaedvlieg et al. Plant Mol. Biol. 37: 715-727, 1998) Rhizobium encoding GFP from jellyfish as a marker Infection thread

28. Bacteria divide as they traverse infection thread

30. Physiology of a legume nodule

31. 6 days 7 days

33. Rhizobium Root Nodules

34. Non-symbiotic nitrogen fixation Cyanobacteria Anabaena Nostoc Aquatic: Terrestrial and rhizosphere-associated: Azospirillum Azotobacter Acetobacter Klebsiella Clostridium

35. The aquatic fern Azolla is the only fern that can fix nitrogen. It does so by virtue of a symbiotic association with a cyanobacterium (Anabaena azollae). -Co+Co A nitrogen-fixing fern

36. Another cyanobacterium on the palm Welfia regia in an epiphyllic relationship It is believed that these bacteria transfer some % of fixed N to the plants through the leaf surfaces

37. Nitrogen Fixation All nitrogen fixing bacteria use highly conserved enzyme complex called Nitrogenase Nitrogenase is composed of of two subunits: an iron-sulfur protein and a molybdenum-iron-sulfur protein Aerobic organisms face special challenges to nitrogen fixation because nitrogenase is inactivated when oxygen reacts with the iron component of the proteins

38. Nitrogenase All nitrogen fixing bacteria use highly conserved enzyme complex called Nitrogenase Nitrogenase is composed of of two subunits: an iron-sulfur protein and a molybdenum-iron-sulfur protein Aerobic organisms face special challenges to nitrogen fixation because nitrogenase is inactivated when oxygen reacts with the iron component of the proteins

39. Nitrogenase Complex Two protein components: nitrogenase reductase and nitrogenase Nitrogenase reductase is a 60 kD homodimer with a single 4Fe-4S cluster Very oxygen-sensitive Binds MgATP 4ATP required per pair of electrons transferred Reduction of N 2 to 2NH 3 + H 2 requires 4 pairs of electrons, so 16 ATP are consumed per N 2

40. Nitrogenase A 220 kD heterotetramer Each molecule of enzyme contains 2 Mo, 32 Fe, 30 equivalents of acid-labile sulfide (FeS clusters, etc) Four 4Fe-4S clusters plus two FeMoCo, an iron-molybdenum cofactor Nitrogenase is slow - 12 e - pairs per second, i.e., only three molecules of N 2 per second

41. Biological N fixation is energetically expensive, 16 ATP/N 2. Note that Molybdenum is a cofactor

42. FixL I O 2 inactivates nitrogenase O2O2 Heme oxidized FixL inactive FixJ nif regulon nifA nif Bacterial membranes Nitrogen fixation genes are repressed by oxygen fix

43. Malate to bacteria NH 4 + to plant nitrogen- fixing bacteroid containing Rhizobium N2NH 4 + Exchange of nutrients during Rhizobium-legume symbiosis TCA ATPADP+Pi

44. Assimilation of Nitrogen by the Host Indeterminate nodules –produce ammonia –exported to the host there by converted to glutamine, glutamate, and aspartate to asparagines –Asparagine is then exported to the shoot Determinate nodules –export xanthine (a purine) formed from glutamate and aspartate –Xanthine is converted in the host to ureides, allantoin and allantoic acid to be exported throughout the plant.

45. Ammonia assimilatory cycle: How nitrogen enters biological pathways NH 4 + Amino acids proteins Amino acids proteins purines pyrimidines  -ketoglutarate glutamate GDH + glutamateglutamine + ATP+ ADP + Pi GS + NH 4 + glutamineglutamate  -ketoglutarate GOGAT + Pathway 1 Pathway 2

49. Alfalfa Nodule

50. 1Enhancing survival of nodule forming bacterium by improving competitiveness of inoculant strains 2Extend host range of crops, which can benefit from biological nitrogen fixation 3Engineer microbes with high nitrogen fixing capacity Current approaches to improving biological nitrogen fixation What experiments would you propose if you were to follow each of these approaches?

51. Activation of nif promoters by NifA: A mechanism similar to RNAP(  54) activation by NtrC  54

52. Rhizobium’s bad brother: Agrobacterium tumefaciens Opines are an Agrobacterium-specific C- source to feed future generations Crown gall on rose and on grapevine

53. Chatt-Type Mechanism for Single Site Catalysis N 2 + 6H + + 6e - → 2NH 3

54. Some nitrogen fixing organisms

55. Rhizobium’s bad brother: Agrobacterium tumefaciens Opines are an Agrobacterium-specific C- source to feed future generations Crown gall on rose and on grapevine

56. END

60. Key metabolic step is conversion of fixed ammonia to organic N Fig. 8.7 N assimilation is reliant on a steady supply of C !!