The Spooky Science of How Undead Spores Reanimate

These cells—and the way they reawaken—can tell biologists quite a lot about life, death, and the gray zone in between.

Here’s a spooky conundrum: Is a spore alive or dead?

Gürol Süel, a biologist at the University of California, San Diego, wouldn’t blame you if you voted for dead: “There’s nothing to detect: no heartbeat, no gene expression. There’s nothing going on,” he says.

But a spore might actually just be dormant—in a deep state of suspended animation meant to outlast inhospitable conditions that can persist for millions of years, until the day the spore “wakes up,” zombie-like, ready to grow. For years, the questions of how spores know when to reanimate, and how they actually do it, have been open ones. A new paper in Science by Süel’s group has helped fill in those blanks—and the answer could have ramifications for everything from the search for life on other planets to methods of fighting dangerous spores, such as those that cause foodborne illness.

Spores are typically single cells with tightly packed innards that can create new organisms. While many plants produce them to spread their seeds, bacteria can also form spores during periods of extreme temperatures, dryness, or nutrient deficiency. The spore cell then essentially hibernates its way through tough times.

Süel’s group was intrigued by the concept of a “mostly dead” cell reviving when the surrounding environment becomes more conducive to survival. “It was clear how spores come back to life if you dump a bunch of good stuff on them,” like large quantities of nutrients, says Süel. Likewise, when the environment is extremely hostile (for example, if no water is available), spores will simply not germinate. But most environments, the team realized, are not so black and white. For instance, “good” signals, like the presence of the nutrient L-alanine, might appear intermittently, then vanish. Would a slumbering spore be able to sense and process such a subtle hint?

Getting an accurate read on its surroundings is important for the spore, because it would be a waste to expend the energy needed to wake up and germinate in an unfriendly environment. That could stymie successful growth, or even lead to death. “You need to come back to life with nice timing, because otherwise you throw away your nice dormancy,” says Kaito Kikuchi, a previous student in Süel’s laboratory and a study coauthor. “You want to make sure you’re throwing away your protections when, and only when, the environment is good enough.”

First, the scientists needed to identify which biological processes the spores could use while they were still hibernating. These processes could not use ATP (adenosine triphosphate, or cellular energy) or rely on cellular metabolism (for example, breaking down sugars), since those mechanisms are shut down during dormancy.

But, the researchers hypothesized, there was an alternative method: The spores might be able to sense small cumulative changes in their environment, until enough signals build up to trigger a sort of wake-up alarm. The mechanism that would induce these changes would be the movement of ions out of the cell—specifically, potassium ions.

These movements can be triggered by positive environmental signals, like the presence of nutrients. When the ions travel out of the cell thanks to passive transport, they generate a difference in potassium concentration inside versus outside the cell. This concentration difference allows the spore to store potential energy. Over time, as the spore continues to sense more positive signals, more ions would move out of the cell. This would also create a corresponding drop in potassium levels, as the ions exit. Eventually, the potassium content in the spore would lower to a certain threshold, signaling that it is safe for the cell to wake up. That would trigger reanimation and germination. 

In other words, says Süel, the spore essentially acts similar to a capacitor, or a device that holds electrical energy. “A capacitor is basically an insulator separating the concentration gradient of charges,” he says. “You can really store a lot of energy in this way, because the cell’s membrane is very thin.”

If this concept sounds familiar, that might be because nature has already used it in another branch of biology: This is similar to how a brain neuron fires. Sodium ions stream into the neuron, causing the cell to become positively charged. Once the charge threshold is reached, an action potential is triggered and the neuron discharges. Potassium ions then stream out of the cell, bringing it back to its resting state.

To test their hypotheses, the scientists developed a mathematical model based on equations that describe how neurons fire—then adapted them to predict how the movement of potassium ions could trigger spore germination. To clarify the role these ions play, the scientists modeled a spore strain that lacked a critical unit in the potassium importer that transports ions into the cell. If germination is triggered by potassium dropping below a certain threshold, they theorized, spores with a broken import pump would bloom faster, because they would have fewer of those ions.

That idea worked in a mathematical model, but they wanted to test it in real life. So the scientists genetically engineered spores of the bacteria Bacillus subtilis so that the pump would not work. Then, they applied a timed dose of the nutrient L-alanine to them and monitored their germination. Forty-two percent of the mutated spores bloomed, compared with only 5 percent of normal ones that were used as a control. “We see that if you knock out the pump, and they don’t have enough potassium inside the spore, they are much more trigger happy and germinate,” Kikuchi says—proving their prediction correct.

Next, the scientists wanted to measure how each dose of nutrients changed the electrochemical potential inside the spore. Their mathematical model had predicted that each dose would increase a spore’s negative electrochemical potential in a step-like pattern. If each dose given to the real spores led to a predictable step up, that would support the team’s hypothesis that the cell uses its electrochemical potential to measure the friendliness of its environment, as a cue for when it’s safe to reanimate.

To visualize this with the Bacillus subtilis spores, the scientists mixed a positively charged fluorescent dye into the liquid surrounding them. The dye stuck to the spores, and the more negatively charged they became, the more dye would attach. So by measuring the spores’ fluorescence, the scientists could quantify how negatively charged each one was. When this fluorescence was graphed over time, a step-like pattern emerged that corresponded to each dose of nutrients—once again proving the prediction correct.

“This work has real potential to give us a whole new handle—specifics—on how germination proceeds,” says Peter Setlow, a spore scientist at the University of Connecticut who was not involved in the study. And that has some real-word use cases, he says, because spores can also be “causative agents for all kinds of nasties.” For example, certain bacterial spores can bury themselves in food, causing major illness when ingested. Germinating spores are much easier to get rid of than dormant ones, because they have shed their protections against chemicals and extreme temperatures. As a result, figuring out how spores wake up may provide insights into how to kill them if needed, Setlow says.

Better understanding of spore dormancy could very well provide insights into new creatures that may seem dead but are not—like potential lifeforms on other planets. In a place like Mars, where the environment is dusty and seemingly barren, sources of life would most likely resemble spores—hidden somewhere cozy, waiting for signals to come back to life. “We’re not going to find a green man walking around,” says Süel. “If anything left is still somewhat alive, it’s probably going to be something like a spore that can survive the hostile environment that Mars has been for the past few millions of years.”

Agata Zupanska, a space plant biologist at the Search for Extraterrestrial Life (SETI) Institute who was not involved in the study, agrees. “I would expect that martian bacteria, if they were there, would likely evolve a similar mechanism,” she says. “Dormancy is good. Evolutionarily, it is very successful.”

She calls spores “a fascinating solution to surviving bad environmental conditions—you have a choice: You can either die or become dormant.” This work, she says, answers the question of “how something with no molecular and energetic tools can monitor the environment and respond to persistently good conditions.”

Before scientists search for spores on Mars, there’s still a lot to do on Earth. Süel wants to keep studying how ions affect major processes in the spore. He thinks that while many biologists focus on gene expression or cell metabolism, something more passive, like the energy generated from ion gradients, could lead to surprising new discoveries. “If we can understand extremely dormant cells on our planet, maybe it’ll give us a better understanding of what to expect” when searching for life in the rest of the universe, Süel says.

The Spooky Science of How Undead Spores Reanimate

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