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).

004 - Bacterial Iron Transport: Coordination Properties of Azotobactin, the Highly Fluorescent Siderophore of Azotobacter vinelandii

In order to fix nitrogen, Azotobacter vinelandii needs a lot of iron for its nitrogenase enzyme. But in environments with oxygen, iron is usually found in insoluble, oxidized forms, so A. vinelandii secretes compounds called siderophores that bind/chelate the iron and make it easier to bring into the cell.

One of these in particular is a very pretty yellow-green compound that also can fluoresce blue under ultraviolet light, called azotobactin. The authors here wanted to study azotobactin properties, so they purified it, basically by filtering out bacterial cells and running the filtrate through columns.

They found that azotobactin has five sites that are involved in binding iron. This doesn't mean it binds five iron atoms though. Actually it seems only to bind one. It does seem to bind more strongly than another siderophore called aerobactin that Escherichia coli produces.

Once the cell takes iron-bound azotobactin back inside, it seems to reduce the complex, convert Fe(III) to Fe(II) and thus release the iron for other purposes (such as in nitrogenase).

The last interesting thing is that azotobactin is most fluorescent when it is not binding iron. Only one of the conformational states it goes through when bound to iron is slightly fluorescent.

Citation: Palanché, T., Blanc, S., Hennard, C., Abdallah, M. A. & Albrecht-Gary, A.-M. Bacterial Iron Transport: Coordination Properties of Azotobactin, the Highly Fluorescent Siderophore of Azotobacter vinelandii. Inorg. Chem. 43, 1137–1152 (2004).