Massed at the starting line, the crowd of runners all looked identical. But this wasn’t your standard 5K. Instead, researchers wanted to test both speed and navigational ability as competitors wound their way through a maze, choosing the right direction at every intersection. At the end of the course, the postdocs Mehdi Salek and Francesco Carrara would be waiting to identify each of the finishers. The postdocs wouldn’t have any medals or a commemorative T-shirt for the winners, however, because their racers weren’t human. They were Escherichia coli bacteria.

That there could be individual winners at all is a notion that has shaken the foundations of microbiology in recent years. Working in the lab of Roman Stocker at the Swiss Federal Institute of Technology Zurich (ETH Zurich), a team of microbiologists and engineers invented this unique endurance event. The cells at the starting line of Stocker’s microbial marathon were genetically identical, which implied, according to decades of biological dogma, that their resulting physiology and behavior should also be more or less the same, as long as all the cells experienced identical environmental conditions. At the DNA level, every E. coli cell had a roughly equal encoded ability to swim and steer through the course. A pack of cells that started the race at the same time would in theory all finish around the same time.

But that’s not what Salek and Carrara found. Instead, some bacteria raced through the maze substantially more quickly than others, largely because of varying aptitude for moving toward higher concentrations of food, a process called chemotaxis. What appeared to Salek and Carrara as a mass of indistinguishable cells at the beginning was actually a conglomerate of unique individuals.

“Bacteria can be genetically identical but phenotypically different,” Carrara said.

This bacterial individuality — known more technically as phenotypic heterogeneity — upends decades of traditional thinking about microbes. Although scientists knew that, for example, antibiotics didn’t always kill every last microbe in a colony of identical clones, both the cause of these differences and the resulting implications remained shrouded in mystery. Now advances in microscopy and microfluidics (the technology Stocker’s lab used to build the bacterial maze) have begun to lift the veil on an important evolutionary process.

“This has been a relatively overlooked phenomenon,” said Hesper Rego, a microbiologist at the Yale School of Medicine. “The idea that microbial populations could evolve heterogeneity and control it using genetics is a really powerful concept.”

From Populations to Individuals

Ever since the days of Robert Koch and Louis Pasteur in the 1870s, microbiologists have typically studied groups of bacteria rather than individuals. Much of this was out of necessity: The technology didn’t exist to allow scientists to do much more with single cells than peer at them through a microscope. Besides, if the bacteria were all identical, then there seemingly wasn’t a need to study every cell. An individual cell deposited on a plate of nutrient-rich jelly would divide and divide until it formed a visible colony of cells, all clones of the original cell. All the bacteria in this colony could be expected to show the same behaviors, physiology and physical appearance — the same phenotype — when placed in identical environments. By and large, they did.

The development of antibiotics in the 1940s revealed a curious anomaly, however. In many cases, antibiotics didn’t annihilate all the bacteria, even in groups of cells that were fully susceptible to the killing power of antibiotics. The surviving cells were considered “persistent.” They just hunkered down and waited out the chemical barrage of penicillin or similar drugs. Initially, scientists thought that persisters might come from a genetically distinctive subpopulation that grew more slowly even before the antibiotic treatments. But when microbiologists looked for genes that could predict which cells would become persisters, they were disappointed.

“There was no such [distinct persistent] subpopulation,” said Laurence van Melderen, a microbiologist at the Free University of Brussels in Belgium. “In every population, you will find some persisters if you look for them.” For scientists, this posed a major quandary: How could identical bacteria have such radically different behaviors?

By the late 1970s, researchers had identified one possible answer. Scientists at the University of California, Berkeley showed that random chance alone could lead to different behaviors even in genetically identical cells. Bacteria with whiplike flagella can swim in a straight line (known as “running”) or lurch in random directions. Swimming cells spend much of their time tumbling about, actively sampling their environment. But to move toward higher concentrations of nutrients and away from toxins and predators, bacteria must use a direct run. When they can no longer sense a gradient, they return to tumbling.

Berkeley microbiologists studying E. coli found that each cell stopped swimming and started tumbling at a different concentration of various chemical attractants, including aspartate and L-serine. Even after considering random statistical variations and any influence from unlikely spontaneous mutations during the experiment, the researchers couldn’t account for the cells’ marked and persistent individual differences in running and tumbling. That mystery, according to Thierry Emonet, a biophysicist at Yale, was “a big deal.”

The study appeared during the heyday of the idea that a single gene made a single protein, which would subsequently elicit a consistent behavior when all the cells were in the same environment. After a century of experimentation on batches of bacteria, scientists were accustomed to slight collective deviations in “identical” traits, but their data still tended to cluster tightly around a mean. The Berkeley scientists, in contrast, found that sensitivity to the attractants was smeared out over a broad concentration, not a single mean. Their paper challenged the general assumption by showing substantial cell-to-cell variation in swimming behavior among the individual bacteria. No longer could phenotypic heterogeneity be shrugged off as a quirk of the bacterial response to antibiotics.

Although the researchers knew that this individuality resulted both from how tightly each cell regulated tumbling and from its response to L-serine, quantifying this variation in specific cells was more challenging. In 2002, glowing E. coli changed all of that.