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).

023 - Genome Sequence of Azotobacter vinelandii, an Obligate Aerobe Specialized To Support Diverse Anaerobic Metabolic Processes

One good way to learn a lot about a bacterial species, or at least to get a lot of hints about what it might do or be capable of doing, is to sequence its genome. So that's what a bunch of people decided to do with Azotobacter vinelandii. It makes sense, since this organism is one of the better-studied ones and has interesting capabilities, such as nitrogen fixation.

The strain they chose was called DJ, a variant of the wild-type strain CA. DJ is supposed to be easier to manipulate genetically than its parent. So they sequenced its entire genome, but focused mainly on the surprising amount of oxygen-sensitive enzymes they found in an obligately aerobic organism.

The method of sequencing, for those who care, was plain shotgun Sanger dye-terminator sequencing after generating a clone library. (Apparently, for some reason, Monsanto did a lot of the work.) I guess this was before the next-generation sequencing technologies were available or affordable. And teams of undergrads did much of the work labeling genes and such.

Overall, the genome is pretty similar to that of pseudomonads, especially Pseudomonas stutzeri (another nitrogen-fixing soil microbe). Some of their genes have been rearranged compared to each other, though, and A. vinelandii has almost 1,000 more genes.

In terms of energy-generating systems, A. vinelandii's genome has all the genes needed for aerobic metabolism but seems to lack any complete system for anaerobic respiration or fermentation. It is well-equipped for aerobic respiration though, which it seems to use to consume large amounts of oxygen that would otherwise damage its nitrogenase and other enzymes. The other mechanism it has to protect its nitrogenase is called the FeSII or Shethna protein, which can temporarily deactivate the nitrogenase when oxygen is too high, protecting it from damage.

The sequence showed the precise location of each set of nitrogenase genes relative to each other. They're somewhat spread out. It also located the mod genes for molybdenum (Mo) transport and the hox genes of the uptake hydrogenase (which are pretty close together). Though it turns out there is a second set of genes similar to the original mod operon elsewhere in the genome, that may be a second Mo transport system. Possibly even a third set right next to the first, but it's not certain what it does.

Somewhat interesting is a set of genes that are similar to something called carbon monoxide dehydrogenase (CODH) that is present in some anaerobic organisms. This can convert CO to CO₂ and H₂, effectively using it as an energy source instead of something toxic. But it's not certain whether this is functional in A. vinelandii at all. It may be related to some genes that seem to be related to soluble hydrogenases in other organisms, but their function isn't clear either.

A. vinelandii, some strains of it at least, is well-known for producing certain polymers: polyhydroxybutyrate (PHB), which can be used to make a kind of bioplastic; and alginate, and kind of mucusy stuff that has various uses too. The strains that make alginate are rather slimy and hard to work with, and supposedly this provides a further barrier against oxygen poisoning, but strains CA and DJ don't make it, and this sequence revealed why: a transposon inserted itself in the middle of a regulatory gene, inactivating it. That's all it takes.
The genes for PHB synthesis seem to be intact though.

Knowing the sequence of an organism is very helpful; if you want to check for new capabilities, you can just check the genome. So this is a good study.

Citation: Setubal, J. C. et al. Genome Sequence of Azotobacter vinelandii, an Obligate Aerobe Specialized To Support Diverse Anaerobic Metabolic Processes. J. Bacteriol. 191, 4534–4545 (2009).

015 - Characterization of genes involved in molybdenum transport in Azotobacter vinelandii

It is known that molybdenum (Mo) represses A. vinelandii's alternative nitrogenases, at least in the wild-type. What is not exactly known is the mechanism of this repression. Concentrations of Mo as low as 10 μM repress the alternatives, but some strains (such as CA6) produce the alternatives even with high concentrations of Mo. The question is, is this because Mo is not being transported into the cell, or because the protein that represses the alternatives is not functioning properly somehow?

So in the current study, they took a strain of A. vinelandii that can't make the primary nitrogenase but still represses the alternatives when Mo is present, and created mutants using a transposon, Tn5, then grew it on medium with Mo but without nitrogen, so that only those that had a mutation in a relevant gene could grow. And they found two Tn5-induced mutants, and also some that had mutated spontaneously to be able to grow in such conditions.

They named the two transposon mutants FL2 and FL4, focusing on them because the transposon insertion allowed them to locate and study the genes of interest. The mutants could grow pretty much just as fast with Mo present as the parent strain could grow with Mo absent (though these rates were all somewhat slow because they all needed to use the alternative nitrogenases, which are less efficient). Actually FL4 grew a bit faster than the parent.

They isolated and sequenced the section of the genome that the transposons had inserted themselves into, and it turned out that it was the mod operon that I've discussed before (012). FL2 had an insertion in modE, the regulatory gene, and FL4 had an insertion near the end of modB. So it seems like these genes are important for Mo-induced repression of alternative nitrogenases.

Citation: Luque, F., Mitchenall, L. A., Chapman, M., Christine, R. & Pau, R. N. Characterization of genes involved in molybdenum transport in Azotobacter vinelandii. Mol. Microbiol. 7, 447–459 (1993).

014 - Molybdenum accumulation and storage in Klebsiella pneumoniae and Azotobacter vinelandii

Since the primary nitrogenase of Azotobacter vinelandii (and other nitrogen-fixing organisms) is so dependent on molybdenum (Mo), such that they turn off expression of the nitrogenase in the absence of Mo, I thought it worthwhile to read about A. vinelandii's system for storing Mo. Klebsiella pneumoniae, another well-studied nitrogen-fixing soil organism (given its intimidating name because it is an opportunistic pathogen, I think), is used as a comparison.

Previous research had shown that A. vinelandii takes up and stores Mo continuously when it is present, whether or not the bacteria need to fix nitrogen. This is a different strategy from other diazotrophs (nitrogen-fixing organisms). In this study, bacteria were starved of Mo but given fixed nitrogen, and then put into media with varying amounts of Mo with or without fixed nitrogen. When fixed nitrogen was absent, nitrogenase activity maxed out at low concentrations of Mo, but the more Mo present, the more accumulated in the cells, much more than needed to make nitrogenase. When fixed nitrogen was present, the cells showed the same pattern of increasing Mo accumulation, though the levels seemed lower.

In contrast, K. pneumoniae seemed not to accumulate any Mo when fixed nitrogen was available, and while it did store Mo when fixing nitrogen, it was more than 10x less than the levels in A. vinelandii in any condition.

The researchers also measured accumulation of Mo over time. A. vinelandii took up 100% of the Mo provided within 1 hour (it was only a low concentration though), but K. pneumoniae didn't even start uptake until 2 hours in, and then took 2 hours to reach only 25% accumulation. Clearly the two organisms have different strategies; Klebsiella's uptake pattern correlated with its nitrogen-fixation pattern.

They tested the effect of oxygen on Mo uptake; oxygen is toxic to nitrogenase, such that K. pneumoniae doesn't fix nitrogen in its presence, and A. vinelandii takes steps to protect its nitrogenase. But A. vinelandii is an obligate aerobe, so it must still fix nitrogen and accumulate Mo in the presence of oxygen. Klebsiella, on the other hand, shuts off its nitrogenase and Mo uptake both when oxygen is present.

Chloramphenicol, an antibiotic that inhibits protein synthesis in bacteria, inhibited K. pneumoniae's Mo uptake too, but surprisingly not A. vinelandii's; the latter actually had more Mo per cell with chloramphenicol than without, because it was unable to multiply, so there was the same amount of Mo divided among fewer cells.

So there must be some protein in A. vinelandii that is able to store lots of molybdenum. Indeed, when transferred to Mo-free medium after accumulating Mo, the bacteria could fix nitrogen at the same rate as when growing in Mo-containing medium, at least for a while. Trying to purify the Mo-containing proteins from each organism, the scientists found that pretty much all the Mo in Klebsiella was contained in the nitrogenase, whereas (depending on the amount available) that only accounted for a fraction of Mo in Azotobacter. There is another protein that is able to store large amounts of the metal, about 14-15 atoms per molecule of storage protein.

As a bonus, they tested A. vinelandii's ability to store tungsten, and found that it was accumulated in the same way as Mo. It's a sneaky element, apparently.

Citation: Pienkos, P. T. & Brill, W. J. Molybdenum accumulation and storage in Klebsiella pneumoniae and Azotobacter vinelandii. J. Bacteriol. 145, 743–751 (1981).

012 - Mutational analysis of genes of the mod locus involved in molybdenum transport, homeostasis, and processing in Azotobacter vinelandii

So Azotobacter vinelandii CA6 has impaired molybdenum (Mo) uptake (003). This paper studies the Mo transport system of A. vinelandii, encoded by the mod genes, modEABC. It seems like ModA is a protein that binds Mo outside the cell, ModB brings them inside across the membrane, and ModC powers this process. ModE's role is unknown at this point.

The scientists generated a number of mutant strains of A. vinelandii, knocking out a given gene while also fusing it with lacZ to quantify its expression. They also discovered another mod gene, modG, adjacent to the others but in the opposite strand direction. It looks similar to half of modE, so the protein may have a similar function.

Another enzyme in A. vinelandii that requires Mo is nitrate reductase; the authors measured activity of this enzyme as a proxy for Mo transport activity. The wild-type's activity rises quickly as concentration of Mo in the environment increases, levels off, then rises quickly again at higher concentrations (supporting the idea of two different Mo transport systems). With mutants of modA, modB, and modC, the pattern was always the same: no activity until the concentration reached a certain point (the same point when the wild-type's activity started rising quickly the second time).

Other results were more puzzling: when modE was knocked out in a way that didn't inhibit expression of the other mod genes, it seemed to have good transport activity at lower Mo concentrations but not at higher; and the opposite when its knockout inhibited the other genes.
Strain CA11.6, which genetically combined the lack of Mo nitrogenase in CA11 (002) with the tungsten-tolerant phenotype of CA6 (003), showed good Mo uptake at low concentrations but not at higher. When the modB gene was specifically knocked out of CA11.6, there was hardly any uptake activity at any concentration. Considering my own research, it's difficult to say what is going on genetically in these cases.

When modG was targeted for knockout, it looked pretty much the same as wild-type activity, except when both modG and modE were deleted, in which case it showed activity at much lower concentrations even than wild-type. Explain that, science!

They also directly measured uptake of a radioactive isotope of molybdenum (⁹⁹Mo) in the wild-type and modA or modB mutants. The rate of transport in the wild-type and modB mutant were pretty much constant, though the latter was slower than the former. In the modA mutant though, there was very little transport. They tried adding nonradioactive compounds (Mo, vanadium, sulfate, or tungsten) to compete with transport of radioactive Mo, and found that only Mo and tungsten inhibited radioactive Mo transport by competition. Evidence that the mod genes transport tungsten in addition to Mo.

Lastly, the scientists tested the nitrogen-fixing abilities of mod mutants. With Mo present, nitrogen-fixing growth of modE and G mutants was similar to wild-type. When it was absent, modG knockout grew more slowly and modE more quickly. A double mutant didn't grow hardly at all in either condition, in normal aerobic conditions, but with lower levels of oxygen it grew as well as the wild-type (both very slowly). It could also grow using vanadium (V) and the V-containing alternative nitrogenase.

The conclusions, I suppose, are that modABC are all important for Mo transport, especially at low concentrations. modE's role is not exactly clear, but it may regulate which Mo transporter system is working at a given time (possibly by repressing one and activating the other at low concentrations, and vice versa at high). modG's role is even less clear.

Citation: Mouncey, N. J., Mitchenall, L. A. & Pau, R. N. Mutational analysis of genes of the mod locus involved in molybdenum transport, homeostasis, and processing in Azotobacter vinelandii. J. Bacteriol. 177, 5294–5302 (1995).