Plant-associated Proteobacteria (and a few outsiders): the good and the bad

Transcription

1 Plant-associated Proteobacteria (and a few outsiders): the good and the bad nitrogenase N 2 NH 3

2 Today s Topics: 1. Rhizobeacae and other nitrogen-fixing genera 2. Nitrogen fixation and why we need it 3. Examples of nitrogen-fixing symbioses in plants 4. Processes of nodulation 5. Non-symbiotic nitrogen-fixing Proteobacteria 6. Cyanobacterial associations 7. Crown gall: the selfish doings of Agrobacterium tumefaciens

3 Prokaryotes Deinococci Spirochetes Greensulfur Bacteroides Thermotoga Green, nonsulfur Chlamydiae Gram positive Cyano bacteria d Desulfoivbrio Proteobacteria g b a E. coli Klebsiella Azotobacter Rhizobium Bradyrhizobium Sinorhizobium Agrobacterium Azospirillum Herbaspirillum

4 Ecology of nitrogen-fixing bacteria

5 Biological nitrogen fixation: N flavodoxin - + 8H MgATP H 2 O nitrogenase 2NH OH + 8 flavodoxin + 16 MgADP + 16H 2 PO 4- + H 2 1. Rare, extremely energy consuming conversion because of stability of triply bonded N 2 2. Produces fixed N which can be directly assimilated into N containing biomolecules

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

7 N 2 O nitrous oxide Denitrification Denitrification N 2 dinitrogen gas (78% of air) The Nitrogen Cycle BIOSPHERE Nitrogen fixation the Haber Process and lightning NH + 4 ammonium NO 3 - nitrate nitrification NO 2 - nitrite nitrification

8 The Nitrogen Cycle N 2 dinitrogen gas (78% of air) Biological nitrogen fixation N 2 O nitrous oxide NO 3 - nitrate nitrification Plants uptake consumption Prokaryotes NO 2 - nitrite assimilation Animals NH + 4 ammonium

9 A growing population must eat! Combined nitrogen is the most common limiting nutrient in agriculture Estimated that 90% of population will live in tropical and subtropical areas where (protein-rich) plant sources contribute 80% of total caloric intake. In 1910 humans consumed 10% of total carbon fixed by photosynthesis, by 2030 it is predicted that 80% will be used by humans.

10 Why chemical fertilizers aren t the answer Consumes 1.4% of total fossil fuels annually Production of nitrogenous fertilizers has plateaued in recent years because of high costs and pollution Estimated 90% of applied fertilizers never reach roots and contaminate groundwater

11 Rhizobium-legume symbioses Host plant Alfalfa Clover Soybean Beans Pea Sesbania Bacterial symbiont Rhizobium meliloti Rhizobium trifolii Bradyrhizobium japonicum Rhizobium phaseoli Rhizobium leguminosarum Azorhizobium caulinodans Complete listing can be found at at: Both plant and bacterial factors determine specificity

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

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

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

15 Physiology of a legume nodule

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

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

18 Nod factor biosynthesis NodM NodC NodB Nod factor R-group decorations determine host specificity Nod Factor: a lipooligosaccharide

19 Rhizobium Attachment and infection Flavonoids (specificity) Nod factor (specificity) Invasion through infection tube Bacteroid differentiation Nitrogen fixation Formation of nodule primordia From Hirsch, New Phyto. 122,

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

21 Bacteria divide as they traverse infection thread

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

23 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

24 Inoculation of a mutated Sinorhizobium strain does not transfer fixed N to the plant wild-type mutant Genes & Development 11:1194, 1997

25 wt glnb10 6 days 7 days wt glnbp5 Genes & Development 11:1194, 1997

26 Azorhizobium caulinodans on Sesbania

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

28 Plant-associated nitrogen fixation: the endophytes and epiphytes Acetobacter diazotropicus Lives as an endophyte of sugarcane and various other monocots and some dicots On sugarcane

29 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).

30 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

31 Symptoms of crown gall

32 Agrobacterium tumefaciens transforms plant cells Transgenes produce OPINES, unique amino acid-like molecules, as well as plant hormones

33 The End

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

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

36 Nitrogen fixation genes are repressed by oxygen O 2 inactivates nitrogenase Bacterial membranes FixL I O 2 Heme oxidized FixL inactive FixJ nifa nif nif fix nif regulon

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

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

40 Exchange of nutrients during Rhizobium-legume symbiosis nitrogenfixing bacteroid containing Rhizobium Malate to bacteria TCA ATP ADP+Pi N2 NH 4 + NH 4 + to plant

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