Microcosm

by Carl Zimmer

  The connection became far stronger when a somber young student arrived at Tatum’s lab at Yale. Joshua Lederberg was only twenty-one years old when he began to work with Tatum, but he had a grand ambition: to find out whether bacteria had sex. As part of his military service during World War II, Lederberg had spent time in a naval hospital on Long Island, where he examined malaria parasites from marines fighting in the Pacific. He had gazed down at the single-celled protozoans, which sometimes reproduced by dividing and sometimes by taking male and female forms and mating. Perhaps bacteria had this sort of occasional sex, and no one had noticed. Others might mock the idea as a fantasy, but Lederberg decided to take what he later called “the long-shot gamble in looking for bacterial sex.”

  When Lederberg heard about Tatum’s work, he realized he could look for bacterial sex with a variation on Tatum’s experiments. Tatum was amassing a collection of mutant E. coli K-12, including double mutants—bacteria that had to be fed two compounds to survive. Lederberg reasoned that if he mixed two different double mutants together, they might be able to pick up working versions of their genes through sex.

  Lederberg started work at Yale in 1946. He selected a mutant strain that could make neither the amino acid methionine nor biotin, a B vitamin. The other strain he picked couldn’t make the amino acids threonine and proline. Lederberg put the bacteria in a broth he stocked with all four compounds so that the mutant microbes could grow and multiply. They mingled in the broth for a few weeks, with plenty of opportunity for hypothetical sex.

  Lederberg drew out samples of the bacteria and put them on fresh petri dishes. Now he withheld the four nutrients they could not make themselves: threonine, proline, methionine, and biotin. Neither of the original mutant strains could grow in the dishes. If their descendants were simply copies of their ancestors, Lederberg reasoned, they would stop growing as well.

  But after weeks of frustration—of ruined plates, of dead colonies—Lederberg finally saw E. coli spreading across his dishes. A few microbes had acquired the ability to make all four amino acids. Lederberg concluded that their ancestors must have combined their genes in something akin to sex. And in their sex they proved that they carried genes.

  Two E. coli having bacterial sex

  In the years that followed, the discovery would allow scientists to breed E. coli like flies and to probe genes far more intimately than ever before. Twelve years later, at the ancient age of thirty-three, Lederberg would share the Nobel Prize in Medicine with Tatum and Beadle. But in 1946, when he picked up his petri dishes and noticed the spots that appeared to be the sexual colonies he had dreamed of, Lederberg allowed himself just a single word alongside the results in his notebook: “Hooray.”

  HOST AND PARASITE

  While Lederberg was observing E. coli having sex, other scientists were observing it getting sick. And they were learning things that were just as important about the nature of life.

  The first scientist to appreciate just how revealing a sick E. coli could be was not a biologist but a physicist. Max Delbrück had originally studied under Niels Bohr and the other pioneers of quantum physics. In the 1930s it seemed as if a few graceful equations could melt away many of the great mysteries of the universe. But life would not submit. Physicists like Delbrück were baffled by life’s ability to store away all of the genes necessary to build a kangaroo or a liverwort in a single cell. Delbrück decided to make life—and in particular, life’s genes—his study.

  “The gene,” Delbrück proposed, “is a polymer that arises by the repetition of identical atomic structures.” To discover the laws of that polymer, he came to the United States, joining Morgan’s laboratory to breed flies. But the physicist in Delbrück despised the messy quirks of Drosophila. He craved another system that could provide him with far more data and was far simpler. As luck would have it, another member of Morgan’s lab, Emory Ellis, was studying the perfect one: the viruses that infect E. coli.

  The viruses that infect E. coli were too small for Delbrück and Ellis to see. As best anyone could tell, they infected their bacterial hosts and reproduced inside, killing the microbes and wandering off to find new victims. The new viruses seemed identical to the old, which suggested that they might carry genes. Delbrück and Ellis set out to chart the natural history of E. coli’s viruses.

  To study the viruses—known as bacteriophages—Delbrück and Ellis could look only for indirect clues. If they added viruses to a dish of E. coli, the viruses invaded the bacteria and replicated inside them. The new viruses left behind the shattered remains of their hosts and infected new ones. Over a few hours spots formed on the dish where their victims formed transparent pools of carnage. “Bacterial viruses make themselves known by the bacteria they destroy,” Delbrück said, “as a small boy announces his presence when a piece of cake disappears.”

  Although the signs of the viruses were indirect, there were a lot of them. Billions of new viruses could appear in a dish in a few hours. The power of Delbrück and Ellis’s system attracted a small flock of young scientists. They called themselves the Phage Church, and Delbrück was their pope. The Phage Church demonstrated that E. coli’s bacteriophages were not all alike. Some could infect certain E. coli strains but not others. By triggering mutations in the viruses, the scientists could cause the viruses to infect new strains. The ability to infect E. coli passed down from virus to virus. Viruses, it became clear, had genes—genes that must be very much like those of their host, E. coli.

  The genes of host and parasite are so similar, in fact, that scientists discovered certain kinds of viruses that could merge into E. coli, blurring their identities. These prophages, as they are called, can invade E. coli and then disappear. A prophage’s hosts behave normally, growing and dividing like their virus-free neighbors. Yet scientists found that the prophages survived within E. coli, which passed them down from one generation to the next. To rouse a prophage, the scientists needed only to expose a dish of infected E. coli to a flash of ultraviolet light. The bacteria abruptly burst open with hundreds of new prophages, which began to infect new hosts, leaving behind the clear pools of destruction. Two had become one, only to become two again.

  THE STUFF OF GENES

  In the merging dance of E. coli and its viruses, the Phage Church discovered clues to some of life’s great questions. And for them there was no greater question than what genes are made of.

  Until the 1950s, most scientists suspected that proteins were the stuff of genes. They had no direct evidence but many powerful hints. Genes exist in all living things, even bacteria and viruses, and proteins appeared to be in all of them as well. Scientists studying flies had located genes in the chromosomes, and chromosomes contain proteins. Scientists also assumed that the molecules from which genes are made had to be complicated, since genes somehow gave rise to all the complexity of life. Proteins, scientists knew, often are staggeringly intricate. All that remained was to figure out how proteins actually function as genes.

  The first major challenge to this vague consensus came in 1944, when a physician announced that genes are not in fact made of protein. Oswald Avery, who worked at the Rockefeller Institute in New York, studied the bacteria Pneumococcus. It comes in both a harmless form and a dangerous one that can cause pneumonia. Earlier experiments had hinted that genes control the behaviors of the different strains. If scientists killed the dangerous strain before injecting it into mice, it did not make the mice sick. But if the dead strain was mixed with living harmless Pneumococcus, an injection killed the mice. The harmless strain had been transformed into pathogens, and their descendants remained deadly. In other words, genetic material had moved from the dead strain to the live one.

  Avery and his colleagues isolated compound after compound from the deadly strain and added each one to the harmless strain. Only one molecule, they found, could make the harmless strain deadly. It was not a protein. It was something called deoxyribonucleic acid, DNA for short.

  Scientists had known of D
NA for decades but didn’t know what to make of it. In 1869, a Swiss biochemist named Johann Miescher had discovered a phosphorus-rich goo in the pus on the bandages of wounded soldiers. The goo came to be known as nucleic acid, which scientists later discovered comes in two nearly identical forms: ribonucleic acid (RNA) and deoxyribonucleic acid. The phosphorus in DNA helps form a backbone, along with oxygen and sugar. Connected to this backbone are four kinds of compounds, known as bases, rich in carbon and nitrogen.

  DNA was clearly important to life, because scientists could find it in just about every kind of cell they looked at. It could even be found in fly chromosomes, where genes were known to reside. But many researchers thought DNA simply offered some kind of physical support for chromosomes—it might wind around genes like cuffs. Few thought DNA had enough complexity to be the material of genes. DNA was, as Delbrück once put it, “so stupid a substance.”

  Stupid or not, DNA is what genes are made of, Avery concluded. But his experiments failed to win over hardened skeptics, who wondered if his purified DNA had actually been contaminated by some proteins.

  It would take another decade of research on E. coli and its viruses to start to redeem DNA’s reputation. While Avery was sifting Pneumococcus for genes, Delbrück’s Phage Church was learning how to see E. coli’s viruses. The viruses were no longer mathematical abstractions but hard little creatures. Using the newly invented electron microscope, Delbrück and his colleagues discovered that bacteriophages are elegantly geometrical shells. After a phage lands on E. coli, it sticks a needle into the microbe and injects something into its new host. The shell remains sitting on E. coli’s surface, an empty husk, while the virus’s genes enter the microbe.

  The life cycle of E. coli’s viruses opened up the chance to run an elegantly simple experiment. Alfred Hershey and Martha Chase, two scientists at the Cold Spring Harbor Laboratory on Long Island, created viruses with radioactive tracers in their DNA. They allowed the viruses to infect E. coli and then pulled off their empty husks in a fast-spinning centrifuge. Hershey and Avery searched for radioactivity and found it only within the bacteria, not the virus shells.

  Hershey and Chase then reversed the experiment, spiking the protein in the viruses with radioactive tracers. Once the viruses had infected E. coli, only the empty shells were radioactive. A decade after Avery’s experiment, Hershey and Chase confirmed his conclusion: genes are made of DNA.

  A virus inserts its DNA into E. coli.

  No one was more excited by the new results than a young American biologist named James Watson. Watson was only twenty when he was initiated into the Phage Church, blasting E. coli’s viruses with X-rays for his dissertation work. He was taught the conventional view that genes are made of proteins, but his own research was drawing his attention to DNA. He saw Hershey and Chase’s experiment as “a powerful new proof that DNA is the primary genetic material.”

  In order to understand how DNA acts as genetic material, however, it was necessary to figure out its structure. Watson was working at the time at the University of Cambridge, where he quickly teamed up with Francis Crick, a British physicist who also wanted to understand the secret of life. Together they pored over clues about DNA and tinkered with arrangements of phosphates, sugars, and bases. In February 1953, they suddenly figured out its shape. They assembled a towering model of steel plates and rods. It was a twisted ladder of sugar and phosphates, with bases for rungs.

  The structure was beautiful, simple, and eloquent. It seemed to practically speak for itself about how genes work. Each phosphate strand is studded with billions of bases, arrayed in a line like a string of text. The text can have an infinite number of meanings, depending on how the bases are arranged. By this means, DNA stores the information necessary for building any protein in any species.

  The structure of DNA also suggested to Watson and Crick how it could be reproduced. They envisioned the strands being pulled apart, and a new strand being added to each. Building a new DNA strand would be simplified by the fact that each kind of base can bond to only one other kind. As a result, the new strands would be perfect counterparts.

  It was a beautiful idea, but it didn’t have much hard evidence going for it. Max Delbrück worried about what he called “the untwiddling problem.” Could a double helix be teased apart and transformed into two new DNA molecules without creating a tangled mess? Delbrück tried to answer the question but failed. Success finally came in 1957, to a graduate student and a postdoc at Caltech, Matthew Meselson and Frank Stahl. With the help of E. coli, they conducted what came to be known as the most beautiful experiment in biology.

  Meselson and Stahl realized that they could trace the replication of DNA by raising E. coli on a special diet. E. coli needs nitrogen to grow, because the element is part of every base of DNA. Normal nitrogen contains fourteen protons and fourteen neutrons, but lighter and heavier forms of nitrogen also exist, with fewer or more neutrons. Meselson and Stahl fed E. coli ammonia laced with heavy nitrogen in which each atom carried a fifteenth neutron. After the bacteria had reproduced for many generations, they extracted some DNA and spun it in a centrifuge. By measuring how far the DNA moved as it was spun, they could calculate its weight. They could see that the DNA from E. coli raised on heavy nitrogen was, as they had expected, heavier than DNA from normal E. coli.

  Meselson and Stahl then ran a second version of the experiment. They moved some of the heavy-nitrogen E. coli into a flask where they could feed on normal nitrogen, with only fourteen neutrons apiece. The bacteria had just enough time to divide once before Meselson and Stahl tossed their DNA in the centrifuge. If Watson and Crick were right about how DNA reproduced, Meselson and Stahl knew what to expect. Inside each microbe, the heavy strands would have been pulled apart, and new strands made from light nitrogen would have been added to them. The DNA in the new generation of E. coli would be half heavy, half light. It should form a band halfway between where the light and heavy forms did. And that was precisely what Meselson and Stahl saw.

  Watson and Crick might have built a beautiful model. But it took a beautiful experiment on E. coli for other scientists to believe it was also true.

  A UNIVERSAL CODE

  The discovery of E. coli’s sex life gave scientists a way to dissect a chromosome. It turned out that E. coli has a peculiar sort of sex, with one microbe casting out a kind of molecular grappling hook to reel in a partner. Its DNA moves into the other microbe over the course of an hour and a half. Élie Wollman and François Jacob, both at the Pasteur Institute in Paris, realized that they could break off this liaison. They mixed mutants together and let them mate for a short time before throwing them into a blender. Depending on how long the bacteria were allowed to mate, the recipient might or might not get a gene it needed to survive. By timing how long it took various genes to enter E. coli, Wollman and Jacob could create a genetic map. It turned out that E. coli’s genes are arrayed on a chromosome shaped in a circle.

  Scientists also discovered that along with its main chromosome E. coli carries extra ringlets of DNA, called plasmids. Plasmids carry genes of their own, some of which they use to replicate themselves. Some plasmids also carry genes that allow them to move from one microbe to another. E. coli K-12’s grappling hooks, for example, are encoded by genes on plasmids. Once the microbes are joined, a copy of the plasmid’s DNA is exchanged, along with some of the chromosome itself.

  As some scientists mapped E. coli’s genes, others tried to figure out how their codes are turned into proteins. At the Carnegie Institution in Washington, D.C., researchers fed E. coli radioactive amino acids, the building blocks of proteins. The amino acids ended up clustered around pellet-shaped structures scattered around the microbe, known as ribosomes. Loose amino acids went into the ribosomes, and full-fledged proteins came out. Somehow the instructions from E. coli’s DNA had to get to the ribosomes to tell them what proteins to make.

  It turned out that E. coli makes special messenger molecules for the job. The first ste
p in making a protein requires an enzyme to clamp on to a gene and crawl along its length. It builds a single-stranded version of the gene from RNA. This RNA can then move to a ribosome, delivering its genetic message.

  How a ribosome reads that message was far from clear, though. RNA, like DNA, is made of four different bases. Proteins are combinations of twenty amino acids. E. coli needs some kind of dictionary to translate instructions written in the language of genes into the language of proteins.

  In 1957, Francis Crick drafted what he imagined the dictionary might look like. Each amino acid was encoded by a string of three bases, known as a codon. Marshall Nirenberg and Heinrich Matthaei, two scientists at the National Institutes of Health, soon began an experiment to see if Crick’s dictionary was accurate. They ground up E. coli with a mortar and pestle and poured its innards into a series of test tubes. To each test tube they added a different type of amino acid. Then Nirenberg and Matthaei added the same codon to each tube: three copies of uracil (a base found in RNA but not in DNA). They waited to see if the codon would recognize one of the amino acids.

  In nineteen tubes nothing happened. The twentieth tube was filled with the amino acid phenylalanine, and only in that tube did new proteins form. Nirenberg and Matthaei had discovered the first entry in life’s dictionary: UUU equals phenylalanine. Over the next few years they and other scientists would decipher E. coli’s entire genetic code.

  Having deciphered the genetic code of a species for the first time, Nirenberg and his colleagues then compared E. coli to animals. They filled test tubes with the crushed cells of frogs and guinea pigs, and added codons of RNA to them. Both frogs and guinea pigs followed the same recipe for building proteins as E. coli had. In 1967, Nirenberg and his colleagues announced they had found “an essentially universal code.”