034 - NifB and NifEN protein levels are regulated by ClpX2 under nitrogen fixation conditions in Azotobacter vinelandii

Biological nitrogen fixation (turning N₂ gas into usable forms for protein and such) is an energetically expensive process, requiring large amounts of resources the cell could devote to other purposes; however, if that's the only available source of fixed nitrogen, it's worthwhile, because the alternative is paralysis, essentially.

However, being such an expensive process also means that cells will try to regulate its use very tightly, making sure only to use it when it is absolutely necessary. This is different for different organisms; some regulate the same way all the time, some photosynthetic microbes turn everything off or on depending on available light, etc.

Assembly of the nitrogenase enzyme is a very complex process that requires complex regulation as well. This study looks at the regulation of the molybdenum-containing nitrogenase, the primary one, especially the nifB and nifEN genes. NifB is a protein that seems to help synthesize the Mo-containing cofactor essential for the nitrogenase; it also seems to work for the vanadium- and iron-containing cofactors of the alternative versions. The cofactor that NifB makes transfers to the NifEN complex, which adds the Mo for the nitrogenase. This NifB process seems to be a key point for regulating the entire process.

The nitrogenases each have an activator that helps regulate them: nifA, vnfA, and anfA, and these all influence nifB production. But there are probably other genes involved. For example, Azotobacter vinelandii has a gene called clpX2 in between two other nitrogenase-related genes; clpX encodes a common protease that breaks down proteins that are deformed or no longer useful, but clpX2, while seemingly related, is different. It's sometimes found in nif gene clusters in other species, and knocking it out doesn't disrupt nitrogen fixation; rather, it may increase it.

This study looks more specifically at ClpX2's role in regulating nitrogen fixation in A. vinelandii. To do this, they made new strains with modified genes involved in this process:

• UW233: NifB only works when chemical called IPTG is present; can't fix nitrogen otherwise
• UW238: nifB is IPTG-inducible; nifENX is deleted
• UW295: nifB is IPTG-inducible; nifA is deleted
• UW318: clpX2 fused to lacZ; produces more yellow color from ONPG when clpX2 expressed
• UW319: clpX2 fused to lacZ and nifA is deleted
• UW322: lacks clpX2 gene

UW233 allowed them to control when NifB was produced. They found that when cells were growing with ammonium (a source of fixed nitrogen), not fixing nitrogen, they accumulated higher levels of NifB. This is probably because the cells consume NifB when fixing nitrogen; after removing IPTG from the cells' medium, they stopped producing NifB, but those growing in ammonium still had fairly high levels of NifB even after a few hours, while those fixing nitrogen lost most of theirs.

In UW238, NifB accumulates to higher levels regardless of whether or not ammonium is present, so the NifENX proteins seem to be involved in NifB's regulation. In UW295 when nifA is missing and all the major nif genes are silent, NifB disappears more quickly in both conditions; it seems that whatever is degrading NifB isn't activated by NifA.

Using UW318, the authors discovered that clpX2 was expressed more when fixed nitrogen was absent and the cells were fixing nitrogen, so ammonium seems to downregulate it. UW319 revealed that NifA was not necessary for clpX2 expression either; in fact, expression was higher when nifA was deleted. Why is unclear. Semi-quantitative RT-PCR confirmed these results.

So then the question is, how is ClpX2 involved in regulation of NifB and NifEN? So of course they deleted the clpX2 gene to get UW322. The main difference in this strain was that levels of NifB and NifEN were higher than usual, much higher; it seems that ClpX2 plays a big role in their turnover.

However, deletion of clpX2 comes with a price. When fixed nitrogen was present, the cells grew fine, but when fixing nitrogen they slowed down a little, and the initial setting up of nitrogen fixing was slower too.

One interesting specific effect requires a bit of explanation: the Mo nitrogenase has two main components, which are the main part that contains the Mo cofactor and does the actual reaction with nitrogen, called the dinitrogenase; and the dinitrogenase reductase, which kinda recharges the dinitrogenase and prepares it for the next round of reactions. Having these present in different ratios can affect the overall rate of the process.

But what the authors found in UW322, with clpX2 missing, was that there was much more of the Mo-cofactor-containing dinitrogenase than there was normally, while levels of dinitrogenase reductase remained the same. So maybe ClpX2 holds in check the production of dinitrogenase somehow, so the ratios of the two components are optimized.

The authors hypothesized that ClpX2 might provide an advantage when iron is scarce, because the proteins it regulates are both involved in directing a lot of the cell's iron into nitrogenase cofactors, so they tested in low-iron conditions. UW322 seemed to have a slightly greater disadvantage when fixing nitrogen with limited iron than when fixing nitrogen with sufficient iron, but I'm not sure it looks that significant. Could be.

So here's the figure they made to explain their results, showing the regulation pathways:

Figure 10, Martinez-Noël et al. 2011

It doesn't really specify how ClpX2 might repress those proteins (i.e. by their degradation probably); nor is it clear how ClpX2 itself is regulated. But it is interesting.

Citation: Martínez-Noël, G., Curatti, L., Hernandez, J. A. & Rubio, L. M. NifB and NifEN protein levels are regulated by ClpX2 under nitrogen fixation conditions in Azotobacter vinelandii. Molecular Microbiology 79, 1182–1193 (2011).