Getting an N Fix

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. nitrogenase N2 NH3 Biological Nitrogen Fixation

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

4. Rhizobium-legume symbioses Host plantBacterial symbiont Alfalfa Rhizobium meliloti Clover Rhizobium trifolii Soybean Bradyrhizobium japonicum Beans Rhizobium phaseoli Pea Rhizobium leguminosarum Sesbania Azorhizobium caulinodans Complete listing can be found at at: http://cmgm.stanford.edu/~mbarnett/rhiz.htm Both plant and bacterial factors determine specificity

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 Fixed nitrogen (ammonia) Fixed carbon (malate, sucrose) rhizobia

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

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

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. 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 Role of Root Exudates

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

17. Correctsignal Incorrect signal • Signals early in infection • Complex handshaking between legume root and rhizobium

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

20. 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 Role of Root Exudates

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

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

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

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

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

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 Aquatic: Cyanobacteria Anabaena Nostoc Terrestrial and rhizosphere-associated: Azospirillum Azotobacter Acetobacter Klebsiella Clostridium

35. A nitrogen-fixing fern -Co +Co 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).

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 N2 to 2NH3 + H2 requires 4 pairs of electrons, so 16 ATP are consumed per N2

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 N2 per second

41. Biological N fixation is energetically expensive, 16 ATP/N2. Note that Molybdenum is a cofactor

42. N2 NH4+ Exchange of nutrients during Rhizobium-legume symbiosis Malate to bacteria nitrogen- fixing bacteroid containing Rhizobium TCA NH4+ to plant ATP ADP+Pi

43. 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.

44. GOGAT glutamate -ketoglutarate glutamine + Ammonia assimilatory cycle: How nitrogen enters biological pathways Amino acids proteins purines pyrimidines Pathway 1 GS NH4+ glutamate glutamine + + ATP + ADP + Pi Pathway 2 GDH NH4+ -ketoglutarate glutamate + Amino acids proteins

48. Alfalfa Nodule

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

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