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

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

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

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

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

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

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

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

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

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

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

Reference:

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

525 - Hydrogen-mediated mannose uptake in Azotobacter vinelandii

This study looked at Azotobacter vinelandii's ability to use hydrogen gas to power its uptake of the sugar mannose.

What They Saw
They grew A. vinelandii CA in Burk broth but with mannose instead of glucose or sucrose, and either hydrogen or argon in the atmosphere (along with nitrogen and oxygen). They used ¹⁴C mannose to observe its uptake via the radioactivity of the isotope.

The increase in radioactivity from mannose activity was a lot higher in cells given hydrogen than those without, up to 5-fold.

They tried inhibiting respiration to see if that was related to this effect, and found that usually by inhibiting respiration, they could inhibit the increased mannose uptake, so it seems to be respiration-dependent rather than some sort of regulatory effect.

So this seems to be another of hydrogen's possible roles in the energy metabolism of Azotobacter.

Reference:

Maier, R. J. & Prosser, J. Hydrogen-mediated mannose uptake in Azotobacter vinelandii. J. Bacteriol. 170, 1986–1989 (1988).

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).

523 - Carboxyl-terminal processing may be essential for production of active NiFe hydrogenase in Azotobacter vinelandii

Based on amino acid prediction from gene sequence, the HoxG alpha subunit of the uptake hydrogenase should be about 66.6 kDa, but in the wild-type it appears smaller. In some mutants with accessory genes knocked out, the size matches this number. So this study tried to figure out if post-translational processing was involved in producing active enzyme. N-terminal modification was already ruled out, as that sequence matches the prediction.

What They Saw
They grew Azotobacter vinelandii CA and purified its hydrogenase, then studied its subunits with mass spectrometry.

They observed that the actual size of the larger subunit was 64.9 kDa, smaller than the 66.6 predicted size. The N-terminal was still the same as predicted, so they concluded that about 15 amino acids had been removed from the C-terminal of the subunit. This appears to be necessary for it to function.

Reference:

Gollin, D. J., Mortenson, L. E. & Robson, R. L. Carboxyl-terminal processing may be essential for production of active NiFe hydrogenase in Azotobacter vinelandii. FEBS Letters 309, 371–375 (1992).

516 - Role of magnesium adenosine 5'-triphosphate in the hydrogen evolution reaction catalyzed by nitrogenase from Azotobacter vinelandii

Nitrogenase evolves hydrogen in a reaction that depends on ATP. The amount of ATP varies from 2 ATP per electron to over 20 (supposedly). The 4Fe-4S cluster in the dinitrogenase reductase donates one electron to the dinitrogenase, binding 2 ATP to do so. It seems like the ATP-powered flow of electrons can determine how many go toward hydrogen and how many toward nitrogen fixation. This study wanted to see if powering this flux was ATP's only role.

What They Saw
They purified enzyme from Azotobacter vinelandii and separated the two components, then mixed them with MgATP in in vitro assays. They observed that there was a burst of rapid ATP hydrolysis at first, which leveled off and remained fairly constant for the rest of the time. It seemed like the rate was the same as the rate of electron transfer between components. They confirmed the rate of about 2 ATP per electron; this makes sense with the dinitrogenase accepting two electrons at a time, one for each of its molybdenum atoms. The rate depended on the concentration of ATP, which makes sense and indicates that ATP is essential. This is also true when measuring the amount of hydrogen produced.

What This Means
They appeared to succeed in their goal of showing that ATP's only role in this reaction was powering the electron transfer, though some ATP can be used up to no purpose in some conditions.

Reference:

Hageman, R. V., Orme-Johnson, W. H. & Burris, R. H. Role of magnesium adenosine 5’-triphosphate in the hydrogen evolution reaction catalyzed by nitrogenase from Azotobacter vinelandii. Biochemistry 19, 2333–2342 (1980).

515 - Hydrogen Uptake and Methylene Blue Reduction Activities of Hydrogenase in Azotobacter agile

This study used tritium (³H, a radioactive isotope of hydrogen) uptake to look at hydrogenase and nitrogenase activity in Azotobacter agile (aka A. agilis I think).

What They Saw
The tritium was ditritium gas, similar to dihydrogen. They purified and fractionated protein from the bacteria and tested the fractions for tritium uptake, methylene blue reduction, and acetylene reduction. They found that acetylene reduction and the other two were found in separate fractions (makes sense; one's nitrogenase and the others hydrogenase). Tritium uptake could be stimulated with ATP somehow.

Tritium uptake and methylene blue reduction mostly went together in terms of fractionation, but there was a little of the latter in some fractions where the former wasn't observed. So there could be something else reducing methylene blue.

They found that carbon monoxide (CO) inhibited tritium uptake, though it was less inhibitory when ATP was present.

What This Means
This supports the idea that nitrogenase produces hydrogen while hydrogenase oxidizes it and reduces electron acceptors such as methylene blue. It's weird that ATP should stimulate that though, and also weird that some fractions had reduction activity but not hydrogen oxidation.

Reference:

Suzuki, T., Maruyama, Y. & Nakamura, M. Hydrogen Uptake and Methylene Blue Reduction Activities of Hydrogenase in Azotobacter agile. Agricultural and Biological Chemistry 43, 2067–2073 (1979).

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).

454 - Activity of the H2-oxidizing hydrogenase in different N2-fixing bacteria

Despite some studies suggesting that hydrogen stimulates hydrogenase, other data suggested it does not. So the people who generated this data did this study on various species, including Azotobacter vinelandii CA, and claimed that low oxygen stimulated hydrogenase activity.

What They Saw
They grew A. vinelandii with ammonium chloride and measured hydrogenase activity with different electron acceptors (oxygen, methylene blue, etc). As A. vinelandii grew, it used up the dissolved oxygen, and hydrogenase activity went up but then back down after the oxygen was gone (when oxygen or iron cyanide were the electron acceptors, it went to zero; otherwise it didn't go all the way to zero). This was all in the presence of ammonium.

What This Means
Based on other studies, I wouldn't expect much activity from hydrogenase in general when growing with fixed nitrogen. I'm not sure how to interpret these results, especially with electron acceptors other than oxygen, but I guess it would make sense if hydrogenase were somewhat downregulated in low-oxygen conditions, even if other acceptors were present.

Reference:

Pinkwart, M., Bahl, H., Reimer, M., Wölfle, D. & Berndt, H. Activity of the H₂-oxidizing hydrogenase in different N₂-fixing bacteria. FEMS Microbiology Letters 6, 177–181 (1979).

453 - Direct mass-spectrometric determination of the relationship between respiration, hydrogenase and nitrogenase activities in Azotobacter chroococcum

This study looked at hydrogen and its relationship to different enzymatic processes in Azotobacter chroococcum.

What They Saw
The organism was grown in continuous culture with limited oxygen, 5% glucose. Samples were removed and sparged with different mixtures of argon, oxygen, and deuterium. Gases are measured by mass spectrometer.

Their figure is pretty confusing, poorly designed, and poorly described, but as far as I can tell, when they added either 80% argon with 20% oxygen or 70% argon, 20% oxygen, and 10% deuterium, the oxygen goes down to near zero within about 5 minutes either way, at which point hydrogen production starts increasing and deuterium uptake slows down or stops. So it seems like oxygen is required for hydrogenase to work.

They tried again with the addition of some carbon monoxide and acetylene to inhibit hydrogenase. This didn't really change the oxygen consumption, but deuterium consumption was a lot lower. Hydrogen evolution was the same.

What This Means
The deuterium consumption is by hydrogenase, of course, and it seems like oxygen is a necessary electron acceptor for it to function (in the absence of something else). But it seems like the increase in hydrogen evolution is not because hydrogenase stopped working, but rather because nitrogenase started, as oxygen stopped interfering. This is something we've seen before.

Oxygen is important for many things: it provides energy by accepting electrons, powering the nitrogenase and allowing more hydrogen production. It accepts electrons from hydrogenase, enabling hydrogen oxidation. It inhibits nitrogenase, reducing hydrogen production. Pretty confusing.

Reference:

Lespinat, P.-A., Gerster, R. & Berlier, Y. Direct mass-spectrometric determination of the relationship between respiration, hydrogenase and nitrogenase activities in Azotobacter chroococcum. Biochimie 60, 339–341 (1978).

448 - Hydrogen-deuterium exchange reactions catalysed by nitrogenase

Previous reports suggested that nitrogenase could convert dideuterium (D₂) to hydrogen-deuterium (HD) by swapping one hydrogen from water with one deuterium atom. This only happened in the presence of nitrogen. This was tested in various organisms with D₂ or D₂O as a source of deuterium.

Azotobacter species didn't make HD unless ATP and electrons were present. They actually made less hydrogen and HD with nitrogen than with argon, contradicting earlier findings. 10% CO did inhibit HD formation but not hydrogen formation. Acetylene and methyl isocyanide inhibited HD completely. Cyanide partially or fully inhibited.

There was more exchange with D₂O than with D₂ for some reason, but this was inhibited more by nitrogen or cyanide.

This seems to have implications for nitrogenase's functions relating to hydrogen.

Reference:

Kelly, M. Hydrogen-deuterium exchange reactions catalysed by nitrogenase. Biochem J 109, 322–324 (1968).

365 - Mechanism of biological nitrogen fixation VII. Molecular H2 and the pN2 function of Azotobacter

They looked at the amount of nitrogen fixed with different concentrations in the atmosphere, in Azotobacter vinelandii and chroococcum cells. They were looking for K_N2: the concentration of nitrogen at which the amount of nitrogen fixed is half the maximum.

With inert gases (argon, helium) or with a partial vacuum, the concentration of nitrogen had to get down below 0.15 atm before the fixation decreased much. It gets to 50% around 0.01 atm. But when there was hydrogen, the rate decreased more quickly; the more, the faster. So hydrogen seems to inhibit the nitrogenase, but only at very high concentrations (over 20% of the atmosphere). This inhibition is competitive and reversible.

Reference:

Wyss, O., Lind, C. J., Wilson, J. B. & Wilson, P. W. Mechanism of biological nitrogen fixation VII. Molecular H₂ and the pN₂ function of Azotobacter. Biochem J 35, 845–854 (1941).

362 - Mutants of Azotobacter chroococcum Defective in Hydrogenase Activity

This study isolated some hydrogenase-negative mutants of Azotobacter chroococcum by chemical mutagenesis and looked at how they behaved.

What They Saw
Almost all of the 16 mutants had almost no hydrogenase activity, as expected. Some had a little, <2% of wild-type. Some more had a little hydrogen-producing activity in the right conditions, usually less than 7% of the wild-type, but one had 40% of wild-type. That one also seemed to have a relatively active soluble hydrogenase (possibly the uptake hydrogenase in soluble form). All of them seemed able to take up nickel.

The one weirdest mutant, MCD-124, showed max activity at a different pH (5.5 instead of 8) and was weird in other ways.

Also, the authors were surprised by the frequency with which they could get hydrogenase mutants. They wondered whether the relevant genes were just more susceptible, or if the growth medium was more favorable to mutants somehow, or if there were just that many necessary genes. But judging from the genome sequence, this isn't quite a sufficient explanation.

Overall, it's hard to know exactly what's going on in this study.

Reference:

Yates, M. G. & Robson, R. L. Mutants of Azotobacter chroococcum Defective in Hydrogenase Activity. J Gen Microbiol 131, 1459–1466 (1985).

358 - Molecular H2 and the pN2 function of Azotobacter

There was a question at this time of whether hydrogen in the air could inhibit nitrogen fixation in Azotobacter vinelandii, and in what conditions this might happen. There were various complicating factors though. This study attempted to do a more controlled investigation using purified enzyme.

What They Saw

They observed that the higher the concentration of nitrogen in the atmosphere, the more nitrogen was fixed in a given time (thus the higher the specific activity was). From this they could calculate the K_N2, by plotting the inverse of the specific activity over the inverse of the concentration and taking the slope of the line. This is an indication of the enzyme's affinity for nitrogen, I think. This is higher than seen from intact cells (0.01).

Then they got to the hydrogen inhibition experiments. As they increased the amount of hydrogen, the nitrogen-fixing activity did seem to decrease, indicating competitive inhibition.

One issue that they didn't control for was the production of hydrogen by nitrogenase itself; this could've influenced the numbers. It could also influence the K_N2 numbers, come to think of it. It's also unclear whether the extracts had any hydrogenase activity, which could influence things in the opposite direction.

Reference:

Strandberg, G. W. & Wilson, P. W. Molecular H₂ and the pN₂ function of Azotobacter. Proc Natl Acad Sci U S A 58, 1404–1409 (1967).

340 - Kinetic studies of the nitrogenase-catalyzed hydrogen evolution and nitrogen reduction reactions

This study looked at the kinetics of hydrogen production from purified nitrogenase; also, the nitrogen fixation reaction.

What They Saw
This nitrogenase was purified from Azotobacter vinelandii; they don't give details on culture conditions, so presumably it's the molybdenum version.

First, they observed that more ATP meant more hydrogen over time. The free phosphate-to-hydrogen ratio was similar at all levels though, so that makes sense. The same pattern was seen for nitrogen fixation, except the proportion of electron flux going to ammonia increased as ATP increased; more hydrogen was produced at lower ATP, relative to ammonia.

They found that whether under nitrogen or argon, the electron flux was the same; this was true over multiple ATP concentrations.

Reference:

Silverstein, R. & Bulen, W. A. Kinetic studies of the nitrogenase-catalyzed hydrogen evolution and nitrogen reduction reactions. Biochemistry 9, 3809–3815 (1970).

335 - ATP-Dependent hydrogen evolution by cell-free preparations of Azotobacter vinelandii

This study looked at hydrogen production in Azotobacter vinelandii strain O, to see what induced it in cell extracts.

What They Saw
Hydrogen production depended on ATP. Argon or hydrogen in the atmosphere didn't matter. The higher the protein concentration, the more hydrogen was produced. At 0ºC in air, the enzyme was pretty stable; over 90% activity was left after 3 days.

Hydrogen oxidation activity was found in separate fractions from the production activity, so they concluded it was a different enzyme.

Comparing extracts from cells grown with urea or no fixed nitrogen, they saw no hydrogen production activity in urea samples, but the hydrogen oxidation activity with urea was lower than that without.

What This Means
We know now that the hydrogen production comes from nitrogenase and the hydrogen oxidation from hydrogenase. It's surprising how air-stable the nitrogenase seemed to be outside the context of the cell, but I guess it was still surrounded by cellular elements in the crude extract. Also I'm not sure how oxygen inactivation affects nitrogenase hydrogen production.

Reference:

Burns, R. C. & Bulen, W. A. ATP-Dependent hydrogen evolution by cell-free preparations of Azotobacter vinelandii. Biochim Biophys Acta 105, 437–445 (1965).

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).

297 - The identification, characterization, sequencing and mutagenesis of the genes (hupSL) encoding the small and large subunits of the H2-uptake hydrogenase of Azotobacter chroococcum

And finally, seeming to complete our journey back in time through the discovery of hydrogenase genetics in Azotobacter chroococcum, this study looks at the structural genes, hupSL.

What They Saw
The sequences were similar to A. vinelandii's hoxKG structural genes. They tried knocking each out, then measuring hydrogen oxidation (with methylene blue) and hydrogen production (with methyl viologen). As expected, hydrogen oxidation in mutants was no higher than negative controls. Surprisingly, they did see hydrogen production in the some of the different mutants with a strong electron donor, but it was less than in the wild-type. Only the mutant with an insertion very close to the start of the hupS gene had no hydrogen production.

What This Means
It seems like a fragment of HupS is sufficient to produce hydrogen with a strong electron donor, but not as much as with both HupS and HupL completely intact.

Reference:

Ford, C. M. et al. The identification, characterization, sequencing and mutagenesis of the genes (hupSL) encoding the small and large subunits of the H₂-uptake hydrogenase of Azotobacter chroococcum. Mol Microbiol 4, 999–1008 (1990).

296 - The Azotobacter chroococcum hydrogenase gene cluster: sequences and genetic analysis of four accessory genes, hup A, hupB, hupY and hupC

So at this point, hupSL (hydrogenase structural genes) and hupDE (accessory genes at the end of the operon) had already been identified. This study found a few more upstream of hupDE.

What They Saw
They sequenced the DNA upstream of hupDE and found four open reading frames, which they called hupABYC. The AB and C were similar to E. coli genes, but the Y wasn't, so they called it Y (for Ynknown, I guess). These were all homologous to A. vinelandii genes though, and in the same order.

Then they tried knocking out each of these. Each knockout was unable to oxidize hydrogen, even in the presence of methylene blue as an electron acceptor.

They also made a fusion of HupL (the structural subunit) and beta-galactosidase, then knocked out hupY or hupB to see if this changed the expression of hupL. Beta-galactosidase activity rose a little bit, like 25% in each, but it didn't seem either was an important regulator.

Reference:

Tibelius, K. H., Du, L., Tito, D. & Stejskal, F. The Azotobacter chroococcum hydrogenase gene cluster: sequences and genetic analysis of four accessory genes, hup A, hupB, hupY and hupC. Gene 127, 53–61 (1993).

295 - Sequences, organization and analysis of the hupZMNOQRTV genes from the Azotobacter chroococcum hydrogenase gene cluster

This study focuses on the hydrogenase genes in Azotobacter chroococcum. They had already found hupSL encoding the structural genes (equivalent to A. vinelandii's hoxKG I guess), and then the accessory genes hupABYCDE further downstream (homologous to A. vinelandii's hypABFCDE). Now, in between, are the hupZMNOQRTV genes, completing the 16-gene operon.

What They Saw
This set of genes seems to be homologous to A. vinelandii's hoxZMLOQRTV string, making the whole operon very similar in both organisms.

As in previous studies, HupZ (and its analog, HoxZ) seems to be an electron carrier in the membrane, possibly a cytochrome. When they knocked it out, they observed similar results to 070: hydrogenase could oxidize hydrogen with methylene blue as an electron acceptor, but not with oxygen, so HupZ seems to be part of the transport chain to oxygen.

Based on comparison with homologs in other organisms, HupM may help the hydrogenase attach to the membrane, or with processing the subunits. It's unclear. The other genes may be involved in processing or metal stuff. HupR may be another electron-carrying protein. Further study is required.

Reference:

Du, L., Tibelius, K. H., Souza, E. M., Garg, R. P. & Yates, M. G. Sequences, organization and analysis of the hupZMNOQRTV genes from the Azotobacter chroococcum hydrogenase gene cluster. Journal of Molecular Biology 243, 549–557 (1994).

190 - Characterization of a Tungsten-Substituted Nitrogenase Isolated from Rhodobacter capsulatus

The three normal nitrogenases have molybdenum and iron, vanadium and iron, or just iron in their central cofactors. This study looked at whether it's possible to have a version that only has tungsten in it though, and if it has any activity, in Rhodobacter capsulatus.

What They Saw
This strain only has Mo and Fe nitrogenases, and they knocked out the latter. So when they removed all Mo from the medium and added W, the only thing it would produce would be Mo nitrogenase with W in it, theoretically.

Adding tungsten almost completely stopped acetylene reduction activity in the wild-type, unless 10x more Mo was added. So tungsten doesn't allow that kind of activity.

Mo also induces production of the Mo nitrogenase (which makes sense), but W also seems to have that effect, though not to the same extent.

Then they purified nitrogenase that had been produced when only W was present (in significant amounts) in the mutant. Purification was the same with this as with Mo in the wild-type, though of course the yield was lower. But the protein had 1 atom of W and none of Mo pretty much, as hoped. Still, it seems like there's only one FeW cofactor, not two, but at least there aren't any FeMos.

This protein still wasn't very active in acetylene reduction, and it didn't seem to be able to fix nitrogen at all. What it could do, though, was produce hydrogen when under an argon atmosphere, so electron flux wasn't totally abolished. And this activity wasn't inhibited by acetylene, unlike in the Mo version. Still, the amount of hydrogen it produced was only 1/4th the amount the normal enzyme could produce in the same conditions.

Finally they wanted to see if rhenium (Re) could take the place of Mo as a FeReco and be functional, since Re is not far from Mo and W (one more proton than W), so they tried growing cells with perrhenate (KReO₄), but it didn't seem to help at all. The cells didn't even appear to be able to assimilate it, so it wasn't possible to test whether the nitrogenase could use it.

What This Means
It's difficult to do this kind of study, because Mo and other metals are almost impossible to eliminate completely from the medium. But they seem to have succeeded as much as possible, and still the nitrogenase only had one FeW cofactor rather than two. But surprisingly it showed some proton reduction activity, though not an exceptional amount. It seems like W in the protein is much more difficult to reduce, an essential step in the catalysis. Maybe the amount of reduction that's possible is only enough for some proton reduction activity.

One interesting speculation is based on an observation that Methanococcus thermolithotrophicus can fix nitrogen in the presence of tungstate at 60ºC, a pretty high temperature, so they wonder whether the nitrogenase with FeWco in R. capsulatus might also have more activity at higher temperature, but they didn't actually test this. Maybe another study.

Reference:

Siemann, S., Schneider, K., Oley, M. & Müller, A. Characterization of a Tungsten-Substituted Nitrogenase Isolated from Rhodobacter capsulatus. Biochemistry 42, 3846–3857 (2003).

162 - Hydrogen evolution: A major factor affecting the efficiency of nitrogen fixation in nodulated symbionts

In the nitrogen fixation process, hydrogen gas is produced. This consumes extra ATP, about 4 per molecule of hydrogen (for reference, a molecule of glucose can be fully oxidized to produce about 30 ATP). This paper looks at this process in legume nodules and its effects on yield.

What They Saw
They got nodules from different kinds of plants, infected with either wild bacteria or commercial strains, and measured hydrogen production in air or argon, and acetylene reduction.

In an in vitro nitrogenase system, activity depended on the presence of ATP, reducing equivalents, and the enzyme. The ratio of hydrogen in argon to hydrogen in air here was about 3.

There was a pretty wide variety of values they saw, but most seemed to cluster around a measure of 50% of the hydrogen produced in argon was produced in air; so the presence of nitrogen reduced the hydrogen production by half. It's not clear how uptake hydrogenases might have affected these numbers though. They got similar results with whole plants. This means that each nitrogen gets 6 electrons to become 2 ammonia, and another 6 electrons go to hydrogen; that seems like a lot.

They also noticed that adding enough acetylene stopped the hydrogen production, so it seemed like hydrogen is not a necessary byproduct of that reaction.

What This Means
Correlation between amounts of acetylene reduced and nitrogen fixed are not necessarily accurate, because of the hydrogen produced as a byproduct. But the hydrogen production seems to be a big waste of efficiency; reducing this could help increase yield, maybe by choosing good nodulating bacteria. They wonder whether the hydrogen could be captured from plants somehow and used for energy.

Reference:

Schubert, K. R. & Evans, H. J. Hydrogen evolution: A major factor affecting the efficiency of nitrogen fixation in nodulated symbionts. Proc Natl Acad Sci 73, 1207–1211 (1976).

159 - Effect of chelating agents on hydrogenase in Azotobacter chroococcum: Evidence that nickel is required for hydrogenase synthesis

This study used chelating (metal-binding) compounds to study the cofactor of hydrogenase in Azotobacter chroococcum.

What They Saw
All the chelators they added (NTA, EDTA, etc.) decreased the hydrogenase activity in batch cultures, though to different extents. NTA was much stronger than EDTA. The effect was not inhibition of already-formed enzyme (since adding chelators to resting cells or extracts didn't affect activity), so it must be from preventing formation of additional enzyme.

They tried adding trace metal salts along with the chelators to see if pure excess of whatever was missing could restore activity. Copper, zinc, and manganese didn't really do anything. Cobalt helped if it was added in fairly large amounts, but the most helpful was nickel. Adding extra iron boosted this effect even more.

Monitoring nickel uptake by adding radioactive nickel, they saw that cyanide completely wiped out uptake (possibly by binding the nickel), but juglone and 2,4-dinitrophenol enhanced it (despite inhibiting respiration). Sodium azide didn't really affect either. The chelators they tested earlier generally seemed to inhibit nickel uptake too, generally in the same proportions as they had inhibited hydrogenase activity.

What This Means
Chelators seem to inhibit hydrogenase, but rather than acting on the enzyme directly, it seems to be by inhibiting its synthesis, and even this mechanism seems to be by inhibiting nickel uptake in most cases, rather than something more direct. Nickel is important for synthesizing the enzyme; it's a part of its essential cofactor. It seems like cobalt might be able to substitute for nickel somewhat though. I wonder if palladium would work too, since it has similar orbital arrangements. But apparently cobalt doesn't help in the absence of chelators or contaminating trace metals, so maybe it only helped here because it distracted the chelators away from nickel (so to speak).

Reference:

Partridge, C. D. & Yates, M. G. Effect of chelating agents on hydrogenase in Azotobacter chroococcum: Evidence that nickel is required for hydrogenase synthesis. Biochem. J. 204, 339–344 (1982).

071 - Two open reading frames (ORFs) identified near the hydrogenase structural genes in Azotobacter vinelandii, the first ORF may encode for a polypeptide similar to rubredoxins

This study looked at the genetics of the two open reading frames (ORFs) near the hydrogenase structural genes (hoxZM).

What They Saw
They sequenced the ORFs and compared to known genes. hoxZ seemed to have most homology with genes encoding proteins called rubredoxin from other species. These are typically small proteins that play roles in electron transport, which makes sense. And that's all.

Reference:

Chen, J. & Mortenson, L. Two open reading frames (ORFs) identified near the hydrogenase structural genes in Azotobacter vinelandii, the first ORF may encode for a polypeptide similar to rubredoxins. Biochim Biophys Acta 1131, 122–124 (1992).

070 - The hoxZ gene of the Azotobacter vinelandii hydrogenase operon is required for activation of hydrogenase

Here they wanted to look into the hoxZ gene more closely. Previous studies suggested that the product might be involved in electron transport for the hydrogenase.

What They Saw
They grew Azotobacter vinelandii DJ (an easy-to-transform strain) and knocked out hoxZ and hoxKG by transformation and screening for hydrogen production.

Comparing the hoxZ mutant to DJ (positive control) and the hoxKG mutant (negative control), they observed an intermediate rate of hydrogen oxidation, so there seemed to still be some activity. DJ consumed nearly all the hydrogen, and hoxKG consumed very little (the graph showed a decrease but it was apparently because gas leaked out of the vial, so it's a good thing they had good controls!).

Then they tried measuring short-term hydrogen oxidation with different electron acceptors: oxygen or methylene blue. DJ quickly oxidized all the hydrogen while reducing oxygen or methylene blue, as expected. Both mutants didn't show activity with either acceptor at first, despite the difference in the previous assay. But then they added sodium dithionite (a powerful reducer of oxygen) and more methylene blue, and the hoxZ mutant showed up to 80% of the activity of DJ. As far as I can tell, the hoxKG didn't show the same effect when they gave it the same treatment, but they don't say that explicitly. But it seems like the hydrogenase needs to be activated somehow, as by dithionite.

These results were confirmed by observing methylene blue color change too; DJ quickly started oxidizing hydrogen, but the hoxZ mutant did too after a longer lag period.

When they isolated membrane-bound hydrogenase from cells (still embedded in membranes), even DJ needed activation with dithionite. hoxKG mutants had no activity in any case, of course. But hoxZ mutant had more activity in the soluble supernatant portion than DJ did, at least when membranes were isolated aerobically; it seemed like lack of hoxZ led to more soluble enzyme. But it had low activity in general so this conclusion was uncertain. Though membrane-bound activity in general was higher when isolated anaerobically, and they didn't measure soluble activity in that case for some reason. So HoxZ may help stabilize hydrogenase in the presence of oxygen.

The increased presence of detached hydrogenase in the mutant was not confirmed by Western blot, so it seems like an artifact.

What This Means
HoxZ seems to have a role in shuttling electrons between hydrogenase and oxygen, though there may be other components involved in this path. It's possible that when HoxZ is missing, another acceptor can take the electrons, but isn't as good at it.

It also may be involved in activating the enzyme (which requires removing oxygen and providing reduction); somehow hydrogen is not enough for this. And it may help stabilize the hydrogenase to keep oxygen from inactivating it, maybe also using its role as electron transporter.

Reference:

Sayavedra-Soto, L. A. & Arp, D. J. The hoxZ gene of the Azotobacter vinelandii hydrogenase operon is required for activation of hydrogenase. J. Bacteriol. 174, 5295–5301 (1992).

067 - Analysis of a gene region required for dihydrogen oxidation in Azotobacter vinelandii

This study looks more closely into the genes discovered in previous studies (518,050,051,052) to be required for hydrogenase in Azotobacter vinelandii.

What They Saw
They knocked out different genes (or the whole operon) in the hyp operons of A. vinelandii CA by inserting resistance + lacZ cassettes, then tested these strains for hydrogen oxidation and expression of the genes (via beta-galactosidase activity). The medium they used had a lot more trace elements than typical Burk medium, including nickel.

They found that when lacZ was inserted in the same direction as the gene, they saw expression in all cases, whether fixing nitrogen or not, but if it was inserted in the opposite direction, they didn't. So apparently the genes are expressed to some extent even when not fixing nitrogen.

When they measured hydrogen oxidation, there was about 6x more when fixing nitrogen though, in the wild-type, and none in the mutants. The growth rates of the mutants were similar to the wild-type though, or so they claim without reporting any details.

Finally they tried growing the strains with extra added nickel, because the hydrogenase is a nickel-containing enzyme. This didn't have much effect on the wild-type, but the hypB mutant actually showed some hydrogenase activity in nitrogen-fixing conditions with the extra nickel, and activity was higher with more nickel added.

What This Means
It seems like extra added nickel can substitute for the lack of HypB, so maybe the enzyme has a role in nickel cofactor processing somehow. This makes sense considering the multiple histidines it contains. It's interesting that nickel didn't help activity in non-fixing conditions; maybe there are multiple hydrogenases, active in different conditions, and hypB is required for all but nickel doesn't help some of them.

Reference:

Chen, J. C., Mortenson, L. E. & Seefeldt, L. C. Analysis of a gene region required for dihydrogen oxidation in Azotobacter vinelandii. Curr. Microbiol. 30, 351–355 (1995).

056 - Properties of Hydrogenase from Azotobacter vinelandii

Another study tries to purify the uptake hydrogenase from Azotobacter vinelandii.

What They Saw
They were unable to get pure, soluble enzyme; they couldn't separate it from the membrane, so it remained in insoluble particles.

They observed that hydrogenase activity of these particles didn't decrease after 18 days of exposure to air, unlike particles from other species. They did lose activity within a few weeks though.

They also observed that the enzyme reduces cytochrome C, possibly as part of its electron transport role. They found that carbon monoxide and cyanide inhibit the enzyme. Azide does not, at least not at pH 8.

What This Means
Some of the observations here may be due to the presence of other membrane-bound proteins, not just hydrogenase.

Reference:

Hyndman, L. A., Burris, R. H. & Wilson, P. W. Properties of Hydrogenase from Azotobacter vinelandii. J. Bacteriol. 65, 522–531 (1953).

054 - Nitrogen fixation by Azotobacter vinelandii in tungsten-containing medium

This study investigates how the alternative (vanadium) nitrogenase interacts with tungsten.

What They Saw
They grew Azotobacter vinelandii UW (aka CA) or a tungsten-tolerant mutant with tungsten and Mo or V, then extracted the nitrogenase.

This mutant was derived by growing UW with W and ammonium over several passages to try to remove all the Mo the cells might be storing, and then plating the cells on plates without ammonium, so they would have to fix nitrogen in the presence of W and absence of Mo to survive. Stuff that grew was tungsten-tolerant: strain LM2.

Then to get nitrogenase, they grew LM2 with tungsten and UW with tungsten and ammonium (not sure why it would produce nitrogenase in that condition, though I guess it might when it ran out of fixed nitrogen).

Both UW and LM2 could grow on medium with Mo, though LM2 was sometimes slower. With W, LM2 grew faster or slower depending on amount of W, while UW did not grow.

They used something called rocket immunoelectrophoresis to measure how much MoFe nitrogenase components each strain produced with W. Each produced about the same amount of dinitrogenase reductase in all conditions, but UW with 1mM W produced about 56% the amount of dinitrogenase as it produced with Mo, and LM2 with 10mM W produced about 7% of the amount that UW produced with Mo. I wonder how accurate this technique is. But they also note that the specific acetylene reduction activity of crude extracts was about 6-8% for each strain grown in W compared to UW in Mo. Does this include alternative nitrogenase activity? They say no. But overall, it seems that both strains produce less Mo nitrogenase with W, LM2 less than UW, but LM2's is relatively more active.

I think what happened next was that they couldn't isolate nitrogenase well from LM2, so they studied it from UW. They had two kinds: typical Mo nitrogenase, and Mo/W nitrogenase that had one FeW-cofactor and one FeMo-cofactor. This latter showed less activity in every way: hydrogen under argon, nitrogen, or acetylene atmospheres, nitrogen reduction, and acetylene reduction. This fits with at least one previous study (045). There is still activity though, even with nitrogen reduction, so it's not clear why the cells can't grow in W; maybe its interaction with other important proteins?

What This Means
It's possible that instead of each molecule of Mo/W protein containing one W and one Mo, half the protein could have all Mo and the other half all W; this would give the same results, but seems a less likely explanation. Of course, both possibilities are pretty weird and confusing.

According to some chemistry stuff they did, it seems like the enzyme can't reduce FeW-cofactors, which could reduce the possible electron flux by half, I think. There are a lot of mysteries here.

Reference:

Hales, B. J. & Case, E. E. Nitrogen fixation by Azotobacter vinelandii in tungsten-containing medium. J. Biol. Chem. 262, 16205–16211 (1987).

045 - Tungsten incorporation into Azotobacter vinelandii nitrogenase

Tungsten is known to cause problems for molybdenum-containing enzymes. This report looks into its effect on Azotobacter vinelandii's Mo nitrogenase.

What They Saw
They grew A. vinelandii OP (aka CA) in Burk without Mo, with added ammonium phosphate. Because it's really hard to get rid of every little bit of Mo, they added lots of tungsten (W) to make sure that they could see it if it got incorporated into enzymes. Some of the W was radioactive.

W didn't inhibit growth when ammonia was present, which makes sense. But it did inhibit it, about the same, with N2, nitrate, or urea. The enzymes that use these N sources all need Mo. When Mo was about 0.1 μM, it took 20 μM W to inhibit growth 50%; when Mo was 10 μM, it took 4 mM W. When just a little ammonia was added, it took about 5000 times more W than Mo to stop growth.

When they purified nitrogenase from these W-grown cells, they actually did see acetylene reduction activity, though not nearly as much as with normal Mo nitrogenase. The W content of extracts was very high, though it seemed to be easily removable. Specifically purifying Fe-W protein and comparing to the Fe-Mo version, all activities seemed relatively low: acetylene reduction, hydrogen production, and ATP hydrolysis.

What This Means
A. vinelandii might treat W the same as it treats Mo: taking up as much as it can and storing what it doesn't incorporate. But it does seem to incorporate some into the Mo nitrogenase. This seems to result in a poorly functional enzyme, but is that enough to stop cells from growing entirely? Maybe W's effects on other enzymes cause some problems too.

Reference:

Benemann, J. R., Smith, G. M., Kostel, P. J. & McKenna, C. E. Tungsten incorporation into Azotobacter vinelandii nitrogenase. FEBS Lett 29, 219–221 (1973).

053 - Purification to homogeneity of Azotobacter vinelandii hydrogenase: a nickel and iron containing αβ dimer

This study purified and characterized the uptake hydrogenase from Azotobacter vinelandii.

What They Saw
As a single unit, not denatured, the hydrogenase ran as a single band on a gel. On a denaturing gel it was two bands though, 31 and 67 kDa, indicating that it had two subunits, a larger and a smaller (HoxG and HoxK). The whole thing is about 98.6 kDa. This contrasts with previous reports that it was a single subunit of 60 kDa (049), but it's not really clear why there's this discrepancy.

They also measured metal contents, and found 6.6 mol iron and 0.68 mol nickel per mol hydrogenase, so they rounded up to 1 mol Ni and 10 mol Fe, 1:10 ratio.

They also contradicted the previous result that the hydrogenase couldn't donate to acceptors with negative potential. But they confirmed the possibility that hydrogenase produces hydrogen when paired with a very reduced donor, except this peaked at a higher pH (6 to 8.5).

Reference:

Seefeldt, L. C. & Arp, D. J. Purification to homogeneity of Azotobacter vinelandii hydrogenase: a nickel and iron containing αβ dimer. Biochimie 68, 25–34 (1986).

518 - Cloning, sequencing and characterization of the [NiFe]hydrogenase-encoding structural genes (hoxK and hoxG) from Azotobacter vinelandii

To continue our journey back in time through the discovery of the uptake hydrogenase genetics in Azotobacter vinelandii, this study describes the sequencing of the hydrogenase structural genes, hoxKG.

What They Saw
They probed A. vinelandii DNA with a chunk from A. chroococcum with its hydrogenase genes, and sequenced a fragment they found. This fragment contained three full open reading frames (ORFs) and possibly the beginning of a fourth. The first two encode the structural subunits, HoxK and HoxG (for hydrogen oxidation), based on comparison to sequences in other organisms.

HoxK seems to undergo some processing, since the final protein is smaller than that predicted by the gene sequence; there might be a signal sequence that localizes the protein to the membrane or something. The structural ORFs overlap each other a little, indicating that they're probably transcribed as a unit (in the same operon) which helps the proportions come out right.

The third ORF, ORF3 (which we know now is hoxZ) was also similar to other organisms, and seemed to be a membrane protein of some sort. Possibly part of the hydrogenase electron transport chain.

Reference:

Menon, A. L., Stults, L. W., Robson, R. L. & Mortenson, L. E. Cloning, sequencing and characterization of the [NiFe]hydrogenase-encoding structural genes (hoxK and hoxG) from Azotobacter vinelandii. Gene 96, 67–74 (1990).

052 - Nucleotide sequences and genetic analysis of hydrogen oxidation (hox) genes in Azotobacter vinelandii

This study looks at the hydrogenase-related genes in Azotobacter vinelandii in between the structural genes and the hyp genes.

What They Saw
They already knew about the structural genes, hoxKG. So they got some overlapping fragments containing those and sequences downstream of them, and sequenced them. They found 5 new open reading frames (ORFs), ORF3-7. They named these hoxZMLOQ. There was also part of an eighth at the end. All of them were homologous to hydrogenase genes in other organisms.

Then they knocked out each gene by inserting kanamycin or kan+lacZ cassettes, though probably some of these insertions affected other genes (polar effects) so it wasn't possible to study each gene individually. Each insertion abolished hydrogen oxidation. All 5 new ORFs seem to be involved in subunit processing, as the unprocessed form was present in higher proportion (or alone) in their knockouts.

The knockouts didn't seem to be impaired in growth, even when fixing nitrogen, though they might show more effect in certain conditions (like carbon limitation).

HoxZ seems to be a membrane protein, possibly a cytochrome, so part of the electron transport chain. HoxL seems like it might be involved in metal-binding somehow.

What This Means
Knowing more of the sequence of the operon helps to study it. The whole thing, hox and hyp, is pretty big, kinda surprising for an enzyme that doesn't seem to help the organism in some conditions. Probably in nature, the likelihood of encountering carbon-limited circumstances is much higher, so having an uptake hydrogenase is important.

Reference:

Menon, A. L., Mortenson, L. E. & Robson, R. L. Nucleotide sequences and genetic analysis of hydrogen oxidation (hox) genes in Azotobacter vinelandii. J Bacteriol 174, 4549–4557 (1992).

051 - The hypE Gene Completes the Gene Cluster for H2-oxidation in Azotobacter vinelandii

As a followup to 050, this study completed the hyp operon by sequencing hypE. They suspected its existence because other sequenced operons always had a hypE after the hypD, so they purified a fragment from Azotobacter vinelandii that hybridized hypD and contained some downstream region.

What They Saw
The open reading frame (ORF) in the downstream region had good homology to hypE genes in other organisms. A few bases of hypD and hypE genes overlap, suggesting that they're translated together so there should be equal amounts present so they can work together equally.

They knocked out hypE by inserting a lacZ and kanamycin resistance cassette, which also let them detect expression (the product of lacZ can break down certain compounds to make color, so color intensity can be measured as a proxy for enzyme activity). It seemed like transcription of all the hox and hyp genes was in the same direction, and knocking out hypE meant that A. vinelandii couldn't oxidize hydrogen anymore.

They also noticed that there was a higher proportion of unprocessed hydrogenase structural component, so HypE might be involved in processing; this seems true in other organisms. It also affects the localization of hydrogenase, soluble or membrane-bound. It may affect pre-protein folding.

What This Means
This study completes the discovery of the whole uptake hydrogenase gene set in A. vinelandii. It's pretty similar to the gene sets in other organisms, including A. chroococcum, and sequencing A. vinelandii's genome confirmed the results.

Reference:

Garg, R. P., Menon, A. L., Jacobs, K., Robson, R. M. & Robson, R. L. The hypE Gene Completes the Gene Cluster for H₂-oxidation in Azotobacter vinelandii. J. Mol. Biol. 236, 390–396 (1994).

050 - Identification of six open reading frames from a region of the Azotobacter vinelandii genome likely involved in dihydrogen metabolism

This study looked into which genes in Azotobacter vinelandii are required for its uptake hydrogenase other than the structural genes, hoxKG.

What They Saw
They sequenced almost 6 kilobases somewhat downstream of the structural genes and found 6 open reading frames (ORFs), the last incomplete, so they sequenced some more to complete it. All 8 seemed to be a single operon. These genes are now called hoxV and hypABFCD (based on homology to E. coli genes).

They predicted that the middle 3 and last ORF (now called hypABF and hypD) would produce proteins good at binding Fe-S clusters. HypB has a histidine-rich region that might be good for binding nickel, and HypF has a zinc finger-like region. They have good homology to genes related to hydrogen metabolism in other organisms. They could be related to hydrogenase regulation or assembly, especially related to Ni and Fe; some (HypBF) have homology to other Ni-related proteins, such as urease.

Reference:

Chen, J. C. & Mortenson, L. E. Identification of six open reading frames from a region of the Azotobacter vinelandii genome likely involved in dihydrogen metabolism. Biochim. Biophys. Acta 1131, 199–202 (1992).

049 - Purification and properties of membrane-bound hydrogenase from Azotobacter vinelandii

Azotobacter has a hydrogenase. Some organisms have hydrogenases that produce hydrogen, or sometimes produce and sometimes break down (reversible). Azotobacter's had not been observed to break down hydrogen (so was considered unidirectional). This study purified the membrane-bound uptake hydrogenase from A. vinelandii and tested its properties.

What They Saw
They measured activity of purified enzyme with an electrode measuring hydrogen oxidation in the presence of methylene blue dye (an electron acceptor).

They measured stability of the enzyme in the presence of oxygen, and found that crude extracts were very stable (and could go for weeks without losing activity), but the more pure the preparation, the less oxygen-tolerant it was: the most pure lost half its activity in 20 minutes at 20% oxygen. This inactivation was irreversible.

With a very good electron donor (methyl viologen), the hydrogenase could produce hydrogen. The highest rate they saw was 3.4 μmol hydrogen per minute per mg protein, which peaked at a fairly low pH (around 4). The rate was almost 0 closer to neutral. Also, the presence of hydrogen in the environment inhibits its production by the enzyme.

Even when electron acceptors were not present, the enzyme could combine one deuterium from D₂ with one hydrogen from water to make HD.

The enzyme seemed to be good at donating electrons to acceptors with positive mid-point potentials but not to negative ones, so it seems to have a higher potential than reversible hydrogenases.

Reference:

Kow, Y. W. & Burris, R. H. Purification and properties of membrane-bound hydrogenase from Azotobacter vinelandii. J Bacteriol 159, 564–569 (1984).

256 - Construction of a recF deletion mutant of Azotobacter vinelandii and its characterization

So we saw that Azotobacter vinelandii seems to have multiple copies of its chromosome in each cell (253,255), but we also seemed to see that it doesn't exhibit the behavior expected of a polyploid organism, at least sometimes (445). Other times it might. One proposed explanation is that it has some mechanism by which it homogenizes its genotype, a type of homologous recombination or "homogenotization."

A possible mechanism for this is the RecF system found in E. coli, which is involved in repairing the genome after recombination. A. vinelandii has a homologous system, so this study investigated the phenotype of cells with it knocked out.

What They Saw
They knocked out recF, recA, or both from A. vinelandii UW (aka CA), by inserting tetracycline resistance cassettes. This was confirmed by Southern blotting. Actually they already had a recA knockout, so they just made that a double-knockout. That was somewhat tricky, since the recombination frequency is much lower than in the wild-type; that's not very surprising, since RecA is probably pretty important for recombination.

They found that knocking out recF seemed to impair recombination too (also not surprising), though not as much as lack of recA. With both gone, the recombination proficiency was even lower. They saw similar results with UV sensitivity (and thus ability to repair DNA damage).

Finally, the question of homogenotization: to test this, they knocked out the nifLA genes (which regulate/activate nitrogenase) by inserting a kanamycin resistance cassette, expecting that if RecA or RecF were involved in homogenotization, the lack of them would mean that it would be easy to isolate cells with kan resistance in some chromosome copies and yet capable of nitrogen fixation (because wild-type nifLA is still present in other copies to turn on the nitrogenase). This approach, again, seems questionable to me due to the presence of multiple nitrogenases. I'm not sure if NifLA are necessary to regulate all three versions, or just the Mo one.

However, they didn't find any transformants from any of the strains (wild-type or recA or recF or double-knockouts) that were both kan-resistant and nitrogen-fixing. So they conclude that these deleted genes aren't involved in so-called homogenotization, since their absence didn't make a difference. Is it possible that the homogenotization happened when cells divided and only those with kan resistance survived? I'm not sure that question was addressed. It doesn't seem known if progeny get just one copy and then make more, or if copies are equally partitioned. I suppose the latter makes more sense, since it is binary fission, as far as we know.

Also, they did say that antibiotics weren't necessary to prevent nitrogen fixation phenotypes, so I guess that addresses my question. And they did more Southern blotting to show that there was a cassette inserted in the nifLA locus in all the transformants, with no wild-type alleles visible.

I'm not convinced that homogenotization is a real thing, but I'm not yet sure how to explain these data. Somehow it seems like A. vinelandii has many copies of its genome, but it's just as possible to transform all of them the same way as to transform just some. Guess I'll keep reading.

Reference:

Badran, H., Sohoni, R., Venkatesh, T. V. & Das, H. K. Construction of a recF deletion mutant of Azotobacter vinelandii and its characterization. FEMS Microbiology Letters 174, 363–369 (1999).

255 - Segregation characteristics of multiple chromosomes of Azotobacter vinelandii

Again on the subject of multiple chromosome copies in Azotobacter vinelandii, they wanted to see if the larger amount of DNA in the cells compared to E. coli actually meant that many more copies of each gene.

What They Saw

They mutagenized A. vinelandii UW (aka CA) with transposons and tried to see if these insertions were present in as many copies as they had observed for other genes (30-40). They tried to find auxotroph mutants by conjugating with E. coli carrying a transposon. They didn't find any A. vinelandii that failed to grow on plates lacking certain amino acids, but they did find some that didn't grow very well unless the amino acids were present. This poor growth got less poor over time in successive generations though, but the cells were still resistant to the selective marker (ampicillin). They interpret this as being related to the proportion of genome copies with an insertion vs. without.

They also saw that mutagenized cells mostly couldn't grow with ampicillin when plated directly, though they could grow on antibiotic-free Burk medium. Then when cells from each of these conditions were transferred again to plates with antibiotic, only some from selective plates grew, while all from non-selective plates grew. The idea is that growing on selective plates, the ability to grow without added amino acids would be lost (since the transposon knocked out that ability in some copies, and those copies would be higher in proportion because of the selection). They should've tried growing cells from selective medium on non-selective medium to see if they got the same result.

And gaining the ability to resist the antibiotic? Shouldn't they have had that from the beginning? This whole set of experiments is unclear.

They also mutagenized cells with a different transposon that conferred tetracycline resistance, isolated DNA from them after growing on different amounts of tet, and probed with radioactive probes, correlating radioactivity to number of copies. They observed more radioactivity in cells grown with greater selective pressure, implying that there were multiple alleles and selection increased the proportion of resistant allele in a population.

I'm still somewhat dubious, but it seems like the data might be fairly solid.

Reference:

Phadnis, S. H., Dimri, G. P. & Das, H. K. Segregation characteristics of multiple chromosomes of Azotobacter vinelandii. J. Genet. 67, 37–42 (1988).

445 - Segregation pattern of kanamycin resistance marker in Azotobacter vinelandii did not show the constraints expected in a polyploid bacterium

This is a paper similar to the last one, by the same authors (254), investigating Azotobacter vinelandii's seeming ability to possess multiple copies of its chromosome, and thus exhibit polyploidy (multiple genotypes in the same locus).

What They Saw
They used A. vinelandii ATCC12837 and knocked out the nifY gene (whose product makes the central Mo cofactor) with a kanamycin resistance insertion, then grew it with or without nitrogen or kanamycin. It shouldn't be able to fix nitrogen with Mo present, though maybe with Mo absent.

Looking at the methods, I couldn't find one of the restriction sites they claim to have used, but it's possible that it was present in their strain and not in mine. All the others seem to be there.

Actually, they transformed cells, plated them out to obtain single colonies on non-selective plates, and then screened these colonies for kanamycin resistance. The idea was that if each cell had multiple copies of its chromosome, only some of the offspring of a resistant cell would have kan resistance, because some would inherit the resistant version of the genome and others wouldn't.

But when they grew more colonies from originally resistant colonies and tested their resistance, all of them were resistant that came from resistant colonies. So once the kan-resistant phenotype was present in a cell, it got passed to all offspring; no evidence of polyploidy.

This study is lacking in some ways: I would've liked to see confirmation of the locus of insertion by sequencing, to make sure it's in the right place. And it would be interesting to see what happened if they selected for nitrogen fixation, as in 254. So the question isn't quite settled.

Reference:

Pulakat, L., Efuet, E. T. & Gavini, N. Segregation pattern of kanamycin resistance marker in Azotobacter vinelandii did not show the constraints expected in a polyploid bacterium. FEMS Microbiology Letters 160, 247–252 (1998).

254 - Isolation and characterization of nifDK::kanamycin and nitrogen fixation proficient Azotobacter vinelandii strain, and its implication on the status of multiple chromosomes in Azotobacter

Others seemed to find that Azotobacter vinelandii had many copies of its chromosome (253). This was done partly by comparison to E. coli, but it seems that A. vinelandii's genome size is similar to E. coli's. And probing for specific genes seemed to show high copy numbers. A. vinelandii cells seem larger (about 12.5x), but is this enough to accommodate 40-80x more DNA? Also, it's possible to knock out genes from the species, which would be difficult if they had many copies of a gene that could substitute for each other. This study investigated.

What They Saw
They grew A. vinelandii OP (aka CA) and did genetic transformations with it, knocking out the nifDK genes (which encode the Mo dinitrogenase) by inserting a kanamycin resistance cassette by homologous recombination.

They plated transformant colonies on plate with or without kanamycin and with or without fixed nitrogen (so four different kinds of plate). The wild-type of course could grow on either plate without kanamycin but neither plate with it. They also saw two kinds of mutant phenotype: one that could grow with fixed nitrogen either with or without kanamycin, and another that could grow on all plates, with or without antibiotic or fixed nitrogen.

They tried to confirm this using PCR across the insertion, and restriction digestions followed by electrophoresis. The primers they report seem to be appropriate for getting the Mo nitrogenase genes.

Trying to figure out what they did, this paper seems to have lots of problems with reporting exactly what kind of digestions/cloning they did... sites they claim to have used don't seem to exist in my copy of the genome, or aren't in the right place, or are in too many places. So who knows what's actually going on genetically with this strain they isolated.

The gel they show from the PCR shows two bands in the kan-resistant, N-fixing mutant, and the bands seem to be the right size to correspond to sequences with and without the resistance cassette insertion. They claim to have sequenced the region but don't report the sequence in the paper.

What This Means
The authors conclude that A. vinelandii is exhibiting behavior suggesting multiple copies of a single gene locus (both with kan resistance and with nitrogen fixation). While it does seem to exhibit both phenotypes (assuming no contamination with multiple strains), I'm not sure the interpretation is clear. A. vinelandii has multiple different enzymes capable of fixing nitrogen, so a mutation in their regulation could explain that phenotype, though it wouldn't explain the bands on the gel. Still, it would be good to see this study replicated; unfortunately, the methods are explained poorly.

Reference:

Suh, M.-H., Pulakat, L. & Gavini, N. Isolation and characterization of nifDK::kanamycin and nitrogen fixation proficient Azotobacter vinelandii strain, and its implication on the status of multiple chromosomes in Azotobacter. Genetica 110, 101–107 (2000).

253 - Multiple chromosomes of Azotobacter vinelandii

Previous studies and some preliminary data supposedly showed that Azotobacter vinelandii cells had up to 40 times as much DNA material as Escherichia coli cells, so this study looks at the form that this excess of DNA takes. Is it all one molecule or are there multiple chromosomes or copies of the same chromosome?

What They Saw
They extracted DNA from A. vinelandii and E. coli and determined the amount per cell (by counting number of cells), finding that E. coli had about 3.4 femtograms per cell (3.4 * 10^-15 g) and A. vinelandii had 135 femtograms. Assuming E. coli's genome is about 4 megabases (actually 4.6, though it depends on the strain) and it has only one copy per cell, A. vinelandii should have about 160 megabases-worth of DNA per cell. A. vinelandii's genome is only about 5.4 megabases, so that's about 30 copies of the genome per cell.

However, they made an artificial mixture of known numbers of copies of a certain gene in A. vinelandii and measured the intensity of radioactivity for the correct band of a Southern blot when probed with a probe labeled with radioactive phosphorus. The amount of radioactivity for DNA extracted from cells was about twice the amount seen with 40 copies of the gene, so they concluded there must be about 80 copies of the gene present. Does this mean there are 80 copies of the chromosome per cell? Not necessarily; there could be multiple copies of the gene per chromosome. However, I don't think that is the case.

They also tried with nitrogenase nifDK genes and got similar amounts of radioactivity. nifH gave multiple bands (presumably because of the alternative nitrogenases), but the main band gave a similar brightness.

To distinguish between a giant chromosome with 80 copies of each gene, and 80 copies of a smaller chromosome (or something in between), they integrated a resistance marker in a particular place in the genome. If it were a single large genome, the marker would probably only integrate once or a few times, whereas after several generations, the cell would make copies of a smaller genome with an integrated marker such that the amount of marker would increase over time. They observed the latter result, suggesting single copies of genes on a chromosome with many copies.

What This Means
The copy number of Azotobacter chromosomes was about 30-40 compared to E. coli but about 80 in terms of specific gene copies. A possible way to reconcile this is that the E. coli cells might actually have had more than one copy too. In any case, this many copies of the genome might make it difficult to stably transform the organism.

Reference:

Nagpal, P., Jafri, S., Reddy, M. A. & Das, H. K. Multiple chromosomes of Azotobacter vinelandii. J. Bacteriol. 171, 3133–3138 (1989).

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).

216 - Effect of Dissolved Oxygen on Growth Yield and Aldolase Activity in Chemostat Culture of Azotobacter vinelandii

This study looked at carbon- or oxygen-limited cultures of Azotobacter vinelandii and the effects on enzymes of the TCA cycle: aldolase, glyceraldehyde-3-phosphate dehydrogenase, isocitrate dehydrogenase, and isocitrate lyase.

What They Saw
They grew A. vinelandii in chemostats with Burk medium with glucose at different agitation speeds and different dilution rates.

At the second-lowest agitation, dissolved oxygen was low and a bit of glucose was residual in the outflow (about 0.44 g/L); this increased as D increased. Growth yield and carbon dioxide production didn't change much as D changed. Things were similar in the lowest agitation, except there was less biomass and presumably more residual sugar.

At the second-highest agitation, residual glucose was very low up to D = 0.2, then went up, so the culture seemed to switch from glucose-limited to oxygen-limited at that point. Biomass increased up to that point too, and then decreased. Growth yield and CO₂ production changed inversely, with yield increasing up to a certain D before 0.2 and then leveling off. Once leveled off, values were similar to those from lower aerations.

So overall for all aerations, as dissolved oxygen increased, growth yield from glucose decreased; growth became less efficient. Carbon dioxide production increased though, indicating that the carbon was being consumed but going toward that gas, complete oxidation.

In terms of enzyme activities, of the four tested enzymes, only aldolase increased as oxygen increased. Below is their model of what effect this has:

Fig 4, Nagai et al. 1971

The rise in aldolase activity meant that carbon was cycling through the pentose cycle more instead of moving on to the TCA cycle (which could lead to greater ATP generation and anabolism), so that explains the increase in CO₂ production and decreased growth yield.

Reference:

Nagai, S., Nishizawa, Y., Onodera, M. & Aiba, S. Effect of Dissolved Oxygen on Growth Yield and Aldolase Activity in Chemostat Culture of Azotobacter vinelandii. J Gen Microbiol 66, 197–203 (1971).

215 - Respiratory protection of nitrogenase in Azotobacter vinelandii

This study is pretty similar to 214, on how oxygen levels influence the respiratory chain of Azotobacter vinelandii.

What They Saw
They grew cells in batch at low aeration, and then increased the aeration to expose cells to excess oxygen.

The results were similar to 214: when aeration increased, cells stopped growing until respiration had ramped up and leveled off. Despite the large increase in respiration, cellular ATP levels dropped 40%. P/O ratios decreased as respiration increased to accommodate the increased oxygen, at least for NADH dehydrogenase.

They tried adding chloramphenicol again, since last time it didn't affect the growth lag, but this time observed that it lessened the increase in respiration about half, and prevented cytochrome a₂ and other dehydrogenase increases.

What This Means
The M/N ratio, meaning maintenance M (moles ATP consumed per weight of cells over time) over phosphorylating efficiency N (P/O ratio x 2), explains doubling of respiratory activity upon increase in oxygen only if M increases or N decreases. It doesn't seem like M does increase, so N seems to be decreasing, because respiration uncouples from phosphorylation.

A. vinelandii seems to have different branches in its respiratory chain, which allows it to tolerate different and suddenly changing levels of oxygen.

Reference:

Jones, C. W., Brice, J. M., Wright, V. & Ackrell, B. A. C. Respiratory protection of nitrogenase in Azotobacter vinelandii. FEBS Letters 29, 77–81 (1973).

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).