062 - Characterization of the iron superoxide dismutase gene of Azotobacter vinelandii: sodB may be essential for viability

This study looked at superoxide dismutase in Azotobacter vinelandii, an Fe-SOD encoded by sodB, and its importance.

What They Saw
Running proteins on a gel testing for SOD activity, they saw two bands: one was Fe-SOD and the other CuZnSOD (which sits in the periplasm). They tried knocking out sodB from A. vinelandii by introducing a kanamycin resistance cassette, and isolated a kan-resistant strain, but it appeared to have two copies of sodB (only one of which was knocked out). They tried increasing the concentration of kanamycin (presumably to force the strain to have multiple copies of the resistance gene), and got one that grew slowly at 100x more kanamycin than I use, but they couldn't get rid of the SOD. Seems like it's essential.

This also supports the idea that A. vinelandii can have multiple copies of its chromosome, since they saw multiple PCR products from the same locus, with and without the resistance marker. The genome only has one copy of sodB, so there must be multiple genome copies.

Reference:

Qurollo, B. A., Bishop, P. E. & Hassan, H. M. Characterization of the iron superoxide dismutase gene of Azotobacter vinelandii: sodB may be essential for viability. Can. J. Microbiol. 47, 63–71 (2001).

017 - Mo-independent nitrogenase 3 is advantageous for diazotrophic growth of Azotobacter vinelandii on solid medium containing molybdenum

Everyone now knows that Azotobacter vinelandii has three nitrogenases, and that the Mo-containing one is the best. The iron-only nitrogenase is the least efficient but requires the fewest different metals (only iron, obviously, compared to iron plus another for the other two). But does A. vinelandii ever encounter situations in nature where it can't find molybdenum? It seems likely.

In this study, the scientists tested the difference in nitrogen fixation between cells growing on agar plates and cells growing in liquid broth. This is important because in liquid, nutrients are constantly being mixed and cells can all experience the same concentration of them, pretty much; whereas on agar, nutrients don't move around much (they're trapped in the gel) so the concentration around the bacteria decreases as cells use them up.

They tried growing two strains, wild-type CA and strain CA70 which lacks the genes for the iron-only nitrogenase, to see if one or the other grew more quickly on each type of medium. It turned out that, with the same total concentration of Mo in each, on agar CA outgrew CA70 more and more over time, while in liquid the numbers of cells of each strain remained about the same. And when they tested the strains on agar with different concentrations of Mo, CA outgrew CA70 more and more the lower the concentration of Mo was.

To confirm this, they used a strain (CA73) that had a fusion between anfH (one of the iron nitrogenase genes) and lacZ (makes an enzyme that can break down a compound into color, used to determine amount of a gene produced). They tested this on agar or in liquid with different concentrations of Mo, and found that expression of anfH was much higher on agar than in liquid for mid-range concentrations of Mo (on the low end, A. vinelandii expressed anfH in both conditions, while on the high end, it didn't need to express anfH in either).

Finally, they used a 2-D gel to confirm the presence of iron nitrogenase subunits.

So it seems that, when A. vinelandii is growing on agar, it can sometimes deplete the Mo present in the agar to the point that it needs to switch over to use the iron nitrogenase. In the soil, where A. vinelandii is found naturally, it is probably a common occurrence to encounter areas of low Mo and have to switch.

Citation: Maynard, R. H., Premakumar, R. & Bishop, P. E. Mo-independent nitrogenase 3 is advantageous for diazotrophic growth of Azotobacter vinelandii on solid medium containing molybdenum. J. Bacteriol. 176, 5583–5586 (1994).

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).

002 - Nitrogen fixation in molybdenum-deficient continuous culture by a strain of Azotobacter vinelandii carrying a deletion of the structural genes for nitrogenase (nifHDK)

Previously, another study (001) suggested the presence of an alternative nitrogenase system in Azotobacter vinelandii, but it was not conclusive. Another, later study confirmed this hypothesis by creating a strain completely lacking the genes encoding the primary, molybdenum-containing nitrogenase (nifHDK genes), so there's no way that strain could be using the primary nitrogenase. But this strain could still fix nitrogen and grow when molybdenum (Mo) was not present in its environment, so clearly it had to have some kind of alternative enzyme.

So in this study, the scientists wanted to figure out if this alternative enzyme had the same characteristics as the Mo-containing one, and if not, how they differed. And one of the best ways to determine the characteristics of metabolic pathways, such as nitrogen fixation, is to use continuous culture!

Continuous culture is a technique for maintaining cells in a constant state so they keep growing indefinitely. At its most basic, what it requires is a container in which the cells grow, with fresh culture medium (liquid containing all the nutrients the cells need) flowing into the container at a constant rate, while liquid and cells inside the container are constantly being removed at the same rate to keep the volume inside the container constant. If done right, the culture of cells will eventually reach a point when their population density, consumption of nutrients, growth rate, and all other metabolic characteristics all remain constant over time. This is called "steady state." By measuring the characteristics of the cells' metabolism at steady state in one condition (for example, with a high concentration of sugar), and then changing the condition (reducing the concentration of sugar) and allowing the cells to reach a new steady state, measuring the new characteristics, and comparing the two, it is possible to determine how the cells' metabolism works.

In this study, the scientists used continuous culture to grow A. vinelandii strain CA11, the one lacking the genes nifHDK for the Mo nitrogenase. They grew it in Mo-free medium, and used several techniques to confirm that it was indeed still fixing nitrogen (for example, depriving it of N₂ gas for a time and observing its lack of growth; or more directly measuring the incorporation of a heavier isotope of nitrogen from ¹⁵N₂ gas).

Then they measured CA11's steady-state characteristics at a number of different dilution rates. Dilution rate is a measure of how quickly new medium is flowing into the culture and old culture volume is being removed, so basically the rate the cells are being diluted. As you might expect, the faster the dilution rate, the more quickly the cells have to grow to maintain their population density; otherwise they would be diluted more and more until none were left. Fortunately, higher dilution rate also means that fresh nutrients are being added more quickly, so growing faster is usually not a problem. But there is a point at which cells just can't grow any faster, called the maximum growth rate, so if the dilution rate is higher than this point, the cells can't keep up, and the population density decreases.

In this study, at different dilution rates, the authors measured the population density in a number of different ways: optical density (how much light passes through a volume of culture; the more densely-packed the cells, the less light passes through, so the higher the optical density); protein content (the amount of protein in a volume of culture; usually correlates with number of cells, but in some conditions cells will have more protein per cell than in other conditions); dry weight (the weight of a volume of culture after all the water is removed; usually correlates well with number of cells, but sometimes fewer larger cells can weigh as much as more smaller cells); nitrogen content (correlates well with protein content, since protein contains nitrogen); and number of colony-forming units (by spreading a known volume of cells onto a nutrient agar plate and counting the number of colonies that grow on the plate, you can get an idea of how many living cells were present in a given volume of culture). They found that with all these measures, there were fewer cells at higher dilution rates, but not much else was noteworthy about the experiment.

In a second experiment, they measured specific activities of nitrogen fixation at the steady states of different dilution rates. Normally, in the wild-type strain, the Mo nitrogenase takes one molecule of N₂, converts it to two molecules of NH₃, and also gives off one molecule of H₂ as a byproduct. There's another enzyme called the uptake hydrogenase that takes the H₂ produced by nitrogenase and oxidizes it for energy, similar to how cells oxidize sugar for energy. This recovers some energy the nitrogenase uses, which would otherwise be wasted. Nitrogenase requires a lot of energy, so it's worthwhile.

So the scientists measured the amount of hydrogen produced by CA11 to see if its alternative nitrogenase produced more or less hydrogen than the Mo-containing version. (They could do this because there was a chemical in the medium that happened to inhibit the uptake hydrogenase, so the hydrogen was released into the headspace of the culture vessel.)

They also measured the nitrogen-fixing activity of the nitrogenase more directly, both by measuring amounts of nitrogen and another way called the acetylene reduction assay. Nitrogenase is not a very picky enzyme; its main substrate is two nitrogens connected by a triple bond, but it will also transform most other molecules that consist of two atoms connected by a triple bond, including carbon monoxide and acetylene (C₂H₂, aka ethyne). So in the acetylene reduction assay, acetylene is added to a container with the enzyme, the enzyme (if present and active) converts it into ethylene (C₂H₄, aka ethene), which can be quantified to measure the enzyme's activity.

They found that as dilution rate increased, nitrogenase activity tended to increase also, producing more of all products (hydrogen, fixed nitrogen, and ethylene). They knew the hydrogen was produced by the nitrogenase because when they added ammonium (which represses nitrogenase activity; the cells aren't going to waste energy fixing nitrogen if there is already fixed nitrogen available), the hydrogen production ceased. They also found that, at mid-range dilution rates, the alternative nitrogenase produced about three H₂ molecules for each ammonia (this ratio decreased at higher and lower dilution rates), which compared to the Mo-containing nitrogenase (1 hydrogen for each ammonia) is less efficient.

The scientists tried adding Mo to see what would happen. They found that, for the wild-type strain that still possessed the Mo-containing nitrogenase, adding Mo made it grow a lot more, but it actually inhibited the growth of CA11.

So it seems that the alternative nitrogenase is less efficient than the Mo-containing one, so the bacteria prefer to use the latter.

Citation: Bishop, P. E., Hawkins, M. E. & Eady, R. R. Nitrogen fixation in molybdenum-deficient continuous culture by a strain of Azotobacter vinelandii carrying a deletion of the structural genes for nitrogenase (nifHDK). Biochem J 238, 437–442 (1986).

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).