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Would the host want anyone to starve?

This week’s paper (Ratcliff, et al., in press) is from my lab.

The relationship between a legume plant and the rhizobium bacteria living in its root nodules is usually beneficial to both. Rhizobia convert nitrogen from the atmosphere into a form their plant host can use to make proteins. Rhizobia benefit from infecting plants because they reproduce more inside nodules than they would in the soil. They can also acquire certain resources in nodules. In particular, they can accumulate large amounts of energy-rich PHB – up to 50% of their own weight!

But there can also be conflicts of interest between rhizobia and their host plants. Resources used to make PHB could have been used, instead, to acquire more nitrogen for the plant. Therefore, mutant rhizobia that don’t make PHB provide their hosts with more nitrogen (Cevallos, et al., 1996). So why do most rhizobia make PHB? Our hypothesis has been that the rhizobia themselves benefit from having more PHB. This contrasts with an earlier hypothesis that rhizobia store PHB so that they can use the energy for the benefit of the plant. Consistent with our hypothesis, mutants defective in PHB metabolism reproduce less (Cai, et al., 2000). However, mutations can have complex interacting effects, making it hard to be sure that differences in PHB were the only cause of differences in reproduction. So Will Ratcliff decided to compare rhizobia that were genetically identical but had different amounts of PHB per cell.
centrifuge.jpg

He started by supplying rhizobia with a mix of resources that created favorable conditions for reproduction (by growing, then dividing) and also accumulating PHB. He started with a single cell, so all the resulting rhizobia were genetically identical, mostly. (A few mutations probably occurred, even in a few days, but conditions were similar to those under which the rhizobia had long grown in the lab, so any mutants would not be likely to increase by natural selection.)

So the rhizobia were almost all genetically identical, but did they vary in PHB per cell? To find out, Will added Nile red, which binds to PHB and fluoresces. Then he ran his cells through a flow cytometer. As each cell passed through a laser beam, its Nile-red fluorescence was measured, giving an estimate of PHB per cell. Analyzing many thousands of cells – this takes several seconds – showed that there was indeed a great deal of variation in PHB per cell.

The next stage was to separate the rhizobium cells into low-, medium-, and high-PHB groups. Will did this using a centrifuge, which spins to create a strong gravity-like field inside a tube. The tubes contained the rhizobia, plus a liquid with particles that separate in the field so that the liquid becomes denser with depth. Under these conditions, the rhizobia float up or down until they reach a depth where their density matches that of the liquid. (Similarly, someone with less body fat could float at the same depth in salt water as someone with more body fat floats in fresh water.)

Next, he starved each of the three rhizobium groups separately, for 160 days. We predicted that the high-PHB cells would survive longer. This turned out to be true. Not only that, but over the first month they actually reproduced. There was almost no food in their culture liquid – low-PHB cells did not reproduce – so we assume the energy for reproduction came from the PHB they started with.
starve.jpg
There’s more, both in the paper and in terms of interesting results since then, but these results were enough to answer our main question. As we predicted, rhizobium cells can benefit from having more PHB. Therefore, the previously published tradeoff between hoarding more PHB versus supplying the plant with more nitrogen does indeed create a conflict of interest between plant and rhizobia. They also have shared interest, of course. Plants with more nitrogen can grow bigger and support more rhizobia. But what if this benefit is shared with competing strains of rhizobia infecting the same individual plant? We have shown previously (West, et al., 2002) that this could select for rhizobial cheaters, who invest in their own current or future reproduction (by hoarding more PHB, say) rather than in acquiring nitrogen for the plant. Cheaters would spread, we predicted, unless plants impose sanctions that decrease their survival or reproduction. So Toby Kiers, previously in my lab, checked for sanctions and found them (Kiers, et al., 2003). There are still plenty of unanswered questions, both for rhizobia and for other root-associated beneficial microbes. We will discuss some of these questions in a review coming out next year (Kiers and Denison, in press) and try to answer some of them in our ongoing research.

References

Cai, G.Q., Driscoll, B.T., Charles, T.C., 2000. Requirement for the Enzymes Acetoacetyl Coenzyme A Synthetase and Poly-3-Hydroxybutyrate (PHB) Synthase for Growth of Sinorhizobium meliloti on PHB Cycle Intermediates. Journal of Bacteriology 182, 2113-2118.

Cevallos, M.A., Encarnación, S., Leija, A., Mora, Y., Mora, J., 1996. Genetic and physiological characterization of a Rhizobium etli mutant strain unable to synthesize poly-ß-hydroxybutyrate. Journal of Bacteriology 178, 1646-1654.

Kiers, E.T., Denison, R.F., in press. Host sanctions, cooperation, and the stability of plant-rhizosphere mutualisms. Annual Review of Ecology, Evolution, and Systematics .

Kiers, E.T., Rousseau, R.A., West, S.A., Denison, R.F., 2003. Host sanctions and the legume-rhizobium mutualism. Nature 425, 78-81.

Ratcliff, W.C., Kadam, S.V., Denison, R.F., in press. Polyhydroxybutyrate supports survival and reproduction in starving rhizobia. FEMS Microbiology Ecology .

West, S.A., Kiers, E.T., Simms, E.L., Denison, R.F., 2002. Sanctions and mutualism stability: why do rhizobia fix nitrogen? Proceedings of the Royal Society of London B, Biological Sciences 269, 685-694.

Comments

Kudos! Very cool.

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