195 - Levels and activities of nitrogenase proteins in Azotobacter vinelandii grown at different dissolved oxygen concentrations

Obviously oxygen levels have a big effect on nitrogen-fixing Azotobacter. This study looked into specific effects on levels of different proteins related to nitrogen fixation, at different oxygen levels.

What They Saw
They grew A. vinelandii OP (aka CA) in chemostats with 3 g/L sucrose at different oxygen levels (or dilution rates). They measured nitrogenase activity and purified nitrogenase components as well as flavodoxin and FeSII protein.

Unlike in previous studies (111, 165, 183), increasing the oxygen levels didn't seem to reduce nitrogenase activity (in steady state), at least not in the range they tried (except maybe a little at low levels). At all different levels, nitrogenase activity (i.e. acetylene reduction) correlated only with dilution rate.

With Western blots, they found that levels of different nitrogenase proteins (and others) didn't really vary much across different oxygen levels; they were always about 10% of total protein. And if the activity doesn't vary, this means the proportion of active enzyme is constant too. The enzyme activity does match previous numbers though (106).

Trying to grow cells in ammonia, the two components of nitrogenase disappeared, flavodoxin decreased, but FeSII remained constant.

They tried measuring nitrogenase levels at different dilution rates and oxygen levels. At the lowest D, nitrogenase increased as oxygen increased, but it remained pretty constant at higher rates (as shown before). In contrast to the earlier data though, levels didn't seem to increase consistently with increasing D; it could be that the same quantity of enzyme is less active at lower D, probably related to the flow of electrons to the enzymes.

Then they tried inhibiting protein synthesis with chloramphenicol, at either low or high oxygen. The culture started to wash out, of course. Levels of the four proteins didn't change much with oxygen or with time passed after addition of the antibiotic, but nitrogenase activity decreased greatly over time (while respiratory activity didn't change much). This wasn't due to damage to the nitrogenase components; nitrogenase extracts had just as much activity as cells grown without chloramphenicol. Somehow the activity is inhibited.

What This Means
As suggested before, it seems like the absolute presence/concentration of oxygen doesn't determine its toxicity so much as the ratio of oxygen to availability of energy and reducing equivalents. So if there's enough energy and electrons available to nitrogenase, it can keep going up to high levels of oxygen.

Reference:

Dingler, C., Kuhla, J., Wassink, H. & Oelze, J. Levels and activities of nitrogenase proteins in Azotobacter vinelandii grown at different dissolved oxygen concentrations. J Bacteriol 170, 2148–2152 (1988).

178 - Dependence of nitrogenase switch-off upon oxygen stress on the nitrogenase activity in Azotobacter vinelandii

This study looks at what it takes for oxygen stress to induce Azotobacter vinelandii to shut off its nitrogenase.

What They Saw
They grew A. vinelandii OP (aka CA) in chemostats, fixing nitrogen, with limited carbon (3 g/L of sucrose, acetate, or citrate). They stressed the culture with oxygen by increasing the aeration for 6-minute periods. They measured acetylene reduction, and nitrogen fixation directly (by fixed nitrogen increase). They also measured oxygen levels going in and coming out, to determine consumption.

As usual, with sucrose, they observed that respiration rates increased as oxygen levels rose. At a given oxygen level, respiration also increased as the dilution rate D increased. The amount of respiration increase wasn't the same at different dilution rates though, even with the same change in oxygen. The oxygen maintenance requirement increases as oxygen increases, but not linearly (the rate of increase goes down).

With acetate or citrate, the oxygen maintenance coefficient (and respiration at a given oxygen level) was much lower than with sucrose. Also as D increased, respiration with citrate increased linearly, but with acetate it leveled off at some point.

The rate of nitrogen fixation depended only on D, and increased linearly with D. The carbon source didn't affect it.

When they did the oxygen challenges, they found that up to D = 0.15 h^-1, increasing the oxygen shut off nitrogenase completely. Above that D, the shut-off was less severe. With a less severe challenge, there was less shut-off at the same D too, as expected. Substrate didn't seems to matter.

They couldn't measure a change in respiration from oxygen stress directly, because it was too short, but they knew that cultures grown in acetate or citrate couldn't increase their respiration because they had already consumed all the substrate. There was still residual sucrose though, but the amount didn't seem to change with oxygen challenge, so they concluded that respiration didn't suddenly increase.

Finally they tried controlling nitrogenase activity by giving cells small amounts of ammonium, not enough to repress nitrogen fixation, just reduce it. So giving cells 1 mM ammonium when D = 0.16 resulted in the same nitrogenase activity as when D = 0.06 with no ammonium. And they found that an equivalent oxygen challenge led to the same amount of nitrogenase shut-off.

What This Means
So the rate of oxygen consumption doesn't affect how severe the nitrogenase shut-off is, only the rates of substrate feeding and nitrogenase activity, and more so the latter.

This is kinda weird, because if respiration is how the cells protect nitrogenase from oxygen by removing it (respiratory protection), then higher respiration should correlate with higher nitrogenase activity, but it doesn't seem to here. Also, oxygen level and oxygen consumption should correlate linearly, but they don't, especially considering different carbon substrates.

The authors propose that, instead of respiratory protection, the cells' redox state is what matters: nitrogenase requires a reduced state to function, and more oxygen leads to a more oxidized state. Reduction is made possible by the carbon substrate, which provides energy and electrons; at higher dilution rates, more reduction is possible, so the nitrogenase activity can be higher. That also explains why at higher D, the same oxygen challenge leads to less nitrogenase shut-off, because the change in the redox state is less severe. That way, the cells don't need to create an anaerobic environment in their cytoplasm, just maintain a low redox potential and good flow of electrons.

Respiratory protection as a concept is still useful, since it is still true that the cells' respiration increases as oxygen increases when fixing nitrogen, to allow nitrogenase to function; it's just the details that have been challenged here.

Reference:

Kuhla, J. & Oelze, J. Dependence of nitrogenase switch-off upon oxygen stress on the nitrogenase activity in Azotobacter vinelandii. J. Bacteriol. 170, 5325–5329 (1988).

106 - Control of dinitrogen fixation in ammonium-assimilating cultures of Azotobacter vinelandii

What They Wanted to Know

As mentioned last time (105), as the carbon-to-nitrogen ratio of culture medium increases (and the carbon becomes a lot more available than fixed nitrogen), Azotobacter vinelandii biomass stays fairly level for a time, and then starts increasing; it's like two different phases. This depends on the oxygen exposure somewhat (at low oxygen, biomass increases more linearly; at high oxygen, it doesn't increase much at all, at least at the C/N ratios tested), but is a definite phenomenon at some levels.

The hypothesis is that, when there's not much more carbon than fixed nitrogen, there's not enough nitrogen to produce much more biomass (nitrogen is limiting), and there's not enough carbon to make the cells start fixing nitrogen (because that takes a lot of energy; so carbon is also limiting). But as carbon increases, the cells start up their nitrogenase, and nitrogen stops being limiting, so biomass increases.

In this paper, Bühler, Oelze, and colleagues wanted to see if this was actually what was happening in the cells, by testing nitrogenase activity directly.

What They Did

As before, they grew A. vinelandii CA in a chemostat, but this time they measured nitrogenase activity by acetylene reduction (nitrogenase can reduce acetylene/ethyne to ethylene/ethene, which is easy to measure). They also measured total nitrogen and protein contents of the culture, corrected for added ammonium. And to make triple-sure, they did Western blots on samples of culture, using antibodies targeting nitrogen-fixing proteins.

What They Observed

As in 105, protein/nitrogen content remained fairly constant at a mid-range oxygen level up to a point as sucrose increased, and beyond that point, it increased proportionally along with dry weight.

For nitrogenase activity, they saw that the higher the oxygen, the higher the C/N ratio had to be before the cells had detectable nitrogenase activity (and the lower the peak activity at the highest carbon level). After nitrogenase started, it increased up to a certain C/N level, then leveled off. 

They give a formula for how to calculate the C/N ratio when nitrogenase starts working. And based on that, they figured out that cells started fixing nitrogen when the ammonium they were given was not enough for production of biomass from the sucrose they were given. Which makes sense. That happens at about 14 mmol ammonium per gram of protein.

Finally, they wanted to figure out whether nitrogenase proteins needed to be synthesized from scratch in ammonium-grown cells, or whether they were already present to some extent, just not active. So they used Western blots to look at nitrogenase proteins from cells at various C/N ratios. The lowest ratio showed no nitrogenase activity and no visible nitrogenase protein on the blot; mid-range showed slight activity and the faintest of bands; and the highest showed good activity and solid, visible bands. Flavodoxin proteins, related to electron transport to nitrogenase, showed up at all ratios, interestingly.

What This Means

It appears from this that cells have to produce nitrogenase proteins from scratch as C/N ratios increase, but I'm not sure it's clear that inactive versions of the proteins would show up on the blot. Maybe the antibodies they used to detect the active versions don't work well on inactive versions. It's possible.

The other possibility is that A. vinelandii does keep inactive nitrogenase around for short periods, but eventually breaks it down, and the cells in this study were kept too long in nitrogen-sufficient conditions, so they had to re-synthesize nitrogenase. This would make sense too.

In order to explain the nitrogenase regulation, the authors say it's tempting to say the need for respiratory protection is why cells don't fix nitrogen until C/N ratios are high enough, but clarify that another explanation could be that the cells just have enough fixed nitrogen until a certain point. A tricky conclusion to a tricky series of studies. Perhaps I will revisit later.

Citation: Bühler, T. et al. Control of dinitrogen fixation in ammonium-assimilating cultures of Azotobacter vinelandii. Archives of Microbiology 148, 247–251 (1987).

105 - Control of respiration and growth yield in ammonium-assimilating cultures of Azobacter vinelandii

What They Wanted to Know

As discussed in the previous posts, Bühler, Oelze, and others knew that Azotobacter vinelandii could fix nitrogen at high oxygen levels, but weren't sure how: the respiratory protection hypothesis, that the cells increased their respiration to consume all the oxygen, only seemed to make sense at relatively low levels of oxygen (below 30% saturation); respiratory activity doesn't increase very much beyond a certain point.

Also, since nitrogenase requires a large amount of energy, it's possible the respiration might just be increasing to provide for it. In 089, this lab showed that increasing the fixed nitrogen provided to A. vinelandii led to lower respiratory activity. So, similar to 090 which looked at substrate use efficiency in nitrogen-fixing conditions, here they wanted to see how the efficiency changed when fixed nitrogen was provided.

What They Did

As in previous, they grew A. vinelandii OP (aka CA) in chemostats. They added various levels of sucrose as a substrate and ammonium chloride as fixed nitrogen. They also added sodium citrate, 0.05g/L, to keep the iron they provided from precipitating.

They measured respiratory activities in different states based on oxygen consumption, and also cell dry weights and protein contents, as well as residual sucrose and ammonium.

What They Observed

At the lowest oxygen level (5%), biomass increased almost linearly with increasing sucrose. But as oxygen increased, biomass stayed constant at lower sucrose levels (up to 13-20 mM), and then rose linearly but not as steeply as at low oxygen. At high oxygen (60%), biomass barely rose at all. This was all with the same amount of ammonium added. So, at a given sucrose level, more oxygen meant less biomass. This is consistent with previous studies (090).

They also tried keeping the sucrose constant and varying the amount of ammonium, which affected the carbon-to-nitrogen ratio. They saw similar patterns of biomass vs. C/N ratio, with shifts in the biomass increase at higher ratios, as they had seen when varying the sucrose.

Residual sucrose and ammonium were always very low, so it was all being consumed, and thus limiting. Ratios of dry weight to protein contents were always constant, so there didn't seem to be any nutrient storage going on, even at high sucrose levels.

In nitrogen-limited states at low C/N ratios, yields of biomass were higher, though they decreased as sucrose increased. This is sorta the opposite of what was seen in purely nitrogen-fixing cultures (090), where yield increased as dilution rate (and thus, amount of sucrose) increased. They leveled off when cultures started fixing nitrogen. Even with ammonium though, higher oxygen meant lower yields.

Similarly, respiratory activity increased as C/N ratio increased, up until nitrogen-fixing started; the higher the oxygen, the higher the respiration. Though at a given C/N, higher oxygen always meant higher respiration, unlike in previous studies where it leveled off, though maybe the ranges of sucrose concentrations were different. Also, they measured both respiratory capacity and actual respiration, and cells always seemed to be using only about 50% of their capacity.

What This Means

The way to understand this is that at low sucrose levels, there's only enough ammonium to support a certain amount of biomass production, and not enough sucrose to make it worth turning on nitrogenase, but as the sucrose increases (or ammonium decreases), it becomes more worthwhile.

So C/N ratios seem to control respiratory capacity and activity. That kinda explains why respiration might level off at higher C/N ratios, when nitrogen-fixing activity has started: cells fix as much nitrogen as the carbon level permits, keeping the C/N ratio constant, so the respiration level is constant also. I guess. Look for more discussion about that in the next post.

One last cool thing about this paper: they give the ratios of the main components of A. vinelandii cells, based on the thesis of one H.W. van Verseveld in 1979. The composition is C₆H_10.8N_1.5O_2.9. Useful for calculating molar yields.

Given this, it appeared that the cells converted between 20-30% of the sucrose they consumed into biomass, getting rid of the rest of it, at the lowest oxygen level (5%). At 60% oxygen saturation, they only assimilated 5-10%. Overall, the results aren't really consistent with respiratory protection of nitrogenase, since these were cells grown with ammonium. Interesting.

Citation: Bühler, T. et al. Control of respiration and growth yield in ammonium-assimilating cultures of Azotobacter vinelandii. Arch. Microbiol. 148, 242–246 (1987).

090 - Dependency of growth yield, maintenance and Ks-values on the dissolved oxygen concentration in continuous cultures of Azotobacter vinelandii

What They Wanted to Know

Considering Azotobacter vinelandii's high respiration rates at higher oxygen concentrations, it seemed like its energy metabolism was uncoupled from respiration, meaning that it was respiring without getting as much energy from it, proportionally. This meant that its maintenance energy, the amount of food it needs just to remain the same, should be pretty high in some conditions. So Kuhla and Oelze wanted to examine maintenance requirements and growth yields in more depth, at different levels of oxygen.

What They Did

Similar to previous posts, they grew A. vinelandii OP (aka CA) in a chemostat, limiting its carbon diet and controlling its oxygen exposure, under nitrogen-fixing conditions. They adjusted dilution rates (by adjusting the rate that fresh medium flowed into the reactor) and oxygen saturations.

They also tested different carbon substrates: grams per liter of sucrose, glucose, or acetate. They measured cell dry weights in different conditions, and protein content, nitrogen content, and residual substrates.

What They Observed

First they ruled out the possibility of nutrient storage as a confounding factor, by increasing the amount of food the bacteria received. This didn't really affect the nitrogen content of the cells much, so they didn't seem to be storing it for later. So protein content is a good proxy for total biomass.

Protein content increased as dilution rate increased, not surprising because it meant they were getting more food. At the lowest oxygen level, though, this increase leveled off before too long (around 0.1 h^-1), but even after leveling off the values were still much higher than at any other oxygen level, so they might've just run out of oxygen. Speaking of oxygen, as the saturation increased, the protein content decreased at a given dilution rate.

Growth yields followed almost exactly the same pattern as protein content. So the higher the oxygen, the less efficiently cells used the substrate.

They used a couple methods to calculate maintenance requirements in different conditions (including consumption vs. growth rate from 199 and 1/Y vs. 1/D from 065), but they gave similar results, at least for sucrose and glucose. As expected, maintenance increased as oxygen increased, at least for sucrose, but the maintenance on acetate at mid-range oxygen seemed relatively low and the yield relatively high, so it seems like acetate is used more efficiently.

What This Means

Oxygen above a certain level seems to place extra demands on a cell, preventing it from converting as much of the substrate it consumes into biomass. Exactly what these demands are is unclear though, but growing on acetate seems to reduce the demands somewhat, at least proportional to the amount of carbon consumed.

The authors calculated that, theoretically, A. vinelandii could convert up to 26.2% of the carbon it consumed into biomass, when fixing nitrogen. Presumably this would be significantly higher if not for the requirement to fix nitrogen.

Speaking of respiratory protection again, the expectation would be that maintenance requirements and oxygen levels would be linearly related, but this was only true at lower oxygen levels, suggesting that at higher oxygen, respiration isn't as good at removing oxygen.

Citation: Kuhla, J. & Oelze, J. Dependency of growth yield, maintenance and K_s-values on the dissolved oxygen concentration in continuous cultures of Azotobacter vinelandii. Arch. Microbiol. 149, 509–514 (1988).