What They Wanted to Know
Pau et al. knew that Azotobacter vinelandii had three versions of nitrogenase, including one with no heterometal (Mo or V), only iron. All of these had similar requirements for energy and conditions. They're all similar in structure too, except that the alternatives both have an extra subunit.
So Pau and colleagues wanted to purify the iron-only dinitrogenase from A. vinelandii and analyze its structure and such.
What They Did
They used a strain of A. vinelandii with the genes for Mo and V nitrogenases deleted, so the only one it could produce was the iron-only one. Since this strain couldn't fix nitrogen in the presence of Mo (it represses the alternatives), they selected for a mutant that didn't have this limitation: RP306. They grew large amounts of this strain (in a 400-L fermenter) and purified the nitrogenase from it. Then they analyzed the enzymatic activity and chemical structure of protein and metallic cofactor.
What They Saw
The parent of strain RP306 couldn't grow by fixing nitrogen when molybdenum (Mo) was higher than 5nM in the medium, but RP306 actually grew better as Mo increased, up to 20nM.
Since the V nitrogenase has an extra subunit (δ) encoded by the vnfG gene, and the Fe nitrogenase has a homologous gene, anfG, Pau et al. thought that it might encode a δ subunit also. So they analyzed the subunits of the dinitrogenase with SDS-PAGE, and did indeed see a third small subunit as expected, whose amino acid sequence corresponds to the sequence of the anfG gene.
In terms of metal content, the dinitrogenase seemed to have about 24 atoms of iron and 18 of sulfur, which corresponded well to previous work. Not surprisingly, it had negligible V, but surprisingly it had 1 atom of Mo. So they analyzed it with electron paramagnetic resonance or EPR spectroscopy, which gives different curves depending on the chemical composition, and it seemed like the iron-only nitrogenase actually had a Mo-containing cofactor! Though it seemed like only one of the two cofactors in the dinitrogenase contained Mo. They were able to extract this cofactor, observed that it had a Mo-to-Fe ratio of 1:4.3, and could insert into a cofactor-less Mo nitrogenase from Klebsiella pneumoniae and make it active.
This Fe dinitrogenase with a Mo cofactor could reduce acetylene, but only to ethylene, not to ethane like regular V and Fe nitrogenases could produce. This activity, or any other, was only present when the enzyme was paired with the iron-only version of dinitrogenase reductase, not with the other versions.
With other substrates (N2) or no substrate (just argon), this Fe nitrogenase didn't perform as well as the Mo nitrogenase. With argon, it produced 350 nmol hydrogen per minute per mg of enzyme, compared to 2220 from the Mo version; with nitrogen, it produced about 100 times less ammonia than the Mo version, but twice as much hydrogen as ammonia. This is about 4 times as much as expected from the Mo version, which produces one hydrogen per nitrogen fixed. So about 57% or 4/7ths of its electron flux goes to hydrogen, compared to 25% of the Mo nitrogenase's. They also saw some ethane produced from acetylene somehow, especially when the ratio of dinitrogenase reductase to dinitrogenase was higher; at least half the electron flux went to ethane.
What This Means
Apparently the allegedly iron-only nitrogenase can incorporate Mo-containing cofactor, at least partially, and this affects its activity. I wouldn't expect this to happen much in nature, since in the presence of Mo the Fe nitrogenase wouldn't be produced, so it's not clear what this really means in terms of enzyme activity. It seems important to exclude Mo from the medium when studying the real activity of the Fe nitrogenase though.
From other results, it seems like the cofactors, despite their differences in metal content, can substitute for each other in the holoenzymes, though the resulting activity changes (not surprisingly). The cells rely on regulation of genes that produce the proteins and cofactors to keep things running the way they should be, rather than specificity of cofactor for protein. But it's probably usually not disastrous if there are a few mix-ups. The activity is best with the right match, but it still works somewhat with some mismatches.
Citation: Pau, R. N., Eldridge, M. E., Lowe, D. J., Mitchenall, L. A. & Eady, R. R. Molybdenum-independent nitrogenases of Azotobacter vinelandii: a functional species of alternative nitrogenase-3 isolated from a molybdenum-tolerant strain contains an iron-molybdenum cofactor. Biochem. J. 293, 101–107 (1993).
Showing posts with label Eady. Show all posts
Showing posts with label Eady. Show all posts
Wednesday, November 5, 2014
Monday, September 30, 2013
002 - Nitrogen fixation in molybdenum-deficient continuous culture by a strain of Azotobacter vinelandii carrying a deletion of the structural genes for nitrogenase (nifHDK)
Previously, another study (001) suggested the presence of an alternative nitrogenase system in Azotobacter vinelandii, but it was not conclusive. Another, later study confirmed this hypothesis by creating a strain completely lacking the genes encoding the primary, molybdenum-containing nitrogenase (nifHDK genes), so there's no way that strain could be using the primary nitrogenase. But this strain could still fix nitrogen and grow when molybdenum (Mo) was not present in its environment, so clearly it had to have some kind of alternative enzyme.
So in this study, the scientists wanted to figure out if this alternative enzyme had the same characteristics as the Mo-containing one, and if not, how they differed. And one of the best ways to determine the characteristics of metabolic pathways, such as nitrogen fixation, is to use continuous culture!
Continuous culture is a technique for maintaining cells in a constant state so they keep growing indefinitely. At its most basic, what it requires is a container in which the cells grow, with fresh culture medium (liquid containing all the nutrients the cells need) flowing into the container at a constant rate, while liquid and cells inside the container are constantly being removed at the same rate to keep the volume inside the container constant. If done right, the culture of cells will eventually reach a point when their population density, consumption of nutrients, growth rate, and all other metabolic characteristics all remain constant over time. This is called "steady state." By measuring the characteristics of the cells' metabolism at steady state in one condition (for example, with a high concentration of sugar), and then changing the condition (reducing the concentration of sugar) and allowing the cells to reach a new steady state, measuring the new characteristics, and comparing the two, it is possible to determine how the cells' metabolism works.
In this study, the scientists used continuous culture to grow A. vinelandii strain CA11, the one lacking the genes nifHDK for the Mo nitrogenase. They grew it in Mo-free medium, and used several techniques to confirm that it was indeed still fixing nitrogen (for example, depriving it of N2 gas for a time and observing its lack of growth; or more directly measuring the incorporation of a heavier isotope of nitrogen from 15N2 gas).
Then they measured CA11's steady-state characteristics at a number of different dilution rates. Dilution rate is a measure of how quickly new medium is flowing into the culture and old culture volume is being removed, so basically the rate the cells are being diluted. As you might expect, the faster the dilution rate, the more quickly the cells have to grow to maintain their population density; otherwise they would be diluted more and more until none were left. Fortunately, higher dilution rate also means that fresh nutrients are being added more quickly, so growing faster is usually not a problem. But there is a point at which cells just can't grow any faster, called the maximum growth rate, so if the dilution rate is higher than this point, the cells can't keep up, and the population density decreases.
In this study, at different dilution rates, the authors measured the population density in a number of different ways: optical density (how much light passes through a volume of culture; the more densely-packed the cells, the less light passes through, so the higher the optical density); protein content (the amount of protein in a volume of culture; usually correlates with number of cells, but in some conditions cells will have more protein per cell than in other conditions); dry weight (the weight of a volume of culture after all the water is removed; usually correlates well with number of cells, but sometimes fewer larger cells can weigh as much as more smaller cells); nitrogen content (correlates well with protein content, since protein contains nitrogen); and number of colony-forming units (by spreading a known volume of cells onto a nutrient agar plate and counting the number of colonies that grow on the plate, you can get an idea of how many living cells were present in a given volume of culture). They found that with all these measures, there were fewer cells at higher dilution rates, but not much else was noteworthy about the experiment.
In a second experiment, they measured specific activities of nitrogen fixation at the steady states of different dilution rates. Normally, in the wild-type strain, the Mo nitrogenase takes one molecule of N2, converts it to two molecules of NH3, and also gives off one molecule of H2 as a byproduct. There's another enzyme called the uptake hydrogenase that takes the H2 produced by nitrogenase and oxidizes it for energy, similar to how cells oxidize sugar for energy. This recovers some energy the nitrogenase uses, which would otherwise be wasted. Nitrogenase requires a lot of energy, so it's worthwhile.
So the scientists measured the amount of hydrogen produced by CA11 to see if its alternative nitrogenase produced more or less hydrogen than the Mo-containing version. (They could do this because there was a chemical in the medium that happened to inhibit the uptake hydrogenase, so the hydrogen was released into the headspace of the culture vessel.)
They also measured the nitrogen-fixing activity of the nitrogenase more directly, both by measuring amounts of nitrogen and another way called the acetylene reduction assay. Nitrogenase is not a very picky enzyme; its main substrate is two nitrogens connected by a triple bond, but it will also transform most other molecules that consist of two atoms connected by a triple bond, including carbon monoxide and acetylene (C2H2, aka ethyne). So in the acetylene reduction assay, acetylene is added to a container with the enzyme, the enzyme (if present and active) converts it into ethylene (C2H4, aka ethene), which can be quantified to measure the enzyme's activity.
They found that as dilution rate increased, nitrogenase activity tended to increase also, producing more of all products (hydrogen, fixed nitrogen, and ethylene). They knew the hydrogen was produced by the nitrogenase because when they added ammonium (which represses nitrogenase activity; the cells aren't going to waste energy fixing nitrogen if there is already fixed nitrogen available), the hydrogen production ceased. They also found that, at mid-range dilution rates, the alternative nitrogenase produced about three H2 molecules for each ammonia (this ratio decreased at higher and lower dilution rates), which compared to the Mo-containing nitrogenase (1 hydrogen for each ammonia) is less efficient.
The scientists tried adding Mo to see what would happen. They found that, for the wild-type strain that still possessed the Mo-containing nitrogenase, adding Mo made it grow a lot more, but it actually inhibited the growth of CA11.
So it seems that the alternative nitrogenase is less efficient than the Mo-containing one, so the bacteria prefer to use the latter.
Citation: Bishop, P. E., Hawkins, M. E. & Eady, R. R. Nitrogen fixation in molybdenum-deficient continuous culture by a strain of Azotobacter vinelandii carrying a deletion of the structural genes for nitrogenase (nifHDK). Biochem J 238, 437–442 (1986).
So in this study, the scientists wanted to figure out if this alternative enzyme had the same characteristics as the Mo-containing one, and if not, how they differed. And one of the best ways to determine the characteristics of metabolic pathways, such as nitrogen fixation, is to use continuous culture!
Continuous culture is a technique for maintaining cells in a constant state so they keep growing indefinitely. At its most basic, what it requires is a container in which the cells grow, with fresh culture medium (liquid containing all the nutrients the cells need) flowing into the container at a constant rate, while liquid and cells inside the container are constantly being removed at the same rate to keep the volume inside the container constant. If done right, the culture of cells will eventually reach a point when their population density, consumption of nutrients, growth rate, and all other metabolic characteristics all remain constant over time. This is called "steady state." By measuring the characteristics of the cells' metabolism at steady state in one condition (for example, with a high concentration of sugar), and then changing the condition (reducing the concentration of sugar) and allowing the cells to reach a new steady state, measuring the new characteristics, and comparing the two, it is possible to determine how the cells' metabolism works.
In this study, the scientists used continuous culture to grow A. vinelandii strain CA11, the one lacking the genes nifHDK for the Mo nitrogenase. They grew it in Mo-free medium, and used several techniques to confirm that it was indeed still fixing nitrogen (for example, depriving it of N2 gas for a time and observing its lack of growth; or more directly measuring the incorporation of a heavier isotope of nitrogen from 15N2 gas).
Then they measured CA11's steady-state characteristics at a number of different dilution rates. Dilution rate is a measure of how quickly new medium is flowing into the culture and old culture volume is being removed, so basically the rate the cells are being diluted. As you might expect, the faster the dilution rate, the more quickly the cells have to grow to maintain their population density; otherwise they would be diluted more and more until none were left. Fortunately, higher dilution rate also means that fresh nutrients are being added more quickly, so growing faster is usually not a problem. But there is a point at which cells just can't grow any faster, called the maximum growth rate, so if the dilution rate is higher than this point, the cells can't keep up, and the population density decreases.
In this study, at different dilution rates, the authors measured the population density in a number of different ways: optical density (how much light passes through a volume of culture; the more densely-packed the cells, the less light passes through, so the higher the optical density); protein content (the amount of protein in a volume of culture; usually correlates with number of cells, but in some conditions cells will have more protein per cell than in other conditions); dry weight (the weight of a volume of culture after all the water is removed; usually correlates well with number of cells, but sometimes fewer larger cells can weigh as much as more smaller cells); nitrogen content (correlates well with protein content, since protein contains nitrogen); and number of colony-forming units (by spreading a known volume of cells onto a nutrient agar plate and counting the number of colonies that grow on the plate, you can get an idea of how many living cells were present in a given volume of culture). They found that with all these measures, there were fewer cells at higher dilution rates, but not much else was noteworthy about the experiment.
In a second experiment, they measured specific activities of nitrogen fixation at the steady states of different dilution rates. Normally, in the wild-type strain, the Mo nitrogenase takes one molecule of N2, converts it to two molecules of NH3, and also gives off one molecule of H2 as a byproduct. There's another enzyme called the uptake hydrogenase that takes the H2 produced by nitrogenase and oxidizes it for energy, similar to how cells oxidize sugar for energy. This recovers some energy the nitrogenase uses, which would otherwise be wasted. Nitrogenase requires a lot of energy, so it's worthwhile.
So the scientists measured the amount of hydrogen produced by CA11 to see if its alternative nitrogenase produced more or less hydrogen than the Mo-containing version. (They could do this because there was a chemical in the medium that happened to inhibit the uptake hydrogenase, so the hydrogen was released into the headspace of the culture vessel.)
They also measured the nitrogen-fixing activity of the nitrogenase more directly, both by measuring amounts of nitrogen and another way called the acetylene reduction assay. Nitrogenase is not a very picky enzyme; its main substrate is two nitrogens connected by a triple bond, but it will also transform most other molecules that consist of two atoms connected by a triple bond, including carbon monoxide and acetylene (C2H2, aka ethyne). So in the acetylene reduction assay, acetylene is added to a container with the enzyme, the enzyme (if present and active) converts it into ethylene (C2H4, aka ethene), which can be quantified to measure the enzyme's activity.
They found that as dilution rate increased, nitrogenase activity tended to increase also, producing more of all products (hydrogen, fixed nitrogen, and ethylene). They knew the hydrogen was produced by the nitrogenase because when they added ammonium (which represses nitrogenase activity; the cells aren't going to waste energy fixing nitrogen if there is already fixed nitrogen available), the hydrogen production ceased. They also found that, at mid-range dilution rates, the alternative nitrogenase produced about three H2 molecules for each ammonia (this ratio decreased at higher and lower dilution rates), which compared to the Mo-containing nitrogenase (1 hydrogen for each ammonia) is less efficient.
The scientists tried adding Mo to see what would happen. They found that, for the wild-type strain that still possessed the Mo-containing nitrogenase, adding Mo made it grow a lot more, but it actually inhibited the growth of CA11.
So it seems that the alternative nitrogenase is less efficient than the Mo-containing one, so the bacteria prefer to use the latter.
Citation: Bishop, P. E., Hawkins, M. E. & Eady, R. R. Nitrogen fixation in molybdenum-deficient continuous culture by a strain of Azotobacter vinelandii carrying a deletion of the structural genes for nitrogenase (nifHDK). Biochem J 238, 437–442 (1986).
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