015 - Characterization of genes involved in molybdenum transport in Azotobacter vinelandii

It is known that molybdenum (Mo) represses A. vinelandii's alternative nitrogenases, at least in the wild-type. What is not exactly known is the mechanism of this repression. Concentrations of Mo as low as 10 μM repress the alternatives, but some strains (such as CA6) produce the alternatives even with high concentrations of Mo. The question is, is this because Mo is not being transported into the cell, or because the protein that represses the alternatives is not functioning properly somehow?

So in the current study, they took a strain of A. vinelandii that can't make the primary nitrogenase but still represses the alternatives when Mo is present, and created mutants using a transposon, Tn5, then grew it on medium with Mo but without nitrogen, so that only those that had a mutation in a relevant gene could grow. And they found two Tn5-induced mutants, and also some that had mutated spontaneously to be able to grow in such conditions.

They named the two transposon mutants FL2 and FL4, focusing on them because the transposon insertion allowed them to locate and study the genes of interest. The mutants could grow pretty much just as fast with Mo present as the parent strain could grow with Mo absent (though these rates were all somewhat slow because they all needed to use the alternative nitrogenases, which are less efficient). Actually FL4 grew a bit faster than the parent.

They isolated and sequenced the section of the genome that the transposons had inserted themselves into, and it turned out that it was the mod operon that I've discussed before (012). FL2 had an insertion in modE, the regulatory gene, and FL4 had an insertion near the end of modB. So it seems like these genes are important for Mo-induced repression of alternative nitrogenases.

Citation: Luque, F., Mitchenall, L. A., Chapman, M., Christine, R. & Pau, R. N. Characterization of genes involved in molybdenum transport in Azotobacter vinelandii. Mol. Microbiol. 7, 447–459 (1993).

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).

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).

001 - Evidence for an alternative nitrogen fixation system in Azotobacter vinelandii

Azotobacter vinelandii is a well-studied microbe, discovered in 1903. It is most well-known for its nitrogen-fixing abilities (thus its name, "azoto" = nitrogen), the ability to convert nitrogen gas (N₂) into "fixed" nitrogen forms, such as ammonia (NH₃) and then into useful stuff like protein or nucleic acids. The enzyme that performs this reaction (called "nitrogenase") is almost always sensitive to/inactivated by oxygen, but A. vinelandii has ways of protecting its nitrogenase such that it can fix nitrogen even when oxygen is present; it is an obligate aerobe (i.e. it requires oxygen to grow). So that's the introduction.

In this particular study, the hypothesis was that the well-studied nitrogenase at the time, which contained a molybdenum (Mo) cofactor, was not the only nitrogenase that A. vinelandii possessed. That meant that when Mo was scarce or the enzyme was otherwise inactivated, the bacteria in some circumstances could still fix nitrogen (and, because fixed nitrogen is required for growth, could continue to proliferate) using its alternative enzyme.

In order to test this hypothesis, the researchers had a number of mutant strains of A. vinelandii (the wild-type strain being named CA), some of which couldn't fix nitrogen under some conditions, and others that had other phenotypes. These mutants were called CA1, CA2, etc.

The behavior the scientists were looking for in particular was tolerance to tungsten (W). Tungsten is similar to molybdenum in its atomic structure, just a bit bigger, so it sorta imitates Mo enough that when it's present in large enough concentrations, A. vinelandii incorporates W into its nitrogenase instead of Mo, but this form of the enzyme is unable to fix nitrogen. So the wild-type strain, CA, is unable to fix nitrogen or grow when too much W is present. However, some of the mutant strains could grow.

Another bit of evidence was the observation that all of the strains, even CA, could grow and fix nitrogen when neither tungsten nor molybdenum was present. The scientists used a technique called 2-D (two-dimensional) gels to observe changes in concentrations of all proteins in the cells individually. This process involves separating the proteins based on their polarity first in one direction, then separating them perpendicular to that direction based on their size, so this should allow them to see whether a protein is present in one condition but not in another. Indeed, they observed some proteins that were present only when the cells were fixing nitrogen in the presence of tungsten or absence of molybdenum! These seemed to be the components of the alternative nitrogenase.

So the model the authors propose for regulation of this alternative nitrogenase in the wild-type is, when tungsten or Mo is present, it's turned off (probably because it is less efficient than the Mo-containing nitrogenase, so preference is given to the latter when Mo is present), but when those metals are absent, it's turned on. The mutants can fix nitrogen in the presence of tungsten because somehow the repression of the alternative nitrogenase is not active in them.

So that's interesting. What was not known was the nature of this alternative enzyme, what metal it might contain instead of Mo, how the regulatory mechanisms functioned exactly, or whether the alternative system was completely independent genetically or just a modification of the Mo-containing one. But at least they had good evidence that the alternative exists.

Citation: Bishop, P. E., Jarlenski, D. M. & Hetherington, D. R. Evidence for an alternative nitrogen fixation system in Azotobacter vinelandii. Proc. Natl. Acad. Sci. 77, 7342–7346 (1980).