N Cycle Exam 4 - Final Exam

Question 1

What causes the inactivation of nitrogenase?

Answer 1

O2, large amounts of available nitrogen, low carbon and energy sources, and nitrite binding to leghemoglobin.

Question 2

What is the ratio of bound O2 to Free O2?

Answer 2

10,000:1

Question 3

How do aerobic bacteria protect nitrogenases from O2 inactivation?

Answer 3

1. Rapid removal of O2 by respiration
   Example: Azotobacter vinelandii (large rod shaped free living nitrogen fixing bacteria and aerobic)
2. Production of slime layers that retard O2
   diffusion from the atmosphere
   Example: Azotobacter vinelandii produce a thick slime layer consisting of alginate. It is thicker when there are higher conc of O2
3. Conformation protection
   Example: Azotobacter produces a protein that binds to nitrogenase and changes the conformation of the complex when O2 levels are too high, so nitrogen fixation stops and the complex is protected. When conc levels are low again, N2 fixation starts back up.
4. Production of specialized cells
   Example: N2 fixation only occurs in special cells. These include heterocysts in cyanobacteria, which have photosystem I which produce ATP without producing O2 (unlike photosystem II). Another example is N2 fixation occurring in vesicles in Frankia. These vesicles have thick wall with lipids that slow down O2 diffusion. Frankia are actinobacteria that are filamentous, and the vesicles form at the end of the filament.
5. Separation of N2 fixation and oxygen-evolving processes
   -Temporal and spatial. Temporal = fix N2 in dark, so no O2 produced. Spatial = cells that fix nitrogen but don’t have photosystem II, so no O2 produced.
6. Leghemoglobin binds free O2
   -Bound O2 to free O2 ratio = 10,000:1

Question 4

What are the types of nonsymbiotic nitrogen fixing bacteria?

Answer 4

-Free-living heterotrophs
-Free-living autotrophs
-Associate N2-fixing bacteria

Question 5

Describe Free-living Heterotrophs.

Answer 5

-Need organic carbon as their carbon and energy source.
-Some are aerobes, anaerobes, microaerophilic bacteria, or facultative aerobes
-Free-living bacteria fix LESS nitrogen than symbiotic nitrogen fixing bacteria.

Question 6

Free-living Heterotrophs:
Give examples of the four different free-living heterotrophs (aerobes, anaerobes, microaerophilic bacteria, and facultative aerobes).

Answer 6

Aerobes: Azotobacter (doesn’t grow if pH below 6), Beijerinckia(grows pH 3-9), Derxia (grows in pH 5-9)
Microaerophilic bacteria: Azospirillum (can form a loose association with some plants)
Facultative aerobes: Klebsiella & Bacillus
Anaerobes: Clostridium, Desulfovibrio, Methanosarcina

Question 7

Describe Free-living autotrophs.

Answer 7

-Can fix nitrogen and are free-living.
-Carry out photosynthesis and use inorganic carbon as carbon source and light as their energy sources

Question 8

Free-living autotrophs:
Give an example of a free-living autotroph, and describe that autotroph.

Answer 8

Cyanobacteria.
-Fix MORE nitrogen than free-living bacteria because use light as their energy source, so it is not limited.
-Are unicellular
-Can be filamentous heterocystous or filamentous nonheterocystous (usually found in marine envirn)
-If they form heterocysts, then the heterocyst has a thick cell wall that is rich in glycolipids, slowing down O2 diffusion.
-Examples of filamentous heterocystous: Nostoc and Anabaena

Question 9

Describe Associate N2-Fixing Bacteria.

Answer 9

-Form loose association w/ plants.
-Place themselves close to plants in the rhizosphere and some in the plant roots
-Nonobligative (don’t have to be there) relationship between plants and bacteria

Question 10

Associate N2-fixing bacteria:
What are the different places where bacteria associate?

Answer 10

-Grasses and grain crops: bacteria here fix more nitrogen than free-living heterotrophs. Plants provide carbon source to bacteria, and then bacteria can fix more nitrogen, which helps the plants.
-Leaf surface: Not a lot of nutrients here, but enough to feed bacteria. Ex: Klebsiella & Bacillus
-Roots: Some bacteria are present in the surface layer of plant root cells. Ex: Azospirllum

Question 11

What is the associate N2-fixing bacteria that forms a close association with sugar cane?

Answer 11

Acetobacter diazotrophicus
-Found in rhizosphere, root tissue, and in vascular elements of sugar cane. When sugar cane produces sugar, these bacteria get it first. Then, bacteria fix nitrogen, which the plants benefits from.

Question 12

Describe nitrogen fixation in a landrace of maize.

Answer 12

Found association of N2 fixing bacteria with corn. These indigenous plants have aerial roots, and a gel material around it. Bacteria live here and fix nitrogen.
-29-82% of the plant nitrogen is derived from the atmosphere
-These plants live in N-depleted soils**

Question 13

Describe Symbiotic N2-fixing bacteria.

Answer 13

Association is obligate, so there has to be fixed partners.
A major symbiotic relationship is the legume-rhizobia symbiosis:
Legumes (beans, peanuts, soybeans) have 7000 genera and 14,000 species. They fix way more nitrogen than free-living heterotrophs and autotrophs.
Examples: Rhizobium, Bradyrhizobium, and Azorhizobium

Question 14

How many genera and species are there of Rhizobia? What kind of bacteria are there here? What family are most in?

Answer 14

-13 genera and >90 species
-Alpha and beta-proteobacteria
-Most in Rhizobiaceae family of alpha-proteobacteria

Question 15

What are the common genera of rhizobia?

Answer 15

1. Azorhizobium
2. Bradyrhizobium
3. Mesorhizobium
4. Rhizobium
5. Ensifer

Question 16

What are the different types of symbiotic N-fixing bacteria?

Answer 16

-Legume rhizobia symbiosis
-Actinorhizal symbiosis
-Symbiosis involving cyanobacteria
-Phyllosphere N2 fixation

Question 17

Actinorhizal symbiosis:
What bacteria is involved here? What kind of plants are present in this symbiosis?

Answer 17

-Actinobacteria is involved (Frankia)
-25 genera of nonleguminous plants: together they fix nitrogen.

Question 18

Actinorhizal symbiosis:
What does Frankia form a relationship with? Describe this bacteria. How much nitrogen is fixed?

Answer 18

-Forms a relationship with trees and shrubs. For example, alder and bayberry trees are woody plants that form this symbiosis.
-Filamentous and most are G+
-Fixes a significant amount of nitrogen, way more than free-living

Question 19

Actinorhizal symbiosis:
What are anthocyanins?
How do Frankia form this symbiosis, and what does this lead to?

Answer 19

-Anthocyanins cause the red/pink color in nodules (NOT LEGHEMOGLOBIN, only for legumes).
-Frankia form this symbiosis by root hair infection or intercellular invasion (enter through root cortical cells). This leads to nodule formation.

Question 20

Symbiosis involving cyanobacteria:
What does cyanobacteria form a symbiotic relationship with?

Answer 20

1) Lichens: together they can fix more nitrogen then if it was just cyanobacteria by itself
2) Azolla-Anabaena symbiosis: Azolla is the water fern and Anabaena is the cyanobacterium. Together they fix a significant amount of nitrogen because the number of heterocysts increases.

Question 21

Describe Legume-Rhizobia Symbiosis.

Answer 21

Legumes and rhizobians by themselves do not fix nitrogen. Once they form a symbiotic relationship, they can fix a lot of nitrogen.

Question 22

Legume-Rhizobia Symbiosis:
What are the stages of nodule formation (clover and beans)?

Answer 22

1. Recognition and attachment
2. Root hair curling
3. Invasion
4. Formation of the infection thread
   From here, can split off to either:
   formation of bacteroids (where N2 fixation occurs), or rapid root cell division
   *Note: bacteroids are surrounded by periobacteriod membranes and Leghemoglobin is present in between the bacteroid and the periobacteroid membrane, regulating the O2 concentration in root nodules.
5. Formation of mature nodules

Question 23

What are flavones?

Answer 23

Plants produce a group of chemicals called flavones. These are aromatic compounds. They attract bacteria to the plant and help support the growth of the bacteria near the plant roots.

Question 24

In the first first stage of root nodule formation, there are two important proteins produced by rhizobia involved. What are they?

Answer 24

In the recognition and attachment stage, once the attachment becomes permanent, Rhicadhesin (calcium binding protein) and Lectin (carbohydrate binding protein).

Question 25

In peanut root nodule formation, where do rhizobia enter?

Answer 25

Through cracks in the walls.
-Root nodules usually form where the lateral roots come out, it has a crack there and then the bacteria can enter there.

Question 26

Legume-Rhizobium Symbiosis:
What are the molecular changes during nodule formation?

Answer 26

-nod (italicized) genes control nodulation process
-Morphological changes associated with nodule formation are parallel w/ molecular changes.

Question 27

Where are nod (italicized) genes present?

Answer 27

On Sym plasmid.

Question 28

Do rhizobium nodulate all plants?

Answer 28

No

Question 29

What are examples of nod genes in rhizobia and what do they do?

Answer 29

-nodD controls transcription of other nod genes
-nodE and nodL are involved in host range
-nodABC controls production of oligosaccharides (nod factors)

Question 30

What is a nod factor?

Answer 30

An important chemical compound that is a plant morphogen that causes morphological change of plants. It has a backbone of acetylglucosamine (~3-6 of these).
-Nod factors at low concentration can cause root hair curling, plant cell division and nodule formation.
-Can be different side chains, even though same backbone. So, rhizobia can have different not factors

Question 31

Legume-Rhizobia Symbiosis:
Effective and ineffective nodules. What indicates effectiveness and ineffectiveness?

Answer 31

-Pink color produced by leghemoglobin indicates effectiveness.
-White, green, or brown color indicates ineffectiveness.
-Some are ineffective b/c some rhizobium strains are not effective in terms of fixing nitrogen (can still from nodules, but won’t fix nitrogen)

Question 32

Will all infected root hairs form nodules?

Answer 32

NO! Less than 5% do.
Not all nodules that are formed are infected, either.

Question 33

What intensity is correlated to the amount of nitrogen fixed?

Answer 33

Color intensity. Bright red vs light red inside nodule, bright red is fixing more nitrogen.

Question 34

Legume-Rhizobia Symbiosis (Nodule physiology):
In terms of the nitrogen that is formed, the first product formed is __________.
What excretes this?

Answer 34

Ammonia (NH3).
Bacteroids excrete ammonia and give it to the plants and then the plants synthesize different kinds of nitrogen containing compounds (can be glutamine, asparagine, and ureides). These organic compounds get transported to different parts of the plants in order to meet nitrogen demand.
Take home: Plants export organic N to different parts for growth.

Question 35

The N forms exported are?

Answer 35

Glutamine, Asparagine (Amino acids)
Ureides (urea derivatives): Allantoin & Allantoic acid

Question 36

What are the factors affecting nodule formation and nitrogen fixation by legume-rhizobia symbiosis?

Answer 36

-pH: Rhizobium usually prefer neutral pH. Below 5, N2 fixation will not occur.
-Temperature: rand 10-37 degrees C. 25 degrees optimum.
-Mineral nutrition: P, Mo, Fe, Ni. N2 fixation is energy intensive and needs ATP. Phosphorous (P) is the P in ATP. Mo and Fe are important elements in nitrogenase complex. Ni is present in dinitrogenase.

Question 37

Legume-Rhizobium Symbiosis:
Inoculation… If a field has never had a legume, should you inoculate?

Answer 37

Yes, should inoculate. If there has been legume planting in the past 3-4 years, then don’t have to. Still beneficial to inoculate every year. This is mostly because rhizobium does not survive well in the soil from previous years.

Question 38

Legume-Rhizobium Symbiosis:
Changes in the rates of N2 fixation as a function of time and CO2 concentration.

Answer 38

When it is time for legumes to fill their seed pod, N2 fixations slows, and when the legume starts fully flowering, N2 fixation stops b/c there is no more carbon to send to the root nodule. It is carbon expensive for symbiosis to happen. Plants prioritize producing seeds when they get to reproductive stage.

Question 39

What kind of organisms fix the least amount of nitrogen?
Cyanobacteria are what kind or organisms, and use what as their energy source?

Answer 39

Free-living organisms (especially free-living heterotrophs b/c this is carbon expensive).
Autotrophs, so use light as energy source (not limited) and they can fix more nitrogen because of this.

Question 40

Free-living heterotrophs fix the least amount of nitrogen. Cyanobacteria produce a bit more than them. What is the next big category that fixes more nitrogen than cyanobacteria?

Answer 40

The Symbiotic relationships produce more than cyanobacteria (especially Azolla-cyanobacterial symbiosis, fixes a shit ton).
Generally, legumes fix the next highest amount of nitrogen. One legume that sticks out is Alfalfa, with a max nitrogen fixation of 600 kg/ha/yr.