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).