457 - Hydrogenase and Nitrogen Fixation by Azotobacter

This study looked at hydrogenase in different Azotobacter species (A. vinelandii, A. chroococcum, A. agile whatever that is).

What They Saw
They looked at different kinds and amounts of fixed nitrogen and their effect and different gases in the atmosphere. Many experiments used ammonium phosphate or other forms of ammonium, and they thought maybe the drop in pH seen as ammonium was consumed led to decreased hydrogenase activity, but actually even when they used forms that didn't allow a pH drop, they still saw the same decrease, suggesting that it's the fixed nitrogen itself that leads to decreased activity. Which makes sense.

They found, consistent across species, that ammonium led to the biggest activity decrease, about 60-80%; nitrate as little as 20%; and glutamate hardly at all. I think these cultures were not adapted to these compounds though.

So they tried adapted cultures too. They found that the more fixed nitrogen they added, the less hydrogenase activity they saw. Adaptation didn't matter with ammonium, but cultures adapted to nitrate had more of a decrease in activity. Apparently they didn't test glutamate.

Then they compared cells with various nitrogen sources grown in air or in a hydrogen-oxygen mixture. They didn't test cells without a nitrogen source in this gas mixture though, maybe because they couldn't grow. Anyway, the hydrogenase was always more active in air with no fixed nitrogen than with any kind of fixed nitrogen (as seen before), and with H₂-O₂ the activity seemed even lower, even than with the same fixed nitrogen source in air. Activity was almost zero in nitrate-adapted cells given nitrate. This seems odd; previous studies seemed to show that hydrogen stimulated hydrogenase activity.

What This Means
I'd say other studies showing stimulation by hydrogen were more convincing, but at least this one was consistent showing an adaptation effect and down-regulation in the presence of fixed nitrogen.

Reference:

Lee, S. B. & Wilson, P. W. Hydrogenase and Nitrogen Fixation by Azotobacter. J. Biol. Chem. 151, 377–385 (1943).

365 - Mechanism of biological nitrogen fixation VII. Molecular H2 and the pN2 function of Azotobacter

They looked at the amount of nitrogen fixed with different concentrations in the atmosphere, in Azotobacter vinelandii and chroococcum cells. They were looking for K_N2: the concentration of nitrogen at which the amount of nitrogen fixed is half the maximum.

With inert gases (argon, helium) or with a partial vacuum, the concentration of nitrogen had to get down below 0.15 atm before the fixation decreased much. It gets to 50% around 0.01 atm. But when there was hydrogen, the rate decreased more quickly; the more, the faster. So hydrogen seems to inhibit the nitrogenase, but only at very high concentrations (over 20% of the atmosphere). This inhibition is competitive and reversible.

Reference:

Wyss, O., Lind, C. J., Wilson, J. B. & Wilson, P. W. Mechanism of biological nitrogen fixation VII. Molecular H₂ and the pN₂ function of Azotobacter. Biochem J 35, 845–854 (1941).

358 - Molecular H2 and the pN2 function of Azotobacter

There was a question at this time of whether hydrogen in the air could inhibit nitrogen fixation in Azotobacter vinelandii, and in what conditions this might happen. There were various complicating factors though. This study attempted to do a more controlled investigation using purified enzyme.

What They Saw

They observed that the higher the concentration of nitrogen in the atmosphere, the more nitrogen was fixed in a given time (thus the higher the specific activity was). From this they could calculate the K_N2, by plotting the inverse of the specific activity over the inverse of the concentration and taking the slope of the line. This is an indication of the enzyme's affinity for nitrogen, I think. This is higher than seen from intact cells (0.01).

Then they got to the hydrogen inhibition experiments. As they increased the amount of hydrogen, the nitrogen-fixing activity did seem to decrease, indicating competitive inhibition.

One issue that they didn't control for was the production of hydrogen by nitrogenase itself; this could've influenced the numbers. It could also influence the K_N2 numbers, come to think of it. It's also unclear whether the extracts had any hydrogenase activity, which could influence things in the opposite direction.

Reference:

Strandberg, G. W. & Wilson, P. W. Molecular H₂ and the pN₂ function of Azotobacter. Proc Natl Acad Sci U S A 58, 1404–1409 (1967).

056 - Properties of Hydrogenase from Azotobacter vinelandii

Another study tries to purify the uptake hydrogenase from Azotobacter vinelandii.

What They Saw
They were unable to get pure, soluble enzyme; they couldn't separate it from the membrane, so it remained in insoluble particles.

They observed that hydrogenase activity of these particles didn't decrease after 18 days of exposure to air, unlike particles from other species. They did lose activity within a few weeks though.

They also observed that the enzyme reduces cytochrome C, possibly as part of its electron transport role. They found that carbon monoxide and cyanide inhibit the enzyme. Azide does not, at least not at pH 8.

What This Means
Some of the observations here may be due to the presence of other membrane-bound proteins, not just hydrogenase.

Reference:

Hyndman, L. A., Burris, R. H. & Wilson, P. W. Properties of Hydrogenase from Azotobacter vinelandii. J. Bacteriol. 65, 522–531 (1953).

010 - Formation of the nitrogen-fixing enzyme system in Azotobacter vinelandii

Apparently, it had previously been shown that ammonium repressed nitrogen fixation in Azotobacter vinelandii, and even when fixing nitrogen, cells would immediately take up ammonium when it was given, but would not immediately start fixing nitrogen if they ran out of ammonium. They wanted to look at this lag period more closely.

What They Did

They grew A. vinelandii OP (aka CA) in Burk's nitrogen-free medium, and actually this is the paper most people later cited as the best recipe for Burk's, the standard medium for growing A. vinelandii.

So they grew the cells, sometimes with ammonium acetate or potassium nitrate as fixed nitrogen sources, sometimes with chloramphenicol to prevent protein synthesis. They also did enzyme activity assays with nitrogenase, using ¹⁵N₂. And determined protein content of cells.

What They Observed

The first figure, taken from Strandberg's master's thesis, shows that when A. vinelandii is grown in a nitrogen-free atmosphere with ammonium, growth eventually levels off; if N₂ is then added, cells start growing again after a short lag, 30-60 minutes. But if ammonium is added instead, there's no lag; the cells start growing again immediately. If N₂ was present the whole time, the cells switch to nitrogen-fixing pretty quickly when ammonium runs out, with a small decrease in growth rate.

A better demonstration for this lag was nitrogenase activity assays: it showed right when nitrogen fixation activity started, about 1 1/4 hours after ammonium ran out. Though oxygen levels and temperature possibly weren't ideal. It could be as little as 45 minutes later.

Another interesting observation was that when ammonium ran out and cells were in an environment of 40% oxygen (with the rest 60% helium or hydrogen), they didn't start producing nitrogenase, but they did start when oxygen was only 20%. The hydrogen level didn't seem to matter.

One problem they encountered was that there were small amounts of nitrogen in their gas tanks of oxygen, helium, and hydrogen, which could've been enough to affect the results. They tried to make purer oxygen by electrolysis (splitting water), though there was still a bit of nitrogen; still, it wasn't clear whether nitrogenase production was induced by the presence of nitrogen or merely repressed by ammonium. My guess would be the latter, since cells wouldn't normally encounter N₂-free environments in nature. But regulation can be complicated.

They noticed a slight rise in turbidity even after ammonium ran out, but speculated it could be due to color change that cells go through (from reddish brown to dark brown) when fixing nitrogen. The small amount of nitrogen in the gas flow was enough to get cells to produce nitrogenase, but not enough for them to use it. But cell-free extracts didn't show different absorbance for the two kinds of cells, despite the visible difference.

When they added chloramphenicol, an antibiotic that inhibits protein synthesis, obviously this inhibited nitrogenase formation. If the enzyme was already present, in vitro, the antibiotic didn't inhibit it. But it did inhibit it in cells, possibly because ammonium built up with no way to use it, repressing nitrogenase.

They tried adding 150 mg N (as ammonium) per liter to a culture of nitrogen-fixing cells, and saw that nitrogenase activity dropped off within about 3 hours. Not as fast as I would expect. They interpreted this to mean that the enzyme is not inhibited immediately, just diluted out as the cells stop producing it while continuing to multiply; but it seems to be inactivated faster than just by dilution, so there might be some inactivation or degradation going on.

Citation: Strandberg, G. W. & Wilson, P. W. Formation of the nitrogen-fixing enzyme system in Azotobacter vinelandii. Can. J. Microbiol. 14, 25–31 (1968).

009 - A Non-Gummy Chromogenic Strain of Azotobacter vinelandii

A popular wild-type Azotobacter vinelandii strain at the time was Wisconsin strain O, which was sometimes difficult to work with because it became "gummy," that is, kind of slimy and mucusy. This was due to alginate production, which helped production nitrogenase from oxygen.

So Bush and Wilson were trying to isolate a stable, non-gummy strain that would be easier to study. Eventually they found one that made dense, slime-free colonies on Burk agar plates, and once sure they had a pure culture, they called it A. vinelandii OP (now also called CA). This strain still produced its nice, yellow-green azotobactin pigment for iron-gathering.

Even in liquid, OP didn't become gummy, like other strains did. So they had found something useful. Later studies, especially sequencing the genome (023), revealed that a transposon had knocked out an alginate regulatory protein in OP.

Citation: Bush, J. A. & Wilson, P. W. A Non-Gummy Chromogenic Strain of Azotobacter vinelandii. Nature 184, 381–381 (1959).