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Redrawn from www.asahi-net.or.jp/~it6i-wtnb/BNF.html Nitrogenase enzyme complex Physical association of nif genes in Klebsiella pneumoniae Nitrogenase.

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Presentation on theme: "Redrawn from www.asahi-net.or.jp/~it6i-wtnb/BNF.html Nitrogenase enzyme complex Physical association of nif genes in Klebsiella pneumoniae Nitrogenase."— Presentation transcript:

1 Redrawn from Nitrogenase enzyme complex Physical association of nif genes in Klebsiella pneumoniae Nitrogenase MoFe protein Fe protein Electron transport Assembling Fe-Mo-Cofactor Regulator H D K T Y E NX U SVWZM F L A B QJ     

2 Redrawn from NtrC in absence of N-compounds NtrB ADPATP nifLA operon nitrC binding site  54 = nitrA binding site nif structural genes  54 = nitrA binding site nifA binding site P Regulation of nitrogen fixation (K. pneumoniae)

3 Nitrogen present, no transcription Function of NtrA,  54, the nitrogen  factor

4 Nitrogen absent, NtrB phosphorylates NtrC, which activates RNA polymerase Function of NtrA,  54, the nitrogen  factor P

5 Redrawn from NtrC in absence of N-compounds NtrB ADPATP nifLA operon nitrC binding site  54 = nitrA binding site nif structural genes  54 = nitrA binding site nifA binding site P Regulation of nitrogen fixation (K. pneumoniae)

6 Redrawn from NifA NifL transcription NtrC in absence of N-compounds NtrB ADPATP nifLA operon  54 = nitrA binding site NtrC P N-compound regulation of NifLA operon nif structural genes  54 = nitrA binding site nifA binding site

7 Redrawn from NifA NifL transcription NtrC in absence of N-compounds NtrB ADPATP nifLA operon  54 = nitrA binding site nif structural genes  54 = nitrA binding site N-compound regulation of NifLA operon NtrC P

8 Redrawn from NifA NifL in presence of O 2 or N-compounds transcription NtrC in absence of N-compounds NtrB ADPATP nifLA operon  54 = nitrA binding site nif structural genes  54 = nitrA binding site nifA binding site Oxygen and N-compound regulation of nif structural genes via nifL NtrC P

9 Measuring N 2 fixation rates

10 Acetylene reduction assay Football has been filled with acetylene Glass jars contain the plant samples being evaluated Sterile vacutainers (normally used to collect blood) are used to take the gas sample following incubation Several hundred samples can be taken each day

11 Acetylene reduction assay A typical trace following gas chromatography The greatest peaks are of residual acetylene Those next to them the ethylene peak

12 Hydrogen evolution assay Reduction of dinitrogen to ammonia N 2 + 8H + + 8e -  2NH 3 + H 2 H 2 is evolved at ratio of 1 molecule per 2 molecules of N 2 reduced –So, can use hydrogen sensor to measure H 2 evolution to quantify N 2 fixation

13 Hydrogen evolution assay Reduction of dinitrogen to ammonia N 2 + 8H + + 8e -  2NH 3 + H 2 H 2 is evolved at ratio of 1 molecule per 2 molecules of N 2 reduced –So, can use hydrogen sensor to measure H 2 evolution to quantify N 2 fixation

14 Hydrogen evolution assay

15 The operation of nitrogenase. The iron protein (Fe) takes electrons from central metabolism electron carriers and transfers them to the molybdenum iron protein (MoFe) expending a fair amount of ATP. N 2 is converted to ammonia and the electrons in H 2 are recycled by hydrogenase. (D. Benson)

16 Stable isotope assays GCMass separationCombustion detector ion source magnet

17 mass 29 ( 15 N 14 N) ion source magnet mass 30 ( 15 N 2 ) mass 28 ( 14 N 2 ) Mass Separation

18 Stable isotope lab

19 Table 15-3 (pg. 380 of text)

20 Lifestyles of N 2 fixing bacteria (diazotrophs) Free living Living in consortia –e.g. stromatolites, soil crusts Plant associative (living in rhizosphere) Symbiotic

21 Middle Proterozoic formations of the Hakatai Shale in Grand Canyon National Park. Lens cap is 55 mm. Living stromatolites Diazotrophic bacteria in consortia

22 Diazotrophic bacteria in consortia soil crusts

23 Cyanobacteria in soil crusts

24 Diazotrophic bacteria in consortia

25 Cyanobacteria Photosynthetic and dinitrogen fixing –heterocysts separate the two functions Anabaena Microcystis Nostoc Free-living

26 Cyanobacteria Oldest known fossils –3.5 bybp (oldest rocks are 3.8 bypb) filamentous Palaeolyngbya colonial chroococcalean

27 Cyanobacteria heterocysts Heterocyst

28 Symbiotic N 2 -fixation: Azolla - Anabena S. Navie

29 Symbiotic N 2 -fixation: Azolla - Anabena

30 Rice-Azolla-Fish, China Azolla to feed cows, Thailand Rice-Azolla-Ducks, Korea Takao Furuno Symbiotic N 2 -fixation: Azolla - Anabena

31 © Paul Cox Cycas micronesica

32 Cycad root nodules

33 © Paul Cox Cycas micronesica  -N-methylamino-L-alanine (BMAA)

34 Guam flying fox (Pteropus mariannus) bio-magnification  -N-methylamino-L-alanine (BMAA)

35 Flying Fox with Prunes and Cream Sauce 6 flying foxes (in case you are wondering, these are bats) 1 pound prunes 1 ¼ cup white wine salt, pepper 1/4 cup flour 2 oz. butter 1 tbsp red currant jelly 1 cup thick cream Remove the flesh from the flying foxes. Either plunge the animals in boiling water for a while, then skin them and remove the flesh from the bones, or roast the animals for a little over an open fire, remove, and when cool, break open down the backbone and remove the flesh from the skin. Soak the prunes overnight in 1 cup of the wine, then heat for about ten minutes in the wine before using. Season the flying fox meat with salt and pepper and roll in flour. Saute in butter over a low heat until brown. Add the rest of the wine, cover and cook another 20 minutes. Add the juice from the prunes, and transfer the prunes onto a serving dish. Cook the meat in the prune juice, uncovered, for another 10 minutes, then place on the serving dish with the prunes. The preparation of this recipe requires an ingredient which is now a protected species.

36 Spinal cord Amyotrophic Lateral Sclerosis  -N-methylamino-L-alanine Mimics glutamate and acts as agonist at glutamate receptor

37 Frankia in alder root nodules Actinorhizal symbioses

38 Frankia vesicles Frankia root nodules Spores & hyphae

39 Colletia Dicaria Ceanothus

40

41 Myrica faya Native to Canary Islands Actinorhizal root nodules

42 Invasive in Hawai’i (no native N 2 - fixing pioneers) Myrica faya

43 Legumes & N-fixing bacteria © Simms Soil-dwelling rhizobia infect legume roots

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

45 Legume & N-fixing bacteria Rhizobia engulfed into nodule cells Differentiate into bacteroids © Simms

46 Illustration: M.S. Hargrove Photo: D. Hume Photo and illustration: R. F. Denison Leghemoglobin

47 Rhizobium Agrobacterium Sawada et al Sinorhizobium Ensifer Mesorhizobium, Bradyrhizobium Nitrobacter, Afipia Methylobacterium Sinorhizobium Devosia Azorhizobium Ralstonia Burkholderia Rickettsia Bartonella Aminobacter, Phyllobacterium   -Rhizobial symbioses have evolved ~10 times -Nested parasites & non-symbionts Brucella Proteobacteria

48 Symbiotic plasmid of Rhizobium etli Víctor González et al. Genome Biology (6):R36

49 The nodulation genes nodABCDIJ are represented in blue The nitrogen-fixation genes nifHDKNEXAB, fixABCX and fdxBN are represented in yellow plasmid 42d M. loti MAFF plasmid NGR234a M. loti MAFF B. japonicum S. meliloti pSymA Víctor González et al. Genome Biology (6):R36

50 Victor Kunin et al. Genome Res. 2005; 15: Figure 3. Three-dimensional representation of the net of life

51

52 Hazards of symbiotic life (or an animal dispersal agent?) Clover Root Curculio Sitona hispidula (Fabricus)


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