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).