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).

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).

165 - The Behaviour of Azotobacter chroococcum in Oxygen- and Phosphate-limited Chemostat Culture

This study looked at Azotobacter chroococcum behavior when limited in oxygen or phosphate.

What They Saw
They grew A. chroococcum in chemostats, similar to previous studies (163). In their allegedly oxygen-limited culture, biomass levels fell as dilution rate increased, while carbon dioxide production fell and then rose again for some reason; they claim it's just random, and that CO₂ was actually fairly constant (or blame it on consistency of electricity in Britain). They also observed that as dilution rate decreased, the amount of PHB in the cells rose. They could tell the cells were oxygen-limited because when more oxygen was added, it disappeared pretty quickly.

When cells were phosphate-limited, extra oxygen was pretty toxic; the cells couldn't handle it. Plating cells out after this challenge revealed that there was very little viability, if any. In contrast, cells that had been oxygen-limited didn't seem to mind extra oxygen much, especially when they were grown on medium with fixed nitrogen.

What This Means
It fits pretty well with previous results. PHB seems to be an electron sink, useful when cells don't have enough oxygen to receive all their electrons.

The fact that oxygen didn't reduce cells' viability when plated on medium with fixed nitrogen suggests that the cells' problem with extra oxygen is lack of ability to fix nitrogen immediately, rather than loss of viability. Then the cells ramp up respiration to consume the extra oxygen—respiratory protection.

Reference:

Lees, H. & Postgate, J. R. The Behaviour of Azotobacter chroococcum in Oxygen- and Phosphate-limited Chemostat Culture. J Gen Microbiol 75, 161–166 (1973).

108 - The Beneficial Effect of Hydrogenase in Azotobacter chroococcum Under Nitrogen-Fixing, Carbon-Limiting Conditions in Continuous and Batch Cultures

Since hydrogen is an energy-rich gas, and nitrogenase produces hydrogen, one would expect that diazotrophs that can re-oxidize the hydrogen they produce (using uptake hydrogenases) would have a competitive advantage over those that lack an uptake hydrogenase. However, results of previous studies of this question, in this and other organisms, have been mixed (019,065).

This study is another comparison of Azotobacter chroococcum strains, one with and three without an uptake hydrogenase, in a variety of conditions.

What They Saw
As the dilution rate increased in carbon-limited nitrogen-fixing conditions, the wild-type strain's growth yield remained relatively constant, while the three mutants' yields were noticeably lower at most rates. There was no noticeable difference when fixed nitrogen was provided. When oxygen or sulfate were limiting, there didn't seem to be much difference between strains.

When the strains were mixed together in equal densities in continuous culture, the mutants seem to overtake the wild-type a couple times at lower dilution rates, but the wild-type always took over at higher rates. With ammonium added, there was no consistent pattern.

What This Means
The mutants in this study were obtained by random mutagenesis, so the lack of hydrogenase wasn't necessarily the only difference from wild-type. Still, the three strains seemed to give some fairly consistent results.

The advantage seen in the wild-type occurred in low-density populations or high dilution rates, so it seems like the hydrogenase helps recover energy (or protect nitrogenase from oxygen) rather than preventing inhibition by hydrogen itself.


Reference:
Aguilar, O. M., Yates, M. G. & Postgate, J. R. The Beneficial Effect of Hydrogenase in Azotobacter chroococcum Under Nitrogen-Fixing, Carbon-Limiting Conditions in Continuous and Batch Cultures. J. Gen. Microbiol. 131, 3141–3145 (1985).

163 - Effect of Oxygen on Growth of Azotobacter chroococcum in Batch and Continuous Cultures

This is another chemostat study of Azotobacter chroococcum and the effects of oxygen on various physiological parameters.

They grew the bacteria on mannitol B medium (apparently Burk medium but with mannitol in place of sucrose) or B₆ (B, modified for chemostats by adding some trace elements and nitrilotriacetic acid (to keep everything in solution). They measured oxygen with an electrode and regulated the gases flowing through.

A nutrient was considered limiting if decreasing its feed decreased bacterial growth proportionally, except for nitrogen gas, whose limitation was diagnosed by exclusion (i.e. no other nutrient addition could increase growth).

They measured dry weights and mannitol consumed.

What They Saw
In initial batch cultures, inoculated A. chroococcum grew only those with low aeration (or low oxygen exposure) when fixing nitrogen, but given ammonium it could grow in up to 40% oxygen with high aeration.

Then they tried continuous culture with different oxygen levels, between about 1% and 60% of the gas flow (with the rest nitrogen), at D = 0.2 h^-1. They saw the highest yield of biomass remain fairly constant between 10 to almost 60% oxygen. Nitrogenase efficiency (fixed N per substrate consumption) was highest at lowest oxygen, dropped some up to 20%, and then dropped very low at 30% or higher.

Dalton and Postgate 1968, fig 1

Respiratory activity plateaued between 20 and 50%.

Then they tried limiting the carbon at 20% oxygen. This led to inhibitory oxygen levels quickly when oxygen was increased (by raising agitation from 680 to 1100rpm or raising oxygen from 20 to 50%). So carbon limitation led to oxygen hypersensitivity, when fixing nitrogen. The same was true of phosphate limitation.

What This Means
It supports the idea of respiratory protection (respiring carbon quickly and inefficiently to reduce oxygen to protect oxygen-sensitive nitrogenase); when carbon is unavailable, the cells are more sensitive to oxygen when fixing nitrogen. Also the efficiency increases as oxygen levels decrease.

Reference:
Dalton, H. & Postgate, J. R. Effect of Oxygen on Growth of Azotobacter chroococcum in Batch and Continuous Cultures. J. Gen. Microbiol. 54, 463–473 (1968).