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).