MIC 105 MT 1 lecture 5

Nitrogen

• 3rd-most common

element in most

organisms

• N2 gas = 79% of

atmosphere

• Earth's crust contains N

in ammonium salts, but

is almost entirely

inaccessible to

microbes

The Nitrogen Triangle

• We can simplify

nitrogen cycling into

the conversions

between three (ok, 4)

key molecules

• NO2/NO3: oxidized

forms

• NH4: reduced form

• N2: Oxidation # = 0

most reduced form of N

NH3 and NH4+

Most oxidized form of N

NO3-, HNO3

which type of N is incorporated into cells

NH3 NH4

what is the most abundant form of N

N2

Denitrification

NO3--> N2

Nitrogen fixation

N2 -> NH4+

Nitrification/Comammox

NH4+-> NO3-

Assimilatory Nitrate reduction

NO3--> NH4+

N≡N bond is

very stable

Nitrogen Fixation bacteria

proteobacteria: Azomonas, acidithiobacillus, purple bacteria, rhizobium

firmicutes:Clostridium, Heliobacteria

Cyanobacteria:gloeothece

Archaea:methanosarcina, methanocaldococus

chlorobi: green sulfure bacteria

actinobacteria: frankia

Nitrogenase

key enzyme

complex, reduces N2 to NH3.

• very oxygen sensitive and is

rapidly and irreversibly

inactivated by oxygen.

N2 fixation energy consumption

N2 + 8e- + 8H+ + ~18 ATP -> 2 NH3 + H2 + ~18 ADP + ~20Pi

how many electrons are needed to fix N

8, 6 for the reaction and 2 to make H2 as the product

N2 fixation gene cluster (Klebsiella)

~20 genes are required for nitrogenase production,

homologous in all known organisms.

N2 evolution

N2 fixation evolved once, was likely present in an early ancestor, lost

from some lineages, and acquired by others through horizontal gene

transfer.Anaerobes: e.g., Clostridium, green sulfur (Chlorobi), etc.?

No Oxygen = No Problem!

Nitrogenase production is highly regulated:

only made under

anoxic nitrogen-limiting conditions.

how Microaerophiles deal with O2

intracellular O2 is very low because it is used as fast as it diffuses in.

Respiration rate keeps O2 concentration low enough to protect nitrogenase.

Azotobacter

obligate aerobes capable of N2

fixation under full oxygen atmosphere

High respiration rate, faster than oxygen

diffusion.

Polysaccharide capsule retards O2 diffusion

into the cell

how Cyanobacteria fix N with O2 present

seperates photosynthesis and N2 fixation in two ways: temporally(time)(unicelluar cyanobacteria), Spatially (filamentous cyanobacteria)

Filamentous cyanobacteria

(Nostoc; Anabaena), form heterocysts

heterocysts

nitrogen-fixing cells, terminally

differentiated, depend on the

adjacent cells for

carbohydrate to generate

reductant.

Genes for nitrogenase, glutamine

synthetase (converts glutamate +

ammonia to glutamine), for increased

respiration rate (e.g. cytochrome oxidase)

are derepressed in heterocysts.

Anabaena

• Heterocysts do not fix CO2 (acquire C from adjacent cells)

• Do not produce O2 (PSII is downregulated)

• high respiration rate

• surrounded by a thick cell wall containing glycolipids and polysaccharides that limits

diffusion of O2.

rhizobia

group of symbiotic N2 fixers, Gram- motile rods, mostly members of the alpha Proteobacteria (and a few Betas). They are aerobic heterotrophs.

rhizobia are only capable of N2 fixation when

in symbiotic association with plants. form root nodules on legumes (plants that bear seeds in pods).

Nodule formation involves

both bacterium- and plant-specific gene expression and

cellular differentiation.

which bacteria is soy beans associated with when orming nodules

Bradyrhizobium japonicum

root nodule formation stages

recognition of correct partner and attachment of the bacterium to the root hair

invasion of the rhizobia into the plant root

diffrentiation of plant cells into nodule tissues and bacterial differentiation into nitrogen fixing bacteroids.

how do the plant and bacterium recognize eachother?

plant roots secrete flavonoids into the rhizosphere(layer of soil surrounding roots)

flavonoids

chemoattractants for rhizobia, and induce rhizobia to produce nod factors (determines host microbe specificity)

nod factors

stimulate root hair curling and producrion of a tube through the root hair called the infection thread for rhizobia to travel through, they also stimulate plant cells to divide, fomring nodules

leghemoglobin

red iron containing O2 binding protein produced when the plant and rhizobia are in association

Sym plasmid

Genes involved in nodule

formation and nitrogen

fixation

Nutrient exchange within the nodule

Fixed nitrogen provided to

the plant.

Rhizobium gets

C-source and a protected

environment.

Frankia

Gram+ filamentous actinomycete

symbiosis with Alder trees and other plants.

Alders

'pioneer' trees that can colonize nutrient poor soil, possibly due to associations

with Frankia, which provides fixed N2.

what can frankia do thats similar to azotobacter

fix nitrogen at full oxygen tension and when in symbiosis

how does frankia fix oxygen in O2 environments?

sequester nitrogenase in terminal swellings that have thick walls that retard gas diffusion, protecting nitrogenase from O2

Potential application of N2 fixation

H2 gas production for biofuel

fate of NH4

easily taken up by microbes and plants, and binds well to soil particles even through rain.

how does NH4 feed into the central metabolism

through generation of glutamine

nitrifying bacteria phyla

proteobacteria, nitrospirae

Nitrification

oxidation of ammonia to nitrate, known to be carried out by 2 physiologically distinct groups

ammonia oxidizers

ammonia to nitrite (NO2)

NH3 + 11/2O2 -> NO2- + H2O

eg nitrosococcus

nitrite oxidizers

convert nitrite to nitrate

NO2- + H2O -> NO3- + 2 H+ + 2 e-

eg nitrobacter

how do these oxidizers fix CO2

they are aerobic chemolithoautotrophs that use the calvin cycle to fix CO2

who is a AOB

beta and gamma proteobacteria and the nitrospira lineage

Ammonia monooxygenase

(AMO; membrane bound) converts ammonia to

hydroxylamine, oxygen is substrate.

NH3 + O2 + 2H+ + 2e--> NH2OH + H2O

evolutionarily related to MMO (CH4 -> CH3OH)

Hydroxylamine

oxidoreductase

(periplasmic) catalyzes NH2OH -> NO2-Energy conserved

as PMF

who is a NOB

α, β, γ and δ Proteobacteria and the

Nitrospira lineage

Nitrite oxidoreductase

catalyzes NO2--> NO3-

Energy conserved as PMF

Ammonia-Oxidizing Bacteria and Nitrite-Oxidizing Bacteria

Obligate aerobes

Widespread in soil and water, especially when ammonia is high, e.g., during protein

degradation [ammonification] and in sewage treatment plants

Nitrite, which is toxic and mutagenic, does not accumulate in the environment.

Tight mutualistic symbiosis between AOB and NOB.

Ammonia oxidizing Archaea

Chemolithoautotrophic ammonia oxidizing marine Archaea

First identified by metagenomic sequencing: found 16S gene on same DNA fragment as amo gene

ammonia-oxidizing archeaon example

Nitrosopumilus maritimus part of Thaumarchaeota

Ammonia oxidizing Archaea habitat

ocean and soil, can grow on nh3 concentration 100x lower than AOB

how do AOA fix CO2?

use 3-hydroxypropionate/4-hydroxybutyrate cycle

"Comammox

complete ammonia oxidation to nitrate by single organisms

genome Nitrospira

had genes for ammonia monooxygenase, hydroxylamine oxidoreductase, and nitrite oxidoreductase (comammox)

Nitrification & Comammox importance/ applications

Detrimental for agriculture. Removes usable nitrogen (NH4

+) from agricultural fields and

introduces it into streams and ponds, which causes algal blooms.

NH4

+ binds to negatively charged soil particles and is available for uptake and utilization.

Nitrate (NO3

-; also usable N source) is very water soluble and readily leached from soils in

rainwater runoff.

Result: more fertilizer must be used.

Nitrapyrin, an inhibitor of nitrification (specifically AMO), is added to ammonia fertilizer

on ag fields

Anammox

Anaerobic ammonia oxidation

NH4+ + NO2--> N2

Oxidization of ammonia with nitrite as the

electron acceptor to yield N2 gas under anoxic

conditions

who uses anammox

planctomycetes

Planctomycetes

Unconventional cell envelope: Gram negative, many have

S-layer

Internal compartments, including a membrane-bound structure containing the

nucleoid

aerobic organoheterotrophs

anammox bacteria are

obligately anaerobic

chemolithoautotrophs that fix CO2 using the

reductive acetyl-CoA pathway

anammoxosome

compartment in anammox surrounded by a bilayer membrane

Hydrazine is toxic and mutagenic (DNA damaging);

compartmentalize to protect cellular components; ladderanes keep

permeability low

metabolism is slow (reaction cycles 15 times to fix one CO2

molecule; generation time of 10-30 days!), so any leakage of

metabolites would be significant

ladderanes

Unique lipids with ring structures in anammoxosome membrane

reduce membrane

permeability

AAO

NH4+ + NO2--> N2 + 2 H2O

Toxic hydrazine (N2H4) is an intermediate

Key enzymes of AAO

NIR - Nitrite reductase

HZS - hydrazine synthase

HDH - hydrazine dehydrogenase

Denitrification

NO3--> NO2--> NO -> N2O -> N2

conversion of nitrate to gaseous nitrogen compounds

nitrate respiration

Bacteria carrying out have membrane-bound or periplasmic enzymes to

couple substrate oxidation to nitrate reduction to generate PMF, which allows ATP

synthesis.

Key enzyme of nitrate respiration

itrate reductase: NO3--> NO2-

Genes encoding nitrate reductase are only expressed anaerobically, as O2 is the preferred

terminal electron acceptor for respiration

Applications/ importance of Denitrification

Detrimental for agriculture: converts NO3

-, a good nitrogen source, into nitrogen gas.

Nitrogen oxides (NO, N2O) = greenhouse gases and contribute to global warming.

Good for wastewater treatment:

Nitrates linked to reproductive

problems, cancer, and 'blue

baby syndrome', in which nitrite

decreases the O2 carrying

capacity of hemoglobin.

Denitrification & Anammox importance

Both are beneficial in wastewater treatment.

Remove usable sources of nitrogen, so water released into rivers and streams has fewer

algal blooms, reduction in anaerobic 'dead zones' which result in fish death.