by Carl Zimmer
Imagine that a microbe encounters a new kind of food that its ancestors had never tasted. All of the enzymes it uses for feeding have been honed by natural selection for feeding on other molecules. That doesn’t necessarily mean the microbe can’t eat the new food. Enzymes are actually not all that finely tuned. An enzyme that can slice up one molecule very efficiently may slice up other kinds of molecules, too, albeit more slowly and clumsily. If mutations give a microbe more copies of the gene, it may be able to eat more of the new food.
Ichiro Matsumura, a biologist at Emory University, used E. coli to demonstrate just how promiscuous enzymes can be. Matsumura and his colleagues created 104 strains of E. coli, each missing a gene that is absolutely essential to the survival of the microbe. They then created thousands of plasmids, each carrying several copies of another E. coli gene . After adding these plasmids to the crippled strains, they waited to see if the plasmid genes would be able to pinch hit for the essential gene Matsumura had knocked out. Matsumura found that he could revive 21 out of the 104 strains.
Matsumura’s experiment exposed a hidden versality in E. coli that may let it adapt to new conditions. Other species may depend on the same potential in their DNA. As mutations make extra copies of those genes, they can do an even better job of feeding on a new food, or detoxifying some poison, or coping with unprecedented heat. In time, one of the copies of the gene may evolve into a far more efficient form. The other genes may then fade away.
Gene amplification may be a creative force, but it can also put us in mortal danger. Like E. coli, the cells in our bodies sometimes mutate. On very rare occasions, mutations in our cells put them on the road to becoming cancerous. They no longer obey the controls that keep the growth of normal cells in check. As they continue to divide and mutate, new mutations help them become more aggressive and better able to evade the immune system. Like E. coli starving for lactose, these cells face many challenges, and any mutation that helps them overcome these is favored by natural selection. Mutations can create extra copies of genes, which can allow tumor cells to grow faster or escape chemotherapy. Some of these extra genes can evolve new functions of their own that make the tumor even more dangerous.
Sometimes E. coli is a little too much like the elephant for the elephant’s comfort.
A GIFT OF GENES
World War II, like all wars, provided E. coli with a ripe opportunity for slaughter. Its dysentery-causing strains, then known as Shigella, stormed across battlefields and invaded cities, killing beyond counting. At the end of the war, Shigella retreated from countries that rebuilt their sewers and water supplies. However, in places where water remained dirty—much of Africa, Latin America, and Asia—Shigella continued to thrive. The one exception to the rule was Japan. Japan cleaned up its water, and for two years dysentery rates fell. But then, inexplicably, Shigella surged back. There were fewer than 20,000 cases in 1948 but more than 110,000 in 1952.
Japanese microbiologists had been very familiar with Shigella ever since Kiyoshi Shiga discovered it in 1897. During the postwar outbreak of Shigella, they gathered thousands of samples of the bacteria from patients and searched for the cause of its resurgence. Antibiotic resistance, they discovered, was on the rise. At first, microbiologists discovered Shigella strains resistant to sulfa drugs. Within a few years, resistance to tetracycline also emerged, then resistance to streptomycin and chloramphenicol.
At first the spread of resistant Shigella followed the pattern mapped out in other bacteria, with mutations giving rise to powerful new genes that gave individual microbes a reproductive edge. But then something startling happened. Shigella strains emerged that were resistant to all the antibiotics. Their transformation was sudden: if doctors gave a victim of Shigella a single type of antibiotic, the bacteria often became resistant not only to that drug but to other antibiotics the patient had never taken.
To make sense of this strangeness, Japanese scientists turned to Joshua Lederberg’s discovery of sex in E. coli a few years earlier. Lederberg had shown that on rare occasion the bacteria could transmit some of their genes to unrelated bacteria. In his experiments, ringlets of DNA—plasmids—moved from one microbe to another, dragging parts of the chromosome with them. Lederberg and other researchers had also discovered that prophages—those quiet viruses—could shuttle genes as well. A roused virus sometimes accidentally copied genes from its host into its own DNA and carried them to other bacteria. Lederberg and other scientists won Nobel Prizes for their discoveries, but for years most scientists considered this “infective heredity” only a convenient laboratory tool. It was not an important part of the natural world. They were wrong, and the dysentery outbreaks in Japan offered the first proof.
Tsutomo Watanabe at Keio University in Tokyo and other Japanese scientists explored the possibility that infective heredity was behind the rise of resistant Shigella. They proved that E. coli K-12 and Shigella could trade resistance genes. Experiments on patients infected with Shigella brought similar results. Watanabe concluded that the heavy use of antibiotics had spurred the evolution of resistance genes, either in Shigella or in another species of bacteria that lived in the gut. On rare occasion, a resistant microbe passed its genes to another species. These genes, later research would show, were carried on plasmids.
With each new antibiotic that Japanese doctors began to use on their patients, new resistance genes evolved, and their plasmids also spread among the bacteria of Japan. Sometimes a microbe would wind up infected with two plasmids at once, each carrying a gene for resistance to a different drug. The two plasmids swapped genetic material, producing a new ring of DNA carrying two resistance genes instead of one. Natural selection now favored the new plasmids even more, because they allowed bacteria to survive either drug. And over time the plasmids kept picking up other resistance genes. Eventually they made Shigella impervious to anything doctors tried to throw at it.
Few scientists outside Japan knew of these discoveries until 1963, when Watanabe wrote a long article in English for the journal Bacteriological Review. Western scientists were taken aback. They followed up with experiments of their own and confirmed that Watanabe was onto something big. Genes can shuttle between bacteria by many routes. Plasmids deliver some of them, but viruses deliver them as well. They accidentally incorporate some host genes into their own genome, which the viruses then carry to new hosts that they infect. Sometimes bacteria simply slurp up the DNA that spills out when other microbes die. These resistance genes can shuttle between individuals of the same species, and they sometimes leap from one species to another.
Horizontal gene transfer, as this genetic leaping is now known, works best in places where bacteria are packed in tight quarters. Many genes shuttle between microbes inside our bodies, as well as inside the bodies of chickens and other livestock that are fed antibiotics. Even houseflies that pick up E. coli can become a gene market. Horizontal gene transfer allows genes to leapfrog from microbe to microbe across staggering distances. In the jungles of French Guiana, scientists have found antibiotic-resistant E. coli in the guts of Wayampi Indians, who have never taken the drugs. In a survey of E. coli living in the Great Lakes, another team of scientists discovered resistance genes in 14 percent of them.
Horizontal gene transfer not only spreads resistance genes around but also speeds up their evolution. Once a gene evolves some resistance to antibiotics, it can benefit not just its original host but other bacteria that take it up. And once in its new host, the gene can continue to undergo natural selection and become even more effective. Microbes can assemble arsenals to defend themselves against antibiotics, gathering weapons from the community of bacteria rather than just inheriting them from their ancestors.
Biologists were slow to recognize just how important the Shigella outbreak in Japan was. Horizontal gene transfer was helping to create a medical disaster, one that is continuing to unfold. At first biologists did not see much evidence of horizontal gene transfer beyond resistance to antibiotics. In the
1990s, scientists began to compare the entire genome of E. coli with that of other bacteria and make a careful search for traded genes. And when they did, our understanding of the history of life changed for good. Horizontal gene transfer, we now know, is no minor trickle of DNA. It is a flood. And it played a big part in making E. coli what it is today.
Eight
OPEN SOURCE
A YOUNG SPECIES
E. COLI IS TRAILED BY thousands of personal historians. They chronicle the birth of sickening new strains in Omaha and Osaka. They trawl streams, lakes, and the guts of kangaroos. They carefully observe the peculiar ways of mutant strains. As the mutants are passed from lab to lab, frozen in stock centers and thawed for new experiments, scientists draw family trees to track their dynasties. Aside from ourselves, we have chronicled no other species so thoroughly.
The written history of E. coli is now far too big for any single person to read in a lifetime. But it is both vast and shallow. It begins only in 1885, with Theodor Escherich’s first sketches of bunches of rods. Archaeologists can offer a few clues to E. coli’s pre-Escherich existence. In 1983, English peat cutters discovered the body of a 2,200-year-old man preserved in a bog near Manchester. The man had been ritually killed: someone had clubbed him on the head, slit his throat, wrapped a cord tightly around his neck, and then pushed him into the bog. The acidic waters preserved his corpse and even its contents. In his stomach, scientists found barley and mistletoe. And in his intestines they found the DNA of E. coli.
There’s no reason to think that the bog man was the first human ever to carry E. coli. There is every reason to think that its history reaches much farther back. Bacteria have an ancient fossil record. Individual microbes left their marks on rocks as least 3.7 billion years ago. Ocean reefs built by bacteria 3 billion years ago still stretch for miles across Africa and Canada. E. coli does not do such a good job of forming fossils, because of its tenuous existence. But what E. coli lacks in fossils it more than makes up for in the historical record that it carries in its DNA. That genetic record rolls out before us like a carpet, back across millions of years to the origins of E. coli as a species, back farther to a time before life dwelled on dry land, back to the origins of cells, to the earliest days of life itself.
To read this record, it’s necessary to become a genealogist of bacteria. When a mutation arises in an E. coli, it will be passed down to its offspring. That mutation can sometimes serve as a genetic marker, revealing to scientists a group of bacteria that are closely related to one another. It’s these genetic markers that public health workers use when an outbreak of nasty E. coli occurs, in order to trace the pathogens to their source. Other scientists use these markers to draw branches on E. coli’s family tree. They have a long way to go before they finish drawing it, but they’ve already filled in enough branches to learn some profound things about the bacteria.
All living strains of E. coli descend from the first members of the species. Scientists have a rough idea of when those earliest E. coli lived. In 1998, Jeffrey Lawrence of the University of Pittsburgh and Howard Ochman of the University of Arizona estimated when the ancestors of E. coli and the ancestors of its close relative Salmonella enterica split off from each other. Lawrence and Ochman tallied the differences in the species’ DNA. When two species branch off from a common ancestor, they acquire mutations at a roughly regular rate. Lawrence and Ochman estimated their common ancestor lived about 140 million years ago. In 2006, Ochman and several other colleagues tackled E. coli’s origins from another direction: they surveyed E. coli strains and estimated when their common ancestor lived. They concluded that the species was already well established 10 million to 30 million years ago. E. coli is much older than the English bog man, in other words, but it is not a living fossil. It is about as primitive as a primate.
E. coli’s ancestors split from those of Salmonella at a time when dinosaurs dominated the land. Pterosaurs flew overhead, along with birds that still had teeth in their beaks and claws on their wings. The typical mammal at the time was a squirrel-like creature. Around 65 million years ago this picture began to change dramatically. Pterosaurs and the big dinosaurs became extinct, probably in part thanks to an asteroid that crashed into the Gulf of Mexico. After the crash, mammals diversified into flying bats, enormous elephant-like browsers, cat-and doglike carnivores, seed-gnawing rodents, tree-scampering primates. Birds took on their modern forms as well. Mammals and birds share more than survival, however. Their ancestors independently evolved the ability to control their body temperature. Their guts became a desirable habitat for bacteria, including the ancestors of E. coli. Warm-blooded animals need to eat a lot of food to fuel their metabolism, and that rich diet can support a menagerie of microbes. The constant warmth of their guts allows the enzymes of microbes to work quickly and efficiently. It may be no coincidence that the rise of E. coli coincides with the rise of its current hosts.
The early E. coli produced the vast diversity of lineages that live inside us today, some harmless, some even beneficial, some that ravage the brain or ruin the kidneys, and some that are adapted to life outside warm bodies altogether. Life has often exploded into this sort of diversity when it has gotten the opportunity. But E. coli’s explosion is different: scientists can dissect it gene by gene.
VENN GENOMES
Two strains, K-12 and O157:H7, are enough to provide a sense of how diverse E. coli is as a species. K-12 is so harmless that scientists make no efforts to protect themselves from it; instead, they have to protect it from fungi and bacteria. If K-12 is a lapdog, O157:H7 is a wolf. It injects molecules into our cells, disrupts our intestines, makes us bleed, loads us with toxins, shuts down our organs, and sometimes kills us. Each microbe relies on a network of genes and proteins to thrive in its particular ecological niche, and those networks are very different from one another. As different as they are, though, K-12 and O157:H7 have a common ancestor, which scientists estimate lived 4.5 million years ago—at a time when our ancestors were upright-walking apes.
In 2001, scientists got their first good look at how a single microbe could give rise to two such different organisms. It was in that year that two teams of scientists—one Japanese, the other American—independently published the complete genome of O157:H7. Scientists could then compare it, gene for gene, with the genome of K-12, which had been published four years earlier. No one could quite predict what would be found.
In the 1970s, scientists had begun comparing small fragments of DNA from different strains of E. coli. The fragments were nearly identical from strain to strain, both in their genetic sequence and in their position on the chromosome. Scientists could even find the corresponding fragments in E. coli’s relative Salmonella enterica. Many scientists assumed that E. coli’s entire genome would follow this pattern. They thought E. coli’s evolutionary history was tidy. An ancestral microbe had given rise to many lineages, some of which evolved into today’s strains. Mutations cropped up in each lineage, a few of which were favored by natural selection, driving their cousins to extinction. Horizontal gene transfer might have imported a few genes from other species, but many scientists assumed that had been a rare event, Lederberg’s and Watanabe’s work notwithstanding.
But when scientists were finally able to compare the genomes of K-12 and O157:H7, that’s not what they found. Vast amounts of DNA in each strain had no obvious counterpart in the other. E. coli O157:H7 has 5.5 million base pairs of DNA, while K-12 has only 4.6 million. About 1.34 million base pairs in O157:H7 cannot be found in K-12, and more than half a million base pairs in K-12 have no counterpart in O157:H7. A map of the genes in each genome offered a similar picture. K-12 has 4,405 genes, 528 with no counterpart in O157:H7. Some 1,387 genes in O157:H7 cannot be found in K-12.
Each genome is like a circle in a Venn diagram. The overlap between K-12 and O157:H7 represents a core of shared genes, inherited from a common ancestor. After the two lineages diverged, they acquired new genes from other microbes—not just gen
es for resistance to antibiotics, but hundreds of other genes that came to make up a quarter of their genomes.
A year after the publication of O157:H7’s genome, scientists published the genome of a third strain. Known as CFT073, it lives harmlessly in the intestines, but if it gets into the bladder it can cause painful infections. The scientists discovered that its genome formed a third overlapping circle on the E. coli Venn diagram. CFT073 shares some genes with K-12 that it doesn’t share with O157:H7. And it shares some genes with O157:H7 that it doesn’t share with K-12. But scientists could not find any counterpart for 1,623 of its genes in the other two strains. At the center of the new Venn diagram was the new core of E. coli genes. Of all the E. coli genes scientists had now identified, only 40 percent could be found in all three strains. The core was shrinking.
As I write this, scientists have sequenced more than thirty E. coli genomes; a vast number of other strains are left to examine. With every new strain, scientists continue to discover dozens, even hundreds of genes found in no other E. coli strain. Each strain also carries hundreds of genes that it shares with some other strains. The list of genes shared by every E. coli is getting shorter, while the list of genes found in at least one strain is getting longer. Scientists call this total set of E. coli genes the pangenome. It’s up to 11,000 genes now, and at the current rate it will probably become larger than the 18,000 or so genes in the human genome.
The discovery of the E. coli pangenome called for a radical rethinking of how the microbe evolved. Tidy is precisely the wrong word to describe the history of E. coli over the past 30 million years. From the earliest days of its existence, a steady surge of new DNA has entered its genomes. Some of those genes moved from one strain of E. coli to another, while some of them came from other species.
Foreign DNA has taken several routes into E. coli’s genome. Plasmids, those tiny ringlets of DNA, brought some. Viruses that infect E. coli brought more. In some cases, viruses have brought only one or two genes. In other cases, they have brought dozens. These gene cassettes, as they’re sometimes called, are not random collections of DNA. They often contain all the instructions necessary to build a complex structure, such as a syringe for injecting toxins. Once these genes become part of the genome of a strain of E. coli, the microbes pass them down to their descendants. Ordinary natural selection can fine-tune the genes for the microbe’s particular way of life. Sometimes the genes slip away to a new host.
