065 - Hydrogenase does not confer significant benefits to Azotobacter vinelandii growing diazotrophically under conditions of glucose limitation

Remember the uptake hydrogenase? A byproduct of nitrogen-fixation is hydrogen gas, which allegedly would be a waste of energy, except Azotobacter vinelandii has an enzyme that oxidizes it and converts it into ATP to recoup some of the energy cost of nitrogen-fixation. That's the common story, anyway. But in science you can't always rely on common stories, so Kerstin Linkerhägner and Jürgen Oelze decided to test that hypothesis.

Actually Yates and others had already tested to see if hydrogenase-knockout mutants of Azotobacter chroococcum were impaired, and found that wild-type strains outcompeted them, at least in some conditions such as when carbon was limiting in the medium (019). But these results may be questionable because the wild-type could consume both its own hydrogen and that produced by the mutant when they were mixed together. This wouldn't often be the case in the wild though.

So Linkerhägner and Oelze grew A. vinelandii strain DJ (relatively wild-type) and a hydrogenase-knockout strain in carbon-limited chemostats. They tried two different concentrations of oxygen, low and high. In terms of biomass, the biomass levels from DJ at each oxygen concentration over a range of dilution rates matched rather well with the results these authors saw in 066 (two years later), though not perfectly. Interestingly, the mutant strain matched its parent perfectly. As D increased, the amount of biomass they produced increased. Presumably this would've leveled off at the lower oxygen concentration if they had tested higher D values, as in 066, but they stopped.

Their next figure is somewhat conceptually challenging, but I'll try to break it down:

Figure 2, Linkerhägner and Oelze 1995

It shows the inverse of the yield, Y (which is, in this case, grams of biomass produced per mole of substrate [glucose] consumed) on the y-axis, and the inverse of the dilution rate D on the x-axis. The different symbols are the two strains and two oxygen concentrations, but the strains are virtually identical, so all you need to know is that the top line is higher oxygen and the bottom is lower.

So what does this mean? Well, since Y is biomass per glucose, 1/Y must be moles of glucose consumed for each gram of biomass produced. Seems about right; 0.1 moles per gram, i.e. 18 g glucose per gram of biomass.

Since D is the rate at which the cells in the chemostat get diluted by in-flowing fresh medium, 1/D is called the retention time: the amount of time, in hours, for one full volume of culture to be replaced with fresh medium. So if there is 1 liter of culture in the reactor (for example; the paper doesn't clearly say), then 1/D is the time it takes for 1 liter of fresh medium to flow in and 1 liter of culture to be removed at the same rate.

So overall, what this graph is saying is that as the time taken to dilute out the culture increases, the cells consume more glucose to produce the same amount of biomass. In other words, as medium is added and removed more slowly, the cells' use of substrate for growth is less efficient.

What's the point of this graph? Well, you'll notice that the points make linear patterns that converge to a single Y-intersect. This intersect is at a 1/D of 0, meaning that it takes zero time to replace a single volume of culture; so D = infinity. Obviously this is impossible, and no cells could grow fast enough to keep up, so this is just a theoretical maximum. But the Y-value is non-zero; it's about 1/Y = 0.05 moles glucose per gram biomass. When you invert this to get Y = 20 g biomass per mole glucose, this is, in fact, the theoretical maximum yield for these cells in this medium. The most biomass they can produce from 180 g glucose is 20 g. 

The other thing valuable about this graph is the slope of the lines. Since slope is change in y over change in x, the value here is effectively 1/Y over 1/D, or D/Y, which is to say, the sucrose consumed per increase in biomass per hour. Somehow, from this, it's possible to derive the cells' maintenance coefficient, which is related to the minimum amount of substrate that the cells need to persist at all, even without growing. You can't decrease the amount of food you give them below a certain amount, because they will not be able to maintain their basic life support functions, let alone grow and divide.

I'm not sure how to derive the maintenance coefficient from this graph (have to do some more studying), but it's clear that, at the higher level of oxygen, the cells' yield drops more dramatically as the dilution rate slows down than at the lower level of oxygen. So higher oxygen decreases their efficiency of substrate utilization. According to the authors, the cells require 1 mmol glucose to maintain each g of protein at the lower oxygen, and 16 mmol at the higher. Big difference.

Since both strains were identical in the same conditions, neither seemed to have an advantage in theoretical yield or maintenance requirements over the other.

There's more to the paper. They measured the levels of each adenine nucleotide (ATP, ADP, and AMP) in each strain at three different dilution rates, and didn't see any significant differences between the strains.

They also estimated respiratory activities by measuring the oxygen and hydrogen going into and coming out of the reactor.

Figure 3, Linkerhägner and Oelze 1995

For hydrogen, not surprisingly, the wild-type produced much less hydrogen in all conditions than the hydrogenase-knockout strain. Hydrogen increased as D increased (since more glucose was being fed in at higher dilution rates). Oxygen being high or low didn't affect anything in the mutant, but the parent produced about 10x more hydrogen at higher oxygen (though it's hard to see here); probably this is because oxygen inhibits the hydrogenase. This confirms that the mutant is losing hydrogen and the wild-type is not, as expected. (Interestingly, while the widely-accepted figure for hydrogen produced per nitrogen fixed is 1 mole per mole, the mutant strain here consistently gave an amount averaging around 2 moles per mole. Not sure what to make of that.)

Both strains produced the same amounts of fixed nitrogen, increasing with increasing D but independent of dissolved oxygen. So hydrogenase wasn't protecting the nitrogenase from oxygen at all, as some have thought.

Finally, Linkerhägner and Oelze calculated the amount of oxygen consumed as the wild-type oxidized the hydrogen it produced. Not surprisingly, respiration in general was higher at higher dilution rates and also at the higher level of oxygen. The parent and mutant strains' respiration rates were pretty similar; the calculated amount that respiration with hydrogen contributed to total respiration was quite low, around 3% at the lower oxygen and 0.5% at the higher. This is somewhat surprising, because the maintenance requirement is higher at higher oxygen, so the hydrogenase should be more helpful. On the other hand, it wouldn't work as well because of the oxygen.

Since the hydrogenase contributes so little to the cells' respiration, it seems like it doesn't help the cells very much in terms of recouping energy costs of nitrogenase. Maybe the hydrogen respiratory chain is more efficient than the normal one, generating more ATP for the same amount of proton motive force, but the authors cite some papers allegedly suggesting that it isn't.

So here's another example of these authors challenging the paradigm of the time, saying that hydrogenase isn't very helpful for the cells' energy metabolism. Their data seems pretty convincing, but I could be missing something. It'd probably be wise to check out papers that cite this paper and 066, to see what other researchers have to say about these results.

Citation: Linkerhägner, K. & Oelze, J. Hydrogenase does not confer significant benefits to Azotobacter vinelandii growing diazotrophically under conditions of glucose limitation. J. Bacteriol. 177, 6018–6020 (1995).

066 - Nitrogenase activity and regeneration of the cellular ATP pool in Azotobacter vinelandii adapted to different oxygen concentrations

Nitrogenase is sensitive to oxygen, which tends to react with and inactivate it and many other enzymes. So often nitrogen-fixing organisms do so only when oxygen is absent. But Azotobacter vinelandii is a very aerobic organism, and can consume large quantities of oxygen relative to other species, especially when fixing nitrogen. How it protects its nitrogenase has been a question of interest for researchers.

There are a couple of generally accepted suggestions: 1) it consumes all the oxygen around it quickly by respiration decoupled from energy production, a process called respiratory protection; or 2) it can deactivate nitrogenase temporarily when overwhelmed with oxygen, and reactivate it when oxygen levels are under control again.

But the authors of the current study, Kerstin Linkerhägner and Jürgen Oelze, question the first of these mechanisms for several reasons (respiration rates and oxygen levels don't always correlate, oxygen can enter the cell without inactivating nitrogenase, etc), and propose their own: that nitrogenase can protect itself from oxygen by reducing it to water, as long as it has the energy needed to do so in the form of ATP. This they call autoprotection.

To test this hypothesis, they grew A. vinelandii wild-type and other strains in chemostats with different levels of oxygen and other nutrients. I'll try to describe each experiment and its possible interpretations.

Experiment 1
First the wild-type was grown in carbon-limited conditions, with different levels of oxygen and different dilution rates (D). They measured rates of respiration per cell and concentrations of protein per volume of culture (as a proxy for biomass).

What they saw was, at the lowest levels of dissolved oxygen, biomass increased slightly over lower D values, when cells were growing more slowly, but then leveled off; at higher levels of oxygen, biomass increased constantly over the range of D values. In all cases, though, higher oxygen meant lower biomass.

Respiration increased over the range of D values also, at all concentrations of oxygen, but higher oxygen meant higher respiration.

Glucose was determined to be fully and equally consumed in all these conditions, so it was truly limiting. Nitrogen fixation (as measured by fixed nitrogen per cell) stayed constant over different levels of oxygen, and increased as D increased.

At the highest level of oxygen, levels of ATP per cell increased with increasing D, while levels of ADP and AMP (spent ATP) stayed pretty constant.

Linkerhägner and Oelze's interpretation: Generally at higher dilution rates, cells' use of energy becomes more efficient, but high levels of oxygen inhibits this in A. vinelandii. So at the lowest level, its efficiency maxed out and the biomass stopped increasing, but at higher oxygen levels, efficiency never reached its maximum potential.

My interpretation: Since oxygen must have some manner of inhibitory effect on biomass production, probably by diverting resources away from growth. The increased respiration must not be providing extra resources for growth. It cannot be determined whether the extra respiration removes oxygen directly, or generates extra ATP for nitrogenase to use for oxygen removal. Since increased respiration did mean increased consumption of oxygen in this case, it seems to go against what L and O suggest in their introduction (that respiration and oxygen levels don't correlate). The possibility of extra ATP for use by nitrogenase is not supported, but I suppose not ruled out yet.
They didn't measure change in adenine nucleotides over different oxygen levels for some reason, so it's hard to say how that changed. Over increasing dilution rates at the highest level of oxygen, both ATP (but not ADP) and fixed nitrogen increased; I'm not sure what this means.

Experiment 2
Then A. vinelandii was grown in phosphate-limited or phosphate-sufficient conditions, with two different levels of oxygen, to control the maximum possible amounts of ATP present.

As supplied phosphate increased, biomass increased (not surprising); respiration rates dropped; and levels of adenosine nucleotides increased. The same trends were observed as levels of dissolved oxygen decreased while holding phosphate levels constant. Nitrogen-fixing remained constant over all conditions.

Linkerhägner and Oelze's interpretation: Somehow these results support their hypothesis.

My interpretation: With sufficient phosphate, despite increases in respiration at higher levels of oxygen, levels of ATP dropped (ADP was fairly constant). With limiting phosphate, higher oxygen meant higher respiration but constant ATP and ADP (presumably the cells were making all they could manage given the limited phosphate). But since nitrogen fixation seemed equal in all conditions, limited phosphate and lower ATP didn't really seem necessary for its protection from oxygen.

ATP per Nitrogen Fixed
When L and O plotted amounts of ATP per cell against fixed nitrogen per cell over all these conditions, the points all ended up in a rather linear relationship:

Figure 3, Linkerhägner and Oelze 1997

This graph also included data from two other strains of A. vinelandii: MK5, which lacks the branch of the respiratory chain thought to be involved in decoupled oxygen consumption for respiratory protection; and hoxKG, which lacks the uptake hydrogenase. The former was grown with very low levels of oxygen, and the latter at the highest level from previous experiments.

ATP Regeneration per Oxygen Consumed
Supposedly it is possible to calculate ATP regeneration rate by multiply dilution rate by cellular ATP content. When L and O did this and plotted the values against the corresponding oxygen consumption rates, the points from the glucose-limited cultures showed linear relationships, but from phosphate-limited cultures the ATP regeneration seemed constant over different levels of oxygen consumption.

For the glucose-limited points, cells in low-oxygen conditions regenerated ATP much faster at lower rates of oxygen consumption than those in high-oxygen conditions. Phosphate-limited cells regenerated ATP pretty slowly even when consuming lots of oxygen.

Nitrogen fixed vs. ATP Regeneration
Finally they plotted amount of rate of fixing nitrogen per cell (D times nitrogen concentration) over rates of ATP regeneration in each condition, and the linear relationship incorporated all the points from all conditions, including with the mutant strains as in Figure 3. So higher rates of nitrogen fixation correlate with higher rates of ATP regeneration.

Overall discussion
In carbon-limited cultures, energy is limited. How this affects things is complex, since everything in the cell requires energy, not just growth, and growth requires other processes than just energy generation (for example, nitrogen fixation).

Interpretation of these results seems to depend a whole lot on previous studies that are likely to be as complex as this one.

At this point, my brain kinda hurts. Physiology is complicated. I'm not sure I buy their conclusions though; I think if I want to understand it better, I'll need to read more about what results to expect from oxygen sensitivity.

Citation: Linkerhägner, K. & Oelze, J. Nitrogenase activity and regeneration of the cellular ATP pool in Azotobacter vinelandii adapted to different oxygen concentrations. Journal of Bacteriology 179, 1362–1367 (1997).

034 - NifB and NifEN protein levels are regulated by ClpX2 under nitrogen fixation conditions in Azotobacter vinelandii

Biological nitrogen fixation (turning N₂ gas into usable forms for protein and such) is an energetically expensive process, requiring large amounts of resources the cell could devote to other purposes; however, if that's the only available source of fixed nitrogen, it's worthwhile, because the alternative is paralysis, essentially.

However, being such an expensive process also means that cells will try to regulate its use very tightly, making sure only to use it when it is absolutely necessary. This is different for different organisms; some regulate the same way all the time, some photosynthetic microbes turn everything off or on depending on available light, etc.

Assembly of the nitrogenase enzyme is a very complex process that requires complex regulation as well. This study looks at the regulation of the molybdenum-containing nitrogenase, the primary one, especially the nifB and nifEN genes. NifB is a protein that seems to help synthesize the Mo-containing cofactor essential for the nitrogenase; it also seems to work for the vanadium- and iron-containing cofactors of the alternative versions. The cofactor that NifB makes transfers to the NifEN complex, which adds the Mo for the nitrogenase. This NifB process seems to be a key point for regulating the entire process.

The nitrogenases each have an activator that helps regulate them: nifA, vnfA, and anfA, and these all influence nifB production. But there are probably other genes involved. For example, Azotobacter vinelandii has a gene called clpX2 in between two other nitrogenase-related genes; clpX encodes a common protease that breaks down proteins that are deformed or no longer useful, but clpX2, while seemingly related, is different. It's sometimes found in nif gene clusters in other species, and knocking it out doesn't disrupt nitrogen fixation; rather, it may increase it.

This study looks more specifically at ClpX2's role in regulating nitrogen fixation in A. vinelandii. To do this, they made new strains with modified genes involved in this process:

• UW233: NifB only works when chemical called IPTG is present; can't fix nitrogen otherwise
• UW238: nifB is IPTG-inducible; nifENX is deleted
• UW295: nifB is IPTG-inducible; nifA is deleted
• UW318: clpX2 fused to lacZ; produces more yellow color from ONPG when clpX2 expressed
• UW319: clpX2 fused to lacZ and nifA is deleted
• UW322: lacks clpX2 gene

UW233 allowed them to control when NifB was produced. They found that when cells were growing with ammonium (a source of fixed nitrogen), not fixing nitrogen, they accumulated higher levels of NifB. This is probably because the cells consume NifB when fixing nitrogen; after removing IPTG from the cells' medium, they stopped producing NifB, but those growing in ammonium still had fairly high levels of NifB even after a few hours, while those fixing nitrogen lost most of theirs.

In UW238, NifB accumulates to higher levels regardless of whether or not ammonium is present, so the NifENX proteins seem to be involved in NifB's regulation. In UW295 when nifA is missing and all the major nif genes are silent, NifB disappears more quickly in both conditions; it seems that whatever is degrading NifB isn't activated by NifA.

Using UW318, the authors discovered that clpX2 was expressed more when fixed nitrogen was absent and the cells were fixing nitrogen, so ammonium seems to downregulate it. UW319 revealed that NifA was not necessary for clpX2 expression either; in fact, expression was higher when nifA was deleted. Why is unclear. Semi-quantitative RT-PCR confirmed these results.

So then the question is, how is ClpX2 involved in regulation of NifB and NifEN? So of course they deleted the clpX2 gene to get UW322. The main difference in this strain was that levels of NifB and NifEN were higher than usual, much higher; it seems that ClpX2 plays a big role in their turnover.

However, deletion of clpX2 comes with a price. When fixed nitrogen was present, the cells grew fine, but when fixing nitrogen they slowed down a little, and the initial setting up of nitrogen fixing was slower too.

One interesting specific effect requires a bit of explanation: the Mo nitrogenase has two main components, which are the main part that contains the Mo cofactor and does the actual reaction with nitrogen, called the dinitrogenase; and the dinitrogenase reductase, which kinda recharges the dinitrogenase and prepares it for the next round of reactions. Having these present in different ratios can affect the overall rate of the process.

But what the authors found in UW322, with clpX2 missing, was that there was much more of the Mo-cofactor-containing dinitrogenase than there was normally, while levels of dinitrogenase reductase remained the same. So maybe ClpX2 holds in check the production of dinitrogenase somehow, so the ratios of the two components are optimized.

The authors hypothesized that ClpX2 might provide an advantage when iron is scarce, because the proteins it regulates are both involved in directing a lot of the cell's iron into nitrogenase cofactors, so they tested in low-iron conditions. UW322 seemed to have a slightly greater disadvantage when fixing nitrogen with limited iron than when fixing nitrogen with sufficient iron, but I'm not sure it looks that significant. Could be.

So here's the figure they made to explain their results, showing the regulation pathways:

Figure 10, Martinez-Noël et al. 2011

It doesn't really specify how ClpX2 might repress those proteins (i.e. by their degradation probably); nor is it clear how ClpX2 itself is regulated. But it is interesting.

Citation: Martínez-Noël, G., Curatti, L., Hernandez, J. A. & Rubio, L. M. NifB and NifEN protein levels are regulated by ClpX2 under nitrogen fixation conditions in Azotobacter vinelandii. Molecular Microbiology 79, 1182–1193 (2011).

025 - Siderophores Produced by Nitrogen-Fixing Azotobacter vinelandii OP in Iron-Limited Continuous Culture

One thing that's noteworthy about A. vinelandii is that when growing on agar plates or broth tubes of the medium commonly used for it (Burk's), after a certain time it produces this neon green pigment that diffuses through the medium. It's neon green under white light, but under ultraviolet light it fluoresces light blue!

This colorful feature is due to a molecule called a siderophore, which the bacteria secrete to scavenge (i.e. chelate) insoluble oxidized iron in the medium and bring it into the cell. Iron is an important element for A. vinelandii, especially for fixing nitrogen; all of its nitrogenases require it.

In the present study, the researchers wanted to find out how A. vinelandii responds to insufficient iron in its environment, and the function of proteins and compounds it produces. They used continuous culture to do this, analyzing steady states at different concentrations of iron.

The scientists grew A. vinelandii strain OP (aka CA) not with Burk's but with a medium called B₆, which also works I guess. Obviously this didn't always include the same amount of iron that it normally contained. They actually cleaned their medium vessels and reactor with EDTA (a chelator that binds tightly to metals) to get rid of all traces of iron.

So they grew OP at four different concentrations of iron, and at steady state for each concentration, measured the concentration of cells (dry weight), quantities of different potential chemical siderophores (extraction and separation by electrophoresis), and quantity of yellow-green fluorescent protein (fluorescence measurement). They also measured the total amount of chelated (bound) metals from all the siderophores.

What they saw was not surprising: at higher levels of iron, there was a higher concentration of cells in the culture and a lower amount of chelation going on. This makes sense because the more iron there is, the less need there is for the bacteria to produce special chelators to scavenge it. The numbers they got were very consistent, differing only about 5% even between different chemostat runs.

Regarding the levels of specific siderophores, one was clearly the predominant one compared to the others. This one went down dramatically as iron increased, dropping all the way to zero when iron was sufficient; the others dropped also but still had low levels even in iron-sufficient conditions.

A few other things to note is that, according to the authors, the lack of fixed nitrogen in the medium prevented contamination of the chemostat, and since the reactor was made of Teflon, no bacterial growth built up on the walls (which presumably would happen on a different material).

So this study shows pretty well which siderophore is important for iron scavenging in A. vinelandii, and that iron deficiency (in nitrogen-fixing conditions at least) impairs its growth.

Citation: Fekete, F. A., Spence, J. T. & Emery, T. Siderophores Produced by Nitrogen-Fixing Azotobacter vinelandii OP in Iron-Limited Continuous Culture. Appl. Environ. Microbiol. 46, 1297–1300 (1983).

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).

020 - Comparative characterization of H2 production by the conventional Mo nitrogenase and the alternative "iron-only" nitrogenase of Rhodobacter capsulatus hup- mutants

As I've mentioned before, hydrogen gas is a byproduct of nitrogen fixation (which makes sense; nitrogenase adds protons and electrons to N₂ to form NH₃, and in the process some protons and electrons stick to each other, forming H₂. And the different kinds of nitrogenase have different efficiencies—that is, different proportions of protons and electrons that come off as hydrogen instead of actually useful stuff. The molybdenum-containing nitrogenase is most efficient, with one H₂ per N₂ fixed, and the others have higher ratios.

So this study intended to compare different nitrogenases to find out how much hydrogen they produced. It was done with Rhodobacter capsulatus, not Azotobacter, but the enzymes are similar. R. capsulatus is a type of phototrophic bacterium that possesses the primary Mo nitrogenase and also the iron-only alternative, as well as an uptake hydrogenase.

In order to get around the confounding effects of an uptake hydrogenase, which would significantly reduce the amount of measurable hydrogen given off by all nitrogenases, the scientists used a hydrogenase-negative strain they had generated using a transposon (jumping gene). Besides this strain and its parent, they had a strain with the Mo nitrogenase and Mo transport genes deleted (so it could use only the iron nitrogenase), and a hydrogenase-negative mutant of this strain.

The strains were each grown in broth and then exposed to an atmosphere of argon or nitrogen, sometimes mixed with acetylene or oxygen. The purpose of argon is that, when the nitrogenase enzyme lacks any other substrate (nitrogen, acetylene, etc), it will still work but just devote all its protons and electrons to producing hydrogen gas, producing a lot more than in any other condition. Adding acetylene measured the enzyme activity converting acetylene to ethylene (and a little ethane too, in the case of the iron nitrogenase), and adding oxygen measured its effect on the enzymes. Then after some time for the reaction to occur, concentrations of ethylene, ethane, and hydrogen in the headspace were measured.

In an argon atmosphere with nothing else, the hydrogenase-negative Mo nitrogenase strain produced the most hydrogen. In the parent strain that was hydrogenase-positive, the hydrogenase consumed about 1/4 the hydrogen produced. The hydrogenase-negative strain using the iron nitrogenase produced about 1/2 the hydrogen of the top producer, and when present, hydrogenase consumed about 1/2 its hydrogen, resulting in 1/4 the amount of the top producer. So like this:

Under argon:

• nif+ hup-: 100%
• nif+ hup+: 75%
• nif- hup-: 50%
• nif- hup+: 25%

This may seem odd because the iron nitrogenase is supposed to produce more hydrogen (relative to other substrates), but that is not the only difference between the enzymes; the Mo nitrogenase's rate of production (productivity) is also higher, such that it produces more of any product in a given time. Since these reactions were measured after 1 hour, the Mo nitrogenase was able to produce more hydrogen in that time than the alternative, though they might have produced the same amount (or the iron version more) if allowed to consume all their substrate.

The story of hydrogen production is somewhat different in a nitrogen gas atmosphere:

• nif+ hup-: 62%
• nif+ hup+: 5%
• nif- hup-: 100%
• nif- hup+: 3%

Not surprisingly, when nitrogen is present for the enzymes to fix, the alternative nitrogenase produces a lot more hydrogen than the Mo nitrogenase. However, even in the hydrogenase-negative iron nitrogenase strain's case, the hydrogen produced is a bit more than 1/4 of that produced by the top producer under argon. When hydrogenase is present, it is able to consume most of the hydrogen. Similar results are obtained when acetylene is added to an argon atmosphere.

When acetylene was added, the hydrocarbon results were as expected also. Presence or absence of hydrogenase didn't make much difference regarding ethylene and ethane produced. Mo nitrogenase produced much more ethylene than iron nitrogenase, and hardly any ethane; while the iron nitrogenase produced about 17 times more ethane than the Mo nitrogenase, and 11 times less ethylene. Total products for Mo nitrogenase were also about 11 times more than for iron nitrogenase. More efficient, I say.

In terms of protons and electrons, these results suggest that 80% of Mo nitrogenase's electrons go toward nitrogen fixation (that is, 1 hydrogen for every nitrogen fixed, as I said), but only 45% of the alternative's electrons (so, it makes 3-4 hydrogens for each nitrogen).

Oxygen had different effects on the different enzymes also. In the hydrogenase-negative strains, higher levels of oxygen inhibited each, but the alternative nitrogenase's activity dropped to below 40% with very small increases in oxygen levels, whereas the decrease in Mo nitrogenase activity was almost linear with increasing O₂, retaining as much activity as the alternative at more than five times the level of oxygen. So the iron nitrogenase seems to be about 4 times more sensitive to oxygen. Understandably alternative.

So, at least in this species, the iron nitrogenase is 3-4 times less efficient in terms of electrons going to hydrogen, about 11 times slower, and 4 times more sensitive to oxygen than the Mo nitrogenase.

Citation: Krahn, E., Schneider, K. & Müller, A. Comparative characterization of H₂ production by the conventional Mo nitrogenase and the alternative ‘iron-only’ nitrogenase of Rhodobacter capsulatus hup^- mutants. Appl. Microbiol. Biotechnol. 46, 285–290 (1996).

019 - The Effect of Nutrient Limitation on the Competition between an H2-uptake Hydrogenase Positive (Hup+) Recombinant Strain of Azotobacter chroococcum and the Hup- Mutant Parent in Mixed Populations

Since Azotobacter has this uptake hydrogenase enzyme that seems to recover the energy lost as hydrogen gas from nitrogen fixation, it is worth testing to see if this hydrogenase actually does provide a benefit to the bacteria. This study was done in A. chroococcum, not A. vinelandii, but they're related enough that we should be able to generalize the data gathered, with caution.

The hydrogenase works by taking H₂'s electrons and passing them through the electron transport chain to oxygen to generate a membrane potential (like voltage) that provides energy for the production of ATP (the cell's energy currency, that it uses to power many of its reactions). This can be an important process for crop production, since some crops (legumes) are colonized with bacteria that fix nitrogen for them, but at the time it was unclear whether the uptake hydrogenase in this system was actually helpful for the crop at all. So the authors decided to study the question in an easier system: free-living Azotobacter.

In this study, the strains under investigation were a mutant strain, offspring of the wild-type, that lacked hydrogenase, called MCD103; and another strain derived from MCD103, called MCD503, that did have a hydrogenase because they crossed MCD103 with a plasmid containing wild-type hydrogenase genes, resulting in MCD503, which was the same as MCD103 in every way except the hydrogenase (presumably).

These strains were grown together in continuous culture/chemostats, fixing nitrogen. If one had a growth advantage over the other, it would come to dominate the culture in time. They measured proportions of the strains in two ways: first, by plating them out on agar and using a technique called "scrying" (which seemed to consist of exposing the colonies to H₂ in a sealed box with an indicator present, such that those that had hydrogenase would stay white and those that didn't would turn blue-black) and then counting the colonies of each kind. Second, by plating and transferring individual colonies to a 96-well plate, then exposing all of them to radioactive hydrogen (tritium, ³H₂) and measuring which ones retained radioactivity (indicative of consuming the hydrogen with their hydrogenase). These sound like rather painful and burdensome procedures; nowadays people would probably just do gene sequencing or transcript analysis to see how many copies of hydrogenase genes/transcripts were present over time. I suppose the techniques in this paper may give a more direct measure though.

So anyway, about what they found. When sucrose (sugar) was the limiting nutrient (that is, when the cells were consuming all the sugar they were given and could've consumed even more), the hydrogenase-positive strain MCD503 came to dominate the culture over time, regardless of the proportions of the strains at the beginning of the experiment. Even when initially there were 99 hydrogenase-negative cells for every one hydrogenase-positive cell, before too long they saw the amount of hydrogen produced falling quickly as hydrogenase activity increased. The domination happened faster at higher dilution rates, which makes sense because faster-growing cells would be able to tolerate these better. When fixed nitrogen (ammonium) was added to the cultures, neither strain dominated the other consistently. So it seems that hydrogenase is important when fixing nitrogen.

When nutrients other than sucrose were limiting, though, the situation was not always the same. When phosphate was limiting, MCD503 still dominated, though not as well as with sucrose limitation. However, when oxygen was limiting, the strain missing its hydrogenase (MCD103) was dominant! Even when MCD503 started as 78% of the cells present, it fell to less than 20% before stabilizing. So it seems that hydrogenase-negative strains can deal with low oxygen better.

When sulfate was limiting, MCD503 declined slowly, but it declined more quickly when iron was limiting (makes some sense because the hydrogenase requires iron in its cofactor).

It was interesting to note, also, that MCD503 (hydrogenase-positive) consumed its own hydrogen but also that produced by MCD103 (hydrogenase-negative). Might've contributed to its faster growth in some conditions, and this is consistent with how the domination slows down as more and more of the population is MCD503 (thus there is less hydrogen produced by its competitor to steal).

Speculations about explanations for the findings, as far as I understand them: carbon-limiting results make sense because H₂ oxidation adds to the energy recovery and makes up for some of the limitation.
Phosphate-limiting results make sense because hydrogen could help increase consumption of oxygen to protect nitrogenase and hydrogenase, which are sensitive to it.
Oxygen-limiting results make sense because hydrogen oxidation might take precedence over other kinds in the electron transport chain due to greater affinity.
And sulfate- and iron-limiting results make sense because hydrogenase requires sulfur in addition to iron, so the cells would devote some of their nutrients to this enzyme instead of other, more important ones; while hydrogenase-negative strains wouldn't have this disadvantage.

So it seems that the hydrogenase is helpful in some circumstances and harmful in others. Interesting results.

Citation: Yates, M. G. & Campbell, F. O. The Effect of Nutrient Limitation on the Competition between an H₂-uptake Hydrogenase Positive (Hup⁺) Recombinant Strain of Azotobacter chroococcum and the Hup^- Mutant Parent in Mixed Populations. Microbiology 135, 221–226 (1989).

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).

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).

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).