111 - Localization and activities of nitrogenase, glutamine synthetase and glutamate synthase in Azotobacter vinelandii grown in oxygen-controlled continuous culture

Previous research showed that A. vinelandii forms intracellular membrane structures in certain oxygen conditions (098). This could function to protect the sensitive nitrogenase from oxygen, except that one study seemed to disprove any such enzyme localization; this study was inadequate though.

So Röckel, Oelze and colleagues decided to test this hypothesis better, using oxygen-controlled continuous culture. They also tested localization of glutamine and glutamate synthases (which incorporate nitrogenase-fixed nitrogen into biomass).

What They Saw

First, the more oxygen saturation, the less nitrogenase activity (and protein content in general) they observed. In cell-free extracts, nitrogenase was only found in the soluble fraction (and thus wasn't membrane-bound). Glutamine synthase also seems all soluble.

With glutamate synthase, on the other hand, as oxygen increased, membrane-bound activity increased while soluble activity decreased.

What This Means

It's not impossible that the soluble-seeming enzymes may have a weak attachment to membranes that couldn't be resolved in this study, but it didn't seem like the membranes affected them much. The increase in membrane-bound glutamate synthase might be a form of stabilization with increasing oxygen.

Reference:

Röckel, D., Hernando, J. J., Vakalopoulou, E., Post, E. & Oelze, J. Localization and activities of nitrogenase, glutamine synthetase and glutamate synthase in Azotobacter vinelandii grown in oxygen-controlled continuous culture. Archives of Microbiology 136, 74–78 (1983).

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

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

What They Wanted to Know

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

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

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

What They Did

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

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

What They Observed

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

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

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

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

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

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

What This Means

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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