Wednesday, September 24, 2014

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

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

What They Wanted to Know

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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