Monday, March 23, 2015

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.

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

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