Bacterial Physiology: Nitrogen Metabolism and Fixation

Principles of Inorganic Metabolism

Microorganisms utilize inorganic molecules, such as derivatives of sulfur, nitrogen, and iron, in diverse ways to support both energy metabolism and biosynthesis. These metabolic pathways are categorized into three primary types:

• Assimilatory Inorganic Metabolism: This involves the reduction of inorganic compounds specifically for their incorporation into organic cellular material. The goal is biosynthesis rather than energy generation.

• Dissimilatory Pathways: In these pathways, inorganic compounds serve as terminal electron acceptors in respiration. The focus is on the generation of energy rather than the accumulation of the compound within the cell.

• Oxidative Pathways: These involve the oxidation of inorganic compounds to serve as a source of electrons and energy for the cell.

Inorganic Nitrogen Assimilation

Nitrogen assimilation typically follows a two-stage process to transform inorganic nitrogen into forms that can be utilized for organic synthesis.

Nitrate Reduction to Ammonia

Nitrate ($NO_3^-$) is stepwise reduced to ammonia ($NH_3$/$NH_4^+$) through a series of intermediates:

1. Nitrate ($NO_3^-$) is reduced to Nitrite ($NO_2^-$) using $2e^-$ and $2H^+$.

2. Nitrite is further reduced, passing through a Nitroxyl intermediate ($[NOH]$).

3. Further reduction leads to Hydroxylamine ($NH_2OH$).

4. Finally, Ammonia ($NH_4^+$) is produced. This stage involves the consumption of $6e^-$ and multiple protons ($H^+$), releasing water ($H_2O$) as a byproduct.

Ammonia Incorporation: The GS/GOGAT Pathway

Once ammonia is formed, it is incorporated into the amino acids glutamine and glutamate. This process, known as the GS/GOGAT pathway, provides the foundational nitrogen sources for all other nitrogen-containing organic compounds in the cell.

1. Glutamine Synthetase (GS): This enzyme adds ammonia to a molecule of glutamate to produce Glutamine. This step is energy-intensive and requires the hydrolysis of $ATP$.

2. Glutamate Synthase (GOGAT): This enzyme takes the substrates Glutamine and $\alpha$-ketoglutarate ($akg$) to produce two molecules of glutamate. This step requires reducing power, where $NADPH$ is oxidized to $NADP^+$.

3. These resulting glutamate and glutamine molecules serve as the primary nitrogen donors for the synthesis of nucleotides, amino acids, and other cellular components.

The Global Nitrogen Cycle

Microorganisms are the primary drivers of the nitrogen cycle, mediating transitions between various oxidation states of nitrogen in the environment:

• Nitrogen Fixation: Atmospheric nitrogen ($N_2$) is converted into ammonium ($NH_4^+$) by nitrogen-fixing soil bacteria and bacteria in legume root nodules.

• Assimilation: Plants and microbes take up nitrogen for use in biological molecules.

• Ammonification: Decomposers (aerobic and anaerobic bacteria and fungi) convert organic nitrogen from dead matter back into ammonium ($NH_4^+$).

• Nitrification: Nitrifying bacteria oxidize ammonium ($NH_4^+$) into nitrites ($NO_2^-$) and then into nitrates ($NO_3^-$).

• Denitrification: Denitrifying bacteria reduce nitrates ($NO_3^-$) back into atmospheric nitrogen ($N_2$).

Biological Nitrogen Fixation

Nitrogen fixation is the biological conversion of atmospheric nitrogen ($N_2$) into ammonia ($NH_3$). This capability is not universal among bacteria but is restricted to specific groups.

Symbiotic Nitrogen Fixation

Certain bacteria establish symbiotic relationships with plants to fix nitrogen in exchange for carbon sources:

• Rhizobium and Bradyrhizobium form symbioses with leguminous plants such as soybeans, alfalfa, and clover.

• Anabaena azollae is a cyanobacterium that forms a symbiotic relationship with the water fern Azolla.

Free-Living Nitrogen Fixation

Many soil and aquatic bacteria are capable of fixing nitrogen independently without a host plant.

The Nitrogenase System

Nitrogen fixation is performed by the multicomponent nitrogenase enzyme complex. The overall chemical reaction is as follows:

$N_2 + 8\,H^+ + 8\,e^- + 16\,ATP \rightarrow 2\,NH_3 + H_2 + 16\,ADP + 16\,Pi$

Enzyme Components

The nitrogenase complex consists of two primary oxygen-sensitive proteins:

• Component I (Dinitrogenase): Also known as the molybdenum-iron (MoFe) protein. It provides the site where $N_2$ binds and is reduced to $NH_3$. During this process, Component I becomes oxidized and must be reduced again to continue the cycle.

• Component II (Dinitrogenase Reductase): Also known as the iron (Fe) protein. This protein donates electrons to reduce Component I. Component II itself is reduced by electron donors such as Ferredoxin.

Oxygen Sensitivity

Nitrogenase is highly sensitive to oxygen ($O_2$), which irreversibly inactivates the enzyme. Bacteria that fix nitrogen must employ specific strategies to create an oxygen-free or microaerobic environment. Examples include the symbiotic Sinorhizobium meliloti and heterocystous cyanobacteria.

Symbiotic Mechanism in Sinorhizobium meliloti

Sinorhizobium meliloti forms a specialized symbiosis with alfalfa (leguminous plants) when nitrogen levels in the environment are limiting.

Chemical Signaling and Infection

1. Flavonoid Secretion: When plants are starved for nitrogen, they secrete flavonoids.

2. Nod Factor Secretion: Bacteria recognize the flavonoids and respond by secreting Nod factors.

3. Root Hair Curling: Nod factors trigger the plant's root hair to curl, trapping the bacterial cells.

4. Infection Thread (IT): The trapped bacteria divide and form an infection thread, which allows them to invade the plant root tissue.

Nodule Formation

• The plant stimulates tissue growth to form a nodule, which is a specialized microaerobic organ housing the bacteria.

• Leguminous Hemoglobin: These nodules often appear pink because they contain leghemoglobin, which binds oxygen to keep concentrations low enough for nitrogenase to function while still providing oxygen for bacterial respiration.

• Nutrient Exchange: The bacteria provide the plant with fixed nitrogen, while the plant provides the bacteria with carbohydrates (sugars) generated through photosynthesis.

Nitrogen Fixation in Cyanobacteria: Heterocysts

Filamentous cyanobacteria protect their nitrogenase by differentiating approximately 5% to 10% of their cells into specialized cells called heterocysts.

Characteristics of Heterocysts

• Oxygen Exclusion: Heterocysts are surrounded by a thick cell wall that acts as a permeability barrier to $O_2$. They degrade Photosystem II (PSII), the complex responsible for producing $O_2$ during photosynthesis.

• Energy Production: They retain Photosystem I (PSI) to produce $ATP$ via cyclic photophosphorylation.

• Metabolic Specialization: They do not fix $CO_2$ (the Calvin cycle is inactive). Instead, they receive fixed carbon (carbohydrates) from adjacent vegetative cells and, in return,提供 fixed nitrogen (glutamine) to the vegetative cells.

• Terminal Differentiation: Once a cell becomes a heterocyst, it can no longer divide.

Spatial Arrangement

Heterocysts typically appear every ~10 cells along a filament. This spatial arrangement is optimized to facilitate the efficient exchange of fixed nitrogen and carbon products throughout the entire filament.

Questions & Discussion

Concept Quiz: Which of the following statements is true of assimilatory metabolism?

• A. Compounds generated are excreted.

• B. Compounds generated are used as a source of electrons.

• C. Compounds generated are incorporated into cell material.

• D. Compounds generated are used as electron acceptors.

• Answer: C.

Concept Quiz: How is nitrate assimilated in bacterial systems?

• A. Atmospheric nitrogen is reduced to ammonia.

• B. It is reduced to ammonia and incorporated into nucleotides.

• C. It is oxidized to ammonia and incorporated into ribonucleotides.

• D. It is reduced to ammonia and incorporated into amino acids (Glu and Gln).

• Answer: D.

Concept Quiz: Which of the following statements is NOT true of nitrogen fixation?

• A. It is a property of all bacteria.

• B. It is an oxygen sensitive process.

• C. It is an energy intensive process.

• D. $N_2$ is reduced via a series of redox reactions.

• Answer: A.

Concept Quiz: Match each term with the most appropriate description.

• Nod factor: Secreted by the bacteria to cause the root hairs to curl.

• Flavonoid: Secreted by plants when starved for nitrogen.

• Infection thread: Formed by bacterial cell divisions.

• Nodule: Organ made by the plant to house bacteria.