Wednesday, January 7, 2015

021 - Nitrogen fixation system of tungsten-resistant mutants of Azotobacter vinelandii

People had known at this point that tungsten was poisonous to nitrogenase in Azotobacter vinelandii. It seemed to replace molybdenum in the primary nitrogenase, rendering it non-functional for nitrogen fixation. But then Paul Bishop and colleagues discovered some strains that could grow in the presence of tungsten, using alternative nitrogenases (001). So Riddle and colleagues wanted to characterize such strains and their weird nitrogenases.

What They Did
The scientists first isolated some tungsten-tolerant mutants of their own. This is pretty simple; just grow cells (in this case, A. vinelandii ATCC 12837) in the presence of tungsten, and some tolerant mutants are likely to grow. They chose one and called it WD2 (for Tungsten-Derived 2). For some reason they grew their bacteria at 35ºC, which seems odd because usually Azotobacter is grown at 30ºC, but whatever.

Then they confirmed WD2's ability to grow and fix nitrogen in the presence of tungsten, compared to the parent, did acetylene reduction assays to measure nitrogenase activity, and ran 2-D gels to compare proteomics of the cells in different conditions. Finally they used electron spin resonance (ESR) and other tests to see the chemical characteristics of the cells' enzymes.

What They Observed
They compared the growth of the wild-type and WD2 in broth with or without tungsten. WD2's growth in tungsten was in between the growth of the wild-type in tungsten-free broth and the wild-type in broth with tungsten. Wild-type with tungsten didn't grow hardly at all, as expected. So tungsten inhibits WD2's growth a little bit, compared to the wild-type. To be fair, the amount of tungsten they used was 1000 times more than the minimum needed to inhibit nitrogen fixation in the wild-type.

Then they grew WD2 with various combinations of Mo and W. The growth curves were all pretty similar, not clearly significantly different, but the one with Mo and no W grew best and the one with W and no Mo was worst. Others were in between as expected (more Mo = better, more W = worse), so it seems like a good trend.

They measured acetylene reduction (as a proxy for nitrogen in nitrogenase activity assays) with the wild-type and WD2 (with tungsten), with various concentrations of acetylene in the atmosphere. In each case, activity (as measured by ethylene produced) was highest early in the exponential growth phase. It was not clear (to my eyes) that different amounts of acetylene or the different strains had significant effects on activity. It seemed like perhaps the higher concentrations permitted more activity later in the growth phase. WD2 in W broth showed only 17% the activity of the wild-type in regular broth.

2-D gels showed different profiles for WD2 in regular Burk broth vs. broth with tungsten, though it's always tough to interpret gel images on their own (as two of the same rarely look alike). It did seem like WD2 and the wild-type with ammonium showed similar profiles, and WD2 with tungsten or even with Mo was different, indicating different proteins for tungsten-tolerant nitrogen fixation. ESR spectra of the different proteins were different too.

With heat-extracted proteins from Mo-grown wild-type or W-grown WD2 cells, the wild-type had about 1 nmol Mo per mg protein, whereas WD2 had negligible, but had 18 nmol W. Acetylene reduction activity was much different too: wild-type had more than 10x the activity of WD2, at least in terms of ethylene produced. Spectra were very different too, especially in the band associated with the Mo-iron cofactor.

What This Means
Overall, WD2's results seem to agree with those of others (001), that mutants of wild-type A. vinelandii can fix nitrogen and grow in the presence of tungsten. And the 2-D gels apparently look similar between these two groups of researchers.

Some of the results (growth curves with different Mo:W ratios) seem to indicate that WD2 was using Mo even when tungsten was present. Others (2-D gels) seemed to indicate otherwise.

Based on the acetylene reduction assay, cells might have lower affinity for acetylene later in their growth phase.

Not much new, but it corroborates some data.

1. Riddle, G. D., Simonson, J. G., Hales, B. J. & Braymer, H. D. Nitrogen fixation system of tungsten-resistant mutants of Azotobacter vinelandii. J. Bacteriol. 152, 72–80 (1982).

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