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Sleep as a survival strategy

This week's paper, Bacterial persistence and bet hedging in Sinorhizobium meliloti, was just published in Communicative and Integrative Biology. It's a brief but important follow-up to a paper in Current Biology, which I've already discussed.

Bacterial persisters are a serious medical problem. An infection that appears to have been cured by antibiotics sometimes "springs back to life." Evolutionary biologists have focused on cases where the renewed infection is caused by an antibiotic-resistant mutant, a classic example of evolution by human-imposed selection. Sometimes, however, the resurgent bacteria are still susceptible to the original antibiotic, yet bounce back after one or more treatments. What gives?

Many antibiotics only kill bacteria that are actively growing. So, if a few cells go dormant, these persisters may survive until the antibiotic breaks down, even if they aren't otherwise resistant.

Will Ratcliff recently reported that Sinorhizobium meliloti bacteria, best known as the nitrogen-fixing, root-nodule symbiont of alfalfa, can also make dormant cells. When S. meliloti cells divide, under starvation conditions, the elder daughter inherits most of the accumulated wealth (energy-rich polyhydroxybutyrate or PHB) and the younger daughter goes off to seek her fortune. You can see this unequal allocation of PHB in the Nile-red-stained image of a dividing cell, below right.PHB.jpg
This apparent bet-hedging strategy is much more organized and more common than the random, one-in-a-thousand process that seems typical of human pathogens. A rhizobial population that starts with the usual normal distribution of PHB (above left) divides, initially, into one with roughly equal numbers of persisters and growers (above center).

Our original paper showed that about 70% of the high-PHB S. meliloti persisters are still alive after 528 days without food. So the well-known ability of rhizobia to survive in soil for months or years between legume hosts may not depend on their ability to out-compete other soil bacteria for limited food supplies.

But how relevant is this work with rhizobia, which benefit their legume hosts by providing them with nitrogen, to the antibiotic-resistant bacterial persisters that cause disease? In this new paper, Will Ratcliff showed that...

AntibioticPersisters.jpg
...the rhizobial persisters are resistant to the antibiotic, ampicillin. He generated mixtures of high-PHB persisters and low-PHB growers in two different ways, either by growing them differently or using centrifugation to separate high- and low-PHB cells (all of the same genotype). Either way, ampicillin killed more cells in mixtures that were mostly low-PHB growers than in mixtures that were mostly high-PHB persisters, as shown above.

Will also followed up on the apparently lower protein synthesis in the persisters, as seen in the earlier figure as less green-fluorescent protein (GFP) fluorescence in the high-PHB persister daughter cell. He showed that the eldest daughter ("old-pole") cells had consistently less GFP fluorescence, consistent with the lower protein synthesis expected in dormant or semidormant cells.

We are continuing work on this system, exploring the conditions under which persisters may, by consuming fewer resources, free resources for their clonemates nearby. S. meliloti is directly important as the main nitrogen source for alfalfa, which occupies more land than most other crops. But we also hope that insights from this work may lead to more-effective control of infections caused by other bacterial persisters.

This work is being supported by National Science Foundation grant NSF/DEB-0918897.

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