Tuesday, October 1, 2013

003 - Phenotypic characterization of a tungsten-tolerant mutant of Azotobacter vinelandii

One tungsten-tolerant strain from 001 in particular caught the attention of the researchers. Azotobacter vinelandii strain CA6 just happened to mutate spontaneously to be able to fix nitrogen in the presence of tungsten (W).

Later research showed that A. vinelandii possesses three nitrogenase system, actually: the primary, molybdenum-containing one, and two alternatives: one with vanadium instead of molybdenum, and a third with iron. The third is least efficient, but iron is most likely to be available, so it is the most versatile.

But CA6 was still interesting, because somehow it was able to overcome the repressive effect that molybdenum (Mo) and W have on the alternative nitrogenases. So in order to study it, among other things, the scientists made a number of recombinant strains of A. vinelandii, to test the functions of different nitrogenase genes.

They tried growing wild-type strain CA and mutant strain CA6 with different concentrations of W. All tested concentrations of W inhibited CA, and above 1 μM (0.184mg W per liter) all concentrations inhibited it the same amount. With CA6, however, no amount of W seemed to affect its growth. However, when Mo was present (and no W), CA grew about twice as fast as CA6.

To figure out why, they deleted the genes for the alternative nitrogenases to create strain CA6.1.71 (sounds like software versions, heh). Obviously this couldn't fix nitrogen or grow without Mo present for its primary nitrogenase. But when Mo was present, it could grow just as fast as the wild-type, showing that the difference in growth rate is probably because CA6 wastes its energy producing less efficient nitrogenases instead of focusing on the efficient primary one.

They also made some genetic fusions of nitrogenase genes with a gene called lacZ, which codes for an enzyme that breaks the bond between the two sugar molecules of lactose, resulting in one molecule of glucose and one galactose. The purpose of this is that this enzyme also breaks the bond in a molecule called o-nitrophenyl-β-galactoside (ONPG), which releases a molecule of galactose but also o-nitrophenyl, which is a bright yellow color. So when you add ONPG to liquid containing the enzyme, you can tell how much enzyme is present by how yellow the liquid becomes. And by fusing lacZ to other genes, you can get an idea of how much those other genes are expressed in the cell.

So this way, they found that, in the wild-type strain CA, Mo-nitrogenase genes are expressed when Mo or W are present (not surprisingly), and alternative nitrogenase genes are only expressed when Mo or W is absent. In CA6, the iron-nitrogenase is produced with or without Mo or W; only vanadium represses it. And the vanadium nitrogenase in both is expressed only when vanadium is present. They confirmed these results with 2-D gels (described in 001).
(Side note: vnfH, vanadium dinitrogenase reductase, is expressed in CA whenever Mo or W is absent, whether or not V is present; in CA6, it is always expressed regardless of the metals in question.)

One possible reason for the difference between CA and CA6 is the latter's ability to take Mo into its cells; if its uptake of Mo is impaired, that could result in the observed phenotype. So the scientists tested that. They found that, not only was CA6's Mo uptake slower than CA's, but it ceased to take up more above a certain concentration, whereas for CA, the more that was available, the more CA took up. It seemed like there were two separate Mo-uptake systems, one that worked better in low concentrations and one in higher, and CA6 lacked the latter. However, there was still enough Mo present in CA6 that it should have repressed the alternative nitrogenases, so this explanation didn't quite work; there must be something else. These observations just add to the mystery of A. vinelandii CA6.

Citation: Premakumar, R., Jacobitz, S., Ricke, S. C. & Bishop, P. E. Phenotypic characterization of a tungsten-tolerant mutant of Azotobacter vinelandii. J. Bacteriol. 178, 691–696 (1996).

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