217 - Short-Term Effect of Ammonium Chloride on Nitrogen Fixation by Azotobacter vinelandii and by Bacteroids of Rhizobium leguminosarum

The question here is how fixed nitrogen regulates nitrogenase in Azotobacter vinelandii. Adding ammonium above a certain concentration immediately shuts off nitrogen fixation, but the organism doesn't have the DRAT/DRAG enzyme system for ADP-ribosylation of the nitrogenase for posttranscriptional regulation that other organisms have.

This shut-off in A. vinelandii is not due solely to repression of nitrogenase synthesis, since that would not show an effect so quickly. To test alternative mechanisms, this paper took cells grown without fixed nitrogen, suspended them in buffer with a carbon source (sucrose or succinate) and oxygen, and measured acetylene reduction before and after adding ammonium. They also measured nucleotide phosphate levels and respiration.

As expected, adding ammonium greatly lowered nitrogen fixation. The ratio of ATP to ADP seemed mostly to increase though, so lack of ATP didn't seem to cause the inhibition. So what's left? Maybe lack of reducing equivalents used to reduce nitrogen.

Then they did a very confusing and poorly explained experiment (Fig 2) showing uptake of ammonium, which doesn't seem very surprising or informative. However, apparently adding a compound that dissipates membrane potential (valinomycin) caused the opposite effect (loss of ammonium), and another (nigericin) does the opposite (enhancing uptake), so that's somewhat interesting.

They measured proton motive force by a gradient of lipophilic cations, such as tetraphenylphosphonium, across the membrane. They also used a weak acid, 5,5-dimethyloxazolidine-2,4-dione to measure the pH gradient. The sum of these measurements was the proton motive force, in mV.

What they saw was that increasing amounts of ammonium chloride decreased the total proton motive force, but not the pH gradient part or the internal pH of the cells. So the electrical gradient was decreased. This could be because taking up a lot of ammonium, a cation, affects the charge of the membrane. I wonder if they could've tested this further using a different cation though.

Anyway, that's pretty interesting. Nitrogen fixation requires a fairly delicate redox balance, but this can be beneficial for the cells if they use imbalance to regulate their metabolism.

Reference:

Laane, C., Krone, W., Konings, W., Haaker, H. & Veeger, C. Short-Term Effect of Ammonium Chloride on Nitrogen Fixation by Azotobacter vinelandii and by Bacteroids of Rhizobium leguminosarum. Eur J Biochem 103, 39–46 (1980).

524 - In vivo and in vitro nickel-dependent processing of the [NiFe] hydrogenase in Azotobacter vinelandii

This study looked at Azotobacter vinelandii's hydrogenase again, its post-translational processing, and whether nickel influenced this process.

What They Saw
The normal Azotobacter medium (Burk's) has enough contaminating nickel that adding it is unnecessary. But when they added a chelator (nitrilotriacetate) to bind it up, the hydrogenase activity decreased by 80% without affecting growth. This inhibition was lessened by adding nickel.

Nickel availability seemed to affect which form of the alpha subunit was present: the larger, unprocessed form, or the smaller, mature form. With nickel available, only the smaller form was seen; when it was bound up, only the larger. But when excess nickel was added, following the proteins over time showed that gradually the population shifted from larger to smaller as the nickel was used. These two forms are found in different places: the smaller is bound to the membrane (as it should be), and the larger is soluble.

Inhibiting protein synthesis, such as with chloramphenicol, and then adding nickel led to a similar increase in activity as a control without an inhibitor, up to 70 minutes; so for this period, increasing activity wasn't due to protein synthesis. But after this point, the inhibited cultures stopped increasing while the uninhibited continued. The processing of the large form into the small continued regardless of inhibition. So it seems that nickel is important partially for processing and partially for stimulating protein synthesis.

In vitro, ATP or GTP was important for processing. Membranes and oxygen (or lack thereof) were not important. No divalent cation could substitute for nickel: zinc inhibited processing completely, and cobalt or calcium some too. The only protease inhibitor that prevented processing was 1,10-phenanthroline, which inhibits metal-activated proteases.

What This Means
It seems that nickel and processing are both essential for hydrogenase activity, and apparently they are interrelated. It's possible that the processing is regulated by the presence of nickel; without the metal, there isn't much point. Or maybe processing without nickel available will lead to nonfunctional product that can't be fixed. Alternatively, the protease that does the processing could require nickel. It's hard to distinguish these possibilities though. Anyway, it seems like when nickel is absent, the hydrogenase subunits are present but in a premature form, waiting for nickel. How poetic.

Reference:

Menon, A. L. & Robson, R. L. In vivo and in vitro nickel-dependent processing of the [NiFe] hydrogenase in Azotobacter vinelandii. J. Bacteriol. 176, 291–295 (1994).

457 - Hydrogenase and Nitrogen Fixation by Azotobacter

This study looked at hydrogenase in different Azotobacter species (A. vinelandii, A. chroococcum, A. agile whatever that is).

What They Saw
They looked at different kinds and amounts of fixed nitrogen and their effect and different gases in the atmosphere. Many experiments used ammonium phosphate or other forms of ammonium, and they thought maybe the drop in pH seen as ammonium was consumed led to decreased hydrogenase activity, but actually even when they used forms that didn't allow a pH drop, they still saw the same decrease, suggesting that it's the fixed nitrogen itself that leads to decreased activity. Which makes sense.

They found, consistent across species, that ammonium led to the biggest activity decrease, about 60-80%; nitrate as little as 20%; and glutamate hardly at all. I think these cultures were not adapted to these compounds though.

So they tried adapted cultures too. They found that the more fixed nitrogen they added, the less hydrogenase activity they saw. Adaptation didn't matter with ammonium, but cultures adapted to nitrate had more of a decrease in activity. Apparently they didn't test glutamate.

Then they compared cells with various nitrogen sources grown in air or in a hydrogen-oxygen mixture. They didn't test cells without a nitrogen source in this gas mixture though, maybe because they couldn't grow. Anyway, the hydrogenase was always more active in air with no fixed nitrogen than with any kind of fixed nitrogen (as seen before), and with H₂-O₂ the activity seemed even lower, even than with the same fixed nitrogen source in air. Activity was almost zero in nitrate-adapted cells given nitrate. This seems odd; previous studies seemed to show that hydrogen stimulated hydrogenase activity.

What This Means
I'd say other studies showing stimulation by hydrogen were more convincing, but at least this one was consistent showing an adaptation effect and down-regulation in the presence of fixed nitrogen.

Reference:

Lee, S. B. & Wilson, P. W. Hydrogenase and Nitrogen Fixation by Azotobacter. J. Biol. Chem. 151, 377–385 (1943).

308 - Hydrogen-mediated enhancement of hydrogenase expression in Azotobacter vinelandii

This study looked at whether added hydrogen could stimulate hydrogenase activity in Azotobacter vinelandii.

What They Saw
They grew cells with or without ammonium, then added argon or hydrogen to their headspace, and measured whole-cell or purified hydrogenase activity. Oxygen or methylene blue were electron acceptors.

With ammonium, there was a little activity, but adding hydrogen gas increased it about 2.5 to 5 times. As a control, injecting the same amount of argon didn't change anything. In nitrogen-fixing cells, adding hydrogen didn't affect activity.

As with others, activity increased over time in the culture, even corrected by biomass; the hypothesis was that excess carbon inhibits it somehow.

If they added an mRNA or protein synthesis inhibitor (rifampin or chloramphenicol) before adding the hydrogen, activity didn't increase with either case, so it seemed like the regulation was transcriptional.

Also, since the effect was the same with methylene blue (which doesn't require electron transport chain components to act as electron acceptor), it seemed that the regulation was at the hydrogenase directly rather than a related component.

Comparing a couple of Mo nitrogenase-deficient strains (CA11 and CA30) to their parent, they saw that hydrogen didn't affect hydrogenase activity in CA much (in nitrogen-fixing conditions), but it did increase the activity a lot in the mutants. The hydrogenase protein abundance increased too. But in conditions with ammonium, CA and CA11 behaved pretty similar.

Reference:

Prosser, J., Graham, L. & Maier, R. J. Hydrogen-mediated enhancement of hydrogenase expression in Azotobacter vinelandii. J. Bacteriol. 170, 1990–1993 (1988).

307 - The Relationship Between Hydrogenase and Nitrogenase in Azotobacter chroococcum: Effect of Nitrogen Sources on Hydrogenase Activity

This study looked at the influence of different sources of nitrogen on the activity of nitrogenase and hydrogenase in Azotobacter chroococcum.

What They Saw
They grew cells in batch or continuous culture with sodium nitrate, ammonium acetate or chloride, or dinitrogen gas. Cultures were either carbon- or sulfate-limited. Dissolved oxygen was kept above zero. Nitrogenase activity was measured by acetylene reduction and hydrogenase by methylene blue (or by adding H-T with radioactive tritium and measuring radioactivity of resulting water when oxygen was the electron acceptor).

There was about twice as much hydrogenase activity when cells were fixing nitrogen in batch than when they had either ammonium or nitrate. They cite other results in A. chroococcum and A. vinelandii that showed higher activity with nitrate than ammonium though, but still less than when fixing nitrogen. This might be because cells have to adapt to use nitrate, and they'll be fixing nitrogen before that happens. But care is necessary because activity changes over the course of a batch culture, increasing throughout exponential phase even when standardized by protein concentration.

In continuous cultures, they started growing with ammonium, then switched to nitrogen-free, watched what happened with hydrogenase and nitrogenase, and then pulsed a limited amount of ammonium. As expected, nitrogenase activity started up and rose to a plateau, then immediately stopped when ammonium was added, and restarted when it was removed. Hydrogenase activity showed a similar pattern, though more delayed, and it never went all the way to zero.

In sulfate-limited culture, hydrogenase activity was lower even when fixing nitrogen, about the same as with ammonium in carbon limitation, and when going from fixing to non-fixing (with ammonium), the activity declined a bit but then went back up to about the same level. Results were similar going from non-fixing to fixing. So with sulfate limitation, nitrogen source doesn't matter much.

Also with nitrogen-fixing cells in sulfate limitation, when they increased the dilution rate, hydrogenase activity decreased but nitrogenase increased. The decrease wasn't linear, though; it leveled off.

They also tested whether adding hydrogen to the atmosphere of an ammonium-grown culture would influence hydrogenase activity, and it did! Hydrogenase activity doubled. This was not the case with sulfate limitation though, only carbon limitation. They didn't test nitrogen-fixing cells.

What This Means
The continuous culture experiments helped overcome the constantly changing activity in batch cultures.

That hydrogenase activity lags behind nitrogenase activity increase when ammonium runs out could be explained by the last observation: maybe nitrogenase starts producing hydrogen (as it does) and this stimulates hydrogenase activity.

They reasoned from the data that excess carbon might inhibit hydrogenase activity somehow (like catabolite repression). I'm not sure that makes sense, but it seems possible, and does fit with the data from batch cultures, sulfate limitations, and increasing dilution rates. Interesting.

Reference:

Partridge, C. D. P., Walker, C. C., Yates, M. G. & Postgate, J. R. The Relationship Between Hydrogenase and Nitrogenase in Azotobacter chroococcum: Effect of Nitrogen Sources on Hydrogenase Activity. J Gen Microbiol 119, 313–319 (1980).

225 - O2-repression of nitrogenase synthesis in Azotobacter chroococcum

Klebsiella pneumoniae only fixes nitrogen when oxygen levels are very low (or absent), so oxygen represses nitrogenase synthesis. This study looks at whether the same is true in Azotobacter chroococcum; it seems unlikely, since this organism is known for its very aerobic nitrogen fixation; however, it is possible to stress this organism with oxygen such that it may shut down nitrogenase, at least temporarily.

What Robson Saw
He grew A. chroococcum in chemostats with Burk medium with mannitol, with or without ammonium for nitrogen, and stressed them with oxygen either by moving ammonium-grown cells to ammonium-free medium or by suddenly increasing aeration in nitrogen-fixing cells. He looked at levels of nitrogenase proteins (by labeling with radioactive sulfur isotopes) and nitrogenase activity.

At initial low oxygen levels, activity increased and radioisotype-labeled protein levels were relatively high (indicating high levels of nitrogenase protein synthesis). Upon oxygen shock, activity went to zero and protein synthesis levels dropped a lot. When the stress was relieved, both measures increased again.

With ammonium removed and then re-added, things were similar: nitrogenase activity went up and synthesis gradually increased to a plateau, but decreased when more ammonium was added. It did take a relatively longer time for nitrogenase activity to pick up after ammonium was removed, about 80 minutes.

Along with nitrogenase, flavodoxin and the small protein that protects nitrogenase from oxygen by temporarily inactivating it both showed up in radiolabeling. The former matched nitrogenase synthesis patterns, but the latter was fairly constant.

What This Means
It seems that oxygen stress can repress synthesis of nitrogenase even in Azotobacter, though radiolabeling might not be the best method for studying this question. This adds another layer to Azotobacter's protection of its enzymes from oxygen.

Reference:

Robson, R. L. O₂-repression of nitrogenase synthesis in Azotobacter chroococcum. FEMS Microbiology Letters 5, 259–262 (1979).

214 - The Respiratory System of Azotobacter vinelandii 2. Oxygen Effects

This study looks at how oxygen levels influence the respiratory chain in Azotobacter vinelandii.

What They Saw
They grew cells with high or low aeration (based on volume of culture in the same size flask), then isolated respiratory membranes and measured P/O ratios.

The cells grew and quickly used up all the dissolved oxygen. With high aeration, they grew much faster and leveled off once the oxygen was gone, and with low aeration the growth was slower (not even really logarithmic) but continued long after the oxygen was gone. Respiratory activities were 2-5x higher when cells were growing logarithmically with excess oxygen than when oxygen was limited.

If they suddenly increased the aeration when oxygen had run out, respiratory activity increased back up to high levels (1000 μl/h/mg dry weight). Cells didn't start growing again until it had leveled off. This lag was the same when chloramphenicol (which inhibits protein synthesis) was present, suggesting that it wasn't due to the synthesis of new enzymes for respiration.

When oxygen was being consumed, cytochrome and oxidase levels were pretty constant (except cyt o oxidase, which increased), but when oxygen ran out, levels of c₄, c₅, and b₁ increased quickly and a₂ more slowly. Cytochrome o oxidase and a₂ oxidase also increased a lot, a₁ less so. The increase of o was fastest and greatest.

P/O ratios were similar to those seen before at maximum, but they didn't reach this maximum until oxygen was mostly used up late in the logarithmic growth phase, at least for NADH dehydrogenase.

With low aeration, cytochrome levels were pretty constant (since oxygen ran out almost immediately), increasing just a little, except for cyt a₂ oxidase which went up pretty constantly. P/O ratios were pretty level too, and fairly high, similar to when oxygen ran out in high aeration.

What This Means
This kind of pattern fits in well with those seen in other obligate aerobes. Cytochromes probably increase during oxygen limitation to try to compensate for the limitation. But the low P/O ratios at high oxygen makes sense in light of respiratory protection; respiration is uncoupled from energy generation.

Reference:

Ackrell, B. A. C. & Jones, C. W. The Respiratory System of Azotobacter vinelandii 2. Oxygen Effects. Eur. J. Biochem. 20, 29–35 (1971).

099 - Oxygen and Hydrogen in Biological Nitrogen Fixation

Oxygen is pretty toxic to nitrogen fixation enzymes, so organisms or the people studying them need to take steps to protect them. They lose more than half their activity within minutes exposed to air. The dinitrogenase reductase is more sensitive than the dinitrogenase itself, at least the Mo version. Some can retain some activity even up to an hour in air. But the FeMo-cofactor, when extracted, is even more sensitive than the dinitrogenase reductase. Overall, it seems that the metal-sulfur centers are the most sensitive parts.

And yet, there are nitrogen-fixing species that are obligate aerobes, or even oxygenic. How do they do it?

Azotobacter has been shown to increase its respiration while its growth efficiency decreases as oxygen increases, seeming to waste the oxygen: this has been called "respiratory protection." The mechanism for this is not simple though; it involves carefully regulated shifts in respiratory components throughout the whole catabolic system.

Azotobacter also has the ability to reversibly inactivate its nitrogenase if respiratory protection is not possible (such as in carbon-limited conditions, or upon a sudden increase in oxygen). This seems to depend on FeSII protein (aka Shethna), though it is suggested that there may be other mechanisms.

When oxygen is too high and cells' supply of fixed nitrogen runs out, production of nitrogenase may be regulated (no sense making an enzyme when it can't function). This regulation may be done by the products of nifAL genes.

Azotobacter also produces gummy alginate which might have a role in protection from oxygen, but non-gummy strains (such as CA) have been isolated that don't seem especially oxygen-sensitive. I wonder if they have higher rates of respiration though, or if they might be more sensitive in carbon-limited conditions.

Nitrogenase also produces hydrogen gas, whether or not it's reducing anything else. This reaction seems separate from the nitrogen fixation reaction, since some things can inhibit the latter without inhibiting the former. Acetylene seems to inhibit hydrogen production though. Nitrogen can't compete with hydrogen for electrons completely, even with pure pressurized nitrogen; the enzyme always produces at least 1 mol hydrogen for each mol nitrogen gas fixed.

Of course, this hydrogen usually doesn't just escape; Azotobacter and other diazotrophs recapture it with their uptake hydrogenase. The exact purpose this serves is not clear though.

Reference:

Robson, R. L. & Postgate, J. R. Oxygen and Hydrogen in Biological Nitrogen Fixation. Ann Rev Microbiol 34, 183–207 (1980).

183 - Studies on the mechanism of electron transport to nitrogenase in Azotobacter vinelandii

Later investigators criticized the previous study (182) as too simplistic, not explaining the total potential of the nitrogenase system. Azotobacter seems to make three flavodoxins, not just azotoflavin; flavodoxin II seems like the important one but it wasn't known how it got reduced; and ferredoxin might not be involved at all.

So this study grew A. vinelandii with and without ammonium, then observed the differences in its redox systems.

What They Saw
They grew cells in a chemostat with ammonium, and then removed samples and washed with nitrogen-free medium to remove the fixed nitrogen. They measured nitrogenase activity of these samples and labeled newly formed proteins with radioactive sulfur compounds.

They observed that nitrogenase activity correlated well with rate of respiration in different conditions, so they wondered if the two might be linked.

After they removed the fixed nitrogen from cells that had been growing with it, they observed nitrogenase activity within 20 minutes. Then the activity increased linearly over time for at least 40 minutes in this condition. On protein gels, they observed the nitrogenase proteins produced quickly, within 5 minutes, and flavodoxin II showed up some time later. There are some others of uncertain identity, and some interesting ones showing up only in the membrane protein fraction.

What This Means
The linear relationship between respiration and nitrogenase activity has a number of possible explanations. Extra respiration could mean higher membrane potential or ATP levels, so more energy for nitrogenase, or there could be more enzymes (or enzymes that are more active) to transport electrons to nitrogenase. The former seems unlikely, since Azotobacter uncouples respiration from energy generation at higher oxygen levels, so extra respiration doesn't necessarily mean more energy. And extra enzymes seems unlikely too, based on the protein results.

So it seems like increased nitrogenase activity might be due to increased transport of electrons to the enzyme, though it's not clear how that happens.

Reference:

Klugkist, J., Haaker, H. & Veeger, C. Studies on the mechanism of electron transport to nitrogenase in Azotobacter vinelandii. European Journal of Biochemistry 155, 41–46 (1986).

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).

156 - Poly-β-hydroxybutyrate biosynthesis and the regulation of glucose metabolism in Azotobacter beijerinckii

This study was intended to study carbon metabolism in Azotobacter and the formation of PHB.

What They Saw
The enzymes they studied (glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, 6-phosphogluconate dehydratase, 3-deoxy-2-oxo-6-phosphogluconate aldolase, citrate synthase, and isocitrate dehydrogenase) all seemed to be inhibited by NADH and/or NADPH.

What This Means
PHB synthesis consumes reduced nucleotides such as NADH, which could reduce their inhibitory effects when oxygen is limited.

Reference:

Senior, P. J. & Dawes, E. A. Poly-β-hydroxybutyrate biosynthesis and the regulation of glucose metabolism in Azotobacter beijerinckii. Biochem. J. 125, 55–66 (1971).

026 - Siderophore-mediated uptake of iron in Azotobacter vinelandii

Iron is an important element for aerobes, and Azotobacter vinelandii is definitely aerobic. Many oxygen-related enzymes contain the metal, as do the nitrogenases and the uptake hydrogenase, among others. Iron is pretty common in the soil where the bacteria live, but it is generally found in its insoluble form thanks to oxygen, so it's not very bioavailable.

What They Wanted to Know

O. Knosp, M. von Tigerstrom, and W.J. Page knew that A. vinelandii makes several iron-binding enzymes called siderophores (as noted before, 025), but it wasn't yet proven that these siderophores were actually involved with the cells' iron transport; they might have bound the iron but not helped the bacteria to take it up.

What They Did

They grew A. vinelandii strain CA and a capsule-forming strain, ATCC 12837, in nitrogen-free Burk buffer with or without sufficient iron (in the form of iron sulfate heptahydrate). Interestingly, they removed residual iron from the iron-free medium by autoclaving it, letting the iron salts precipitate, as they tend to do, and then filtering them out. Clever!

Then they let the cells sit for a bit in a broth with a radioactive isotope of iron, ⁵⁵Fe, sometimes adding some purified siderophores or crude culture supernatants. They measured how much of this iron the cells took up by measuring radioactivity of washed cells.

What They Observed
When cells had been growing with iron, giving them ⁵⁵Fe resulted in 67% of the radioactivity sticking to a filter (along with the cells). Washing the stuck cells with buffer containing non-radioactive iron removed most of the radioactivity, while washing with iron-free buffer didn't.

When cells had been growing without iron, about 44-55% of the ⁵⁵Fe stuck to the filter; the longer the incubation, the more got stuck.

Though actually when no cells were present, some of the iron still stuck to the filter, retaining about 36% of the radioactivity. A little sodium citrate (a chelator) increased that to almost 50%, while a lot of citrate or a little nitrilotriacetate (another chelator) reduced it. Citrate also prevented iron from binding to cells non-specifically.

Then they measured iron uptake more directly, again with ⁵⁵Fe, with or without initial iron starvation and/or the presence of citrate. Iron-starved cells with citrate took up iron far faster when citrate was present, in supernatant from iron-starved cells (presumably filled with siderophores). Otherwise, iron-starved cells took up iron slightly faster than iron-sufficient cells in buffer without citrate or in supernatant from iron-sufficient cells.

Adding cyanide to cells prevented iron uptake, so it must have been mostly active uptake. Incubating cells in siderophore-free buffer helped to increase uptake, the longer the better.

Two of the three tested siderophores, azotobactin (a delightful neon green molecule) and azotochelin, helped iron uptake in iron-starved cells, though not as much as iron-starved cell supernatant with added sodium citrate. The third, 2,3-dihydroxybenzoic acid (DHBA) didn't seem to do any better than lack of any siderophore.

They also found that iron uptake wasn't affected much by the nitrogen-fixing status; cells grown in ammonium still took it up at the same rate. And the alginate-producing strain ATCC 12837 took up iron at the same rate as CA, except when they were held on ice, in which case the former took up twice as much, possibly because the iron bound to the capsule.

The amount (and ratio) of siderophores produced fluctuated a lot between cultures, despite controlled conditions, but this didn't seem to affect iron uptake because there was always enough siderophore for the amount of iron present.

Adding one siderophore when the other was present already didn't increase the uptake rate, and together they didn't account for all of the uptake activity.

To make sure they weren't damaging siderophores by purifying them, they tried adding acid and then neutralizing it (which happened in the purification too). This reduced the iron uptake 60%. But even adding this acidified/neutralized supernatant to untreated supernatant reduced the uptake somewhat; the process seemed to generate some kind of inhibitor.

Adding HCl (acid) and then NaOH (base) generates NaCl, salt: this could have an effect. So they just added some salt, and found that it also inhibited the uptake, as did other salts. Azotobactin was more sensitive than azotochelin to high salt concentrations.

The complexes the siderophores form with iron still seemed to form still seemed to form in the presence of high salt, nor did medium salt seem to affect A. vinelandii growth.

What This Means
Nothing in the iron-sufficient cell supernatant accounted for the radioactivity stuck to the filter with iron-sufficient cells, because it didn't help increase uptake.

It's kinda weird that DHBA didn't seem to affect iron uptake, since it does seem to bind iron and inhibit A. vinelandii's production of other siderophores. It's possible that the citrate masked its effect.

It's possible that there is another siderophore they didn't know about or test for, which could explain why adding more siderophores didn't seem to increase iron uptake. It was also possible that the purification damaged the siderophores, and they did seem to show such a thing might be possible.

This was a tricky paper. The regulation and use of iron seems to be a complex topic.

Reference:

Knosp, O., von Tigerstrom, M. & Page, W. J. Siderophore-mediated uptake of iron in Azotobacter vinelandii. J Bacteriol 159, 341–347 (1984).

203 - Encystment and alkylresorcinol production by Azotobacter vinelandii strains impaired in poly-β-hydroxybutyrate synthesis

As mentioned before, Azotobacter species can make some useful polymers, such as poly-β-hydroxybutyrate (PHB), a kind of bioplastic. A. vinelandii also makes some other potentially useful polymers: alginate, a kind of slimy polysaccharide; and compounds called alkylresorcinols. 

This last is involved in encystment, when the cells change into a more resistant, dormant state, called a cyst. Alginate is involved in that too, actually, but alkylresorcinols are lipids that replace phospholipids in the membrane. And PHB accumulates in granules in the cyst, possibly as food storage for when the cyst germinates.

What They Wanted to Know
Considering that PHB seems important for the encystment process, or at least shows up in cysts, Segura and colleagues wondered if mutating the phb genes in A. vinelandii might affect the cells' encystment.

What They Did
The scientists sequenced the region of genome containing the phb operon, and compared the open reading frames they found to known sequences. Of the genes they found, they made strains of A. vinelandii with two different genes knocked out, phbC (which makes PHB synthase) and phbB (acetoacetyl-CoA reductase), by inserting stuff into the genes; and characterized these mutants, testing their PHB production, aklylresorcinol production, and encystment. Measuring the PHB followed the usual methods, with boiling chloroform and concentrated sulfuric acid; sounds like tons of fun.

What They Observed
In the sequence they got, there were six open reading frames (ORFs). By comparing the sequences to other known PHB-related genes (such as from 174), they identified the ORFs from A. vinelandii as the PHB-producing operon phbBAC, along with the regulator-producing gene phbR. Another of the six was like phbP from Ralstonia eutropha, making a granule-associated protein, and next to that an ORF similar to phbF in R. eutropha, seemingly a putative regulator for PhbP.

Then they knocked out phbB and phbC, though not in the same strain. Neither of these mutants produced detectable levels of PHB. The phbB knockout had over 90% reduction in acetoacetyl-CoA reductase activity (makes sense) and also much less activity from PhbA or PhbC; it seemed like the mutation had polar effects on the operon. The phbC mutant only had much reduction in PHB synthase, about 95%, which makes sense, though the other enzymes were affected a little too (~40%), maybe because of unstable mRNA.

Then they induced encystment, apparently with n-butanol. Neither mutant seemed impaired; phbB knockout actually seemed to encyst more. And with a different induction method, they saw the same results, even in regular Burk medium. Obviously they didn't contain PHB granules, but this didn't seem to be a problem: their viability was the same or even higher than wild-type cysts.

Regarding alkylresorcinol production, A. vinelandii produces them when PHB or n-butanol replace glucose as a carbon source. But the authors tested the mutant strains first in regular Burk, since they apparently could form cysts in that; turns out they also were able to produce alkylresorcinols, unlike the wild-type, especially the phbB knockout, which also had greater alginate production (possibly contributing to its higher viability).

Under an electron microscope, the mutants' cysts didn't have PHB granules (of course), and in the phbB knockout strain, the exine of the cysts seemed thicker than other strains', probably due to extra alginate and alkylresorcinols.

What This Means
The phb operon is the one involved in PHB production in A. vinelandii too. Knocking out phbC seems to produce a cleaner phenotype, with less effect on the cells other than lack of PHB production. But it seems like lack of PHB channels more carbon through the lipid metabolism pathway. It doesn't seem to affect encystment much, at least not negatively, but this may only be because of the unnatural lab environment in which the cells are growing.

All this extra production of alkylresorcinol and alginate may be due to accumulation of acetyl-CoA that would normally go toward PHB. The mutant lacking PHB synthase may accumulate hydroxybutyrate instead; it's not clear what effects that might have.

Reference: Segura, D., Cruz, T. & Espín, G. Encystment and alkylresorcinol production by Azotobacter vinelandii strains impaired in poly-β-hydroxybutyrate synthesis. Arch Microbiol 179, 437–443 (2003).

248 - NAD-, NMN-, and NADP-dependent modification of dinitrogenase reductases from Rhodospirillum rubrum and Azotobacter vinelandii

What They Knew
Nitrogen fixation is a demanding process, using a lot of energy, so bacteria regulate it tightly, shutting it off whenever fixed nitrogen is available already. One diazotroph, Rhodospirillum rubrum, regulates its nitrogenase by ADP-ribosylating the dinitrogenase reductase component, using a protein called Dinitrogenase Reductase ADP-ribosyl Transferase, or DRAT. This takes ADP-ribose from NAD when ammonium is present. Another protein, Dinitrogenase Reductase-Activating Glycohydrolase (DRAG), reverses the process.

What They Wanted to Know
Ponnuraj and colleagues studied the nitrogenases of R. rubrum and Azotobacter vinelandii to see how specifically each interacted with the DRAT/DRAG system and various ADP-ribosyl donors.

What They Did
They took different ADP-ribosyl containing molecules (NAD, NADP, NADH, NMN), combined each with A. vinelandii's dinitrogenase reductase (DNR) and R. rubrum's DRAT, then ran them with SDS-PAGE along with samples lacking the molecules, to see which molecules could be used to donate ADP-ribosyl. They also exposed some of each sample to DRAG and ran that alongside to see if it could remove the modification.
Results from this were confirmed with another test, seeing if modified or de-modified DNR could function with dinitrogenase to reduce acetylene.

To see how small a modification works to inactivate the system, they removed a phosphate from phosphoribosylated DNR and tested it again.

In addition to testing A. vinelandii's DNR, they tested R. rubrum's too.

To see more specifically what was going on with A. vinelandii's DNR, they used MALDI-TOF mass spectroscopy.

What They Observed
Based on gels and activity assays, NAD, NMN, and NADP all seemed pretty good at donating to DRAT to inactivate A. vinelandii's DNR. NAAD not so much, or anything else they tried. DRAG seemed able to re-activate DNR with all of them too.

Surprisingly, with R. rubrum's DNR, only NAD seemed to be a good donor. Mass spec results confirmed their expectations about what was going on biochemically.

What This Means
R. rubrum uses DRAT and DRAG to regulate its nitrogenase activity based on whether fixed nitrogen is available already and whether its environment is illuminated or not. This helps save energy, so it doesn't have to break down the whole system and reconstruct it with every little environmental change.

It's not clear how relevant it is for A. vinelandii, though, because that organism doesn't appear to have the genes to produce DRAT/DRAG proteins. It's somewhat interesting that R. rubrum's proteins are able to modify A. vinelandii's nitrogenase, arguably even better than they can with R. rubrum's, but this actually makes some sense, that R. rubrum would have tighter control over its nitrogenase regulation system. Though apparently some in vivo studies suggest it might not be as tight as it seemed here.

This doesn't necessarily mean that A. vinelandii doesn't have a system for post-translational regulation, just that it isn't exactly this one. I haven't found what it is yet, if there is one. And other studies seem to imply that A. vinelandii might not have such tight control (010). I wonder why.

Reference:

Ponnuraj, R. K., Rubio, L. M., Grunwald, S. K. & Ludden, P. W. NAD-, NMN-, and NADP-dependent modification of dinitrogenase reductases from Rhodospirillum rubrum and Azotobacter vinelandii. FEBS Letters 579, 5751–5758 (2005).

010 - Formation of the nitrogen-fixing enzyme system in Azotobacter vinelandii

Apparently, it had previously been shown that ammonium repressed nitrogen fixation in Azotobacter vinelandii, and even when fixing nitrogen, cells would immediately take up ammonium when it was given, but would not immediately start fixing nitrogen if they ran out of ammonium. They wanted to look at this lag period more closely.

What They Did

They grew A. vinelandii OP (aka CA) in Burk's nitrogen-free medium, and actually this is the paper most people later cited as the best recipe for Burk's, the standard medium for growing A. vinelandii.

So they grew the cells, sometimes with ammonium acetate or potassium nitrate as fixed nitrogen sources, sometimes with chloramphenicol to prevent protein synthesis. They also did enzyme activity assays with nitrogenase, using ¹⁵N₂. And determined protein content of cells.

What They Observed

The first figure, taken from Strandberg's master's thesis, shows that when A. vinelandii is grown in a nitrogen-free atmosphere with ammonium, growth eventually levels off; if N₂ is then added, cells start growing again after a short lag, 30-60 minutes. But if ammonium is added instead, there's no lag; the cells start growing again immediately. If N₂ was present the whole time, the cells switch to nitrogen-fixing pretty quickly when ammonium runs out, with a small decrease in growth rate.

A better demonstration for this lag was nitrogenase activity assays: it showed right when nitrogen fixation activity started, about 1 1/4 hours after ammonium ran out. Though oxygen levels and temperature possibly weren't ideal. It could be as little as 45 minutes later.

Another interesting observation was that when ammonium ran out and cells were in an environment of 40% oxygen (with the rest 60% helium or hydrogen), they didn't start producing nitrogenase, but they did start when oxygen was only 20%. The hydrogen level didn't seem to matter.

One problem they encountered was that there were small amounts of nitrogen in their gas tanks of oxygen, helium, and hydrogen, which could've been enough to affect the results. They tried to make purer oxygen by electrolysis (splitting water), though there was still a bit of nitrogen; still, it wasn't clear whether nitrogenase production was induced by the presence of nitrogen or merely repressed by ammonium. My guess would be the latter, since cells wouldn't normally encounter N₂-free environments in nature. But regulation can be complicated.

They noticed a slight rise in turbidity even after ammonium ran out, but speculated it could be due to color change that cells go through (from reddish brown to dark brown) when fixing nitrogen. The small amount of nitrogen in the gas flow was enough to get cells to produce nitrogenase, but not enough for them to use it. But cell-free extracts didn't show different absorbance for the two kinds of cells, despite the visible difference.

When they added chloramphenicol, an antibiotic that inhibits protein synthesis, obviously this inhibited nitrogenase formation. If the enzyme was already present, in vitro, the antibiotic didn't inhibit it. But it did inhibit it in cells, possibly because ammonium built up with no way to use it, repressing nitrogenase.

They tried adding 150 mg N (as ammonium) per liter to a culture of nitrogen-fixing cells, and saw that nitrogenase activity dropped off within about 3 hours. Not as fast as I would expect. They interpreted this to mean that the enzyme is not inhibited immediately, just diluted out as the cells stop producing it while continuing to multiply; but it seems to be inactivated faster than just by dilution, so there might be some inactivation or degradation going on.

Citation: Strandberg, G. W. & Wilson, P. W. Formation of the nitrogen-fixing enzyme system in Azotobacter vinelandii. Can. J. Microbiol. 14, 25–31 (1968).

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).

089 - Whole Cell Respiration and Nitrogenase Activities in Azotobacter vinelandii Growing in Oxygen Controlled Continuous Culture

This paper was basically a sequel to 098, by most of the same authors.

What They Wanted to Know

The question that Post, Kleiner, and Oelze wanted to answer in this paper was in regard to Azotobacter vinelandii's ability to protect its nitrogenases from the damaging effects of oxygen. Azotobacter is an obligate aerobe, so this is always an issue, but how it protects its sensitive enzymes was not clear.

The theory was that A. vinelandii employs respiratory protection, in which it consumes oxygen at so high a rate that oxygen cannot build up to toxic levels in the cell. If oxygen does build up too high, or there isn't enough substrate available to consume it all, A. vinelandii can reversibly change the conformation of its nitrogenase so it is protected, though it cannot fix nitrogen in this state, so the cell effectively goes dormant.

The idea of respiratory protection comes from the observation that A. vinelandii can only fix nitrogen aerobically when there is adequate substrate available to maintain high enough rates of respiration. If there's no substrate from which to get electrons to dump onto oxygen, the system doesn't work. It takes time to increase respiration rates, so this process doesn't cope well with sudden increases in oxygen. This is when conformational change helps. In theory.

What They Did

They grew A. vinelandii OP (aka CA) in a chemostat, limiting its carbon (at two different levels) and controlling oxygen exposure, either with nitrogen gas or ammonium as a source of N. Similar to 098. Oxygen was always kept higher than limiting, so they could measure exactly how much there was; I wonder if that was the best range to observe though, and it means that 0% wasn't really anaerobic.

They measured cell protein contents and nitrogenase and respiratory activities, as well as residual sucrose.

What They Observed

Cell protein levels were always higher in ammonium-grown cells, not surprisingly, and in both N conditions they rose a bit as oxygen rose to about 3% saturation, and then dropped, leveling off at around 30%. At higher carbon, N-fixing cells took a bit longer to level off, at about 50% oxygen.

Protein yield followed a similar pattern, dropping as oxygen increased up to 30%. Carbon level didn't affect N-fixing cells' yield, but ammonium-grown cells had higher yields with lower carbon levels.

The pattern of respiratory activity was similar to the above, but inverted: it rose between 1 and 30% oxygen saturation, then remained pretty constant. Nitrogen status didn't affect it much at the higher carbon level, and was always higher than the lower carbon values, but at the lower carbon it was about double when fixing nitrogen compared to when grown with ammonium.

Nitrogenase activity decreased quickly up to about 3% saturation, then gradually up to 100%. Carbon level didn't matter.

Then they tried holding the oxygen constant at 45% and increasing the dilution rate (how fast new medium flowed into the reactor, diluting out the contents). Respiration increased linearly with dilution rate, as did protein content and nitrogenase activities at first, but at a point (around D = 0.25 h^-1), the protein content dropped off and nitrogenase activity increased greatly.

Finally, instead of gradual increases in oxygen saturation, they adapted cells to one level and then suddenly changed it to a higher level for 7 minutes, then dropped it back. Regardless of the starting saturation or new peak of oxygen, the cells always switched off their nitrogenase activity when exposed to a larger amount of oxygen. They started it up again when the oxygen dropped back down, but not at the same level as before.

What This Means

Cell activity seemed to level off around 30% oxygen saturation, so either that's more than they can use, or their ability to deal with it has peaked and doesn't need to increase any more. However, a sudden large increase does cause them to suddenly shut down, even if they wouldn't have shut down with a gradual increase to the same level, so there's something else going on.

It's interesting to note the lower yields as oxygen increased, indicating that A. vinelandii was sorta wasting the carbon to deal with the oxygen. It wasn't just when fixing nitrogen though, so it might not be specifically to protect the nitrogenase. Hard to say from just this.

Inconsistent with the respiratory protection hypothesis is the large increase in nitrogenase activity at higher dilution rates without a simultaneous increase in respiration, while oxygen remained the same. Also the fairly constant rates of respiration and nitrogenase activity as oxygen increased above 30% to 100%; we would expect respiration to rise and nitrogenase to drop more severely.

So respiratory protection might be important at some levels of oxygen, but possibly not all.

Citation: Post, E., Kleiner, D. & Oelze, J. Whole Cell Respiration and Nitrogenase Activities in Azotobacter vinelandii Growing in Oxygen Controlled Continuous Culture. Arch. Microbiol. 134, 68–72 (1983).

098 - Morphological and Ultrastructural Variations in Azotobacter vinelandii Growing in Oxygen-Controlled Continous Culture

What They Wanted to Know
At the time of this paper, some had noticed that Azotobacter vinelandii seemed to produce extra cell membrane surface area protruding into its cytoplasm in some conditions. It seemed to happen when the cells were fixing nitrogen, to the extent that some thought it related only to nitrogen and not to oxygen/respiration at all, though others disagreed and thought it could happen with high enough cell densities or low aerations, regardless of nitrogen (implying that oxygen was the important factor).

So in this paper, Post, Golecki, and Oelze investigated conditions resulting in membrane formation very specifically, using chemostats with controlled levels of oxygen and nitrogen.

What They Did
They grew A. vinelandii strain OP (aka CA), either with ammonium acetate added as a fixed nitrogen source, or no fixed nitrogen (so it had to fix its own). They used an oxygen probe to measure and control the oxygen dissolved in the medium, as a percentage of saturation from 1-100%. The dilution rate was 0.15 h^-1 for 800mL of culture, stirred at 1000 rpm, so the doubling time for the bacteria was 4.6 hours.

They measured the vesicles/invaginations of cell membrane in each condition by transmission electron microscopy, and cell shrinkage with light microscopy, calculating average cell volume.

What They Found
What they found was that for all nitrogen conditions, cells got bigger as oxygen levels increased, up to about 1.6-fold. At all levels, nitrogen-fixing cells were about 10% larger than cells growing with fixed nitrogen. Length:width ratio remained the same for all though. The size increase happened mostly between 1% and 25% oxygen saturation.

The result of this increase meant less area on the surface of the cell, which could mean less penetration of oxygen inside, but also less area available for establishing a proton gradient for energy generation. However, in nitrogen-fixing cells, the number of membrane vesicles also increased with higher oxygen levels, which meant more membrane surface area for proton gradients. The two effects nearly balanced each other, such that membrane surface area per cell volume increased only 1.5x.

In ammonium-grown cells, by contrast, the amount of membrane increase was lower, so the ratio of membrane area to cell volume dropped a little.

Another contrast is that in nitrogen-fixing cells, the ratio of vesicle membrane area to cytoplasmic membrane area increased almost 3-fold, but in ammonium-grown cells it stayed pretty constant.

What It Means
This means that both nitrogen status and oxygen levels can affect cell size and area of membranes in the cells. Oxygen may affect whether or not vesicles are formed at all, while nitrogen affects the numbers and proportions at a given oxygen level, at least between oxygen saturations of 0 and 25%. But the amount of vesicle area increased as oxygen increased, rather than decreasing (as others had proposed).

It makes some sense that nitrogen-fixing cells would try to increase membrane area while decreasing cell surface area (by increasing volume): that would help increase potential respiration rates while decreasing oxygen penetration, so it'd be easier to protect oxygen-sensitive enzymes. This doesn't really explain why ammonium-grown cells also increased cell volume though. But it's an interesting result.

Citation: Post, E., Golecki, J. R. & Oelze, J. Morphological and Ultrastructural Variations in Azotobacter vinelandii Growing in Oxygen-Controlled Continous Culture. Arch. Microbiol. 133, 75–82 (1982).

185 - Transcriptional Profiling of Nitrogen Fixation in Azotobacter vinelandii

Azotobacter vinelandii is known for its amazing ability to fix nitrogen, converting nitrogen gas into biological forms like protein even in the presence of oxygen. This is apparent even in its name—Azotobacter—which translates approximately to "nitrogen bacteria."

But the nitrogen-fixing process and machinery is pretty complicated and difficult to study in a reductionist fashion. Many components don't work the same outside of the context of the rest of them or outside of the cell itself.

So in this study, scientists (some of whom were involved in sequencing the first A. vinelandii genome) took a broad approach to the subject, by looking at the expression of all the genes in the organism, comparing their expression when the cells were fixing nitrogen compared to when they weren't.

In addition, they examined the expression of genes when the bacteria were grown with or without certain metals needed for the various nitrogenase versions: molybdenum, vanadium, etc. More specifically:

• To study non-nitrogen-fixing cells: they grew cells with ammonium
• Cells using the molybdenum nitrogenase: they grew cells with molybdenum and no ammonium
• Cells using the vanadium nitrogenase: they grew cells with vanadium and no Mo or ammonium
• Cells using the iron-only nitrogenase: they grew cells with iron and no other metals or ammonium

Simple enough.

Then, to measure levels of expression, they extracted RNA from the cells in each condition, converted the RNA sequences to DNA (called cDNA or complementary DNA, converted from RNA messengers), and then these chunks of DNA were sequenced using a high-throughput technology called SOLiD.

SOLiD (or Sequencing by Oligonucleotide Ligation and Detection) is one of the main kinds of next-generation sequencing, alongside Roche's 454 pyrosequencing and Illumina. I had to look it up. Apparently one machine these days can sequence 5 trillion bases per day (for reference, the human genome is about 3.2 billion bases long, so SOLiD could sequence more than 1500 human genomes per day). Of course, this is not cheap: that much sequence data would cost about $500,000.

The way it works is by cutting a piece of DNA into short sequences, binding them to tiny magnetic beads so there's one sequence per bead. Then the beads are mixed into an emulsion of oil so that on average, each bead is encased in a small bit of water in a sea of oil, along with reagents needed for polymerase chain reaction (PCR). This allows the DNA on each bead to be copied many times, all at once yet individually, so that many reactions can be done in the volume of liquid that would normally allow only one. Very cool. This is the same technology used to prepare samples for 454 pyrosequencing.

In pyrosequencing, the sequencing is done with DNA polymerase, which is what is normally used to copy DNA, and each base is added one at a time, so each bead will incorporate only the base that fits with the sequence bound to the bead. When a base is added, a tiny bit of light is given off, which a camera in the machine detects and registers it as the base that was added at that time.
However, SOLiD works a bit differently: instead of DNA polymerase, the enzyme is DNA ligase, which links together two strands of DNA. And instead of single bases, short DNA sequences called probes are added, with the two bases at one end known. When one of these matches the bead's sequence, ligase links it up. The probes are labeled with fluorescent molecules, so the next two bases in a sequence can be determined from the color of the fluorescent probe. Then this is cleaved off and another two bases are added. It's somewhat confusing; this site might help a little.
This system avoids some of the problems that pyrosequencing experiences, such as with accuracy, but has some problems of its own (especially price), so it's useful in some cases and not as much in others.

So what good is sequencing all the cDNA? The number of copies of one cDNA, relative to the copies of others, shows how much the cell is transcribing that gene, which can be an indicator of expression levels (transcribing more may mean that the gene is translated into protein more, so there may be more of that specific protein in the cell). It doesn't always work quite proportionally, since there are mechanisms other than transcription for regulating cellular protein levels, but it can usually provide some interesting data. So they sequenced all the cDNA in the cell and compared the number of copies for each gene to see which ones were present in higher or lower numbers in different growth conditions.

Then after analyzing all this data (a large undertaking in itself) and finding genes that seemed to be expressed at higher or lower levels in the different conditions, the scientists confirmed the most interesting findings using real-time quantitative PCR, which is a more sensitive way of measuring the same information. It works by doing PCR on a gene but adding some kind of fluorescent molecule to detect exactly how much of that sequence is present over time in the PCR. Ideally PCR should double the number of sequences in every round of the reaction, but this doesn't always work exactly, so the most accurate measure is to determine the point at which the fluorescence becomes bright enough that the PCR machine can detect it, and then extrapolate back to figure out how much of the sequence was present at the beginning. It's another way to compare transcription levels.

Ok, finally on to results. The authors found that almost 30% of A. vinelandii's genes were affected when fixing nitrogen compared to when not doing so. Many of these were affected regardless of which nitrogenase the cells were using. Mo nitrogenase growth affected the most genes on its own compared to the other two, but the two alternative nitrogenases (V and Fe) together affected more genes than any nitrogenase on its own. So overall, compared to non-fixing conditions, the using the alternatives affected many more genes than using the Mo version; but compared to each other, using the alternatives didn't change many genes. Apparently using the alternatives involves a large shift in the cell's gene regulation, compared to using the main Mo nitrogenase. I wonder why.

Genes Expressed When Using Mo Nitrogenase
The nif genes that make up the Mo nitrogenase are found in two clusters in the genome, one near the beginning (relative to the origin of replication) and one near the end. Some of these genes form the actual enzyme structure itself, some help to put it together with its metal-containing cofactor and such, and some (especially nifA) regulate the process.

Not surprisingly, the main structural genes increased their expression greatly when the cells switched to nitrogen-fixing mode, between 50 and 150 times higher. The primary dinitrogenase reductase, NifH, increased the most, which fits in with previous observations that a high ratio of this protein to the Mo-containing dinitrogenase allows higher nitrogenase activity.

More surprisingly, other nif genes in the major cluster only increased expression up to about 14 times more. This could be because not much of their proteins is needed, or possibly that they were already expressed at high levels and their regulation is mostly post-transcriptional, so not much change would be visible in transcript levels. In the minor cluster, some genes necessary for making the metal cofactor increased around 20-fold.

Other than these expected increases, lots of other genes changed as well; not surprising, considering that nitrogen fixation is essential for growth in low-nitrogen environments but is also very energy-intensive. The most significant changes were in type IV pilus genes. These pili, little hairlike projections from the cell, are involved in lots of things: motility, sensing the environment, attachment to surfaces, etc. It's not entirely clear what they're doing in this context, but apparently something.

Another important factor for nitrogen fixation is protecting the nitrogenase from oxygen. A. vinelandii seems to do this by consuming a lot of carbon in order to reduce whatever oxygen is present, transforming it to water. Its genome has many electron-transporting proteins such as oxidoreductases and terminal oxidases, some of which did appear to be somewhat upregulated in nitrogen-fixing conditions. This could also be useful for producing more energy to power the nitrogenase. The genes for the uptake hydrogenase, which recovers hydrogen produced by the nitrogenase and regenerates some energy from it, also showed increased expression.

There also seems to be a change in some genes associated with iron and sulfur organization, something else that is important for nitrogenase because it contains multiple atoms of these elements. Also, not very surprisingly, the genes related to molybdenum gathering increased also.

Genes Expressed When Using Alternative Nitrogenases
Obviously, the genes that encode the alternative nitrogenases themselves (vnf genes for the vanadium-containing nitrogenase and anf genes for the iron-only nitrogenase) are going to be upregulated when these are in use. The V (vanadium) nitrogenase is used when molybdenum is not present but vanadium is, and the Fe nitrogenase is used when neither of these metals is available.

But these alternative systems don't have alternative versions of all of the necessary enzymes for fixing nitrogen, only the main ones, so they share some of the proteins that the Mo nitrogenases uses. This is especially true of enzymes involved in assembling the nitrogenases and their cofactors, such as NifUSVMB.

When vanadium was present, vnf genes were upregulated, as I said, but in this case, the vnfH gene encoding the vanadium dinitrogenase reductase wasn't as high relative to the other V nitrogenase components as was the case with the molybdenum nitrogenase. Not sure why. There were some other differences, especially that vnf homologs of nif proteins involved in cofactor synthesis were expressed in different proportions, so the process of V-containing cofactor synthesis might be different somehow.

In the case of the iron-only nitrogenase, the nifH and other components' homologs (anfH, etc.) were upregulated in the same ratio as the nif genes, distinct from the vnf homologs: that is, anfH was expressed much higher than anfDK, around four- to five-fold higher.

Of the genes that don't have anf homologs, some nif genes were upregulated (nifUSVMG again), but in other cases the vnf versions were preferred (vnfENXY). vnfH was also upregulated, even though there is a separate anfH. This is in agreement with other previous studies (003), and may be because vnfH has some kind of role in regulating gene expression.

Genes Related to Electron Transport
All of the nitrogenases require electron transport machinery, since the nitrogenase functions by putting electrons (and protons) onto nitrogen gas (N₂) to make ammonia (NH₃). This takes at least eight electrons for each molecule of nitrogen: six for two molecules of ammonia, and two for one molecule of hydrogen as a byproduct. The alternative nitrogenases produce more molecules of hydrogen, so they need even more.

Some of the genes involved are nifF and vnfF, which encode proteins called flavodoxins that transport electrons. They may not be necessary to fix nitrogen, but presumably they're helpful. When the cells were using the Mo nitrogenase, nifF was upregulated, and both were higher when Mo was absent (though vnfF much more so).

Some other genes that seemed involved included rnf1 genes, whose products are membrane-bound and also help to transport electrons to nitrogenase; they also seem to be important for the iron-sulfur cofactor of dinitrogenase reductase. And fix genes also seem important for electron transport. All of these were expressed more when fixing nitrogen in all conditions, but when Mo was absent, fix genes were much higher than rnf1 genes.

Regulatory Genes
Clearly A. vinelandii's nitrogenase system has a lot of regulation going on, so regulatory genes are important. vnfA and anfA are necessary to use the alternative nitrogenases, as is nifA for the primary nitrogenase, and these regulatory genes increased whenever their respective isozyme was in use, though low levels of them were present constantly. Regulation of these genes is likely to be how the cells turn on and off the alternative nitrogenases.

There are a few other homologs of nifA and vnfA that show similar patterns, but may fine-tune the regulation somehow (how is not yet known).

Other Differences in Global Expression
As mentioned, the transcriptional profile when using the Mo nitrogenase is very different from when using V or Fe nitrogenases, probably because the latter are less efficient. The most apparent difference in this study was in the hutU gene, for urocatanase hydratase, which increased greatly when Mo was absent. This gene is necessary to degrade histidine, one of the 20 common amino acids, which makes sense because cells using a less efficient nitrogen-fixing enzyme might want to get nitrogen from other places too, like breaking down some less essential proteins. Similar results have been seen in other diazotrophs.

Also quite interesting, genes for a putative soluble hydrogenase discovered when the genome was sequenced were upregulated, especially when using the Fe nitrogenase. This may be a backup system for recycling the extra hydrogen molecules that these nitrogenases produce, to recover the valuable energy that would otherwise escape.

Some other genes increased also, but the function of their products is as yet unknown. Might be worth investigating.

Comparing expression when using the V nitrogenase vs. the Fe nitrogenase, there was at least one interesting point: there were a few genes near a vnf operon upregulated during V nitrogenase growth, seemingly related to a transporter system, so they're probably a vanadate transporter.

Evolution of Nitrogenase
There's some debate about which came first in history: the Mo nitrogenase or the alternatives. Since the alternatives are less efficient, it would make some sense if they came first and the Mo nitrogenase just improved on them, especially since Mo and possibly V were probably difficult to find before there was much oxygen in the atmosphere. But no one has discovered a species that has alternative nitrogenases and not the Mo nitrogenase, though there are plenty that have only the latter, and not many that have all three. And this study seems to show that the alternatives evolved from the Mo nitrogenase to allow cells to thrive in environments where Mo is absent, which also makes sense.

So these results are very interesting and potentially useful, and seem to tell us a lot about what's going on inside the cells in different conditions!


Citation: Hamilton, T. L. et al. Transcriptional Profiling of Nitrogen Fixation in Azotobacter vinelandii. J. Bacteriol. 193, 4477–4486 (2011).

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).