663 - Azotobacter vinelandii Vanadium Nitrogenase: Formaldehyde Is a Product of Catalyzed HCN Reduction, and Excess Ammonia Arises Directly from Catalyzed Azide Reduction

The V nitrogenase is similar to but distinct from the Mo nitrogenase in various ways. This study tests its activity and patterns with cyanide and azide as substrates; previously this had only been tested with the Mo nitrogenase.

What They Saw
As with the Mo version, cyanide inhibited hydrogen production and the overall electron flux through the V nitrogenase, though less than with the Mo nitrogenase; more cyanide had more effect, up to 75% inhibition of hydrogen and electrons at 50 mM cyanide. Methane formation from cyanide increased at first, and then decreased with more cyanide, while ammonia continued to increase. They didn't measure methylamine in most conditions though, so the electron flux numbers might be off. With low cyanide, there was significant methylamine relative to the methane, about 0.66 to 1, higher than the Mo nitrogenase; this ratio seems to increase to 1:1 as methane decreases.

Interestingly, the enzyme also produces formaldehyde from cyanide. It's not clear whether the Mo version does too and it's too hard to detect, or if it just doesn't. It can be tricky, since other components in the reaction react with it, and cyanide inhibits the Mo enzyme a lot more.

Azide inhibited hydrogen production from the V nitrogenase about 50%. Hydrazine was more of a product from azide than it is for the Mo nitrogenase, relative to dinitrogen and ammonia. Overall activity was less than the Mo version, which is typical, but also because azide seems to reduce the total electron flux in this version. The ammonia seems to come from azide directly, not from the dinitrogen produced, because adding hydrogen gas (which specifically inhibits nitrogen reduction) didn't change the values.

They tried seeing what adding carbon monoxide (CO) might do to affect these reactions. With azide, it rescued hydrogen production, though not the electron flux, and CO didn't entirely prevent some azide reduction. With cyanide, the effects were similar, except the electron flux inhibition was relieved too.

What This Means
It seems that these chemicals have similar effects on the V nitrogenase as they do on the Mo version, but not completely the same. I wonder, though, about the CO assays: CO can be a substrate for the V nitrogenase, reduced mostly to ethylene but also some propylene and methane; how did this affect their assays?

Reference:

Fisher, K., Dilworth, M. J. & Newton, W. E. Azotobacter vinelandii Vanadium Nitrogenase: Formaldehyde Is a Product of Catalyzed HCN Reduction, and Excess Ammonia Arises Directly from Catalyzed Azide Reduction. Biochemistry 45, 4190–4198 (2006).

563 - Construction and Characterization of Hybrid Component 1 from V-Nitrogenase Containing FeMo Cofactor

This study looked at a purified V nitrogenase from Azotobacter vinelandii with the FeMo cofactor instead of FeVco.

What They Saw
They detected only Mo, no V, in the preparation. The activities they saw were pretty weird in some ways: the electron flux going to nitrogen was almost as much as in the Mo nitrogenase (~70%, with the rest going to hydrogen), whereas with the normal FeVco, 50% went to each; in previous studies, V nitrogenase with FeMoco couldn't fix nitrogen at all. On the other hand, with acetylene, almost all electrons went to ethylene in the Mo nitrogenase, whereas only 30-35% did in the V nitrogenase with either cofactor, while 3% went to ethane with FeVco but 10% did with FeMoco, which is consistent with previous studies. So I'm confused.

When they added carbon monoxide, there was inhibition of all nitrogenase versions, as expected.

What This Means
I think something was weird in this study, but I don't know what. It seems very questionable. However, most of the later studies citing this one either accept it without question, or confirm its results, so maybe it's not as questionable as it seems.

Also, it's worth noting that the results only show the electron flux going to each substrate, not the total electron flux, so even if most electrons go to nitrogen in the FeMoco-substituted V nitrogenase, it's still possible that it's fixing a lot less nitrogen overall than either Mo or V versions proper.

Reference:

Moore, V. G., Tittsworth, R. C. & Hales, B. J. Construction and Characterization of Hybrid Component 1 from V-Nitrogenase Containing FeMo Cofactor. J. Am. Chem. Soc. 116, 12101–12102 (1994).

558 - Isolation of a new vanadium-containing nitrogenase from Azotobacter vinelandii

This study is the first to fully purify the vanadium nitrogenase from Azotobacter vinelandii.

What They Saw
They used strain UW (aka CA) and a nifHDK knockout. It didn't seem like they scrubbed the medium for Mo, but they did test the purified product for metals and didn't see a detectable amount of Mo, only V and Fe (in a 1-to-13 ratio), so I guess it's good.

They did acetylene reduction assays on the V and Mo nitrogenases, and found that 90% of the electrons went to acetylene in the former, but only 12% in the latter. It seems like they only measured ethylene production though, so they might've underestimated the total activity by neglecting the ethane produced by the V nitrogenase.

Under argon, the Mo nitrogenase produced 1.6x more hydrogen than the V version. This was the same ratio as that of nitrogen fixed by each version. I'm not sure how much it's possible to compare these enzymes in vitro though. But it seems like the V nitrogenase is better at nitrogen and hydrogen than at acetylene.

As mentioned, ICP emission spectroscopy didn't detect any metals other than V or Fe: no Mo, Cr, Co, Ni, Cu, or W, so that's good.

What This Means
This shows how the Mo and V nitrogenases are similar in some ways and yet different in others. They work in similar ways and are sensitive to similar things, but the products of their reactions and such are different (though possible substrates seem largely the same). By comparing the two, we can learn more about the process of nitrogen fixation in general.

Reference:

Hales, B. J., Case, E. E., Morningstar, J. E., Dzeda, M. F. & Mauterer, L. A. Isolation of a new vanadium-containing nitrogenase from Azotobacter vinelandii. Biochemistry 25, 7251–7255 (1986).

557 - Detection of the in vivo incorporation of a metal cluster into a protein - The FeMo cofactor is inserted into the FeFe protein of the alternative nitrogenase of Rhodobacter capsulatus

This is another study looking at central cofactors from some nitrogenase versions inserted into other apoproteins than usual, but this time in Rhodobacter capsulatus.

What They Saw
They looked at purified enzymes from wild-type R. capsulatus and a nifHDK deletion mutant. The latter should only produce the iron-only nitrogenase, if anything. They had to treat the medium to remove as much Mo as possible so as to be able to control the concentration; this reduced the Mo present from around 1 ppb to less than 0.05 ppb (the detection limit).

They found that adding 10μM Mo to cultures growing with no Mo (and thus producing the Fe nitrogenase) greatly increased the amount of ethane produced from acetylene reduction (up to 40% of the ethylene produced). Without Mo, ethane remained constant at about 2% of ethylene. The amount of ethane increased over 72 hours too, up to 68% of ethylene; however, total activity decreased greatly over that time, down to only about 5% of what it had been before adding Mo. The ethane production rate increased for 24 hours and then decreased more slowly than the ethylene rate. This may be due to repression of the Fe nitrogenase, but the authors claim it is not, because the rate decreases more quickly in late-log cultures with chloramphenicol + Mo than with just chloramphenicol (or neither, which was about the same as with chloramphenicol alone). This shows that no new nitrogenase protein is being made even when chloramphenicol is absent, but adding Mo speeds the loss of it.
They also found that the more Mo they added, the higher the proportion of ethane produced (and also the lower the total acetylene reduction activity).

They tested the sensitivity of the system to oxygen, both with and without Mo: in both cases, more oxygen meant less acetylene reduction, though the system with Mo seemed a bit more sensitive (dropping to almost 0% with 1% oxygen while that without Mo only fell to about 20%), but also they noticed that increased oxygen increased the proportion of ethane produced after Mo was added. So somehow oxygen enhanced the Mo effect.

Rhenium, tungsten, and vanadium did not cause anything similar to the Mo effect. The Mo effect was also absent in mutants unable to produce FeMo cofactor (nifE knockouts), so it seems like the cofactor is part of the system. nifQ knockouts seemed to show the effect only at high concentrations of Mo (0.1-1mM); this gene's product is involved in cofactor synthesis at a different step. Mo uptake wasn't an issue; all strains had the same intracellular concentrations.

Using EPR spectroscopy on purified enzyme, they claim to show that the spectrum for Fe nitrogenase with added Mo is similar to that of the Mo nitrogenase from the wild-type, so it seems like the FeMo cofactor is incorporated into the Fe nitrogenase. I would've liked to see their result for the Fe nitrogenase without added Mo as a control, but I'll have to take their word for it. Though they did do metal analysis that found ratios of Fe to Mo similar to that of the Mo nitrogenase.

The fact that chloramphenicol didn't prevent the Mo effect seemed to show that the proteins required to make FeMoco were present before Mo was added. This was confirmed with lacZ fusions to related genes to observe expression more directly.

What This Means
Is FeMoco actually replacing FeFeco in completed enzymes? Seems more likely that FeMoco is inserting into incomplete apoprotein, but it's hard to distinguish between these possibilities. Considering that the process seems to continue over several days, maybe the FeMoco is actually displacing FeFeco from completed proteins over time. This is supported by the observation of oxygen enhancement of the Mo effect; oxygen seems to make the enzyme more labile.

Reference:

Gollan, U., Schneider, K., Müller, A., Schüddekopf, K. & Klipp, W. Detection of the in vivo incorporation of a metal cluster into a protein - The FeMo cofactor is inserted into the FeFe protein of the alternative nitrogenase of Rhodobacter capsulatus. Eur. J. Biochem. 215, 25–35 (1993).

544 - Nitrogenase from vanadium-grown Azotobacter: Isolation, characteristics, and mechanistic implications

It was known that Mo seemed important for nitrogen fixation in Azotobacter vinelandii. This study looked at substituting V for Mo for nitrogen fixation. It wasn't discovered until about a decade later that there were two separate sets of genes for two versions of nitrogenase with different metals, so here they thought it was a substitution of metals in the same protein.

What They Saw
They grew A. vinelandii OP (aka CA) with Mo or V and extracted and purified its nitrogenase. They said it didn't grow without either Mo or V, which seems weird because it should be able to grow with just iron.

The V nitrogenase activity (measured as hydrogen production) was lower than that of the Mo nitrogenase, about 22% of it, though it's hard to compare in vitro assays. It also seemed less stable and more prone to heat inactivation.

They did detect traces of Mo in the V purification, so it's not clear exactly what's happening. There was about 20x more V than Mo. Both purifications could reduce acrylonitrile, propionitrile, and acetonitrile in addition to the more familiar substrates, and hydrogen was produced at the same time. Hydrogen inhibited nitrogen reduction and carbon monoxide inhibited everything except hydrogen production.

In terms of efficiency, they observed that the Mo nitrogenase allocated 70% of its electrons to nitrogen and only 30% to hydrogen (similar to the typical 75%/25% numbers), while electrons in the V nitrogenase only went to nitrogen 25% of the time, which works out to 6 electrons making 2 ammonia, and another 18 making 9 hydrogen. Other substrates gave different numbers, but the V nitrogenase always had higher flux to hydrogen.

It seemed like CO inhibition of the V version was competitive but it wasn't clear that the same was true of the Mo version. And acrylonitrile reduction was different between them: V nitrogenase produced about twice as much propane as opposed to propylene compared to the Mo version.

What This Means
It seems, from the results, pretty likely that they were studying the V nitrogenase (Vnf) in this study, despite slight contamination with Mo. The CO inhibition pattern, and other activity patterns, support this conclusion.

I didn't know that acrylonitrile and such could be substrates for nitrogenase, but perhaps the cyanide residue is reduced to make it a hydrocarbon, either propylene or propane. Not sure this seems more useful than other substrates.

Overall, it's interesting how much this study revealed that wasn't really known until later studies confirmed it.

Reference:

Burns, R. C., Fuchsman, W. H. & Hardy, R. W. F. Nitrogenase from vanadium-grown Azotobacter: Isolation, characteristics, and mechanistic implications. Biochem Biophys Res Commun 42, 353–358 (1971).

542 - Purification and Characterization of the vnf-encoded Apodinitrogenase from Azotobacter vinelandii

This study looks at the vanadium nitrogenase apoprotein and how it works with its own cofactors or those of the other versions.

What They Saw
Azotobacter vinelandii strains possessing or lacking genes for one or more of the nitrogenases were grown and their protein was extracted. They also extracted central cofactors for each type separately.

Without nifB, a strain can't produce any of the cofactors, so it's easier to get apoproteins. They took vanadium aponitrogenase (Vnf version) and activated it with FeVco. Not surprisingly, they only saw activity in strains containing vnf when vanadium was present and Mo or ammonium was not. Extracts of active strains lost activity when treated with heat or exposed to air for 45 minutes, though the holoenzyme seemed more stable to heat.

When they purified the enzyme as much as possible, the delta subunit (vnfG-encoded) seemed only loosely attached to the others, unlike in A. chroococcum where it purifies together with the others; though it's not clear that conditions were the same. It was necessary for active enzyme though, and seems to be involved in inserting the cofactor into the enzyme, but also something else.

They tried replacing FeVco with FeMoco in the V nitrogenase (or vice versa in the Mo version). With the V version and FeVco, carbon monoxide inhibited about 70% of the acetylene reduction activity but no hydrogen production activity. The Mo nitrogenase with FeVco had a fraction of the V version's acetylene activity but no nitrogen fixation; sadly they didn't test hydrogen production.

With the V nitrogenase and FeMoco, it had a bit (1/6th) of the acetylene reduction activity, which seemed insensitive to CO. Ethane production was proportionally higher (1:4 instead of 1:12). Hydrogen production was reduced about 40%. Nitrogen fixation was pretty much abolished.

With the correct cofactors, the V nitrogenase had about 31% the acetylene reduction activity, 34% of the hydrogen production activity, and 22% of the nitrogen fixation activity of the Mo nitrogenase, but it's not clear if these in vitro assays allow for accurate comparisons.

It didn't seem like FeFeco allowed any activity in the V aponitrogenase.

What This Means
It seems like nitrogen fixation requires a very specific environment, and messing with it in various ways (mutations, different cofactors) messes it up while allowing the enzymes to still do less strict activities.

Reference:

Chatterjee, R., Allen, R. M., Ludden, P. W. & Shah, V. K. Purification and Characterization of the vnf-encoded Apodinitrogenase from Azotobacter vinelandii. J. Biol. Chem. 271, 6819–6826 (1996).

541 - Diversity of Nitrogenase Systems in Diazotrophs

This review looks at different kinds of nitrogen-fixing enzymes, real and theoretical. Azotobacter vinelandii itself has three genetically distinct versions, with different central metals in their central cofactors: molybdenum, vanadium, and iron.

The molybdenum version is most common in nature, and used preferentially in organisms that possess it. It has also been studied the most. No known organism possesses either of the other two versions while lacking this one. Protein sequences of this enzyme in different organisms are remarkably similar to each other. It uses 2 ATP to transfer one electron from the dinitrogenase reductase protein to the dinitrogenase complex, and 6 such transfers reduce one dinitrogen to two ammonia (along with 2 electrons going to hydrogen).

The other versions were discovered when Mo or its enzyme were unavailable yet nitrogen fixation continued. They have an extra subunit of unknown function (perhaps cofactor insertion), but otherwise seem pretty similar in structure and function.

Other than those three, Streptomyces thermoautotrophicus has a novel system: it has Mo in the dinitrogenase and the dinitrogenase reductase equivalent is a manganese-superoxide oxidoreductase with no iron or sulfur. It doesn't do acetylene reduction, but far from being oxygen-sensitive, it depends on oxygen for its activity. The overall stoichiometry is similar to the Azotobacter Mo nitrogenase though, including the hydrogen production, but the minimum ATP requirement is only 4, instead of 16.

Then the authors go into some discussion of other nitrogenases, with the same apoenzymes as those in A. vinelandii but with different central cofactors that may or may not be found in nature. For example, a tungsten-iron cofactor, which has been studied before: it doesn't permit nitrogen fixation, but some proton reduction is possible. Then there were chromium- or manganese-iron cofactor proteins: when extracts were treated with o-phenanthroline and purified as apoproteins, activity could be partially restored with solutions including salts of Mn, V, Mo, Cr, or Re. W failed to give this effect, but the others permitted some acetylene and proton reduction activity. I don't have access to the citations for these claims though.

These in vitro proteins, even if they contain weird metals, also seem to contain Mo in equal proportions to the others. A. vinelandii UW3 lacks nif genes required for the primary nitrogenase, but can use alternatives, and some suggest that the V nitrogenase may use Re or Mn or Cr instead; or maybe the Mo nitrogenase proteins are expressed but use these metals. It doesn't seem very clear, but it's interesting.

Reference:

Zhao, Y., Bian, S.-M., Zhou, H.-N. & Huang, J.-F. Diversity of Nitrogenase Systems in Diazotrophs. J Integr Plant Biol 48, 745–755 (2006).

024 - Essential metals for nitrogen fixation in a free-living N2-fixing bacterium: chelation, homeostasis and high use efficiency

The molybdenum (Mo) nitrogenase in Azotobacter is preferred, of course, but molybdenum is not always easy to find, especially in nature. Vanadium and iron are more common and available, so the V and Fe nitrogenases probably see regular use.

What They Wanted to Know

J.P. Bellenger and colleagues had already showed how bacteria capture and take up the metals they need with metallophores and transporters, but they wanted to figure out how much of each metal the organisms actually need.

What They Did

They studied A. vinelandii strain CA as a wild-type, and several mutants with various nitrogenases deleted: CA1.70 only has the Mo nitrogenase, CA11.70 only the V, and RP1.11 only the Fe. They also tested A. chroococcum.

They grew these with all the nutrients they needed except nitrogen and with varied concentrations of Mo, V, or Fe, measuring growth rate (by optical density), metallophore production, nitrogen fixation (by acetylene reduction and ¹⁵N uptake), intracellular metal/phosphorus, short-term metal uptake (with metal heavy/radioactive isotopes), nitrogenase gene expression (by RT-qPCR of nifD and vnfD), and actual nitrogenase protein levels (by Western blot on NifH).

What They Observed

Growth Rates

Not surprisingly, growth rates for each mutant were lower when given sub-optimal amounts of the metals they needed to fix nitrogen (Fe and sometimes Mo or V). But instead of an exponential growth phase in the curve like one expects, they saw initial fast growth, then a second phase of slower growth before stationary phase. Growth rates were also proportional to intracellular metal levels and nitrogen fixation.

For optimal growth, the Mo-only mutant needed 10^-7 to 10^-6 M Mo initially. The same was true of V and Fe. Levels higher than that seemed somewhat toxic, but extra Fe helped reduce that effect.

Growth rates of the V-only mutant maxed about 15% less than Mo-only (0.23 vs. 0.27 h^-1). Fe-only only got up to 0.12.

Metallophores and Metal Uptake

A. vinelandii produces azotochelin and protochelin to bind useful metals and make them easier to obtain. The authors observed that at the highest Mo/V concentration, these metallophores were much more concentrated (possibly to reduce the metal toxicity). These are produced mostly during early exponential phase (which makes sense).

Though actually, measuring V uptake rates with V either free or bound to metallophores, they found that bound V uptake is slower than uptake of free V. This could be intentional (in a non-anthropomorphic sense); that is, differentially regulated. The maximum rate is always found in intermediate concentrations though. Rates for bound V are very slow at high V levels.

It seemed like intracellular levels of each metal didn't have much effect on levels of other metals; they were mainly regulated based on concentrations in the environment. A. vinelandii keeps taking up Mo as long as it is available (A. chroococcum stops at a much lower value though). This matches with other research on A. vinelandii (014).

With V, A. vinelandii levels increase up to a plateau in intermediate concentrations, but at higher concentrations the levels increase beyond the plateau. Similar with Fe, except Fe-only mutants might have a higher plateau than others. Which makes sense.

Metal Levels and Growth/Nitrogen Fixation
In wild-type A. vinelandii, when growing with V and limited amounts of Mo, the cells start by taking up Mo and growing constantly; when outside Mo runs out, they start taking up V. When that is depleted, the growth rate slows down.

Correspondingly, V-nitrogenase gene expression starts up when Mo runs out, though not all Mo-nitrogenase genes necessarily stop expression at that point.

And finally, overall nitrogenase protein levels stay fairly constant until both Mo and V run out, and then they rise a lot.

What This Means

The two phases can be explained this way: after all the necessary metals have been taken up, no more functional nitrogenase can be made, but what's already there can still fix nitrogen, so the cells can still produce biomass but not at maximum capacity. It's like if you have a big factory that can hold 50 assembly lines but you only have enough equipment for 20; you still produce, but not at your maximum.

The rise in nitrogenase levels after Mo and V run out could indicate that the cells produce extra iron-only nitrogenase when necessary because it will take extra to fix as much nitrogen as they were fixing before, since it's less efficient. That's an interesting, clever regulatory effect, though it isn't entirely clear that's what's happening.

At lower metal concentrations, metallophores seem important in part because they allow the cells to capture metals and make them more available, but also because they make metal uptake easier to regulate so they don't become toxic. This isn't as easy at high levels, as shown by lower growth rates.

The molybdenum and vanadium toxicity could be due to inhibition of iron uptake, causing limitation; the observation that more iron helps alleviate toxicity supports this hypothesis. On the other hand, they didn't really see a drop in iron levels in lower-iron conditions.

They saw Mo storage, but strangely not Fe storage, despite A. vinelandii seeming to have iron-storing mechanisms. It could just be the growth conditions (exponential, nitrogen-fixing).

Overall, it's a lot of data about a pretty complex system. I liked this quote from the conclusions:

"Azotobacter vinelandii thus seems to be well adapted for diazotrophic growth in a soil environment where low availability, large spatiotemporal heterogeneity and strong competition may contribute to metal limitation."

Reference:

Bellenger, J.-P., Wichard, T., Xu, Y. & Kraepiel, A. M. L. Essential metals for nitrogen fixation in a free-living N₂-fixing bacterium: chelation, homeostasis and high use efficiency. Environ. Microbiol. 13, 1395–1411 (2011).

021 - Nitrogen fixation system of tungsten-resistant mutants of Azotobacter vinelandii

People had known at this point that tungsten was poisonous to nitrogenase in Azotobacter vinelandii. It seemed to replace molybdenum in the primary nitrogenase, rendering it non-functional for nitrogen fixation. But then Paul Bishop and colleagues discovered some strains that could grow in the presence of tungsten, using alternative nitrogenases (001). So Riddle and colleagues wanted to characterize such strains and their weird nitrogenases.

What They Did

The scientists first isolated some tungsten-tolerant mutants of their own. This is pretty simple; just grow cells (in this case, A. vinelandii ATCC 12837) in the presence of tungsten, and some tolerant mutants are likely to grow. They chose one and called it WD2 (for Tungsten-Derived 2). For some reason they grew their bacteria at 35ºC, which seems odd because usually Azotobacter is grown at 30ºC, but whatever.

Then they confirmed WD2's ability to grow and fix nitrogen in the presence of tungsten, compared to the parent, did acetylene reduction assays to measure nitrogenase activity, and ran 2-D gels to compare proteomics of the cells in different conditions. Finally they used electron spin resonance (ESR) and other tests to see the chemical characteristics of the cells' enzymes.

What They Observed

They compared the growth of the wild-type and WD2 in broth with or without tungsten. WD2's growth in tungsten was in between the growth of the wild-type in tungsten-free broth and the wild-type in broth with tungsten. Wild-type with tungsten didn't grow hardly at all, as expected. So tungsten inhibits WD2's growth a little bit, compared to the wild-type. To be fair, the amount of tungsten they used was 1000 times more than the minimum needed to inhibit nitrogen fixation in the wild-type.

Then they grew WD2 with various combinations of Mo and W. The growth curves were all pretty similar, not clearly significantly different, but the one with Mo and no W grew best and the one with W and no Mo was worst. Others were in between as expected (more Mo = better, more W = worse), so it seems like a good trend.

They measured acetylene reduction (as a proxy for nitrogen in nitrogenase activity assays) with the wild-type and WD2 (with tungsten), with various concentrations of acetylene in the atmosphere. In each case, activity (as measured by ethylene produced) was highest early in the exponential growth phase. It was not clear (to my eyes) that different amounts of acetylene or the different strains had significant effects on activity. It seemed like perhaps the higher concentrations permitted more activity later in the growth phase. WD2 in W broth showed only 17% the activity of the wild-type in regular broth.

2-D gels showed different profiles for WD2 in regular Burk broth vs. broth with tungsten, though it's always tough to interpret gel images on their own (as two of the same rarely look alike). It did seem like WD2 and the wild-type with ammonium showed similar profiles, and WD2 with tungsten or even with Mo was different, indicating different proteins for tungsten-tolerant nitrogen fixation. ESR spectra of the different proteins were different too.

With heat-extracted proteins from Mo-grown wild-type or W-grown WD2 cells, the wild-type had about 1 nmol Mo per mg protein, whereas WD2 had negligible, but had 18 nmol W. Acetylene reduction activity was much different too: wild-type had more than 10x the activity of WD2, at least in terms of ethylene produced. Spectra were very different too, especially in the band associated with the Mo-iron cofactor.

What This Means
Overall, WD2's results seem to agree with those of others (001), that mutants of wild-type A. vinelandii can fix nitrogen and grow in the presence of tungsten. And the 2-D gels apparently look similar between these two groups of researchers.

Some of the results (growth curves with different Mo:W ratios) seem to indicate that WD2 was using Mo even when tungsten was present. Others (2-D gels) seemed to indicate otherwise.

Based on the acetylene reduction assay, cells might have lower affinity for acetylene later in their growth phase.

Not much new, but it corroborates some data.

Reference:

1. Riddle, G. D., Simonson, J. G., Hales, B. J. & Braymer, H. D. Nitrogen fixation system of tungsten-resistant mutants of Azotobacter vinelandii. J. Bacteriol. 152, 72–80 (1982).

013 - Molybdenum-independent nitrogenases of Azotobacter vinelandii: a functional species of alternative nitrogenase-3 isolated from a molybdenum-tolerant strain contains an iron-molybdenum cofactor

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).

185 - Transcriptional Profiling of Nitrogen Fixation in Azotobacter vinelandii

Azotobacter vinelandii is known for its amazing ability to fix nitrogen, converting nitrogen gas into biological forms like protein even in the presence of oxygen. This is apparent even in its name—Azotobacter—which translates approximately to "nitrogen bacteria."

But the nitrogen-fixing process and machinery is pretty complicated and difficult to study in a reductionist fashion. Many components don't work the same outside of the context of the rest of them or outside of the cell itself.

So in this study, scientists (some of whom were involved in sequencing the first A. vinelandii genome) took a broad approach to the subject, by looking at the expression of all the genes in the organism, comparing their expression when the cells were fixing nitrogen compared to when they weren't.

In addition, they examined the expression of genes when the bacteria were grown with or without certain metals needed for the various nitrogenase versions: molybdenum, vanadium, etc. More specifically:

• To study non-nitrogen-fixing cells: they grew cells with ammonium
• Cells using the molybdenum nitrogenase: they grew cells with molybdenum and no ammonium
• Cells using the vanadium nitrogenase: they grew cells with vanadium and no Mo or ammonium
• Cells using the iron-only nitrogenase: they grew cells with iron and no other metals or ammonium

Simple enough.

Then, to measure levels of expression, they extracted RNA from the cells in each condition, converted the RNA sequences to DNA (called cDNA or complementary DNA, converted from RNA messengers), and then these chunks of DNA were sequenced using a high-throughput technology called SOLiD.

SOLiD (or Sequencing by Oligonucleotide Ligation and Detection) is one of the main kinds of next-generation sequencing, alongside Roche's 454 pyrosequencing and Illumina. I had to look it up. Apparently one machine these days can sequence 5 trillion bases per day (for reference, the human genome is about 3.2 billion bases long, so SOLiD could sequence more than 1500 human genomes per day). Of course, this is not cheap: that much sequence data would cost about $500,000.

The way it works is by cutting a piece of DNA into short sequences, binding them to tiny magnetic beads so there's one sequence per bead. Then the beads are mixed into an emulsion of oil so that on average, each bead is encased in a small bit of water in a sea of oil, along with reagents needed for polymerase chain reaction (PCR). This allows the DNA on each bead to be copied many times, all at once yet individually, so that many reactions can be done in the volume of liquid that would normally allow only one. Very cool. This is the same technology used to prepare samples for 454 pyrosequencing.

In pyrosequencing, the sequencing is done with DNA polymerase, which is what is normally used to copy DNA, and each base is added one at a time, so each bead will incorporate only the base that fits with the sequence bound to the bead. When a base is added, a tiny bit of light is given off, which a camera in the machine detects and registers it as the base that was added at that time.
However, SOLiD works a bit differently: instead of DNA polymerase, the enzyme is DNA ligase, which links together two strands of DNA. And instead of single bases, short DNA sequences called probes are added, with the two bases at one end known. When one of these matches the bead's sequence, ligase links it up. The probes are labeled with fluorescent molecules, so the next two bases in a sequence can be determined from the color of the fluorescent probe. Then this is cleaved off and another two bases are added. It's somewhat confusing; this site might help a little.
This system avoids some of the problems that pyrosequencing experiences, such as with accuracy, but has some problems of its own (especially price), so it's useful in some cases and not as much in others.

So what good is sequencing all the cDNA? The number of copies of one cDNA, relative to the copies of others, shows how much the cell is transcribing that gene, which can be an indicator of expression levels (transcribing more may mean that the gene is translated into protein more, so there may be more of that specific protein in the cell). It doesn't always work quite proportionally, since there are mechanisms other than transcription for regulating cellular protein levels, but it can usually provide some interesting data. So they sequenced all the cDNA in the cell and compared the number of copies for each gene to see which ones were present in higher or lower numbers in different growth conditions.

Then after analyzing all this data (a large undertaking in itself) and finding genes that seemed to be expressed at higher or lower levels in the different conditions, the scientists confirmed the most interesting findings using real-time quantitative PCR, which is a more sensitive way of measuring the same information. It works by doing PCR on a gene but adding some kind of fluorescent molecule to detect exactly how much of that sequence is present over time in the PCR. Ideally PCR should double the number of sequences in every round of the reaction, but this doesn't always work exactly, so the most accurate measure is to determine the point at which the fluorescence becomes bright enough that the PCR machine can detect it, and then extrapolate back to figure out how much of the sequence was present at the beginning. It's another way to compare transcription levels.

Ok, finally on to results. The authors found that almost 30% of A. vinelandii's genes were affected when fixing nitrogen compared to when not doing so. Many of these were affected regardless of which nitrogenase the cells were using. Mo nitrogenase growth affected the most genes on its own compared to the other two, but the two alternative nitrogenases (V and Fe) together affected more genes than any nitrogenase on its own. So overall, compared to non-fixing conditions, the using the alternatives affected many more genes than using the Mo version; but compared to each other, using the alternatives didn't change many genes. Apparently using the alternatives involves a large shift in the cell's gene regulation, compared to using the main Mo nitrogenase. I wonder why.

Genes Expressed When Using Mo Nitrogenase
The nif genes that make up the Mo nitrogenase are found in two clusters in the genome, one near the beginning (relative to the origin of replication) and one near the end. Some of these genes form the actual enzyme structure itself, some help to put it together with its metal-containing cofactor and such, and some (especially nifA) regulate the process.

Not surprisingly, the main structural genes increased their expression greatly when the cells switched to nitrogen-fixing mode, between 50 and 150 times higher. The primary dinitrogenase reductase, NifH, increased the most, which fits in with previous observations that a high ratio of this protein to the Mo-containing dinitrogenase allows higher nitrogenase activity.

More surprisingly, other nif genes in the major cluster only increased expression up to about 14 times more. This could be because not much of their proteins is needed, or possibly that they were already expressed at high levels and their regulation is mostly post-transcriptional, so not much change would be visible in transcript levels. In the minor cluster, some genes necessary for making the metal cofactor increased around 20-fold.

Other than these expected increases, lots of other genes changed as well; not surprising, considering that nitrogen fixation is essential for growth in low-nitrogen environments but is also very energy-intensive. The most significant changes were in type IV pilus genes. These pili, little hairlike projections from the cell, are involved in lots of things: motility, sensing the environment, attachment to surfaces, etc. It's not entirely clear what they're doing in this context, but apparently something.

Another important factor for nitrogen fixation is protecting the nitrogenase from oxygen. A. vinelandii seems to do this by consuming a lot of carbon in order to reduce whatever oxygen is present, transforming it to water. Its genome has many electron-transporting proteins such as oxidoreductases and terminal oxidases, some of which did appear to be somewhat upregulated in nitrogen-fixing conditions. This could also be useful for producing more energy to power the nitrogenase. The genes for the uptake hydrogenase, which recovers hydrogen produced by the nitrogenase and regenerates some energy from it, also showed increased expression.

There also seems to be a change in some genes associated with iron and sulfur organization, something else that is important for nitrogenase because it contains multiple atoms of these elements. Also, not very surprisingly, the genes related to molybdenum gathering increased also.

Genes Expressed When Using Alternative Nitrogenases
Obviously, the genes that encode the alternative nitrogenases themselves (vnf genes for the vanadium-containing nitrogenase and anf genes for the iron-only nitrogenase) are going to be upregulated when these are in use. The V (vanadium) nitrogenase is used when molybdenum is not present but vanadium is, and the Fe nitrogenase is used when neither of these metals is available.

But these alternative systems don't have alternative versions of all of the necessary enzymes for fixing nitrogen, only the main ones, so they share some of the proteins that the Mo nitrogenases uses. This is especially true of enzymes involved in assembling the nitrogenases and their cofactors, such as NifUSVMB.

When vanadium was present, vnf genes were upregulated, as I said, but in this case, the vnfH gene encoding the vanadium dinitrogenase reductase wasn't as high relative to the other V nitrogenase components as was the case with the molybdenum nitrogenase. Not sure why. There were some other differences, especially that vnf homologs of nif proteins involved in cofactor synthesis were expressed in different proportions, so the process of V-containing cofactor synthesis might be different somehow.

In the case of the iron-only nitrogenase, the nifH and other components' homologs (anfH, etc.) were upregulated in the same ratio as the nif genes, distinct from the vnf homologs: that is, anfH was expressed much higher than anfDK, around four- to five-fold higher.

Of the genes that don't have anf homologs, some nif genes were upregulated (nifUSVMG again), but in other cases the vnf versions were preferred (vnfENXY). vnfH was also upregulated, even though there is a separate anfH. This is in agreement with other previous studies (003), and may be because vnfH has some kind of role in regulating gene expression.

Genes Related to Electron Transport
All of the nitrogenases require electron transport machinery, since the nitrogenase functions by putting electrons (and protons) onto nitrogen gas (N₂) to make ammonia (NH₃). This takes at least eight electrons for each molecule of nitrogen: six for two molecules of ammonia, and two for one molecule of hydrogen as a byproduct. The alternative nitrogenases produce more molecules of hydrogen, so they need even more.

Some of the genes involved are nifF and vnfF, which encode proteins called flavodoxins that transport electrons. They may not be necessary to fix nitrogen, but presumably they're helpful. When the cells were using the Mo nitrogenase, nifF was upregulated, and both were higher when Mo was absent (though vnfF much more so).

Some other genes that seemed involved included rnf1 genes, whose products are membrane-bound and also help to transport electrons to nitrogenase; they also seem to be important for the iron-sulfur cofactor of dinitrogenase reductase. And fix genes also seem important for electron transport. All of these were expressed more when fixing nitrogen in all conditions, but when Mo was absent, fix genes were much higher than rnf1 genes.

Regulatory Genes
Clearly A. vinelandii's nitrogenase system has a lot of regulation going on, so regulatory genes are important. vnfA and anfA are necessary to use the alternative nitrogenases, as is nifA for the primary nitrogenase, and these regulatory genes increased whenever their respective isozyme was in use, though low levels of them were present constantly. Regulation of these genes is likely to be how the cells turn on and off the alternative nitrogenases.

There are a few other homologs of nifA and vnfA that show similar patterns, but may fine-tune the regulation somehow (how is not yet known).

Other Differences in Global Expression
As mentioned, the transcriptional profile when using the Mo nitrogenase is very different from when using V or Fe nitrogenases, probably because the latter are less efficient. The most apparent difference in this study was in the hutU gene, for urocatanase hydratase, which increased greatly when Mo was absent. This gene is necessary to degrade histidine, one of the 20 common amino acids, which makes sense because cells using a less efficient nitrogen-fixing enzyme might want to get nitrogen from other places too, like breaking down some less essential proteins. Similar results have been seen in other diazotrophs.

Also quite interesting, genes for a putative soluble hydrogenase discovered when the genome was sequenced were upregulated, especially when using the Fe nitrogenase. This may be a backup system for recycling the extra hydrogen molecules that these nitrogenases produce, to recover the valuable energy that would otherwise escape.

Some other genes increased also, but the function of their products is as yet unknown. Might be worth investigating.

Comparing expression when using the V nitrogenase vs. the Fe nitrogenase, there was at least one interesting point: there were a few genes near a vnf operon upregulated during V nitrogenase growth, seemingly related to a transporter system, so they're probably a vanadate transporter.

Evolution of Nitrogenase
There's some debate about which came first in history: the Mo nitrogenase or the alternatives. Since the alternatives are less efficient, it would make some sense if they came first and the Mo nitrogenase just improved on them, especially since Mo and possibly V were probably difficult to find before there was much oxygen in the atmosphere. But no one has discovered a species that has alternative nitrogenases and not the Mo nitrogenase, though there are plenty that have only the latter, and not many that have all three. And this study seems to show that the alternatives evolved from the Mo nitrogenase to allow cells to thrive in environments where Mo is absent, which also makes sense.

So these results are very interesting and potentially useful, and seem to tell us a lot about what's going on inside the cells in different conditions!


Citation: Hamilton, T. L. et al. Transcriptional Profiling of Nitrogen Fixation in Azotobacter vinelandii. J. Bacteriol. 193, 4477–4486 (2011).

020 - Comparative characterization of H2 production by the conventional Mo nitrogenase and the alternative "iron-only" nitrogenase of Rhodobacter capsulatus hup- mutants

As I've mentioned before, hydrogen gas is a byproduct of nitrogen fixation (which makes sense; nitrogenase adds protons and electrons to N₂ to form NH₃, and in the process some protons and electrons stick to each other, forming H₂. And the different kinds of nitrogenase have different efficiencies—that is, different proportions of protons and electrons that come off as hydrogen instead of actually useful stuff. The molybdenum-containing nitrogenase is most efficient, with one H₂ per N₂ fixed, and the others have higher ratios.

So this study intended to compare different nitrogenases to find out how much hydrogen they produced. It was done with Rhodobacter capsulatus, not Azotobacter, but the enzymes are similar. R. capsulatus is a type of phototrophic bacterium that possesses the primary Mo nitrogenase and also the iron-only alternative, as well as an uptake hydrogenase.

In order to get around the confounding effects of an uptake hydrogenase, which would significantly reduce the amount of measurable hydrogen given off by all nitrogenases, the scientists used a hydrogenase-negative strain they had generated using a transposon (jumping gene). Besides this strain and its parent, they had a strain with the Mo nitrogenase and Mo transport genes deleted (so it could use only the iron nitrogenase), and a hydrogenase-negative mutant of this strain.

The strains were each grown in broth and then exposed to an atmosphere of argon or nitrogen, sometimes mixed with acetylene or oxygen. The purpose of argon is that, when the nitrogenase enzyme lacks any other substrate (nitrogen, acetylene, etc), it will still work but just devote all its protons and electrons to producing hydrogen gas, producing a lot more than in any other condition. Adding acetylene measured the enzyme activity converting acetylene to ethylene (and a little ethane too, in the case of the iron nitrogenase), and adding oxygen measured its effect on the enzymes. Then after some time for the reaction to occur, concentrations of ethylene, ethane, and hydrogen in the headspace were measured.

In an argon atmosphere with nothing else, the hydrogenase-negative Mo nitrogenase strain produced the most hydrogen. In the parent strain that was hydrogenase-positive, the hydrogenase consumed about 1/4 the hydrogen produced. The hydrogenase-negative strain using the iron nitrogenase produced about 1/2 the hydrogen of the top producer, and when present, hydrogenase consumed about 1/2 its hydrogen, resulting in 1/4 the amount of the top producer. So like this:

Under argon:

• nif+ hup-: 100%
• nif+ hup+: 75%
• nif- hup-: 50%
• nif- hup+: 25%

This may seem odd because the iron nitrogenase is supposed to produce more hydrogen (relative to other substrates), but that is not the only difference between the enzymes; the Mo nitrogenase's rate of production (productivity) is also higher, such that it produces more of any product in a given time. Since these reactions were measured after 1 hour, the Mo nitrogenase was able to produce more hydrogen in that time than the alternative, though they might have produced the same amount (or the iron version more) if allowed to consume all their substrate.

The story of hydrogen production is somewhat different in a nitrogen gas atmosphere:

• nif+ hup-: 62%
• nif+ hup+: 5%
• nif- hup-: 100%
• nif- hup+: 3%

Not surprisingly, when nitrogen is present for the enzymes to fix, the alternative nitrogenase produces a lot more hydrogen than the Mo nitrogenase. However, even in the hydrogenase-negative iron nitrogenase strain's case, the hydrogen produced is a bit more than 1/4 of that produced by the top producer under argon. When hydrogenase is present, it is able to consume most of the hydrogen. Similar results are obtained when acetylene is added to an argon atmosphere.

When acetylene was added, the hydrocarbon results were as expected also. Presence or absence of hydrogenase didn't make much difference regarding ethylene and ethane produced. Mo nitrogenase produced much more ethylene than iron nitrogenase, and hardly any ethane; while the iron nitrogenase produced about 17 times more ethane than the Mo nitrogenase, and 11 times less ethylene. Total products for Mo nitrogenase were also about 11 times more than for iron nitrogenase. More efficient, I say.

In terms of protons and electrons, these results suggest that 80% of Mo nitrogenase's electrons go toward nitrogen fixation (that is, 1 hydrogen for every nitrogen fixed, as I said), but only 45% of the alternative's electrons (so, it makes 3-4 hydrogens for each nitrogen).

Oxygen had different effects on the different enzymes also. In the hydrogenase-negative strains, higher levels of oxygen inhibited each, but the alternative nitrogenase's activity dropped to below 40% with very small increases in oxygen levels, whereas the decrease in Mo nitrogenase activity was almost linear with increasing O₂, retaining as much activity as the alternative at more than five times the level of oxygen. So the iron nitrogenase seems to be about 4 times more sensitive to oxygen. Understandably alternative.

So, at least in this species, the iron nitrogenase is 3-4 times less efficient in terms of electrons going to hydrogen, about 11 times slower, and 4 times more sensitive to oxygen than the Mo nitrogenase.

Citation: Krahn, E., Schneider, K. & Müller, A. Comparative characterization of H₂ production by the conventional Mo nitrogenase and the alternative ‘iron-only’ nitrogenase of Rhodobacter capsulatus hup^- mutants. Appl. Microbiol. Biotechnol. 46, 285–290 (1996).

017 - Mo-independent nitrogenase 3 is advantageous for diazotrophic growth of Azotobacter vinelandii on solid medium containing molybdenum

Everyone now knows that Azotobacter vinelandii has three nitrogenases, and that the Mo-containing one is the best. The iron-only nitrogenase is the least efficient but requires the fewest different metals (only iron, obviously, compared to iron plus another for the other two). But does A. vinelandii ever encounter situations in nature where it can't find molybdenum? It seems likely.

In this study, the scientists tested the difference in nitrogen fixation between cells growing on agar plates and cells growing in liquid broth. This is important because in liquid, nutrients are constantly being mixed and cells can all experience the same concentration of them, pretty much; whereas on agar, nutrients don't move around much (they're trapped in the gel) so the concentration around the bacteria decreases as cells use them up.

They tried growing two strains, wild-type CA and strain CA70 which lacks the genes for the iron-only nitrogenase, to see if one or the other grew more quickly on each type of medium. It turned out that, with the same total concentration of Mo in each, on agar CA outgrew CA70 more and more over time, while in liquid the numbers of cells of each strain remained about the same. And when they tested the strains on agar with different concentrations of Mo, CA outgrew CA70 more and more the lower the concentration of Mo was.

To confirm this, they used a strain (CA73) that had a fusion between anfH (one of the iron nitrogenase genes) and lacZ (makes an enzyme that can break down a compound into color, used to determine amount of a gene produced). They tested this on agar or in liquid with different concentrations of Mo, and found that expression of anfH was much higher on agar than in liquid for mid-range concentrations of Mo (on the low end, A. vinelandii expressed anfH in both conditions, while on the high end, it didn't need to express anfH in either).

Finally, they used a 2-D gel to confirm the presence of iron nitrogenase subunits.

So it seems that, when A. vinelandii is growing on agar, it can sometimes deplete the Mo present in the agar to the point that it needs to switch over to use the iron nitrogenase. In the soil, where A. vinelandii is found naturally, it is probably a common occurrence to encounter areas of low Mo and have to switch.

Citation: Maynard, R. H., Premakumar, R. & Bishop, P. E. Mo-independent nitrogenase 3 is advantageous for diazotrophic growth of Azotobacter vinelandii on solid medium containing molybdenum. J. Bacteriol. 176, 5583–5586 (1994).

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).

003 - Phenotypic characterization of a tungsten-tolerant mutant of Azotobacter vinelandii

One tungsten-tolerant strain from 001 in particular caught the attention of the researchers. Azotobacter vinelandii strain CA6 just happened to mutate spontaneously to be able to fix nitrogen in the presence of tungsten (W).

Later research showed that A. vinelandii possesses three nitrogenase system, actually: the primary, molybdenum-containing one, and two alternatives: one with vanadium instead of molybdenum, and a third with iron. The third is least efficient, but iron is most likely to be available, so it is the most versatile.

But CA6 was still interesting, because somehow it was able to overcome the repressive effect that molybdenum (Mo) and W have on the alternative nitrogenases. So in order to study it, among other things, the scientists made a number of recombinant strains of A. vinelandii, to test the functions of different nitrogenase genes.

They tried growing wild-type strain CA and mutant strain CA6 with different concentrations of W. All tested concentrations of W inhibited CA, and above 1 μM (0.184mg W per liter) all concentrations inhibited it the same amount. With CA6, however, no amount of W seemed to affect its growth. However, when Mo was present (and no W), CA grew about twice as fast as CA6.

To figure out why, they deleted the genes for the alternative nitrogenases to create strain CA6.1.71 (sounds like software versions, heh). Obviously this couldn't fix nitrogen or grow without Mo present for its primary nitrogenase. But when Mo was present, it could grow just as fast as the wild-type, showing that the difference in growth rate is probably because CA6 wastes its energy producing less efficient nitrogenases instead of focusing on the efficient primary one.

They also made some genetic fusions of nitrogenase genes with a gene called lacZ, which codes for an enzyme that breaks the bond between the two sugar molecules of lactose, resulting in one molecule of glucose and one galactose. The purpose of this is that this enzyme also breaks the bond in a molecule called o-nitrophenyl-β-galactoside (ONPG), which releases a molecule of galactose but also o-nitrophenyl, which is a bright yellow color. So when you add ONPG to liquid containing the enzyme, you can tell how much enzyme is present by how yellow the liquid becomes. And by fusing lacZ to other genes, you can get an idea of how much those other genes are expressed in the cell.

So this way, they found that, in the wild-type strain CA, Mo-nitrogenase genes are expressed when Mo or W are present (not surprisingly), and alternative nitrogenase genes are only expressed when Mo or W is absent. In CA6, the iron-nitrogenase is produced with or without Mo or W; only vanadium represses it. And the vanadium nitrogenase in both is expressed only when vanadium is present. They confirmed these results with 2-D gels (described in 001).
(Side note: vnfH, vanadium dinitrogenase reductase, is expressed in CA whenever Mo or W is absent, whether or not V is present; in CA6, it is always expressed regardless of the metals in question.)

One possible reason for the difference between CA and CA6 is the latter's ability to take Mo into its cells; if its uptake of Mo is impaired, that could result in the observed phenotype. So the scientists tested that. They found that, not only was CA6's Mo uptake slower than CA's, but it ceased to take up more above a certain concentration, whereas for CA, the more that was available, the more CA took up. It seemed like there were two separate Mo-uptake systems, one that worked better in low concentrations and one in higher, and CA6 lacked the latter. However, there was still enough Mo present in CA6 that it should have repressed the alternative nitrogenases, so this explanation didn't quite work; there must be something else. These observations just add to the mystery of A. vinelandii CA6.

Citation: Premakumar, R., Jacobitz, S., Ricke, S. C. & Bishop, P. E. Phenotypic characterization of a tungsten-tolerant mutant of Azotobacter vinelandii. J. Bacteriol. 178, 691–696 (1996).

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).

001 - Evidence for an alternative nitrogen fixation system in Azotobacter vinelandii

Azotobacter vinelandii is a well-studied microbe, discovered in 1903. It is most well-known for its nitrogen-fixing abilities (thus its name, "azoto" = nitrogen), the ability to convert nitrogen gas (N₂) into "fixed" nitrogen forms, such as ammonia (NH₃) and then into useful stuff like protein or nucleic acids. The enzyme that performs this reaction (called "nitrogenase") is almost always sensitive to/inactivated by oxygen, but A. vinelandii has ways of protecting its nitrogenase such that it can fix nitrogen even when oxygen is present; it is an obligate aerobe (i.e. it requires oxygen to grow). So that's the introduction.

In this particular study, the hypothesis was that the well-studied nitrogenase at the time, which contained a molybdenum (Mo) cofactor, was not the only nitrogenase that A. vinelandii possessed. That meant that when Mo was scarce or the enzyme was otherwise inactivated, the bacteria in some circumstances could still fix nitrogen (and, because fixed nitrogen is required for growth, could continue to proliferate) using its alternative enzyme.

In order to test this hypothesis, the researchers had a number of mutant strains of A. vinelandii (the wild-type strain being named CA), some of which couldn't fix nitrogen under some conditions, and others that had other phenotypes. These mutants were called CA1, CA2, etc.

The behavior the scientists were looking for in particular was tolerance to tungsten (W). Tungsten is similar to molybdenum in its atomic structure, just a bit bigger, so it sorta imitates Mo enough that when it's present in large enough concentrations, A. vinelandii incorporates W into its nitrogenase instead of Mo, but this form of the enzyme is unable to fix nitrogen. So the wild-type strain, CA, is unable to fix nitrogen or grow when too much W is present. However, some of the mutant strains could grow.

Another bit of evidence was the observation that all of the strains, even CA, could grow and fix nitrogen when neither tungsten nor molybdenum was present. The scientists used a technique called 2-D (two-dimensional) gels to observe changes in concentrations of all proteins in the cells individually. This process involves separating the proteins based on their polarity first in one direction, then separating them perpendicular to that direction based on their size, so this should allow them to see whether a protein is present in one condition but not in another. Indeed, they observed some proteins that were present only when the cells were fixing nitrogen in the presence of tungsten or absence of molybdenum! These seemed to be the components of the alternative nitrogenase.

So the model the authors propose for regulation of this alternative nitrogenase in the wild-type is, when tungsten or Mo is present, it's turned off (probably because it is less efficient than the Mo-containing nitrogenase, so preference is given to the latter when Mo is present), but when those metals are absent, it's turned on. The mutants can fix nitrogen in the presence of tungsten because somehow the repression of the alternative nitrogenase is not active in them.

So that's interesting. What was not known was the nature of this alternative enzyme, what metal it might contain instead of Mo, how the regulatory mechanisms functioned exactly, or whether the alternative system was completely independent genetically or just a modification of the Mo-containing one. But at least they had good evidence that the alternative exists.

Citation: Bishop, P. E., Jarlenski, D. M. & Hetherington, D. R. Evidence for an alternative nitrogen fixation system in Azotobacter vinelandii. Proc. Natl. Acad. Sci. 77, 7342–7346 (1980).