190 - Characterization of a Tungsten-Substituted Nitrogenase Isolated from Rhodobacter capsulatus

The three normal nitrogenases have molybdenum and iron, vanadium and iron, or just iron in their central cofactors. This study looked at whether it's possible to have a version that only has tungsten in it though, and if it has any activity, in Rhodobacter capsulatus.

What They Saw
This strain only has Mo and Fe nitrogenases, and they knocked out the latter. So when they removed all Mo from the medium and added W, the only thing it would produce would be Mo nitrogenase with W in it, theoretically.

Adding tungsten almost completely stopped acetylene reduction activity in the wild-type, unless 10x more Mo was added. So tungsten doesn't allow that kind of activity.

Mo also induces production of the Mo nitrogenase (which makes sense), but W also seems to have that effect, though not to the same extent.

Then they purified nitrogenase that had been produced when only W was present (in significant amounts) in the mutant. Purification was the same with this as with Mo in the wild-type, though of course the yield was lower. But the protein had 1 atom of W and none of Mo pretty much, as hoped. Still, it seems like there's only one FeW cofactor, not two, but at least there aren't any FeMos.

This protein still wasn't very active in acetylene reduction, and it didn't seem to be able to fix nitrogen at all. What it could do, though, was produce hydrogen when under an argon atmosphere, so electron flux wasn't totally abolished. And this activity wasn't inhibited by acetylene, unlike in the Mo version. Still, the amount of hydrogen it produced was only 1/4th the amount the normal enzyme could produce in the same conditions.

Finally they wanted to see if rhenium (Re) could take the place of Mo as a FeReco and be functional, since Re is not far from Mo and W (one more proton than W), so they tried growing cells with perrhenate (KReO₄), but it didn't seem to help at all. The cells didn't even appear to be able to assimilate it, so it wasn't possible to test whether the nitrogenase could use it.

What This Means
It's difficult to do this kind of study, because Mo and other metals are almost impossible to eliminate completely from the medium. But they seem to have succeeded as much as possible, and still the nitrogenase only had one FeW cofactor rather than two. But surprisingly it showed some proton reduction activity, though not an exceptional amount. It seems like W in the protein is much more difficult to reduce, an essential step in the catalysis. Maybe the amount of reduction that's possible is only enough for some proton reduction activity.

One interesting speculation is based on an observation that Methanococcus thermolithotrophicus can fix nitrogen in the presence of tungstate at 60ºC, a pretty high temperature, so they wonder whether the nitrogenase with FeWco in R. capsulatus might also have more activity at higher temperature, but they didn't actually test this. Maybe another study.

Reference:

Siemann, S., Schneider, K., Oley, M. & Müller, A. Characterization of a Tungsten-Substituted Nitrogenase Isolated from Rhodobacter capsulatus. Biochemistry 42, 3846–3857 (2003).

054 - Nitrogen fixation by Azotobacter vinelandii in tungsten-containing medium

This study investigates how the alternative (vanadium) nitrogenase interacts with tungsten.

What They Saw
They grew Azotobacter vinelandii UW (aka CA) or a tungsten-tolerant mutant with tungsten and Mo or V, then extracted the nitrogenase.

This mutant was derived by growing UW with W and ammonium over several passages to try to remove all the Mo the cells might be storing, and then plating the cells on plates without ammonium, so they would have to fix nitrogen in the presence of W and absence of Mo to survive. Stuff that grew was tungsten-tolerant: strain LM2.

Then to get nitrogenase, they grew LM2 with tungsten and UW with tungsten and ammonium (not sure why it would produce nitrogenase in that condition, though I guess it might when it ran out of fixed nitrogen).

Both UW and LM2 could grow on medium with Mo, though LM2 was sometimes slower. With W, LM2 grew faster or slower depending on amount of W, while UW did not grow.

They used something called rocket immunoelectrophoresis to measure how much MoFe nitrogenase components each strain produced with W. Each produced about the same amount of dinitrogenase reductase in all conditions, but UW with 1mM W produced about 56% the amount of dinitrogenase as it produced with Mo, and LM2 with 10mM W produced about 7% of the amount that UW produced with Mo. I wonder how accurate this technique is. But they also note that the specific acetylene reduction activity of crude extracts was about 6-8% for each strain grown in W compared to UW in Mo. Does this include alternative nitrogenase activity? They say no. But overall, it seems that both strains produce less Mo nitrogenase with W, LM2 less than UW, but LM2's is relatively more active.

I think what happened next was that they couldn't isolate nitrogenase well from LM2, so they studied it from UW. They had two kinds: typical Mo nitrogenase, and Mo/W nitrogenase that had one FeW-cofactor and one FeMo-cofactor. This latter showed less activity in every way: hydrogen under argon, nitrogen, or acetylene atmospheres, nitrogen reduction, and acetylene reduction. This fits with at least one previous study (045). There is still activity though, even with nitrogen reduction, so it's not clear why the cells can't grow in W; maybe its interaction with other important proteins?

What This Means
It's possible that instead of each molecule of Mo/W protein containing one W and one Mo, half the protein could have all Mo and the other half all W; this would give the same results, but seems a less likely explanation. Of course, both possibilities are pretty weird and confusing.

According to some chemistry stuff they did, it seems like the enzyme can't reduce FeW-cofactors, which could reduce the possible electron flux by half, I think. There are a lot of mysteries here.

Reference:

Hales, B. J. & Case, E. E. Nitrogen fixation by Azotobacter vinelandii in tungsten-containing medium. J. Biol. Chem. 262, 16205–16211 (1987).

045 - Tungsten incorporation into Azotobacter vinelandii nitrogenase

Tungsten is known to cause problems for molybdenum-containing enzymes. This report looks into its effect on Azotobacter vinelandii's Mo nitrogenase.

What They Saw
They grew A. vinelandii OP (aka CA) in Burk without Mo, with added ammonium phosphate. Because it's really hard to get rid of every little bit of Mo, they added lots of tungsten (W) to make sure that they could see it if it got incorporated into enzymes. Some of the W was radioactive.

W didn't inhibit growth when ammonia was present, which makes sense. But it did inhibit it, about the same, with N2, nitrate, or urea. The enzymes that use these N sources all need Mo. When Mo was about 0.1 μM, it took 20 μM W to inhibit growth 50%; when Mo was 10 μM, it took 4 mM W. When just a little ammonia was added, it took about 5000 times more W than Mo to stop growth.

When they purified nitrogenase from these W-grown cells, they actually did see acetylene reduction activity, though not nearly as much as with normal Mo nitrogenase. The W content of extracts was very high, though it seemed to be easily removable. Specifically purifying Fe-W protein and comparing to the Fe-Mo version, all activities seemed relatively low: acetylene reduction, hydrogen production, and ATP hydrolysis.

What This Means
A. vinelandii might treat W the same as it treats Mo: taking up as much as it can and storing what it doesn't incorporate. But it does seem to incorporate some into the Mo nitrogenase. This seems to result in a poorly functional enzyme, but is that enough to stop cells from growing entirely? Maybe W's effects on other enzymes cause some problems too.

Reference:

Benemann, J. R., Smith, G. M., Kostel, P. J. & McKenna, C. E. Tungsten incorporation into Azotobacter vinelandii nitrogenase. FEBS Lett 29, 219–221 (1973).

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).

014 - Molybdenum accumulation and storage in Klebsiella pneumoniae and Azotobacter vinelandii

Since the primary nitrogenase of Azotobacter vinelandii (and other nitrogen-fixing organisms) is so dependent on molybdenum (Mo), such that they turn off expression of the nitrogenase in the absence of Mo, I thought it worthwhile to read about A. vinelandii's system for storing Mo. Klebsiella pneumoniae, another well-studied nitrogen-fixing soil organism (given its intimidating name because it is an opportunistic pathogen, I think), is used as a comparison.

Previous research had shown that A. vinelandii takes up and stores Mo continuously when it is present, whether or not the bacteria need to fix nitrogen. This is a different strategy from other diazotrophs (nitrogen-fixing organisms). In this study, bacteria were starved of Mo but given fixed nitrogen, and then put into media with varying amounts of Mo with or without fixed nitrogen. When fixed nitrogen was absent, nitrogenase activity maxed out at low concentrations of Mo, but the more Mo present, the more accumulated in the cells, much more than needed to make nitrogenase. When fixed nitrogen was present, the cells showed the same pattern of increasing Mo accumulation, though the levels seemed lower.

In contrast, K. pneumoniae seemed not to accumulate any Mo when fixed nitrogen was available, and while it did store Mo when fixing nitrogen, it was more than 10x less than the levels in A. vinelandii in any condition.

The researchers also measured accumulation of Mo over time. A. vinelandii took up 100% of the Mo provided within 1 hour (it was only a low concentration though), but K. pneumoniae didn't even start uptake until 2 hours in, and then took 2 hours to reach only 25% accumulation. Clearly the two organisms have different strategies; Klebsiella's uptake pattern correlated with its nitrogen-fixation pattern.

They tested the effect of oxygen on Mo uptake; oxygen is toxic to nitrogenase, such that K. pneumoniae doesn't fix nitrogen in its presence, and A. vinelandii takes steps to protect its nitrogenase. But A. vinelandii is an obligate aerobe, so it must still fix nitrogen and accumulate Mo in the presence of oxygen. Klebsiella, on the other hand, shuts off its nitrogenase and Mo uptake both when oxygen is present.

Chloramphenicol, an antibiotic that inhibits protein synthesis in bacteria, inhibited K. pneumoniae's Mo uptake too, but surprisingly not A. vinelandii's; the latter actually had more Mo per cell with chloramphenicol than without, because it was unable to multiply, so there was the same amount of Mo divided among fewer cells.

So there must be some protein in A. vinelandii that is able to store lots of molybdenum. Indeed, when transferred to Mo-free medium after accumulating Mo, the bacteria could fix nitrogen at the same rate as when growing in Mo-containing medium, at least for a while. Trying to purify the Mo-containing proteins from each organism, the scientists found that pretty much all the Mo in Klebsiella was contained in the nitrogenase, whereas (depending on the amount available) that only accounted for a fraction of Mo in Azotobacter. There is another protein that is able to store large amounts of the metal, about 14-15 atoms per molecule of storage protein.

As a bonus, they tested A. vinelandii's ability to store tungsten, and found that it was accumulated in the same way as Mo. It's a sneaky element, apparently.

Citation: Pienkos, P. T. & Brill, W. J. Molybdenum accumulation and storage in Klebsiella pneumoniae and Azotobacter vinelandii. J. Bacteriol. 145, 743–751 (1981).

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).

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).