Monday, October 7, 2013

014 - Molybdenum accumulation and storage in Klebsiella pneumoniae and Azotobacter vinelandii

Since the primary nitrogenase of Azotobacter vinelandii (and other nitrogen-fixing organisms) is so dependent on molybdenum (Mo), such that they turn off expression of the nitrogenase in the absence of Mo, I thought it worthwhile to read about A. vinelandii's system for storing Mo. Klebsiella pneumoniae, another well-studied nitrogen-fixing soil organism (given its intimidating name because it is an opportunistic pathogen, I think), is used as a comparison.

Previous research had shown that A. vinelandii takes up and stores Mo continuously when it is present, whether or not the bacteria need to fix nitrogen. This is a different strategy from other diazotrophs (nitrogen-fixing organisms). In this study, bacteria were starved of Mo but given fixed nitrogen, and then put into media with varying amounts of Mo with or without fixed nitrogen. When fixed nitrogen was absent, nitrogenase activity maxed out at low concentrations of Mo, but the more Mo present, the more accumulated in the cells, much more than needed to make nitrogenase. When fixed nitrogen was present, the cells showed the same pattern of increasing Mo accumulation, though the levels seemed lower.

In contrast, K. pneumoniae seemed not to accumulate any Mo when fixed nitrogen was available, and while it did store Mo when fixing nitrogen, it was more than 10x less than the levels in A. vinelandii in any condition.

The researchers also measured accumulation of Mo over time. A. vinelandii took up 100% of the Mo provided within 1 hour (it was only a low concentration though), but K. pneumoniae didn't even start uptake until 2 hours in, and then took 2 hours to reach only 25% accumulation. Clearly the two organisms have different strategies; Klebsiella's uptake pattern correlated with its nitrogen-fixation pattern.

They tested the effect of oxygen on Mo uptake; oxygen is toxic to nitrogenase, such that K. pneumoniae doesn't fix nitrogen in its presence, and A. vinelandii takes steps to protect its nitrogenase. But A. vinelandii is an obligate aerobe, so it must still fix nitrogen and accumulate Mo in the presence of oxygen. Klebsiella, on the other hand, shuts off its nitrogenase and Mo uptake both when oxygen is present.

Chloramphenicol, an antibiotic that inhibits protein synthesis in bacteria, inhibited K. pneumoniae's Mo uptake too, but surprisingly not A. vinelandii's; the latter actually had more Mo per cell with chloramphenicol than without, because it was unable to multiply, so there was the same amount of Mo divided among fewer cells.

So there must be some protein in A. vinelandii that is able to store lots of molybdenum. Indeed, when transferred to Mo-free medium after accumulating Mo, the bacteria could fix nitrogen at the same rate as when growing in Mo-containing medium, at least for a while. Trying to purify the Mo-containing proteins from each organism, the scientists found that pretty much all the Mo in Klebsiella was contained in the nitrogenase, whereas (depending on the amount available) that only accounted for a fraction of Mo in Azotobacter. There is another protein that is able to store large amounts of the metal, about 14-15 atoms per molecule of storage protein.

As a bonus, they tested A. vinelandii's ability to store tungsten, and found that it was accumulated in the same way as Mo. It's a sneaky element, apparently.

Citation: Pienkos, P. T. & Brill, W. J. Molybdenum accumulation and storage in Klebsiella pneumoniae and Azotobacter vinelandii. J. Bacteriol. 145, 743–751 (1981).

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