536 - Nitrogenase-catalyzed Ethane Production and CO-sensitive Hydrogen Evolution from MoFe Proteins Having Amino Acid Substitutions in an α-Subunit FeMo Cofactor-binding Domain

To figure out which parts of the nitrogenase protein are important, this study made very specific mutations to amino acids in the protein in Azotobacter vinelandii to see how they affected the catalysis.

What They Saw
They grew cells, wild-type and mutants, with molybdenum, then extracted and tested their nitrogenase. There was a nifEN knockout strain, a nifDK knockout, and others with specific changes in nifD, sometimes combined with nifN knockout.

None of these had nitrogen-fixing activity. All had just as much dinitrogenase reductase activity as the wild-type; some had more. But regarding acetylene reduction, all nifN knockouts had about zero, but the single-mutation strains all had some, though none nearly as much as the wild-type. They each had more ethane production than the wild-type though, so although total reduction and ethylene production were lower, ethane production was higher.

The temperature stability of mutants wasn't all the same either; some were more sensitive to heat. Lowering the temperature below 30ºC also led to a lower proportion of electron flux going to ethane instead of ethylene (in the mutants that produced ethane). No ethane was seen at any temperature in the wild-type. Though these measurements may not have been reliable, so the trend might not be real.

Then they tried adding carbon monoxide (CO) to inhibit the enzymes. The pattern was the same for each strain (they say), but the amount of total inhibition was different; some were less sensitive than the wild-type, some more, some equal to wild-type.

The FeMo cofactor didn't seem to be different in the mutants; extracting it and using it to restore activity to an apoprotein gave the same results from each strain.

After these results on crude extracts, they purified wild-type nitrogenase and the mutants' most stable enzyme (that replaced the glutamine in NifD position 191 with a lysine). Under acetylene, the wild-type enzyme had about the same electron flux with or without 0.2% CO, but more went to ethylene (vs hydrogen) when CO was absent. 3% CO completely inhibited nitrogen fixation (under nitrogen, obviously), but didn't inhibit hydrogen production: about as much was produced with nitrogen and CO as with argon and CO (or argon without CO). Incidentally, this study gave a NH₃ to H₂ ratio of 1.4 to 1 in 100% nitrogen, which is somewhat lower than the normal 2 to 1.

With the mutant, there was at least 4x less electron flux overall. With CO absent, most of it went to hydrogen when acetylene was present, but what did go to acetylene produced some ethane and more ethylene (as usual). With argon or nitrogen, it all went to hydrogen. When CO was present, the electron flux seemed even more reduced, but the patterns of product were similar.

What This Means
The 191 glutamine residue seems involved in the catalysis, positioned near the FeMo cofactor active center as it is. I am curious about several things, considering nitrogenase's already interesting catalytic abilities: what would similar studies of the other nitrogenases show? What kind of activity might result from other modifications? And, does this kind of modification allow for the reduction of new substrates, such as carbon monoxide itself? These would be interesting studies, if they haven't been done already. Apparently the vanadium nitrogenase is less sensitive to CO than the Mo version here, and it has already been shown to reduce CO to hydrocarbons, at least in vitro.

The difference in acetylene reduction could be due to different affinities: in the wild-type, a new acetylene replaces an old as soon as it is reduced to ethylene, whereas in a mutant, the ethylene remains long enough to be reduced further to ethane. It seems like this difference is due to a difference in the enzyme itself, rather than the cofactor; so the enzyme itself affects the catalysis (though I guess that's not surprising).

Reference:

Scott, D. J., Dean, D. R. & Newton, W. E. Nitrogenase-catalyzed Ethane Production and CO-sensitive Hydrogen Evolution from MoFe Proteins Having Amino Acid Substitutions in an α-Subunit FeMo Cofactor-binding Domain. J. Biol. Chem. 267, 20002–20010 (1992).

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).