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).

207 - Transformation of Azotobacter vinelandii with plasmid DNA

One cool thing about Azotobacter vinelandii is that it is naturally competent—that is, in certain conditions it takes up DNA from its environment and sometimes incorporates it into its genome. This is a useful characteristic for a species to have when studied in the lab, because it means a researcher can modify its genes and such to see what they do.

But besides taking up straight pieces of DNA and incorporating them (recombination), it'd also be useful if A. vinelandii could take up and maintain plasmids, which are small circular pieces of DNA, usually with only a few genes on them (for example, a gene of interest and an antibiotic resistance gene as a selectable marker). Plasmids are useful for studying overexpression or complementation of genes, for example, when it's not necessary to incorporate anything into the genome.

So Glick, Brooks, and Pasternak attempted to transform A. vinelandii with several broad-host-range plasmids:

• pRK2501 (IncP-1 group, tetracycline and kanamycin resistance) 
• RSF1010 (IncQ group, sulfonamide and streptomycin resistance)
• pGSS15 (IncQ group, tetracycline and ampicillin resistance)

They selected for transformants using kanamycin, streptomycin, and tetracycline, respectively.

To transform A. vinelandii, it's necessary to use Transformation (TF) medium:

• 1.9718 g/L MgSO₄
• 0.0136 g/L CaSO₄
• 1.1 g/L ammonium acetate
• 10 g/L glucose
• 0.25 g/L KH₂PO₄
• 0.55 g/L K₂HPO₄
• For solid medium, 18 g/L agar

This low-iron, nitrogen-containing medium, developed by Page and von Tigerstrom, induces competence in A. vinelandii.

So Glick et al. picked a colony into TF medium, grew at 30ºC to an optical density (620nm) of less than 0.2, then transferred to fresh TF broth and grew some more. They tested transformation at a variety of optical densities at this point to see which is best, standardizing the density of cells transformed to 1.6 x 10⁸ cells per mL with TF broth.

50 µL of cells mixed with 300 µL fresh TF and 50 µL DNA (~22 µg/mL) sat at 30ºC for 30 minutes. These were spun down and resuspended in 400 µL fresh TF and incubated for another hour.

Then the cells were plated onto regular A. vinelandii agar plates, with or without antibiotics, and grown for 3 days at 30ºC. Plates without antibiotics revealed numbers of viable cells after transformation, and plates with antibiotics (compared to those without) indicated frequency of transformation.

From the results, it seemed like cells grown up to optical densities between 0 and 1 (which took 2-24 hours) could be transformed at very similar efficiencies; maybe a slight negative slope, but hardly noticeable. After the first 5 hours of growth, the culture should take on a yellow-green color as it becomes iron-limited.

Interestingly, transformed colonies on plates without antibiotics could be distinguished from non-transformed colonies; the transformed ones grew a lot bigger and more gooey.

The authors also found that, not surprisingly, the more DNA added to the transformation mix (from 0.1 up to 51 µg), the higher the frequency of transformation. At 51, 44% of the viable cells were transformed, which is not bad.

Also useful to note is that even without antibiotic pressure, transformed cells kept their plasmid around for at least 10 generations (not sure if that means 10 cell divisions or 10 transfers from one culture to another); and that the plasmid remained separate from the genome, rather than integrating or recombining or anything.

So this is useful for those who want to introduce genes into A. vinelandii and do some genetic modification; it's not required to integrate anything into the genome to express new proteins.

Citation: Glick, B. R., Brooks, H. E. & Pasternak, J. J. Transformation of Azotobacter vinelandii with plasmid DNA. J. Bacteriol. 162, 276–279 (1985).