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).

185 - Transcriptional Profiling of Nitrogen Fixation in Azotobacter vinelandii

Azotobacter vinelandii is known for its amazing ability to fix nitrogen, converting nitrogen gas into biological forms like protein even in the presence of oxygen. This is apparent even in its name—Azotobacter—which translates approximately to "nitrogen bacteria."

But the nitrogen-fixing process and machinery is pretty complicated and difficult to study in a reductionist fashion. Many components don't work the same outside of the context of the rest of them or outside of the cell itself.

So in this study, scientists (some of whom were involved in sequencing the first A. vinelandii genome) took a broad approach to the subject, by looking at the expression of all the genes in the organism, comparing their expression when the cells were fixing nitrogen compared to when they weren't.

In addition, they examined the expression of genes when the bacteria were grown with or without certain metals needed for the various nitrogenase versions: molybdenum, vanadium, etc. More specifically:

• To study non-nitrogen-fixing cells: they grew cells with ammonium
• Cells using the molybdenum nitrogenase: they grew cells with molybdenum and no ammonium
• Cells using the vanadium nitrogenase: they grew cells with vanadium and no Mo or ammonium
• Cells using the iron-only nitrogenase: they grew cells with iron and no other metals or ammonium

Simple enough.

Then, to measure levels of expression, they extracted RNA from the cells in each condition, converted the RNA sequences to DNA (called cDNA or complementary DNA, converted from RNA messengers), and then these chunks of DNA were sequenced using a high-throughput technology called SOLiD.

SOLiD (or Sequencing by Oligonucleotide Ligation and Detection) is one of the main kinds of next-generation sequencing, alongside Roche's 454 pyrosequencing and Illumina. I had to look it up. Apparently one machine these days can sequence 5 trillion bases per day (for reference, the human genome is about 3.2 billion bases long, so SOLiD could sequence more than 1500 human genomes per day). Of course, this is not cheap: that much sequence data would cost about $500,000.

The way it works is by cutting a piece of DNA into short sequences, binding them to tiny magnetic beads so there's one sequence per bead. Then the beads are mixed into an emulsion of oil so that on average, each bead is encased in a small bit of water in a sea of oil, along with reagents needed for polymerase chain reaction (PCR). This allows the DNA on each bead to be copied many times, all at once yet individually, so that many reactions can be done in the volume of liquid that would normally allow only one. Very cool. This is the same technology used to prepare samples for 454 pyrosequencing.

In pyrosequencing, the sequencing is done with DNA polymerase, which is what is normally used to copy DNA, and each base is added one at a time, so each bead will incorporate only the base that fits with the sequence bound to the bead. When a base is added, a tiny bit of light is given off, which a camera in the machine detects and registers it as the base that was added at that time.
However, SOLiD works a bit differently: instead of DNA polymerase, the enzyme is DNA ligase, which links together two strands of DNA. And instead of single bases, short DNA sequences called probes are added, with the two bases at one end known. When one of these matches the bead's sequence, ligase links it up. The probes are labeled with fluorescent molecules, so the next two bases in a sequence can be determined from the color of the fluorescent probe. Then this is cleaved off and another two bases are added. It's somewhat confusing; this site might help a little.
This system avoids some of the problems that pyrosequencing experiences, such as with accuracy, but has some problems of its own (especially price), so it's useful in some cases and not as much in others.

So what good is sequencing all the cDNA? The number of copies of one cDNA, relative to the copies of others, shows how much the cell is transcribing that gene, which can be an indicator of expression levels (transcribing more may mean that the gene is translated into protein more, so there may be more of that specific protein in the cell). It doesn't always work quite proportionally, since there are mechanisms other than transcription for regulating cellular protein levels, but it can usually provide some interesting data. So they sequenced all the cDNA in the cell and compared the number of copies for each gene to see which ones were present in higher or lower numbers in different growth conditions.

Then after analyzing all this data (a large undertaking in itself) and finding genes that seemed to be expressed at higher or lower levels in the different conditions, the scientists confirmed the most interesting findings using real-time quantitative PCR, which is a more sensitive way of measuring the same information. It works by doing PCR on a gene but adding some kind of fluorescent molecule to detect exactly how much of that sequence is present over time in the PCR. Ideally PCR should double the number of sequences in every round of the reaction, but this doesn't always work exactly, so the most accurate measure is to determine the point at which the fluorescence becomes bright enough that the PCR machine can detect it, and then extrapolate back to figure out how much of the sequence was present at the beginning. It's another way to compare transcription levels.

Ok, finally on to results. The authors found that almost 30% of A. vinelandii's genes were affected when fixing nitrogen compared to when not doing so. Many of these were affected regardless of which nitrogenase the cells were using. Mo nitrogenase growth affected the most genes on its own compared to the other two, but the two alternative nitrogenases (V and Fe) together affected more genes than any nitrogenase on its own. So overall, compared to non-fixing conditions, the using the alternatives affected many more genes than using the Mo version; but compared to each other, using the alternatives didn't change many genes. Apparently using the alternatives involves a large shift in the cell's gene regulation, compared to using the main Mo nitrogenase. I wonder why.

Genes Expressed When Using Mo Nitrogenase
The nif genes that make up the Mo nitrogenase are found in two clusters in the genome, one near the beginning (relative to the origin of replication) and one near the end. Some of these genes form the actual enzyme structure itself, some help to put it together with its metal-containing cofactor and such, and some (especially nifA) regulate the process.

Not surprisingly, the main structural genes increased their expression greatly when the cells switched to nitrogen-fixing mode, between 50 and 150 times higher. The primary dinitrogenase reductase, NifH, increased the most, which fits in with previous observations that a high ratio of this protein to the Mo-containing dinitrogenase allows higher nitrogenase activity.

More surprisingly, other nif genes in the major cluster only increased expression up to about 14 times more. This could be because not much of their proteins is needed, or possibly that they were already expressed at high levels and their regulation is mostly post-transcriptional, so not much change would be visible in transcript levels. In the minor cluster, some genes necessary for making the metal cofactor increased around 20-fold.

Other than these expected increases, lots of other genes changed as well; not surprising, considering that nitrogen fixation is essential for growth in low-nitrogen environments but is also very energy-intensive. The most significant changes were in type IV pilus genes. These pili, little hairlike projections from the cell, are involved in lots of things: motility, sensing the environment, attachment to surfaces, etc. It's not entirely clear what they're doing in this context, but apparently something.

Another important factor for nitrogen fixation is protecting the nitrogenase from oxygen. A. vinelandii seems to do this by consuming a lot of carbon in order to reduce whatever oxygen is present, transforming it to water. Its genome has many electron-transporting proteins such as oxidoreductases and terminal oxidases, some of which did appear to be somewhat upregulated in nitrogen-fixing conditions. This could also be useful for producing more energy to power the nitrogenase. The genes for the uptake hydrogenase, which recovers hydrogen produced by the nitrogenase and regenerates some energy from it, also showed increased expression.

There also seems to be a change in some genes associated with iron and sulfur organization, something else that is important for nitrogenase because it contains multiple atoms of these elements. Also, not very surprisingly, the genes related to molybdenum gathering increased also.

Genes Expressed When Using Alternative Nitrogenases
Obviously, the genes that encode the alternative nitrogenases themselves (vnf genes for the vanadium-containing nitrogenase and anf genes for the iron-only nitrogenase) are going to be upregulated when these are in use. The V (vanadium) nitrogenase is used when molybdenum is not present but vanadium is, and the Fe nitrogenase is used when neither of these metals is available.

But these alternative systems don't have alternative versions of all of the necessary enzymes for fixing nitrogen, only the main ones, so they share some of the proteins that the Mo nitrogenases uses. This is especially true of enzymes involved in assembling the nitrogenases and their cofactors, such as NifUSVMB.

When vanadium was present, vnf genes were upregulated, as I said, but in this case, the vnfH gene encoding the vanadium dinitrogenase reductase wasn't as high relative to the other V nitrogenase components as was the case with the molybdenum nitrogenase. Not sure why. There were some other differences, especially that vnf homologs of nif proteins involved in cofactor synthesis were expressed in different proportions, so the process of V-containing cofactor synthesis might be different somehow.

In the case of the iron-only nitrogenase, the nifH and other components' homologs (anfH, etc.) were upregulated in the same ratio as the nif genes, distinct from the vnf homologs: that is, anfH was expressed much higher than anfDK, around four- to five-fold higher.

Of the genes that don't have anf homologs, some nif genes were upregulated (nifUSVMG again), but in other cases the vnf versions were preferred (vnfENXY). vnfH was also upregulated, even though there is a separate anfH. This is in agreement with other previous studies (003), and may be because vnfH has some kind of role in regulating gene expression.

Genes Related to Electron Transport
All of the nitrogenases require electron transport machinery, since the nitrogenase functions by putting electrons (and protons) onto nitrogen gas (N₂) to make ammonia (NH₃). This takes at least eight electrons for each molecule of nitrogen: six for two molecules of ammonia, and two for one molecule of hydrogen as a byproduct. The alternative nitrogenases produce more molecules of hydrogen, so they need even more.

Some of the genes involved are nifF and vnfF, which encode proteins called flavodoxins that transport electrons. They may not be necessary to fix nitrogen, but presumably they're helpful. When the cells were using the Mo nitrogenase, nifF was upregulated, and both were higher when Mo was absent (though vnfF much more so).

Some other genes that seemed involved included rnf1 genes, whose products are membrane-bound and also help to transport electrons to nitrogenase; they also seem to be important for the iron-sulfur cofactor of dinitrogenase reductase. And fix genes also seem important for electron transport. All of these were expressed more when fixing nitrogen in all conditions, but when Mo was absent, fix genes were much higher than rnf1 genes.

Regulatory Genes
Clearly A. vinelandii's nitrogenase system has a lot of regulation going on, so regulatory genes are important. vnfA and anfA are necessary to use the alternative nitrogenases, as is nifA for the primary nitrogenase, and these regulatory genes increased whenever their respective isozyme was in use, though low levels of them were present constantly. Regulation of these genes is likely to be how the cells turn on and off the alternative nitrogenases.

There are a few other homologs of nifA and vnfA that show similar patterns, but may fine-tune the regulation somehow (how is not yet known).

Other Differences in Global Expression
As mentioned, the transcriptional profile when using the Mo nitrogenase is very different from when using V or Fe nitrogenases, probably because the latter are less efficient. The most apparent difference in this study was in the hutU gene, for urocatanase hydratase, which increased greatly when Mo was absent. This gene is necessary to degrade histidine, one of the 20 common amino acids, which makes sense because cells using a less efficient nitrogen-fixing enzyme might want to get nitrogen from other places too, like breaking down some less essential proteins. Similar results have been seen in other diazotrophs.

Also quite interesting, genes for a putative soluble hydrogenase discovered when the genome was sequenced were upregulated, especially when using the Fe nitrogenase. This may be a backup system for recycling the extra hydrogen molecules that these nitrogenases produce, to recover the valuable energy that would otherwise escape.

Some other genes increased also, but the function of their products is as yet unknown. Might be worth investigating.

Comparing expression when using the V nitrogenase vs. the Fe nitrogenase, there was at least one interesting point: there were a few genes near a vnf operon upregulated during V nitrogenase growth, seemingly related to a transporter system, so they're probably a vanadate transporter.

Evolution of Nitrogenase
There's some debate about which came first in history: the Mo nitrogenase or the alternatives. Since the alternatives are less efficient, it would make some sense if they came first and the Mo nitrogenase just improved on them, especially since Mo and possibly V were probably difficult to find before there was much oxygen in the atmosphere. But no one has discovered a species that has alternative nitrogenases and not the Mo nitrogenase, though there are plenty that have only the latter, and not many that have all three. And this study seems to show that the alternatives evolved from the Mo nitrogenase to allow cells to thrive in environments where Mo is absent, which also makes sense.

So these results are very interesting and potentially useful, and seem to tell us a lot about what's going on inside the cells in different conditions!


Citation: Hamilton, T. L. et al. Transcriptional Profiling of Nitrogen Fixation in Azotobacter vinelandii. J. Bacteriol. 193, 4477–4486 (2011).