026 - Siderophore-mediated uptake of iron in Azotobacter vinelandii

Iron is an important element for aerobes, and Azotobacter vinelandii is definitely aerobic. Many oxygen-related enzymes contain the metal, as do the nitrogenases and the uptake hydrogenase, among others. Iron is pretty common in the soil where the bacteria live, but it is generally found in its insoluble form thanks to oxygen, so it's not very bioavailable.

What They Wanted to Know

O. Knosp, M. von Tigerstrom, and W.J. Page knew that A. vinelandii makes several iron-binding enzymes called siderophores (as noted before, 025), but it wasn't yet proven that these siderophores were actually involved with the cells' iron transport; they might have bound the iron but not helped the bacteria to take it up.

What They Did

They grew A. vinelandii strain CA and a capsule-forming strain, ATCC 12837, in nitrogen-free Burk buffer with or without sufficient iron (in the form of iron sulfate heptahydrate). Interestingly, they removed residual iron from the iron-free medium by autoclaving it, letting the iron salts precipitate, as they tend to do, and then filtering them out. Clever!

Then they let the cells sit for a bit in a broth with a radioactive isotope of iron, ⁵⁵Fe, sometimes adding some purified siderophores or crude culture supernatants. They measured how much of this iron the cells took up by measuring radioactivity of washed cells.

What They Observed
When cells had been growing with iron, giving them ⁵⁵Fe resulted in 67% of the radioactivity sticking to a filter (along with the cells). Washing the stuck cells with buffer containing non-radioactive iron removed most of the radioactivity, while washing with iron-free buffer didn't.

When cells had been growing without iron, about 44-55% of the ⁵⁵Fe stuck to the filter; the longer the incubation, the more got stuck.

Though actually when no cells were present, some of the iron still stuck to the filter, retaining about 36% of the radioactivity. A little sodium citrate (a chelator) increased that to almost 50%, while a lot of citrate or a little nitrilotriacetate (another chelator) reduced it. Citrate also prevented iron from binding to cells non-specifically.

Then they measured iron uptake more directly, again with ⁵⁵Fe, with or without initial iron starvation and/or the presence of citrate. Iron-starved cells with citrate took up iron far faster when citrate was present, in supernatant from iron-starved cells (presumably filled with siderophores). Otherwise, iron-starved cells took up iron slightly faster than iron-sufficient cells in buffer without citrate or in supernatant from iron-sufficient cells.

Adding cyanide to cells prevented iron uptake, so it must have been mostly active uptake. Incubating cells in siderophore-free buffer helped to increase uptake, the longer the better.

Two of the three tested siderophores, azotobactin (a delightful neon green molecule) and azotochelin, helped iron uptake in iron-starved cells, though not as much as iron-starved cell supernatant with added sodium citrate. The third, 2,3-dihydroxybenzoic acid (DHBA) didn't seem to do any better than lack of any siderophore.

They also found that iron uptake wasn't affected much by the nitrogen-fixing status; cells grown in ammonium still took it up at the same rate. And the alginate-producing strain ATCC 12837 took up iron at the same rate as CA, except when they were held on ice, in which case the former took up twice as much, possibly because the iron bound to the capsule.

The amount (and ratio) of siderophores produced fluctuated a lot between cultures, despite controlled conditions, but this didn't seem to affect iron uptake because there was always enough siderophore for the amount of iron present.

Adding one siderophore when the other was present already didn't increase the uptake rate, and together they didn't account for all of the uptake activity.

To make sure they weren't damaging siderophores by purifying them, they tried adding acid and then neutralizing it (which happened in the purification too). This reduced the iron uptake 60%. But even adding this acidified/neutralized supernatant to untreated supernatant reduced the uptake somewhat; the process seemed to generate some kind of inhibitor.

Adding HCl (acid) and then NaOH (base) generates NaCl, salt: this could have an effect. So they just added some salt, and found that it also inhibited the uptake, as did other salts. Azotobactin was more sensitive than azotochelin to high salt concentrations.

The complexes the siderophores form with iron still seemed to form still seemed to form in the presence of high salt, nor did medium salt seem to affect A. vinelandii growth.

What This Means
Nothing in the iron-sufficient cell supernatant accounted for the radioactivity stuck to the filter with iron-sufficient cells, because it didn't help increase uptake.

It's kinda weird that DHBA didn't seem to affect iron uptake, since it does seem to bind iron and inhibit A. vinelandii's production of other siderophores. It's possible that the citrate masked its effect.

It's possible that there is another siderophore they didn't know about or test for, which could explain why adding more siderophores didn't seem to increase iron uptake. It was also possible that the purification damaged the siderophores, and they did seem to show such a thing might be possible.

This was a tricky paper. The regulation and use of iron seems to be a complex topic.

Reference:

Knosp, O., von Tigerstrom, M. & Page, W. J. Siderophore-mediated uptake of iron in Azotobacter vinelandii. J Bacteriol 159, 341–347 (1984).

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).