Lecture 3: Transcriptional regulation Flashcards
How is NifA used to control nitrogenase? How does NifL work as an oxygen sensor?
Nif is a group of operons encoding genes required for nitrogen fixation.
• Nif operons are σ54 dependent.
• The EBP of the operons is NifA.
• The unmodified form is active.
• The use of nitrogenase is very metabolically costly so transcription only occurs in the absence of a fixed nitrogen source.
• Transcription is repressed if oxygen concentrations are high enough to damage nitrogenase.
• NifA levels are under control of NtrBC system. Transcription of the nifLA operon requires NtrC-phosphate and NRI-P) and σ54.
• NifL is an FAD-containing redox sensor. In its oxidised state, NifL binds to NifA and prevents it from activating nif gene transcription. In its reduced state NifL is sequestered at the membrane.
• Current research suggests that NifL flavin oxidation state is controlled by the ETC, not by oxygen.
What is symbiotic nitrogen fixation? How do the bacteria move into the plant?
Some plants use bacteria in their root nodules in order to perform nitrogen fixation.
• The bacteria infect the root cells. Before infection, they have vacuoles of polyhydroxybutyrate.
• Bacteria enter through the infections thread, a cellulose lined tube that the bacteria used to travel through the root hair into the root cells.
• The bacteria differentiate into bacteroids. Differentiation is driven by nodule-specific cysteine rich (NCR) peptides produced by the plant. NCRs are related to plant anti-microbial peptides.
• Differentiation causes chromosome duplication, leaky membranes and swelling. The bacteroids are enlarged, misshapen, free of PHB and engulfed in a plant-derived membrane.
• A peribacteroid membrane is plant-derived membrane that separates the bacteroids from the plant cytoplasm.
• The nitrogen fixing organelle within the plant cell cytoplasm containing one or multiple bacteroids within the peribacteroid membrane is known as a symbiosome.
How does hydrogenase work?
Diazotrophs produce hydrogen as a side product of the nitrogenase reactions.
• Many nitrogen fixing bacteria use a membrane-bound hydrogenase enzyme.
• The type of hydrogenase which is used is a hydrogen quinone reductase which produces quinol from hydrogen and quinone.
• Hydrogen is used as an electron donor to the respiratory chain.
• Some of the metabolic energy can therefore be recouped.
How can the effects of oxygen be mitigated?
The oxygen concentration of the nodule must be kept very low in order to protect nitrogenase.
• It is kept at around 10 nM compared to around 250 μM at saturation.
• This is down by a combination of high respiratory rates at the bacteroids and the buffering effects of leghaemoglobin.
• The bacteroids synthesises a cytochrome c oxidase (cytochrome cbb3 oxidase) that has a very high oxygen affinity.
What is leghaemoglobin?
Leghaemoglobin is a haem-containing oxygen binding protein which is homologous to mammalian globins.
• LH is found in a very high concentration in root cell cytoplasm.
• It binds oxygen with a very high affinity (KD of around 20 nM). It thus assists in maintaining a minimal oxygen environment by buffering oxygen concs at a low level.
• It facilitates diffusion of oxygen to cyt c oxidase.
• Even in low oxygen concentrations, there is a sufficiently high flux of oxygen to the ETC.
• It increases the effective solubility of oxygen from around 10 nM for dissolved free oxygen to around 1 mM of oxygen stored bound to LH.
• This parallels the role of myoglobin in mammalian cells.
How are plant and bacterial metabolism integrated?
- The homocitrate component of the FeMoco cofactor isn’t made in Rhizobia. It is provided by the plant.
- In the symbiotic state, bacterial pathways for the synthesis of BCAAs are repressed, rendering the bacteroids dependent on the plant to supply these amino acids. The plant can therefore control the development and persistence of bacteroids.
How are nif and fix regulated during symbiosis? Draw a diagram.
There are many different components of the signalling pathway.
• NtrBC is overridden during symbiosis control of nif gene expression. Regulation is determined only by oxygen tension, not nitrogen availability.
• FixLJ is a two-component regulatory system which directly senses oxygen. Haem (in a globin-like domain of FixL) reversibly binds oxygen. The deoxygenated form of FixL acts as a FixJ kinase. The oxygenated form acts as a FixJ phosphatase.
• NifA in symbiotic bacteria has an additional redox-regulated domain.
• Nif genes allow nitrogen fixation in the free-living state.
• Fix genes are additional genes required for nitrogen fixation in the symbiotic state. Genes encode for high affinity cyt c oxidase and electron transfer to ferredoxin.
• Nod genes are required for nodule production and maintenance.
• Sym plasmids carry the nif, nod and fix genes in many Rhizobia.
