025 - Siderophores Produced by Nitrogen-Fixing Azotobacter vinelandii OP in Iron-Limited Continuous Culture

One thing that's noteworthy about A. vinelandii is that when growing on agar plates or broth tubes of the medium commonly used for it (Burk's), after a certain time it produces this neon green pigment that diffuses through the medium. It's neon green under white light, but under ultraviolet light it fluoresces light blue!

This colorful feature is due to a molecule called a siderophore, which the bacteria secrete to scavenge (i.e. chelate) insoluble oxidized iron in the medium and bring it into the cell. Iron is an important element for A. vinelandii, especially for fixing nitrogen; all of its nitrogenases require it.

In the present study, the researchers wanted to find out how A. vinelandii responds to insufficient iron in its environment, and the function of proteins and compounds it produces. They used continuous culture to do this, analyzing steady states at different concentrations of iron.

The scientists grew A. vinelandii strain OP (aka CA) not with Burk's but with a medium called B₆, which also works I guess. Obviously this didn't always include the same amount of iron that it normally contained. They actually cleaned their medium vessels and reactor with EDTA (a chelator that binds tightly to metals) to get rid of all traces of iron.

So they grew OP at four different concentrations of iron, and at steady state for each concentration, measured the concentration of cells (dry weight), quantities of different potential chemical siderophores (extraction and separation by electrophoresis), and quantity of yellow-green fluorescent protein (fluorescence measurement). They also measured the total amount of chelated (bound) metals from all the siderophores.

What they saw was not surprising: at higher levels of iron, there was a higher concentration of cells in the culture and a lower amount of chelation going on. This makes sense because the more iron there is, the less need there is for the bacteria to produce special chelators to scavenge it. The numbers they got were very consistent, differing only about 5% even between different chemostat runs.

Regarding the levels of specific siderophores, one was clearly the predominant one compared to the others. This one went down dramatically as iron increased, dropping all the way to zero when iron was sufficient; the others dropped also but still had low levels even in iron-sufficient conditions.

A few other things to note is that, according to the authors, the lack of fixed nitrogen in the medium prevented contamination of the chemostat, and since the reactor was made of Teflon, no bacterial growth built up on the walls (which presumably would happen on a different material).

So this study shows pretty well which siderophore is important for iron scavenging in A. vinelandii, and that iron deficiency (in nitrogen-fixing conditions at least) impairs its growth.

Citation: Fekete, F. A., Spence, J. T. & Emery, T. Siderophores Produced by Nitrogen-Fixing Azotobacter vinelandii OP in Iron-Limited Continuous Culture. Appl. Environ. Microbiol. 46, 1297–1300 (1983).