by Carl Zimmer
THE MYTH OF THE TANGLED SPAGHETTI
E. coli’s brainy tongue does not fit well into the traditional picture of bacteria as primitive, simple creatures. Well into the twentieth century, bacteria remained saddled with a reputation as relics of life’s earliest stages. They were supposedly nothing more than bags of enzymes with some loose DNA tossed in like a bowl of tangled spaghetti. “Higher” organisms, on the other hand—including animals, plants, fungi—were seen as having marvelously organized cells. They all keep their DNA neatly wound up around spool-shaped proteins and bundled together into chromosomes. The chromosomes are tucked into a nucleus. The cells have other compartments, in which they carry out other jobs, such as generating energy or putting the finishing touches on proteins. The cells themselves have structure, thanks to a skeletal network of fibers crisscrossing their girth.
The contrast between these two kinds of cells—sloppy and neat—seemed so stark in the mid-1900s that scientists used it to divide all of life into two great groups. All species that carried a nucleus were eukaryotes, meaning “true kernels” in Greek. All other species—including E. coli—were now prokaryotes. Before the kernel there were prokaryotes, primitive and disorganized. Only later did eukaryotes evolve, bringing order to the world.
There’s a kernel of truth to this story. The last common ancestor of all living things almost certainly didn’t have a nucleus. It probably looked vaguely like today’s prokaryotes. Eukaryotes split off from prokaryotes more than 3 billion years ago, and only later did they acquire a full-fledged nucleus and other distinctive features. But it is all too easy to see more differences between prokaryotes and eukaryotes than actually exist. The organization of eukaryotes jumps out at the eye. It is easy to see the chromosomes in a human cell, the intricately folded Golgi apparatus, the sausage-shaped mitochondria. The geography is obvious. But prokaryotes, it turns out, have a geography as well. They keep their molecules carefully organized, but scientists have only recently begun to discover the keys to that order.
Many of those keys were first discovered in E. coli. E. coli must grapple with several organizational nightmares in order to survive, but none so big as keeping its DNA in order. Its chromosome is a thousand times longer than the microbe itself. If it were packed carelessly into the microbe’s interior, its double helix structure would coil in on itself like twisted string, creating an awful snarl. It would be impossible for the microbe’s gene-reading enzymes to make head or tail of such a molecule.
There’s another reason why E. coli must take special care of its DNA: the molecule is exquisitely vulnerable to attack. As the microbe turns food into energy, its waste includes charged atoms, which can crash into DNA, creating nicks in the strands. Water molecules are attracted to nicks, where they rip the bonds between the two DNA strands, pulling the chromosome apart like a zipper.
Only in the past few years have scientists begun to see how E. coli organizes its DNA. Their experiments suggest that it folds its chromosome into hundreds of loops, held in place by tweezerlike proteins. Each loop twists in on itself, but the tweezers prevent the coiling from spreading to the rest of the chromosome. When E. coli needs to read a particular gene, a cluster of proteins moves to the loop where the gene resides. It pulls the two strands of DNA apart, allowing other proteins to slide along one of the strands and produce an RNA copy of the gene. Still other proteins keep the strands apart so that they won’t snarl and tangle during the copying. Once the RNA molecule has been built, the proteins close the strands of the DNA again. E. coli’s tweezers also make the damage from unzipping DNA easier to manage. When a nick appears in the DNA, only a single loop will come undone because the tweezers keep the damage from spreading farther. E. coli can then use repair enzymes to stitch up the wounded loop.
E. coli faces a far bigger challenge to its order when it reproduces. To reproduce, it must create a copy of its DNA, pull those chromosomes to either end of its interior, and slice itself in half. Yet E. coli can do all of that with almost perfect accuracy in as little as twenty minutes.
The first step in building a new E. coli—copying more than a million base pairs of DNA—begins when two dozen different kinds of enzymes swoop down on a single spot along E. coli’s chromosome. Some of them pull the two strands of DNA apart while others grip the strands to prevent them from twisting away or collapsing back on each other. Two squadrons of enzymes begin marching down each strand, grabbing loose molecules to build it a partner. The squadrons can add a thousand new bases to a DNA strand every second. They manage this speed despite running into heavy traffic along the way. Sometimes they encounter the sticky tweezers that keep DNA in order; scientists suspect that the tweezers must open to let the replication squadrons pass through, then close again. The squadrons also end up stuck behind other proteins that are slowly copying genes into RNA and must wait patiently until they finish up and fall away before racing off again. Despite these obstacles, the DNA-building squadrons are not just fast but awesomely accurate. In every 10 billion bases they add, they may leave just a single error behind.
As these enzymes race around E. coli’s DNA, two new chromosomes form and move to either end of the microbe. Although scientists have learned a great deal about how E. coli copies its DNA, they still debate how exactly the chromosomes move. Perhaps they are pulled, perhaps they are pushed. However they move, they remain tethered like two links in a chain. A special enzyme handles the final step of snipping them apart and sealing each back together. Once liberated, the chromosomes finish moving apart, and E. coli can begin to divide itself in two.
The microbe must slice itself precisely, in both space and time. If it starts dividing before its chromosomes have moved away, it will cut them into pieces. If it splits itself too far toward either end, one of its offspring will have a pair of chromosomes and the other will have none. These disasters almost never take place. E. coli nearly always divides itself almost precisely at its midpoint, and almost always after its two chromosomes are safely tucked away at either end.
A few types of proteins work together to create this precise dance. When E. coli is ready to divide, a protein called FtsZ begins to form a ring along the interior wall of the microbe at midcell. It attracts other proteins, which then begin to close the ring. Some proteins act like winches, helping to drag the chromosomes away from the closing ring. Others add extra membrane molecules to seal the ends of the two new microbes.
FtsZ proteins form their ring without consulting a map of the microbe, without measuring it with a ruler. Instead, it appears that FtsZ is forced by other proteins to form the ring at midcell. Another protein, called MinD, forms into spirals that grow along the inside wall of the microbe. The MinD spiral can scrape off any FtsZ it encounters attached to the wall. But the MinD spiral itself is fleeting. Another protein attaches to the back end of the spiral and pulls the MinD proteins off the wall one at a time.
A pattern emerges: the MinD spiral grows from one end toward the middle but falls apart before it gets there. The dislodged MinD proteins float around the cell and begin to form a new spiral at the other end. But as the MinD spiral grows toward the middle again, its back end gets destroyed once more. The MinD spiral bounces back and forth, taking about a minute to move from one end of the microbe to the other.
The bouncing MinD spiral scrapes away FtsZ from most of the cell. Only in the middle can FtsZ have any hope of forming the ring. And even there FtsZ is blocked most of the time by the chromosome and its attendant proteins. Only after the chromosome has been duplicated and the two copies are moving away from the middle is there enough room for FtsZ to take hold and start cutting the microbe in two.
E. coli may not have the obvious anatomy of a eukaryote cell, but it has a structure nevertheless. It is a geography of rhythms, a map of flux.
OFF THE CLIFF
E. coli caught Theodor Escherich’s eye thanks to its gift for multiplication—the way a single microbe can give rise to a massive, luxurious growth in a matter of hou
rs. If the bacteria Escherich discovered had continued to reproduce at that rapid rate, they would have soon filled his flasks with a solid microbial mass. In a few days they could have taken over the Earth. But E. coli did something else. It began to grow more slowly, and then, within a day, it stopped.
All living things could, in theory, take over the planet. But we do not wade through forests of puffballs or oceans of fleas. A species’ exponential growth quickly slams into the harsh reality of this finite world. As E. coli’s population grows denser, the bacteria use up oxygen faster than fresh supplies can arrive. Their waste builds up around them, turning toxic. This collision with reality can be fatal. As E. coli runs out of its essential nutrients, its ribosomes get sloppy, producing misshapen protein that attacks other molecules. The catastrophe can ripple out across the entire microbe. To continue to grow under such stress would be suicidal, like driving a car over a cliff.
Instead, E. coli slams on the brakes. In a matter of seconds it stops reading its genes and destroys all the proteins it’s in the midst of building. It enters a zombielike state called the stationary phase. The microbe begins to make proteins to defend against heat, acid, and other insults even as it stops making the enzymes necessary for feeding. To keep dangerous molecules from slipping through its membranes, E. coli closes off many of its pores. To protect its DNA, E. coli folds it into a kind of crystalline sandwich. All of these preparations demand a lot of energy, which the microbe can no longer get from food. So E. coli eats itself, dismantling some of its own energy-rich molecules. It even cannibalizes many of its ribosomes, so it can no longer make new proteins.
The threats faced by a starving E. coli are much like the ones our own cells face as we get old. Aging human cells suffer the same sorts of damage to their genes and ribosomes. People who suffer Alzheimer’s disease develop tangles of misshapen proteins in their brains—proteins that are deformed in much the same way some proteins in starving E. coli are deformed. Life not only grows and reproduces. It also decays.
Although humans and microbes face the same ravages of time, it’s the microbe that comes out the winner. If scientists pluck out a single E. coli in a stationary phase and put it in a flask of fresh broth, it will unpack its DNA, build new proteins, and resume its life with stately grace. Scientists can leave a colony of E. coli in a stationary phase for five years and still resurrect some viable microbes. We humans never get such a second chance.
Three
THE SYSTEM
TURNING ON THE GENE
ONE DAY IN JULY 1958, François Jacob squirmed in a Paris movie theater. His wife, Lise, could tell that an idea was struggling to come out. The two of them walked out of the theater and headed for home.
“I think I’ve just thought up something important,” François said to Lise.
“Tell!” she said.
Her husband believed, as he later wrote, that he had reached “the very essence of things.” He had gotten a glimpse of how genes work together to make life possible.
Jacob had been hoping for a moment like this for a long time. Originally trained as a surgeon, he had fled Paris when the Nazis swept across France. For the next four years he served in a medical company in the Allied campaigns, mostly in North Africa. Wounds from a bomb blast ended his plans of becoming a surgeon, and after the war he wandered Paris unsure of what to do with his life. Working in an antibiotics lab, Jacob became enchanted with scientific research. But he did not simply want to find a new drug. Jacob decided he would try to understand “the core of life.” In 1950, he joined a team of biologists at the Pasteur Institute who were toiling away on E. coli and other bacteria in the institute’s attic.
Jacob did not have a particular plan for his research when he ascended into the attic, but he ended up studying two examples of one major biological puzzle: why genes sometimes make proteins and sometimes don’t. For several years, Jacob investigated prophages, the viruses that disappear into their E. coli host, only to reappear generations later. Working with Élie Wollman, Jacob demonstrated that prophages actually insert their genes into E. coli’s own DNA. They allowed prophage-infected bacteria to mate with uninfected ones and then spun them apart. If the microbes stopped mating too soon, they could not transfer the prophage. The experiments revealed that the prophage consistently inserts itself in one spot in E. coli’s chromosome. The virus’s genes are nestled in among those of its host, and yet they remain silent for generations.
E. coli offered Jacob another opportunity to study genes that sometimes make proteins and sometimes don’t. To eat a particular kind of sugar, E. coli needs to make the right enzymes. In order to eat lactose, the sugar in milk, E. coli needs an enzyme called beta-galactosidase, which can cut lactose into pieces. Jacob’s colleague at the Pasteur Institute, Jacques Monod, found that if he fed E. coli glucose—a much better source of energy for E. coli than lactose—it made only a tiny amount of beta-galactosidase. If he added lactose to the bacteria, it still didn’t make much of the enzyme. Only after the bacteria had eaten all the glucose did it start to produce beta-galactosidase in earnest.
No one at the time had a good explanation for how genes in E. coli or its prophages could be quiet one moment and busy the next. Many scientists had assumed that cells simply churned out a steady supply of all their proteins all the time. To explain E. coli’s reaction to lactose, they suggested that the microbe actually made a steady stream of beta-galactosidase. Only when E. coli came into contact with lactose did the enzymes change their shape so that they could begin to break the sugar down.
Jacob, Monod, and their colleagues at the Pasteur Institute began a series of experiments to figure out the truth. They isolated mutant E. coli that failed to eat lactose in interesting ways. One mutant could not digest lactose, despite having a normal gene for beta-galactosidase. The scientists realized that E. coli used more than one gene to eat lactose. One of those genes encoded a channel in the microbe’s membranes that could suck in the sugar.
Strangest of all the mutants Jacob and Monod discovered were ones that produced beta-galactosidase and permease all the time, regardless of whether there was any lactose to digest. The scientists reasoned that E. coli carries some other molecule that normally prevents the genes for beta-galactosidase and permease from becoming active. It became known as the repressor. But Jacob and his colleagues had not been able to say how the repressor keeps genes quiet.
In the darkness of the Paris movie theater, Jacob hit on an answer. The repressor is a protein that clamps on to E. coli’s DNA, blocking the production of proteins from the genes for beta-galactosidase and the other genes involved in feeding on lactose. A signal, like a switch on a circuit, causes the repressor to stop shutting down the genes.
Another similar repressor might keep the genes of prophages silent as well, Jacob thought. Perhaps these circuits are common in all living things. “I no longer feel mediocre or even mortal,” he wrote.
But when François tried to sketch out his ideas for his wife, he was disappointed.
“You’ve already told me that,” Lise said. “It’s been known for a long time, hasn’t it?”
Jacob’s idea was so elegantly simple that it seemed obvious to anyone other than a biologist. Yet it represented a new way of thinking about life. Genes do not work in isolation. They work in circuits. Over the next few weeks, Jacob tried to explain his idea to his fellow biologists, without arousing much interest. It was not until Monod returned to Paris in the fall that Jacob found a receptive audience. The two of them began to draw circuit diagrams on a blackboard, with arrows running from inputs to outputs.
In the fall of 1958, Monod and Jacob launched a new series of experiments to test Jacob’s circuit hypothesis. The experiments produced the results Jacob expected, but it would take years of research by other scientists to work out many of the details. The lactose-digesting genes are lined up next to each other on E. coli’s chromosome. The repressor protein clamps down on a stretch of DNA at the front end of the genes
, where it blocks the path of gene-reading enzymes. With the repressor in place, E. coli cannot feed on lactose.
The best way to get the repressor away from the lactose-digesting genes is to give E. coli some lactose. Once inside the microbe, the sugar changes shape so that it can grab the repressor. It drags the repressor off E. coli’s DNA, allowing the gene-reading enzymes to make their way through the lactose-digesting genes. E. coli can then make the enzymes it needs to feed on lactose.
But E. coli needs a second signal to ramp up its production of beta-galactosidase: it needs to know that its supply of glucose has run out. The signal is a protein called CRP, which builds up inside E. coli when the microbe begins to starve. CRP grabs on to another stretch of DNA, next to the lactose-digesting genes. It bends the DNA to attract the gene-reading enzymes. Once CRP clamps on, E. coli begins producing lactose-digesting enzymes at top speed. If the repressor is an off switch, CRP is an on switch.
Jacob and his colleagues christened the lactose-digesting genes the lac operon, operon meaning a set of genes that are all regulated by the same switches. As Jacob suspected, operons represent a common theme in the way genes work. Hundreds of E. coli’s genes are arrayed in operons, each controlled by switches. Some operons carry several switches, all of which must be thrown for them to make proteins. A single protein may be able to trigger a cascade of genes, switching on genes for making more switches, allowing E. coli to make hundreds of new kinds of proteins.
