Wednesday, October 2, 2013

012 - Mutational analysis of genes of the mod locus involved in molybdenum transport, homeostasis, and processing in Azotobacter vinelandii

So Azotobacter vinelandii CA6 has impaired molybdenum (Mo) uptake (003). This paper studies the Mo transport system of A. vinelandii, encoded by the mod genes, modEABC. It seems like ModA is a protein that binds Mo outside the cell, ModB brings them inside across the membrane, and ModC powers this process. ModE's role is unknown at this point.

The scientists generated a number of mutant strains of A. vinelandii, knocking out a given gene while also fusing it with lacZ to quantify its expression. They also discovered another mod gene, modG, adjacent to the others but in the opposite strand direction. It looks similar to half of modE, so the protein may have a similar function.

Another enzyme in A. vinelandii that requires Mo is nitrate reductase; the authors measured activity of this enzyme as a proxy for Mo transport activity. The wild-type's activity rises quickly as concentration of Mo in the environment increases, levels off, then rises quickly again at higher concentrations (supporting the idea of two different Mo transport systems). With mutants of modA, modB, and modC, the pattern was always the same: no activity until the concentration reached a certain point (the same point when the wild-type's activity started rising quickly the second time).

Other results were more puzzling: when modE was knocked out in a way that didn't inhibit expression of the other mod genes, it seemed to have good transport activity at lower Mo concentrations but not at higher; and the opposite when its knockout inhibited the other genes.
Strain CA11.6, which genetically combined the lack of Mo nitrogenase in CA11 (002) with the tungsten-tolerant phenotype of CA6 (003), showed good Mo uptake at low concentrations but not at higher. When the modB gene was specifically knocked out of CA11.6, there was hardly any uptake activity at any concentration. Considering my own research, it's difficult to say what is going on genetically in these cases.

When modG was targeted for knockout, it looked pretty much the same as wild-type activity, except when both modG and modE were deleted, in which case it showed activity at much lower concentrations even than wild-type. Explain that, science!

They also directly measured uptake of a radioactive isotope of molybdenum (99Mo) in the wild-type and modA or modB mutants. The rate of transport in the wild-type and modB mutant were pretty much constant, though the latter was slower than the former. In the modA mutant though, there was very little transport. They tried adding nonradioactive compounds (Mo, vanadium, sulfate, or tungsten) to compete with transport of radioactive Mo, and found that only Mo and tungsten inhibited radioactive Mo transport by competition. Evidence that the mod genes transport tungsten in addition to Mo.

Lastly, the scientists tested the nitrogen-fixing abilities of mod mutants. With Mo present, nitrogen-fixing growth of modE and G mutants was similar to wild-type. When it was absent, modG knockout grew more slowly and modE more quickly. A double mutant didn't grow hardly at all in either condition, in normal aerobic conditions, but with lower levels of oxygen it grew as well as the wild-type (both very slowly). It could also grow using vanadium (V) and the V-containing alternative nitrogenase.

The conclusions, I suppose, are that modABC are all important for Mo transport, especially at low concentrations. modE's role is not exactly clear, but it may regulate which Mo transporter system is working at a given time (possibly by repressing one and activating the other at low concentrations, and vice versa at high). modG's role is even less clear.

Citation: Mouncey, N. J., Mitchenall, L. A. & Pau, R. N. Mutational analysis of genes of the mod locus involved in molybdenum transport, homeostasis, and processing in Azotobacter vinelandii. J. Bacteriol. 177, 5294–5302 (1995).

No comments:

Post a Comment