by Carl Zimmer
At first, Xavier and Foster found, the glue-making bacteria lost out to the others because they were diverting energy from their growth. But soon the balance tipped. As the bacteria multiplied, they used up the oxygen around them and could not grow as quickly. The glue makers could escape suffocation because they created a rising mound on which their offspring could grow. They could reach higher concentrations of oxygen, which allowed them to grow faster, which in turn allowed them to build their mounds of glue even higher. As a mound grew, it suffocated the older bacteria underneath while their descendants—and thus their genes—lived on. Meanwhile, the bacteria that did not make glue, trapped in the biofilm with no way to escape, were buried by their competitors. In some ways a biofilm may be less like a city and more like a forest, in which trees become wooden towers in order to reach the sunlight and avoid the shade of their rivals.
Conflict and cooperation strike an uneasy balance whenever many cells come together, whether they are E. coli or the cells of our own bodies. We descend from single-celled ancestors that probably looked a lot like amoebas. At some point our ancestors began to form colonies, which gradually evolved into vast collectives, otherwise known as animals. They communicated with one another as they had before, but now their signals caused them to differentiate into different types of cells, forming tissues and organs. Each time a new animal took shape, most of the cells of its body had to make the ultimate evolutionary sacrifice. They would become part of the body, and in that body they would die. Only sperm and eggs had the slightest chance of their genes surviving.
This is not a simple way to exist. In order to form a full-grown body, most cells must divide many times and then stop. Some kinds of cells must not lose the ability to regenerate themselves, but they must multiply only as much as necessary to heal a wound or build a new intestinal lining. Unfortunately, a dividing cell can mutate, just like a dividing E. coli. In some cases, the mutations will turn the cell into a rebel. It will reproduce rapidly, ignoring the signals that tell it to stop. It will produce a mass of insurgent cells, and within that mass new mutations will arise, producing even more rebellious cells. They will develop new tricks for evading the body’s defenses and for manipulating the body so that it brings them new blood vessels in order to supply them with extra oxygen and nutrients. They become cheaters, just like the cheaters that exploit E. coli’s cooperation. We call their success cancer.
PLACE YOUR BETS
When a starving colony of E. coli gets a supply of lactose, there is only one good decision to make: start manufacturing beta-galactosidase and use it to break the lactose down. Some microbes will make the right choice while others will not. The losers keep their lactose-digesting genes shut off, and they continue to starve.
These microbes are all genetically identical, which means that the same genetic circuitry gives rise to both decisions. If natural selection favors genes that boost the reproduction of E. coli, how could it have produced this sort of confusion? That is a difficult question, one scientists have only begun to take seriously. The answer they have now settled on is this: E. coli is a smart gambler.
Every gambler who comes to a racetrack hopes to place a winning bet. The best way to win is to see into the future and know which horse will come in first. But in the real world, gamblers can only hope to winnow down their choices to a few strong horses. Even with this limited selection, they run the risk of losing money. Some gamblers shield themselves against losses by hedging their bets. They wager on several horses in the same race. If one of their horses wins, they get money. They don’t leave the racetrack with as much money as they would have had they bet only on the winner. But the other bets can act as a good insurance policy. If one of the other horses wins, the gambler can still go home with more money than he came with.
Gamblers aren’t the only people who think about hedging bets. Mathematicians and economists have explored all sorts of variations on the strategy, many of which have been borrowed by stockbrokers, bankers, and other people who make uncertain choices about how financial markets behave. A broker buying stock in a biotechnology company may sell short on a different one, so that no matter which way the market goes she makes money. And evolutionary biologists have now borrowed the mathematics of the marketplace to understand why E. coli clones can act so differently.
E. coli’s gamble consists of choosing a response to a particular situation. In some cases, the choice is clear. A population of microbes should all respond in the same way. But in other cases, it pays for the population to hedge its bets. In other words, it pays for some individuals to respond one way and others to respond in another.
Which way E. coli should bet depends on how much information it can get. If E. coli can get a lot of reliable information, it makes sense to put all its money on one bet. But in other cases, it may be hard to determine the best choice. Conditions may be changing quickly and unpredictably. E. coli may be better off hedging its bet in these cases, allowing individuals to respond in different ways.
Lactose, for example, causes E. coli to hedge its bets. A supply of lactose may allow bacteria to survive when other kinds of sugar have been devoured. But in order to feed on lactose, a microbe first has to clear away all the proteins it was using to feed on other sugars and begin making the proteins it needs to eat lactose. That’s a lot of time and energy for a microbe to invest. The investment may pay off or it may be a waste of effort—the lactose may disappear quickly or a more energy-rich sugar may turn up.
E. coli hedges its bets by using its unpredictable bursts of proteins to create both eager and reluctant individuals. If the colony happens to encounter some lactose, the eager microbes will be ready to seize the moment, while the others respond more slowly. If the surge of lactose never comes, the reluctant microbes will grow faster because they haven’t wasted energy preparing for a feast that never arrived. Either way, the colony benefits.
E. coli hedges many bets, scientists are finding, and some of those bets make us sick. Strains of E. coli that infect the bladder need to make sticky hairs to attach to host cells, but the hairs draw the attention of the immune system. To balance this trade-off, the bacteria hedge their bets by randomly switching on and off the machinery for making the hairs. At any moment some individuals in a colony will sprout hairs and others will remain bald.
Bet hedging may also help E. coli defend itself against antibiotics. Many antibiotics kill E. coli by attacking the proteins the microbes use to grow. When antibiotics encounter a population of susceptible E. coli, they kill it off with staggering swiftness. Or at least they kill most of the microbes off. About 1 percent of the E. coli in a biofilm can survive an attack of antibiotics for hours or days. The survivors can rebuild the biofilm and make a person as sick as before.
This resilient minority carries no special genes for resisting antibiotics. They are genetically identical to their dead relatives. Scientists can isolate the survivors and allow them to multiply to form large colonies. The new colonies will be just as vulnerable to antibiotics. Once again, about 99 percent of the microbes die and 1 percent persist.
Scientists discovered so-called persister bacteria in 1944, and for the next sixty years they remained almost entirely baffled by them. Some researchers suggested that antibiotics drive a few microbes into a mysterious dormant state in which they can escape damage. A team of scientists led by Nathalie Balaban of the Hebrew University of Jerusalem tested this idea in 2004 by building a device to spy on persister cells. The scientists placed E. coli in microscopic grooves just wide enough to hold a single microbe. When an individual E. coli divided, its offspring remained in a neat line. Balaban and her team could watch the lines stretch and measure how quickly lineages of cells grew.
After several generations, the scientists doused the bacteria with a potent antibiotic. Most of the E. coli died, but the persisters remained. Balaban and her colleagues found that the persisters grew far more slowly than normal cells, although they had not stopped growing altogether.
By looking back at their earlier measurements, Balaban discovered that the microbes had become slow-growing persister cells before the antibiotics arrived.
Balaban concluded that every E. coli has a tiny chance at any moment of spontaneously turning into a persister. Once it makes the change, the microbe has a small chance of reverting to a normal fast-growing cell. All the bacteria Balaban studied, persisters and growers alike, were genetically identical, which meant that mutations were not the source of persistence. Yet persisters gave rise to more persisters, as if persisting were a hereditary trait.
Persisters are born of noise. That’s the theory of Kim Lewis, a leading expert on the phenomenon at Northeastern University. Lewis and his colleagues have found a way to compare the proteins produced by persister cells with the ones made by normal E. coli. One of the major differences between the two kinds of bacteria is that persister cells make a lot of toxins. Scientists have long puzzled over these toxins, which lock on to E. coli’s other proteins and stop them from doing their normal jobs. In most of the bacteria, these toxins don’t cause any harm because E. coli also produces their antidotes—antitoxins that grab the toxins before they can interfere with the microbe’s physiology.
It’s these toxins, Lewis argues, that turn E. coli into persisters. Normally E. coli churns out a tiny stream of toxins, along with another tiny stream of antitoxins. But thanks to the noisy workings of its genes, the microbe sometimes hiccups, releasing a burst of toxins. The extra toxins that aren’t disabled by E. coli’s small supply of antitoxins are free to attack proteins. They don’t do any permanent damage, but they do bring E. coli’s growth nearly to a halt. After the outburst, E. coli’s toxins gradually dwindle as E. coli produces more antitoxins. Once its proteins are liberated, it can go back to being a normal microbe again.
This noisy network acts like a roulette wheel, randomly picking out a few individuals to stop growing at any moment. It’s usually a bad thing for an individual microbe to get stuck with extra toxins, because a persister will fall behind the other, fast-growing E. coli. But there’s also a small chance that a disaster will strike while the microbe is a persister. That disaster might come in the form of a pencillin pill, or it might be a naturally produced poison released by another microbe. In either case, the persister will turn out to be the big winner. For the entire population of E. coli, it doesn’t matter which individual wins as long as its individuality-generating genes continue to get passed down to new generations.
SPITEFUL SUICIDE
Persister cells make a sacrifice for their companions, giving up the chance to multiply quickly. But when E. coli produce colicins, the chemical weapons for killing rival strains, they pay a far bigger price. In order to let their kin thrive, they explode in a suicidal blast.
The chemical warfare practiced by E. coli is the dark side of altruism. William Hamilton originally argued that natural selection could favor sacrifice if it meant an individual could help its relatives reproduce more. In 1970, he recognized that natural selection could also favor sacrifice if it meant that nonrelatives suffered—a nasty sort of altruism he called spite. Hamilton always argued that spite was rare and inconsequential, because his equations suggested it would be favored only when populations were very small. But in 2004, Andy Gardner and Stuart West at the University of Edinburgh demonstrated that if unrelated individuals compete fiercely with their immediate neighbors spite can also evolve.
E. coli meets these spiteful standards. It competes in the crowded confines of the intestines for a limited supply of sugar. An individual microbe sacrifices its own reproductive future by committing suicide, but its colicins destroy many competitors, allowing the microbe’s own close relatives to thrive. As with persistence, becoming a colicin maker is a matter of chance. The noisy production of proteins determines which few individuals will respond to starvation by switching on their colicin-producing genes. The burden is shared by all.
Spite, some experiments now suggest, may also drive E. coli to become more diverse. Margaret Riley, a biologist at the University of Massachusetts, Amherst, and her colleagues have observed the evolution of this arms race in experiments on E. coli in both petri dishes and the guts of lab mice. Once in a rare while, an antidote gene may mutate into a more powerful form. Instead of just defending E. coli against its own colicin, it can also defend against the colicins made by other strains. This mutation gives a microbe an evolutionary edge, because it can survive enemy attacks that kill other members of its strain.
This powerful antidote opens the way for another advance. A second mutation strikes the colicin-producing gene, causing it to make a new colicin. Its relatives, which still carry an antidote for the old colicin, are killed off by the mutant toxin. But thanks to its powerful antidote, the microbe that makes the new colicin can survive while its relatives die. Its spite becomes intimate.
The emergence of new colicins drives the evolution of new antidotes in other strains. Likewise, new antidotes drive the evolution of new colicins. But E. coli has to pay a price for this weaponry. It has to use energy to make colicins and antidotes, which are particularly big as bacterial molecules go. A new colicin may be even deadlier than its predecessor, but it may also become a drain on a microbe. If a mutation leaves a microbe unable to make colicins—but still able to resist them—it may be able to channel the extra energy into reproducing. A colicin-free strain will spread, outcompeting the colicin makers.
If colicin makers are driven to extinction, their colicins no longer pose a threat to neighboring bacteria. Now antidotes become a waste of effort, since there is nothing for them to protect E. coli against. Natural selection can begin to favor pacifists—microbes that make neither colicins nor antidotes. Once the pacifists come to dominate the population, colicin producers can invade the population once more, killing off the vulnerable strains and getting the food for themselves. And so the journey comes full circle.
These sorts of cycles emerge spontaneously from evolution. You can think of them as games in which players use different kinds of strategies to compete with other players. In the case of E. coli, a strategy might be to make a particular colicin or to do without colicins and antidotes altogether. In the case of a male elephant seal, strategies might include fighting with other males for the opportunity to mate with females or sneaking off with a female without the big male on the beach noticing. In some cases, one strategy may prove superior to all the others. In other cases, two strategies may coexist. Fighting males and sneaker males can coexist in many species, for example. In still other cases, the success of strategies goes up and down over time.
Scientists sometimes call this cycling evolution a rock-scissors-paper game. In the game, each player can make a fist for a rock, extend two fingers for scissors, or hold the hand flat for paper. A player wins or loses depending on what the other players do. Rock beats scissors, but scissors beats paper, and paper beats rock. If a population of organisms is dominated by one strategy—call it paper—then natural selection will favor scissors. But once scissors takes over, rock is favored, then paper, and so on.
The common side-blotched lizard of coastal California plays a colorful version of rock-scissors-paper. The male lizards have colored throats, which may be orange, yellow, or blue. The orange-throated lizards are big fighters; they establish large territories with several females. The blue-throated lizards are medium sized; they defend small territories, holding just a few females, which they can guard carefully. The yellow-throated males are small and sneak around for mates, taking advantage of the fact that they look like females. Each type of male can outcompete one type but not the other. The yellow-throated males can sneak past the orange-throated males because the territories of the orange-throated males are so big. The yellow-throated males cannot use the same strategy against the blue-throated males because the blue-throated males stay close to their females and are bigger than the yellow-throated males. But the blue-throated males lose against the orange-throated males because the orange
-throated males are bigger.
Over a period of six years, each type of male goes through a population cycle. When the orange-throated males become common, natural selection favors yellow-throated males, which can sneak off with their females. But once yellow-throated males become common, the biggest benefits go to blue-throated males, which can fight off the yellow-throated males and father lots of baby lizards with their few females. And in time, natural selection favors the orange-throated males again.
When scientists at Stanford and Yale discovered the E. coli version of the rock-scissors-paper game in 2003, they suggested that it may turn out to be particularly common. Chemical warfare is a frequent strategy in nature, particularly among organisms that are too small or too immobile to use other sorts of weapons. Trees poison their insect visitors, corals ward off grazers, and humans and other animals produce antibodies to fight off pathogens. The race to develop better poisons and defenses, as well as the added dimension of the rock-scissors-paper game, can foster the evolution of diversity. Scientists have long known that a single strain of E. coli may dominate the gut for a few months, only to later shrink away, making way for a rarer strain. The colicin war may be one force behind this cycle.
E. coli may be able to spontaneously evolve a harmonious food web. But when it comes to weaving Darwin’s tangled bank, war may be just as good as peace.
DEATH COMES TO ALL
Not long ago, E. coli was immortal. That’s not to say it was invulnerable. The bacteria can die in all sorts of ways—devoured by protozoans, starved for years in a famine, or ripped open like a water balloon by the prick of a colicin needle. But decades of gazing at E. coli left scientists convinced that death is not inevitable. Left to its own devices, E. coli remained eternally young. Here was one way, at least, in which E. coli was fundamentally different from us. Our bodies slide into decay on a relatively tight schedule. Our immune system lets more viruses and bacteria invade our bodies unchallenged. Our brains shrink; our bones grow brittle; our skin droops.