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 Pau. Show all posts
Showing posts with label Pau. Show all posts
Wednesday, November 5, 2014
Wednesday, October 9, 2013
015 - Characterization of genes involved in molybdenum transport in Azotobacter vinelandii
It is known that molybdenum (Mo) represses A. vinelandii's alternative nitrogenases, at least in the wild-type. What is not exactly known is the mechanism of this repression. Concentrations of Mo as low as 10 μM repress the alternatives, but some strains (such as CA6) produce the alternatives even with high concentrations of Mo. The question is, is this because Mo is not being transported into the cell, or because the protein that represses the alternatives is not functioning properly somehow?
So in the current study, they took a strain of A. vinelandii that can't make the primary nitrogenase but still represses the alternatives when Mo is present, and created mutants using a transposon, Tn5, then grew it on medium with Mo but without nitrogen, so that only those that had a mutation in a relevant gene could grow. And they found two Tn5-induced mutants, and also some that had mutated spontaneously to be able to grow in such conditions.
They named the two transposon mutants FL2 and FL4, focusing on them because the transposon insertion allowed them to locate and study the genes of interest. The mutants could grow pretty much just as fast with Mo present as the parent strain could grow with Mo absent (though these rates were all somewhat slow because they all needed to use the alternative nitrogenases, which are less efficient). Actually FL4 grew a bit faster than the parent.
They isolated and sequenced the section of the genome that the transposons had inserted themselves into, and it turned out that it was the mod operon that I've discussed before (012). FL2 had an insertion in modE, the regulatory gene, and FL4 had an insertion near the end of modB. So it seems like these genes are important for Mo-induced repression of alternative nitrogenases.
Citation: Luque, F., Mitchenall, L. A., Chapman, M., Christine, R. & Pau, R. N. Characterization of genes involved in molybdenum transport in Azotobacter vinelandii. Mol. Microbiol. 7, 447–459 (1993).
So in the current study, they took a strain of A. vinelandii that can't make the primary nitrogenase but still represses the alternatives when Mo is present, and created mutants using a transposon, Tn5, then grew it on medium with Mo but without nitrogen, so that only those that had a mutation in a relevant gene could grow. And they found two Tn5-induced mutants, and also some that had mutated spontaneously to be able to grow in such conditions.
They named the two transposon mutants FL2 and FL4, focusing on them because the transposon insertion allowed them to locate and study the genes of interest. The mutants could grow pretty much just as fast with Mo present as the parent strain could grow with Mo absent (though these rates were all somewhat slow because they all needed to use the alternative nitrogenases, which are less efficient). Actually FL4 grew a bit faster than the parent.
They isolated and sequenced the section of the genome that the transposons had inserted themselves into, and it turned out that it was the mod operon that I've discussed before (012). FL2 had an insertion in modE, the regulatory gene, and FL4 had an insertion near the end of modB. So it seems like these genes are important for Mo-induced repression of alternative nitrogenases.
Citation: Luque, F., Mitchenall, L. A., Chapman, M., Christine, R. & Pau, R. N. Characterization of genes involved in molybdenum transport in Azotobacter vinelandii. Mol. Microbiol. 7, 447–459 (1993).
Wednesday, October 2, 2013
012 - Mutational analysis of genes of the mod locus involved in molybdenum transport, homeostasis, and processing in Azotobacter vinelandii
So Azotobacter vinelandii CA6 has impaired molybdenum (Mo) uptake (003). This paper studies the Mo transport system of A. vinelandii, encoded by the mod genes, modEABC. It seems like ModA is a protein that binds Mo outside the cell, ModB brings them inside across the membrane, and ModC powers this process. ModE's role is unknown at this point.
The scientists generated a number of mutant strains of A. vinelandii, knocking out a given gene while also fusing it with lacZ to quantify its expression. They also discovered another mod gene, modG, adjacent to the others but in the opposite strand direction. It looks similar to half of modE, so the protein may have a similar function.
Another enzyme in A. vinelandii that requires Mo is nitrate reductase; the authors measured activity of this enzyme as a proxy for Mo transport activity. The wild-type's activity rises quickly as concentration of Mo in the environment increases, levels off, then rises quickly again at higher concentrations (supporting the idea of two different Mo transport systems). With mutants of modA, modB, and modC, the pattern was always the same: no activity until the concentration reached a certain point (the same point when the wild-type's activity started rising quickly the second time).
Other results were more puzzling: when modE was knocked out in a way that didn't inhibit expression of the other mod genes, it seemed to have good transport activity at lower Mo concentrations but not at higher; and the opposite when its knockout inhibited the other genes.
Strain CA11.6, which genetically combined the lack of Mo nitrogenase in CA11 (002) with the tungsten-tolerant phenotype of CA6 (003), showed good Mo uptake at low concentrations but not at higher. When the modB gene was specifically knocked out of CA11.6, there was hardly any uptake activity at any concentration. Considering my own research, it's difficult to say what is going on genetically in these cases.
When modG was targeted for knockout, it looked pretty much the same as wild-type activity, except when both modG and modE were deleted, in which case it showed activity at much lower concentrations even than wild-type. Explain that, science!
They also directly measured uptake of a radioactive isotope of molybdenum (99Mo) in the wild-type and modA or modB mutants. The rate of transport in the wild-type and modB mutant were pretty much constant, though the latter was slower than the former. In the modA mutant though, there was very little transport. They tried adding nonradioactive compounds (Mo, vanadium, sulfate, or tungsten) to compete with transport of radioactive Mo, and found that only Mo and tungsten inhibited radioactive Mo transport by competition. Evidence that the mod genes transport tungsten in addition to Mo.
Lastly, the scientists tested the nitrogen-fixing abilities of mod mutants. With Mo present, nitrogen-fixing growth of modE and G mutants was similar to wild-type. When it was absent, modG knockout grew more slowly and modE more quickly. A double mutant didn't grow hardly at all in either condition, in normal aerobic conditions, but with lower levels of oxygen it grew as well as the wild-type (both very slowly). It could also grow using vanadium (V) and the V-containing alternative nitrogenase.
The conclusions, I suppose, are that modABC are all important for Mo transport, especially at low concentrations. modE's role is not exactly clear, but it may regulate which Mo transporter system is working at a given time (possibly by repressing one and activating the other at low concentrations, and vice versa at high). modG's role is even less clear.
Citation: Mouncey, N. J., Mitchenall, L. A. & Pau, R. N. Mutational analysis of genes of the mod locus involved in molybdenum transport, homeostasis, and processing in Azotobacter vinelandii. J. Bacteriol. 177, 5294–5302 (1995).
The scientists generated a number of mutant strains of A. vinelandii, knocking out a given gene while also fusing it with lacZ to quantify its expression. They also discovered another mod gene, modG, adjacent to the others but in the opposite strand direction. It looks similar to half of modE, so the protein may have a similar function.
Another enzyme in A. vinelandii that requires Mo is nitrate reductase; the authors measured activity of this enzyme as a proxy for Mo transport activity. The wild-type's activity rises quickly as concentration of Mo in the environment increases, levels off, then rises quickly again at higher concentrations (supporting the idea of two different Mo transport systems). With mutants of modA, modB, and modC, the pattern was always the same: no activity until the concentration reached a certain point (the same point when the wild-type's activity started rising quickly the second time).
Other results were more puzzling: when modE was knocked out in a way that didn't inhibit expression of the other mod genes, it seemed to have good transport activity at lower Mo concentrations but not at higher; and the opposite when its knockout inhibited the other genes.
Strain CA11.6, which genetically combined the lack of Mo nitrogenase in CA11 (002) with the tungsten-tolerant phenotype of CA6 (003), showed good Mo uptake at low concentrations but not at higher. When the modB gene was specifically knocked out of CA11.6, there was hardly any uptake activity at any concentration. Considering my own research, it's difficult to say what is going on genetically in these cases.
When modG was targeted for knockout, it looked pretty much the same as wild-type activity, except when both modG and modE were deleted, in which case it showed activity at much lower concentrations even than wild-type. Explain that, science!
They also directly measured uptake of a radioactive isotope of molybdenum (99Mo) in the wild-type and modA or modB mutants. The rate of transport in the wild-type and modB mutant were pretty much constant, though the latter was slower than the former. In the modA mutant though, there was very little transport. They tried adding nonradioactive compounds (Mo, vanadium, sulfate, or tungsten) to compete with transport of radioactive Mo, and found that only Mo and tungsten inhibited radioactive Mo transport by competition. Evidence that the mod genes transport tungsten in addition to Mo.
Lastly, the scientists tested the nitrogen-fixing abilities of mod mutants. With Mo present, nitrogen-fixing growth of modE and G mutants was similar to wild-type. When it was absent, modG knockout grew more slowly and modE more quickly. A double mutant didn't grow hardly at all in either condition, in normal aerobic conditions, but with lower levels of oxygen it grew as well as the wild-type (both very slowly). It could also grow using vanadium (V) and the V-containing alternative nitrogenase.
The conclusions, I suppose, are that modABC are all important for Mo transport, especially at low concentrations. modE's role is not exactly clear, but it may regulate which Mo transporter system is working at a given time (possibly by repressing one and activating the other at low concentrations, and vice versa at high). modG's role is even less clear.
Citation: Mouncey, N. J., Mitchenall, L. A. & Pau, R. N. Mutational analysis of genes of the mod locus involved in molybdenum transport, homeostasis, and processing in Azotobacter vinelandii. J. Bacteriol. 177, 5294–5302 (1995).
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