A Blog by Carl Zimmer

Frankenstein Can’t Come Out And Play Today

In the standard Frankenstein story, a scientist creates an unnatural monster that breaks out of the lab and runs amok. But why should unnatural make something unstoppable? The contrary is possible, too. Imagine a different story: Frankenstein’s monster escapes, realizes that it can’t survive in the outside world, and retreats back to the lab. This story line may not make for a satisfying movie, but it might be a good goal for real life.

The fear of the unstoppable unnatural has been with us ever since scientists began moving genes between species in the 1970s. In a 1973 experiment, researchers transferred a gene from a frog into Escherichia coli. The gut microbe used the frog gene to make a frog protein.

It wasn’t long before researchers figured out how to use genetic engineering to turn microbes into factories. When scientists inserted the gene for human insulin into E. coli, the bacteria were able to manufacture a drug that had previously been harvested from cow pancreases. E. coli became the workhorse of biotechnology, spewing out drugs, vitamins, and industrial materials. (For more on E. coli’s strange yet significant history, see my book Microcosm.)

At first, the prospect of foreign genes in E. coli was terrifying. Some critics warned that insulin-producing bacteria would escape from fermenting tanks, get into people’s bodies, and cause an epidemic of diabetic comas. That never happened, probably because insulin does E. coli no good at all. The human gene is a burden to the microbe, draining off energy and resources it could use to grow.

Nevertheless, it was conceivable that some other creation might turn out to be dangerous. The scientific community responded by laying down guidelines for working with genetically engineered creatures. Most of the guidelines involved creating physical barriers to keep organisms from escaping factories or labs. But scientists have also created biological barriers, by changing the creatures themselves to make it hard for them to survive outside the lab.

For example, scientists who study the plague engineered a safe strain of the bacteria Yersinia pestis that they could work with in their labs. Y. pestis needs iron to survive, and it uses special molecules to scavenge the element from our bodies. To make a safe strain, scientists shut down some of the iron-scavenging genes in the bacteria. The bacteria could still grow in a flask if they got a rich supply of iron. But inside people, where iron is scarce, they would starve.

At least that was the plan. In 2009, a University of Chicago scientist named Malcolm Casabadan got infected by a lab strain of Y. pestis and died of the plague. Unfortunately, neither he nor anyone else knew that he suffered from a genetic disorder called hemochromatosis, which caused him to accumulate high levels of iron in his blood. Investigators concluded that his body probably served the same role as an iron-rich lab flask. Inside him, the hobbled bacteria could grow.

Casabadan didn’t die because the engineered Y. pestis that infected him was unnatural. The problem was that it wasn’t unnatural enough. That is, it could still find a place in the natural world where it could thrive. Some scientists think a better safeguard would be to create life that is fundamentally unnatural–in other words, that cannot possibly survive without our help, because the natural world is alien to it.

Fortunately, this goal does not require scientists to create an utterly alien form of life, complete with some alternate form of heredity to take the place of DNA. Scientists can take advantage of the fact that all living things on Earth are incredibly similar, chemically speaking.

All living things build proteins from about twenty building blocks, called amino acids. By combining the amino acids in different sequences, life can produce a vast range of proteins. But there are hundreds of other kinds of amino acids in nature, and scientists have created many others that are never found in nature.

In theory, living things should be able to use these amino acids to build their proteins, too. They don’t, however, because all living things share a nearly identical code for translating the information in their genes into proteins.

Genes are made of a different set of building blocks, called bases. To build a protein, a cell reads three bases at a time (a codon) and then selects a corresponding amino acid. If a base called guanine appears three times in a row in a gene, for example, a cell will pick out an amino acid called glycine.

For the most part, all living things rely on the same genetic code. That’s why E. coli that acquires a human insulin gene makes insulin, too, instead of collagen or hemoglobin. It’s also why viruses can invade our bodies and use their own genes and proteins to hijack our cells to make new viruses. We all use the same language, and so our programming can be hacked.

About a decade ago, Farren Isaacs, then a postdoctoral researcher in the lab of George Church at Harvard, started tinkering with the genetic code, trying to change the rules. Last year, he and his colleagues reported that they had reassigned one codon in E. coli to an artificial amino acid. (It’s known as p-acetyl-L-phenylalanine, or pAcF for short.) They sprinkled the new codon across the genome of the bacteria, which then made some of its proteins using pAcF.

The recoding had a remarkable effect on the bacteria: they became immune to a virus that specializes on infecting E. coli. By changing the code, the scientists made the bacteria harder to hack. (I wrote about this work in more detail in 2013 in Nautilus.)

While these bacteria could make unnatural proteins, they didn’t depend on the proteins for survival. Growing on a regular diet of natural compounds, they could still thrive. Isaacs went on to Yale, where he continued his research, as have Church and his colleagues at Harvard. And in Nature, each team has now published the next logical experiment in this line of research. Each group has recoded E. coli so that it now depends on an artificial amino acid. Without it, the bacteria cannot build essential proteins, and they die.

Because these bacteria can’t find these artificial amino acids in the outside world, the scientists reasoned that they couldn’t survive on their own. To test that possibility, they transferred the recoded microbes to dishes where they got an ordinary diet. In all but one trial, the scientists found no evidence of the recoded bacteria surviving without their essential amino acid. And in the one trial where they did survive, they barely clung to life, easily outcompeted by ordinary E. coli.

To further reduce the odds of the bacteria surviving on their own, the researchers are now building in other features. Church’s group, for example, is reassigning other codons to other unnatural amino acids, further reducing the odds even more that mutations can rescue the bacteria. Ultimately, they hope to push these creatures into an alternate biological universe, walled off from our own.

What could we do with such creatures? We could potentially use them not just in protected labs, but in the outside world. They might clean up oil spills, for example, surviving as long as we supplied the artificial amino acids they needed to build proteins. When their job was done, we could shut off the supply and they’d die. It’s conceivable that scientists could recode plants as well, creating crops that could only grow with our help.

When I talked to other experts about this research, they were pretty impressed. “It’s a landmark moment,” Tom Ellis of Imperial College told me. “I think that it will have an immediate positive impact,” said Karmella Haynes of Arizona State University.

But when I talked to bioethicist Paul Wolpe of Emory University, he thought it unlikely that we’re home-free when it comes to risks from genetic engineering. In the past, people have introduced animal and plant species to new places with the best of intentions, only to see them cause unanticipated harm. “While I applaud these first steps, caution should be the guide here,” Wolpe said.

I expect that most of the conversations these odd bugs will inspire will be about practical matters–about making valuable stuff and avoiding risks to our health. But this research speaks to something deeper. When we try to figure out the definition of life, we look around at the life we know and look for the features all living things have in common. But scientists have also wondered if life as we know it may take up a tiny portion of the space of all possible forms that life can take.

These altered bacteria tell us that suspicion is likely true. With a few years’ work, they’ve made creatures that are probably unlike anything that ever lived on Earth. And within their universe–the universe of artificial amino acids that exists in Massachusetts, Connecticut, and a few other places on Earth–they are as alive as we are.

A Blog by Carl Zimmer

Can the Microbiome Mutiny?

It’s an ugly fact of life that getting old means getting infections. Old people get attacked more by pathogens, and the damage that these germs cause can speed up the aging process, leading to even more infections. The standard explanation for this vulnerability is that the immune system falters in old age, opening an opportunity for pathogens to invade. But in the journal Biology Direct, Viktor Muller of Eotvos Unversity and his colleagues propose that something else is also going on in the aging body. Maybe the microbiome senses that its host is in bad shape and rises up in rebellion. The scientists call their idea “the Microbiome Mutiny Hypothesis.”

It may seem like a strange notion, but several lines of evidence suggest it’s worth considering. First of all, a lot of the pathogens that attack the elderly come from within. They grow quietly and harmless for decades inside people’s bodies and then switch over to causing dangerous infections later in life. The answer to why old people get infections must address why harmless bacteria turn bad in old age.

To understand this turn, we have to abandon any strict division between “good” germs and “bad” ones. For the germs themselves, these are just two ends of a seamless spectrum. Depending on how they use their host, microbes may cause no harm, a little, or a lot. And the virulence of a microbe–the amount of harm it causes–can itself evolve over time. Under some conditions, natural selection may favor gentle handling. But in other situations, causing deadly disease may be the winning strategy.

A lot of factors go into determining which strategy will be a winner for a given microbe. For some microbes, the best way to multiply may be ripping open host cells and feasting on their contents. This may kill a lot of their hosts, but that may not matter to the microbes, since they can escape to a new host–say, by causing diarrhea that contaminates a water supply. But in other conditions, killing a host may be a bad long-term strategy–if, for example, the odds are low that a microbe will get from one host to the next.

It’s even possible for organisms to evolve the ability to switch between these strategies, using the best strategy for different environments. In a 2013 experiment, Oxford scientists observed this switch evolve before their eyes.

They studied phages, which are viruses that infect bacteria. A phage invades a bacterium and makes new copies of itself. The bacterium eventually ruptures, spilling out the next generation of phages.

The scientists reared phages under unusual conditions–they mixed together a very high concentration of phages with bacteria. As a result, each microbe tended to get infected by more than one phage. The phages would then make copies of themselves at different rates. When the microbe ruptures, out would come a mixture of phages. The faster breeders dominated over the slower ones.

The scientists let the phages evolve in these conditions for 50 days. When they were done, the phages could now adjust their speed. If they found themselves alone in a host cell, they grew slowly. But if they sensed other phages in the cell, they sped up, so as to outcompete their rivals. As a result, their host died faster.

Muller and his colleagues propose that some of the microbes that live in our bodies can also switch from benign to deadly for similar reasons. While we’re healthy, they growing slowly, causing us no harm. But as we approach the end of our lives, the microbiome shifts to a more aggressive strategy.

“Killing the goose that lays the golden eggs might not be such a bad idea if the goose is going to die soon, anyway,” the scientists write.

There’s good evidence that microbial residents can eavesdrop on our health. A pathogen called Pseudomonas aeruginosa, for example, can sense certain molecules our brains release in response to stress. They respond by unleashing a toxin that help them grow–while also damaging our lungs.

Muller and his colleagues offer some ways to test their hypothesis. If they’re right, then infections in old age aren’t just the result of a slack immune system. Instead, bacteria and viruses sense a changed environment and respond by making new molecules, which they use to grow aggressively and cause harm. If scientists disable these molecules, then the pathogens should become tame again.

It would be interesting to see the Microbiome Mutiny hypothesis put to such a test. Conceivably, scientists could someday turn the test into a treatment. Rather than blasting the elderly with broad-spectrum antibiotics, doctors could just disarm the mutiny.

A Blog by Carl Zimmer

Antisocial Medicine

One of the biggest surprises to come out of microbiology in recent years is that bacteria have a social life. Rather than existing as lonely, autonomous creatures, bacteria live in communities, in which they cooperate, compete, and communicate. In the January issue of Scientific American, I have a feature about how some scientists are trying to translate their growing understanding of the social life of bacteria into a new kind of medicine. By preventing microbes from cooperating, we may be able to bring infections to a halt. Better yet, this kind of antisocial medicine may even be able to avoid–or at least slow down–the evolution of drug resistance in bacteria.

Here’s the introduction to my piece:

A Blog by Carl Zimmer

The Central Park Zoo Hidden From View

In 2003, an army of 350 scientists and volunteers swept out across Central Park. Their mission, called a BioBlitz, was to find as many species as possible over the course of 24 hours. At the end of the day, they had compiled a catalog of 836 species of plants and animals.

It’s impressive that Central Park–an 843-acre island in an ocean of Manhattan concrete–can play host to so many species. But that’s hardly a complete inventory of the biodiversity of the place. Along with its plants and animals, Central Park is home to invisible wildlife too.

The ground swarms with invertebrates, fungi, and a wealth of microbes. This underground diversity–especially the microbes–has been very hard to explore, not just in Central Park but around the world. For one thing, you have to dig. For another, you can’t usually can’t tell the species apart with the naked eye. It’s possible to distinguish between the five species of turtles in the Central Park’s Turtle Pond just by looking at them. But if you dig up a patch of dirt by the pond and look at the bacteria it contains, they might well look like just a bunch of rods and spheres. The diversity of microbes is instead a matter of chemistry. They have evolved a staggering range of ways to break down molecules and grow on them.

In recent years, scientists have developed powerful new tools for measuring that diversity. Rather than looking at feathers or stripes, they look at DNA.

Each dot represents a place where scientists scooped up dirt to look for DNA. From Ramirez et al 2014
Each dot represents a place where scientists scooped up dirt to look for DNA. From Ramirez et al 2014

A team of researchers has now used this approach to carry out a sort of MicroBioBlitz in Central Park. They marched their way systematically through the park, and every fifty 50 yards or so, they stopped, bent down, and scooped up some dirt. All told, they gathered 596 scoops. Back at their lab, they threw out everything from those scoops except for the DNA. And then they plucked out just one particular stretch of that DNA. To be more precise, they plucked out different versions of that stretch, each carried by a different species.

The scientists then looked at the sequence of each of those versions. In some cases, the DNA turned out to be identical to a known species, or nearly so. In other cases, the sequence was very different. A peculiar sequence of DNA told the scientists that it came from a species that’s new to science.

The results were, to be blunt, pretty insane. All told, the scientists identified over 167,000 species.* That’s about thirty times more species that all the mammals on Earth–everything from fruit bats to walruses, from musk ox to marmosets.

Diversity. Blue: known phylotypes (similar to species). Gray: unknown. Ramirez et al 2014
Diversity. Blue: known phylotypes (similar to species). Gray: unknown. Ramirez et al 2014

This chart shows just how ignorant we are of the life even in Central Park. In each chart, the blue bar shows number of species in the park that are already known to science. The gray bar shows the ones that don’t match anything we know of. The top chart shows bacteria and another group of microbes called archaea. (Archaea are single-celled microbes that, like bacteria, keep their DNA floating loose inside their cell.) The bottom chart shows our own branch of the tree of life, the eukaryotes. Eukaryotes include not just animals and plants, but fungi, amoebae, and other protozoans. In both cases, the unknown dwarfs the known.

The more you drill down into these numbers, the more insane they get. Even the “known” species in these charts aren’t very well known at all. For the most part, scientists have never seen the organisms from which they come. They’ve only fished out the same DNA segment from another sample.

The geography of Central Park’s microbes is also mind-boggling. It’s not as if all 167,000 species were present in every sample of the soil. Instead, different species showed up in different places. On average, each sample had about 7000 bacteria and archaea, and 1250 eukaryote species. When the scientists compared the diversity in the samples, they found that any pair picked at random shared only 19.3% of their bacterial and archaeal species, and just 13.5% of their eukaryote species. Even neighboring sites were no more similar to each other than ones on opposite ends of the park. And as the scientists scooped up more dirt, they kept finding more species. So the true number of species in Central Park is probably far higher. (It’s also worth noting that there are probably a lot of other microbes living on the trees, in the ponds, and in other places in the park the scientists didn’t even touch.)

Now, it might have turned out that many of the species in Central Park were closely related to each other. There are many, many species of beetles, for example. Perhaps Central Park only had microbial versions of beetle, and no microbial ants or termites.

That’s not how things turned out. Central Park has the microbial ants and the termites, too. In fact, its microbes span much of the tree of life. You’d get a similar span of species if you took the same number of soil samples from around the world, from jungles to deserts.

Some of the results of the study may be the result of Central Park’s own peculiar history. Its soil contains a high level of species that are considered potential human pathogens. In their report, the scientists hasten to note that this doesn’t mean you’re especially at risk of getting sick in the park. But it may be a sign of the presence of lots of people nearby.

For the most part, though, Central Park may just be a typical plot of microbial habitat. Soil, as a rule, is just rife with microbial richness on a scale we can barely understand. It’s loaded with dying plant matter and the remains of dead animals. Its particles and tunnels and other features make soil an incredibly complex environment, where microbes can specialize in all sorts of ways of making a living. It may turn out that the only thing that makes Central Park unusual is the many holes that scientists have dug there.

*I’m going to use the term species here for convenience. Determining exactly what a species is can be tricky for microbes, and the scientists only presented distinct lineages that might or might not be separate species–what they call phylotypes. Here’s a piece I wrote on why this whole problem is so challenging.

A Blog by Carl Zimmer

Taking the Yuck Out of Microbiome Medicine

I can still remember the shock I felt when I heard about fecal microbiota transplants for the first time. It is not the sort of thing you forget.

At a microbiology conference, a scientist was giving a lecture about the microbiome–the microbes that live harmlessly inside of us. She described one unusual case she was involved in where a doctor named Alexander Khoruts used the microbiome to save a patient’s life. The patient had taken antibiotics for a lung infection. While the drugs cleared that infection, they  also disrupted the ecology of her gut, allowing a life-threatening species of bacteria called Clostridium difficile to take over. The pathogen was causing horrific levels of diarrhea. Khoruts couldn’t stop it, because it was resistant to every antibiotic he tried.

So Khoruts decided to use an obscure method: the fecal transplant. He took some stool from the patient’s husband, mixed it with water, and delivered it to her large intestines like a suppository. In a matter of days she was recovering.

Since I first heard about these transplants in 2010, they’ve hit the big time. Last year, a team of Danish and Finnish doctors reported clinical trials in which the transplants 94 percent effective against C. difficile. It appears that some species in the transplant from a healthy gut will grow quickly and outcompete the pathogen, returning a sick person’s intestines to its former state. Scientists have been exploring using fecal transplants for other disorders of the gut, along with conditions beyond the gut, such as diabetes and obesity.

But there are many obstacles left to putting fecal transplants into widespread practice. For one thing, the FDA is very cautious with this kind of living medicine. For another thing, fecal transplants are conceptually crude. Doctors simply give a patient a random sample of hundreds of different species from a healthy person’s gut, assuming that at least some of them will restore the patient to health. When the patients get better, they can’t say precisely why.

And then there is the yuck factor. In 2012, scientists conducting a survey about attitudes towards feccal transplants, politely summed up the problem this way: “patients recognize the inherently unappealing nature of FMT.”

But now there’s a potentially promising development in the quest to harness the microbiome. At an American Gastroenterological Association conference in Chicago this weekend, researchers will be describing how they cured C. difficile not with a fecal transplant, but with a pill full of bacterial spores.

The pill is the work of a small Boston-area company called Seres Health. They came up with a combination of certain harmless microbe species that naturally live in our gut. These species  all form spores, which are rugged enough to survive inside a pill. Once they reach the warm refuge of the gut, they pop out of their spores and multiply. In previous studies, Seres researchers showed they could treat C. difficile infection effectively in mice and hamsters. (Technology Review described the company’s efforts in this article from last December.)

Recently, doctors at the Mayo Clinic, the Miriam Hospital in Providence, and Massachusetts General Hospital ran a clinical trial on people to see if the pills from Seres were safe and effective. They gave the pill to fifteen people. The results were striking: the overall cure rate was 100 percent. (The detailed abstract pdf is here.)

I contacted Khoruts to see what he thought of the study. “It looks very promising,” he told me.

But Khoruts also raised a few caveats. He pointed out that the authors excluded very sick patients from the study because of the risk of adverse events. So the 100 percent cure rate might be higher than it would be in the real world.

Khoruts also pointed out a few potential problems with taking a pill full of spores as opposed to getting stool from a donor. Scaling it up to industrial production will require making sure that the factory stocks don’t get contaminated by strains of bacteria that would harm patients, for example.

In those factory stocks, Khoruts pointed out, the microbes will continue to evolve and adapt to their surroundings. If they become too well adapted to life in a factory, they may not do as well inside of people’s bodies.

Nor does the initial report on these pills actually explain how these particular species are conquering C. difficile. I’m sure that the fifteen people who were cured of these awful bugs aren’t clamoring for a detailed  mechanistic explanation of what happened when they swallowed the pills.

But if scientists are going to rationally design microbiome treatments for a lot of different conditions, they’re going to have to open this microbial black box.


A Blog by Carl Zimmer

The Zoo In the Mouth

There’s a philosophical quandary breeding in your mouth.

Fantail. Photo by Jim Gifford via Flickr/Creative Commons http://flic.kr/p/4pkQSb
Fantail. Photo by Jim Gifford via Flickr/Creative Commons http://flic.kr/p/4pkQSb

Ever since Aristotle, philosophers and scientists have searched for the right way to classify living things. We call living things with feathers “birds,” but we can also divide birds up into smaller groups, like pigeons and storks. We can drill down even further, to different species of pigeons. But it doesn’t feel right to classify birds all the way down to every individual feathered creature on Earth. The fundamental unit of life’s biodiversity has long been the species. Charles Darwin named his book The Origin of Species for a reason.

Darwin threw older notions of species into doubt, challenging the idea that they were fixed since creation or only able to change slightly over time. adjusted over time. In fact, old species give rise to new ones like shoots from a tree. Darwin concluded that where we choose to draw a line to mark the boundary of a species is a matter of convenience.

While Darwin created the modern foundation for biology with his theory, biologists didn’t abandon the word species. It’s still a helpful term for describing how life evolves–even if it’s left scientists arguing about which definition can hold up best against the churning complexity of evolution.

In the 1940s, the biologist Ernst Mayr declared that “species are groups of interbreeding natural populations that are reproductively isolated from other such groups.” In other words, it’s all about sex.

Other scientists didn’t like Mayr’s definition, because reproductive isolation is a squishy term. Stick a squirrel on a remote island, and it can’t reproduce with its fellow squirrels on the mainland. Have you made a new species? Biologists now know that populations gradually become more and more reproductively isolated over thousands or millions of years. Do we have to wait till they’re totally isolated before declaring the two populations new species?

Some scientists suggested that we think of a species in terms as a branch of the tree of life–as the smallest group of organisms that all descend from a common ancestor, and which we can distinguish from other groups. (I wrote a feature a few years ago about these debates over species in Scientific American, which you can read here.)

These definitions work tolerably well for animals and plants. But they’ve turned out to be pretty lousy for microbes. And that’s left scientists in a quandary, because it’s now clear that the vast majority of the genetic diversity on Earth belongs to bacteria, viruses, and other life forms invisible to the naked eye.

bacteria panorama
Jane Hurd/National Geographic
Artwork by Jane Hurd/National Geographic

When microbiologists started to study microbes in the nineteenth century, they went about their business like zoologists or botanists. They’d describe a microbe based on its appearance, what it fed on, and other features they could study in their laboratory. If it seemed different from other microbes that had been previously described, they’d give it an official species name, like Escherichia coli. When later microbiologists would come across a microbe–say, in the blood of a sick patient–they’d identify its species by systematically inspecting its traits, in much the same way a bird-watcher would look at a bird’s plumage and listen to its song.

In the late 1900s, microbiologists were abandoning this method in favor of a new one: identifying species by their DNA. Zoologists and botanists were making the shift, too, but microbiologists were in for a particularly big shock. The diversity of microbes turned out to be mind-bogglingly exuberant. Microbes that had been considered almost identical before the age of DNA turned out to be more genetically different from each other than maple trees and penguins.

As I describe in my book Microcosm, a single species–E. coli–turned out to contain multitudes, from beneficial bacteria that can heal a baby’s dysfunctional gut to all sorts of pathogens that make us sick in frightfully different ways, from making our cells spill their contents to slithering into them like tapeworms.

Making matters even more confusing was the discovery that microbes don’t simply pass down their genes to their offspring the way we do. They are constantly swapping genes with each other, with little respect for any so-called species barrier. Scientists sometimes try to represent this gene traffic by drawing a web of life instead of a tree of life.

Trying to fit microbes into a definition of species based on animals and plants is a bit like trying to herd a flock of flamingos into a school bus. Mayr’s definition is not much help, since microbes don’t have sex like animals do. The branch-of-the-tree-of-life definition is not much help, either.  Theoretically, any microbe that picks up a mutation is distinct from other microbes and should be considered its own species. And if it passes that particular mutation to a distantly related microbe, the whole tree-based approach collapses.

The situation is such a mess that some microbiologists have given up on species altogether. In 2012, W. Ford Doolittle wrote an awesomely titled commentary called, “How Bacterial Species Form and Why They Don’t Exist.”

This confusion can make life hard for microbiologists when reporters call. Like many other journalists, I’ve been reporting a lot on the remarkable explorations scientists have been making of the human microbiome–the collection of germs that call us home.

I’ve repeatedly asked, “So, how many species are in us?”

And each time, the microbiologists I talked to would squirm out of a real answer.

As deeply uncomfortable as microbiologists may be with the whole idea of species, they still need some way to measure the diversity of, say, the microbiome. Instead of species, they typically end up using something species-ish.

When microbiologists were shifting to using DNA, they chose one gene as a way to distinguish different kinds of microbes from each other. The gene, found in all living things, is called 16S rRNA. Microbiologists compared the variations in 16S rRNA genes from individual microbes that belonged to a single species–that is, a species as microbiologists would traditionally identify it.

They found that the gene varied by up to three percent. So they decided that if two microbes were 97% identical in this one gene, they belonged to the same group. Instead of a species, they called this group an operational taxonomic unit.

But it eventually turned out that this 97% cut-off was wrong. A good example of how it can lead scientists astray is the case of two species of bacteria that live on our bodies, Streptococcus pneumoniae and Streptococcus mitis. Back in the barbaric pre-sequencing days, microbiologists decided they were two separate species, deserving of two separate names. When you look at how these two microbes make a living, that decision makes ample sense. S. pneumoniae causes pneumonia, while S. mitis can live harmlessly on teeth.

Yet the 16S rRNA sequences of these two “species” are over 97% identical. In fact, they’re 99% similar. While their 16S rRNA genes have remained nearly identical, some of their other genes have veered off in different directions.

In the face of all this delicious confusion, microbiologists need a more powerful way to sort microbes into groups. But there’s been a limit to how fine they can draw their distinctions. Even the best DNA sequencers are not perfect. Here and there, they will make an error in reading a microbe’s genes. When microbiologists are distinguishing different kinds of bacteria with 97% thresholds, those little errors don’t matter. But if microbiologists want to distinguish microbes by tiny differences, making those errors would be akin to mistaking a wolf for a chihuahua.

A. Murat Eren, a microbial ecologist at Marine Biological Laboratory in Woods Hole, Massachusetts, and his colleagues have come up with a powerful new way to chart the diversity of microbes. They call it oligotyping. They line up the 16S rRNA genes from a group of microbes, and then they look for spots in the gene that have the most differences from one microbe to another. The scientists narrow down their search to the fewest spots that can distinguish the largest number of groups, which they call oligotypes. This approach allows them to avoid depending on tiny, error-prone differences that might only be present on a single microbe’s gene.

Photo by Darwin Bell, via Creative Commons https://flic.kr/p/eHmGJ
Photo by Darwin Bell, via Creative Commons https://flic.kr/p/eHmGJ

Recently, Eren and his colleagues tested out oligotyping on the microbiome. They searched the DNA sequences collected by the Human Microbiome Project, a massive survey of microbial genes from over 200 people. Eren and his colleagues limited their search to just the microbes from people’s mouths. That still left them with over 10 million sequences to sift through.

All told, they found at least 362 oligoytpes of bacteria.* Most of their oligotypes belonged to conventional species, but often the scientists found that a single so-called species contained several distinct oligotypes.

These oligotypes are not just minor variations on a theme. They lead different lives. Our mouths are like jungles, with lots of ecological niches. The environment on a tooth is very different from that on the tongue, which is different in turn from the gums. Eren and his colleagues found that each oligotype dwelled in just one or a few parts of the mouth–and did so consistently from one person to the next.  Some oligotypes only live on teeth, for example, while others live mostly on the tongue. The evidence for all this diversity already existed in the Human Microbiome Project’s databases, but until now it was hiding in plain sight.

There are plenty of solid, practical reasons for inventing a better way to measure the diversity of microbes–even just in our mouths. Some bacteria that live there can potentially make us sick–not just by causing cavities, but by causing diseases in other parts of the body, such as heart disease. Old-fashioned definitions of species can lead scientists to overlook these pathogens, lumping them into the same group as harmless microbes.

But this research is also important for a more basic reason–because it helps us to wrap our minds around the staggering diversity of life on Earth. One way to start appreciating how vast that diversity is to just open our mouths.

*The paper will be posted at some point this week by the journal. The reference is: Eren et al., “Oligotyping analysis of the human oral microbiome,” PNAS. www.pnas.org/cgi/doi/10.1073/pnas.1409644111

A Blog by Carl Zimmer

Evolution Hidden in Plain Sight

It’s hard to believe that Escherichia coli could have any secrets left.

For over a century, scientists have picked the microbe apart–sequencing its genes, cracking its genetic code, running experiments on its metabolism, earning Nobel Prizes off of it, and turning it into, arguably, the most-studied organism in history.

But as deep as scientists dive, they have yet to touch bottom. That’s in part because Escherichia coli is not fixed. It continues to evolve, and even in the most carefully controlled experiments, evolution leaves behind a complicated history.

Twenty-five years ago, Richard Lenski used a single microbe to seed twelve lines of bacteria. He fed each line a meager diet of glucose, and the bacteria have been adapting to this existence in his lab at Michigan State University ever since. (Here I’ve gathered together a few pieces I’ve written over the years about the 58,000-generations-and-counting Long-Term E. coli Evolution Experiment.)

In 2003, Lenski’s team realized that something utterly unexpected happened. One of the hallmarks of Escherichia coli as a species is that when there’s oxygen around, it can’t feed on a compound called citrate. But one day a flask turned cloudy with an explosion of E. coli that were doing just that. The change was so profound that it may mean these bacteria had evolved into a new species.

For the past 11 years, the scientists have been trying to figure out how the bacteria gained this ability to feed on citrate. Thankfully, Lenski decided at the outset of the experiment to freeze some of the evolving bacteria every 500 generations. As a result, he and his colleagues can resurrect ancestral microbes, sequence their genomes, and probe their biology for clues.

After sifting through the frozen history of citrate feeding for a couple years, the scientists discovered an important step in this evolution. It involves a gene called citT.

The citT gene encodes a protein that lets E. coli feed on citrate when oxygen levels get low. The protein sits in the microbe’s membrane and helps pull in citrate molecules from the environment. As it draws citrate in, however, it pumps another molecule–succinate–out. The pushing and pulling of these two molecules helps keep the chemistry of the cell in balance.

A small segment of DNA next to citT serves as a switch. If the microbe detects oxygen, a protein grabs onto the segment and shuts citT down. The microbe no longer feeds on citrate, instead feeding on better sources of energy, such as glucose.

The scientists found that around generation 31,500, a microbe that was copying its DNA in order to divide made a big mistake. It accidentally made an extra copy of a segment of DNA. That segment, it just so happened, contained citT. The microbe inserted the copy next to the original one, so that one of its daughter cells now had two copies of citT.

This sort of gene duplication happens from time to time in all living things. Human DNA regularly gets copied, too. And it can lead to important changes, because the two copies can start to do two different things. And that’s what happened to the E. coli. In Lenski’s experiment, the new copy of citT ended up near a new bit of DNA that controlled genes in a different way. Instead of shutting down genes in the presence of oxygen, it keeps them always switched on. Thanks to this mutation to citT, the bacteria could start feeding on citrate in Lenski’s oxygen-rich lab.

But the scientists found that this mutation was only one part of the story. The citT mutation allowed the bacteria to grow on citrate, but only slowly. Only after 1500 more generations had passed did the citrate-feeding bacteria begin growing quickly enough to dominate their flask.

During those 1500 generations, the scientists found, the bacteria made more copying mistakes, turning the new citT gene into four duplicates. Those extra copies enabled the bacteria to make more citrate-pulling proteins. But other mutations arose from generation 31,500 to 33,000, and the scientists had no way of knowing if they were important as well.

The story also turned out to have an earlier chapter. The scientists went back through the frozen archive to the very beginning and thawed out some microbial ancestors. They inserted the evolved citT genes into the ancestors, and found that the microbes could not feed on citrate. So the evolved citT gene alone was not enough to turn a microbe into a citrate-feeder.

The scientists did the same thing to bacteria from generation 20,000 and got a different result. When those more evolved bacteria got the citT gene, they could feed on citrate. Results like these suggested that early in the evolution of the bacteria, they picked up mutations that would later make it possible for the citT mutation to turn them into citrate feeders.

So, to recap: the scientists now had a story in three parts. Up to 31,500 generations, it was a story of groundwork mutations. Then came the big citT duplication. And after that came refining mutations, leading to world domination by generation 33,000. (The world, in this case, being a shot-glass-sized flask.)

In order to read this story in its full details, the scientists would need to understand the order by which every mutation arose, step by step. And they’d have to understand how each mutation helped produce a new kind of organism.

Despite the carefully controlled conditions of the experiment, this was a fiendishly hard problem. By the time the bacteria had evolved into full-strength citrate feeders at generation 33,000, they had acquired 79 mutations not found in their ancestor. Many of those mutations probably had nothing to do with citrate feeding. They may have helped the early bacteria grow better on glucose. Some might have had no effect on the bacteria one way or the other.

One of the scientists studying the citrate eaters was post-doctoral researcher Jeffrey Barrick. In 2011, he moved to the University of Texas to set up his own lab, and there he continued to study the citrate eaters, developing new methods to tease apart the evolutionary history of the citrate feeders.

He and his colleagues developed a new method of engineering bacteria in order to identify the mutations that were absolutely essential for full-blown citrate feeding. They combined portions of the citrate-feeding genome with that of the ancestral genome and then dropped these hybrids into dishes with only citrate to feed on.

Most starved to death. But a few grew. The scientists then plucked out the surviving hybrids and put parts of their DNA into other ancestral bacteria. Round after round of experimenting let them zero in on the essential segments for growing on citrate. Eventually, they could pinpoint the specific mutations.

Their results were weirdly few.

One result was no big surprise. Barrick and his colleagues found that in order to feed on citrate with maximal gusto, bacteria needed extra copies of the rewired citT genes.

But, as Barrick and his colleagues reported in a recent paper, they found just one other essential mutation.

This mutation affects a gene called dctA. When the scientists inserted the evolved versions of citT and dctA into an ancestral microbe, it became a full-blown citrate feeder. Neither gene on its own could achieve the same result. And no other genes were required for the metamorphosis.

This discovery prompted the scientists to look closely at the dctA gene. It encodes another membrane protein that’s responsible for pumping molecules in and out of the microbe. While citT pumps succinate out of the microbe, dctA pumps it in.

Barrick and his colleagues suspect that the evolution of a new kind of dctA gene allowed the bacteria to keep up a supply of succinate, which they needed on hand in order to feed on citrate. Together, the mutations to citT and dctA turned the mutant microbes into winners.

Which leaves the role of all the other mutations shrouded mystery. In the new study, none of the mutations that came before generation 31,500 proved to be vital for being a full-blown citrate feeder. They didn’t lay the groundwork in any essential way. And yet the previous research clearly indicated that things were afoot before generation 31,500.

Given the new results, Barrick and his colleagues have a few ideas for what was going on before then. It’s possible that some of the early, mysterious mutations were favored by natural selection because they helped the bacteria grow on their regular diet of glucose. And as a side effect, they helped build up a small supply of succinate. That succinate turned out to be a big benefit later on, when citT mutated. Now the bacteria had enough succinate (or some related molecule) to push out as it pulled citrate in. If the citT mutation had arisen before those mutations, the bacteria might not have been able to feed on citrate. And then later on, the dctA mutation arrived, kicking the citrate feeding into overdrive.

I contacted Lenski, who was not a co-author on Barrick’s new study, to see what he thought of the results. “I love the fact that this paper shows just how complex evolution can be,” he replied, “even for one little species in a tiny flask world for just a couple of decades.”

(For more on E. coli’s strange scientific history, see my book Microcosm.)

A Blog by Carl Zimmer

A Long Way Left Up Darwin’s Mountain

One of the things I like about a long-running blog is that I can revisit long-running stories whenever I feel like it. And one of the longest of those stories has been unfolding in a lab at Michigan State University since 1988. That year, a biologist named Richard Lenski began rearing Escherichia coli from a single microbe. The bacteria, which he raised in a dozen separate flasks, all faced the same challenge: endure a starvation diet that their lab-pampered ancestors had not suffered.

The twelve flasks that contain the bacteria in Lenski’s long-term experiment. Photo by Michael Wiser
The twelve lines of E. coli in Lenski's experiment live in these flasks. Photo by Michael Wiser

Every few hours, the bacteria reproduced. Each morning, the scientists took a few drops from each flask and moved these colonists to a fresh flask. Mutations arose, which the descendants inherited. Some helped the bacteria grow faster than their cousins, and natural selection spread them across the population.

It’s been 25 years–and 50,000 generations–since Lenski started the experiment, and it just won’t quit. Here are a few of the pieces I’ve written about it over the years:

–In 2007, I wrote about Lenski and the field of experimental evolution for the New York Times.

–In 2008, I dedicated a chapter to Lenski’s work in my book, Microcosm, which is a biography of E. coli. If Google will cooperate, I’m going to embed the chapter here. The blog post continues below it…

–In 2009, I wrote a magazine feature for BBC Knowledge

–One of Lenski’s lines of bacteria even went off in an unexpected direction, evolving the ability to feed on citrate in the presence of oxygen. This might represent the birth of a new species. I blogged about that research here and here.

Yet there is still more to learn from these bugs. In this week’s Science, Lenski and two members of his lab–Michael J. Wiser and Noah Ribeck–took a close look at the evolution of the bacteria over the course of the entire experiment.

One of the great strengths of this experiment is that Lenski is an obsessive hoarder. He and his team freeze bacteria every 500 generations, filling freezer after freezer with them. They can then thaw out a few of the bacteria and put them in a dish with the latest generation and see how fast they each grow under identical conditions. Think of it as microbial Hunger Games.

These contests allow the scientists to precisely gauge what’s known as relative fitness–a measurement of natural selection. In the early years of the experiment, the fitness of the bacteria rose very fast. Within 5,000 generations, all twelve lines were growing 50% faster than the original microbe Lenski started the experiment with. Then they slowed down. By 20,000 generations, they were 75 percent faster.

These results lead to an obvious question: were the bacteria coming to the end of their increase in fitness? You can think of evolution in these cases like a mountain, with the elevation of any spot on the mountain as the average fitness of a population. Perhaps the mountain the bacteria in Lenski’s lab were climbing had steep slopes at low elevations. But now they were getting to the gentle climb just before reaching the mountain’s peak.

So the scientists thawed out some bacteria and pitted them against each other. Even at 50,000 generations, it turns out, the bacteria are still getting faster. Between generations 40,000 and 50,000, their fitness increased by 3 percent.

The scientists then competed the newest bacteria against ancestors from 41 generations across the past 25 years. They plotted the data on a graph and then looked for the curve that fit the dots best. The data do not indicate the mountain is going to flatten out. Instead, the best curve is generated by a mathematical relationship called a power law. In this model, the bacteria will improve by smaller steps in the future, but they will never stop improving. The power law is so powerful that the scientists can plug data into it only up to generation 20,000  and then accurately predict the next 30,000 generations.

fitness graph
Fitness of E. coli over 50,000 generations. (Ancestor=1) Blue curve from a power law; red curve asymptotic. Wiser et al Science 2013
From Wiser et al, Science 2013. The blue line shows a power law curve. The red line shows a curve that approaches a flat line (asymptotic).

To explain this result, the scientists turned to some of the previous experiments that researchers  have  carried out on Lenski’s bugs. Over the years, they’ve  looked closely at the DNA of some of the bacteria, in order to pinpoint new mutations and track them over the generations. By 20,000 generations, the bacteria had already acquired several dozen beneficial mutations. If the scientists engineered the ancestral bacteria with each of those mutations, they grew faster.

These adaptations arose thanks to the fact that mutations spontaneously occur at random in the bacteria. Most of the mutations are harmful, either killing the bacteria outright or slowing down their grow so that their descendants eventually disappear from their flask. But a few are beneficial.

Of all the possible beneficial mutations, most of them provide a small benefit, while a few provide big ones. After a few thousand generations, most of the big mutations probably occurred, leaving only small ones to continue improving the bacteria. And each time a new mutation arises, it has to interact with all the mutations that came before it. Some of those interactions may actually be harmful to the bacteria, reducing the overall benefit. The more time passes, the more mutations there are that may impose that cost.

Yet the experiment also shows that the bacteria still have the capacity to grow even faster. There’s a creativity in the genome that is inexhaustible–despite the fact that just about every spot in the microbe’s DNA has mutated by now. Again, the interaction between mutations gets the credit. In some cases, mutations can’t be beneficial until a series of other beneficial mutations have first evolved.

The creativity of evolution means that this experiment will keep yielding results for a long time. It’s possible that eventually the bacteria will hit some ceiling imposed by physics. There may be an upper limit to how fast DNA can be copied, for example, or how thick cell walls can get.

But no one knows when the bacteria will hit that wall. Lenski and his colleagues estimate that if they could run the experiment for another million years, the bacteria would keep speeding up. Today it takes one microbe 55 minutes to become two. In a million years (or 2.5 billion generations) it would take 23 minutes.

Lenski’s getting towards retirement, so he won’t be running a million-year experiment. Perhaps someone can create a robotic, solar-powered time capsule to take over. As long as the experiment can run, these bacteria will be ready to surprise.

PS: After 25 years, Lenski has evolved too–he has started to blog. Here’s his own post on the new paper.

A Blog by Carl Zimmer

A Living Drug Cocktail

We know that the 100 trillion microbes in the human body are important to our health. What’s harder to know is how to use them to make us healthy.

Normally, our resident microbes–the microbiome–carry out a number of important jobs for us, from fighting off pathogens to breaking down food for us. If they get disrupted, we  suffer the consequences. Sometimes antibiotics can upset the ecological balance in our bodies so severely, for example, that rare, dangerous species can take over.

For decades, doctors and scientists have searched for microbes that can promote our health by taking up residence in our bodies. They’ve had some modest success in treating people by giving them a single species at a time. (Don’t be fooled by all the so-called probiotic foods and pills you can buy over the counter–few, if any, have ever been scientifically shown to be effective.) Part of the problem has been that scientists haven’t been terribly systematic about searching for microbes. Very often, their most important standard for a probiotic germ is not its healing power, but its ability to survive in food, or packed into a pill.

Scientists have also had some limited success at the other end of the spectrum, by exploiting much of the microbiome’s diversity at all once. For some people with deadly gut infections, for example, the only cure is to get a so-called fecal transplant from a healthy donor. Fecal transplants show a lot of promise, but scientists don’t have a clear idea of how they work. A stool sample is loaded with hundreds of different species, any one of which might be either essential for overthrowing pathogens or just along for the ride.

In Nature today, a team of scientists report taking an important step forward in microbiome-based medicine. They searched for species methodically, in the same way medicinal chemists search for new drugs. They have pinpointed a handful of species living in the human gut that collectively show signs of fighting effectively against some autoimmune diseases.

Scientists have long suspected that the immune system would benefit from microbiome-based medicine. That’s because immune systems depend on the microbiome to develop normally in the first place. As a child’s immune cells grow and divide, they pick up signals from the microbes. Those signals teach the immune system to become tolerant. They can still recognize a dangerous pathogen and kill it, but they spare the beneficial bugs. And they also become less likely to overreact to a harmless molecule or our own tissues.

One important group of immune cell involved in this tolerance are known as Tregs (short for the CD4+ FOXP3+ T regulatory cells). They are abundant in the microbe-packed gut, where they help to broker a truce between host and the germs that live there. Tregs also depend on the microbiome for their very existence. When scientists rear mice without any germs at all, the animals develop very few Tregs. And as a result their immune system becomes prone to raging out of control.

A number of experiments have hinted that abnormal levels of Tregs are behind some autoimmune gut diseases, such as colitis, which causes chronic inflammation in the large intestines and diarrhea. This raises the possibility that a treatment for colitis would be to bring Tregs back to normal levels. Perhaps among all the bacteria in the gut, scientists could find the ones that sent the signals to the Tregs.

Over the past few years, Kenya Honda of the University of Tokyo and his colleagues have been hunting for those species. They started by testing out subsets of the microbiome to find groups of species that could foster the growth of Tregs. Raising healthy mice, they collected the mouse droppings, which contained lots of bacteria. They treated the droppings with chloroform, which kills most bacteria. The only microbes that survived were species that make spores tough enough to withstand the chemical.

When the scientists gave those spore-forming bacteria to germ-free mice, the level of Tregs in the animals went up. That didn’t happen when the scientists gave the mice other kinds of bacteria instead.

This discovery didn’t pinpoint exactly which bacteria were fostering Tregs. But it certainly narrowed down the line-up of suspects dramatically. And it also prompted Honda and his colleagues to start acting more like a drug company testing promising new compounds. In fact, Honda co-founded a company called Vedanta Biosciences for that express purpose.

Their goal now became finding a species, or a group of species, that live in humans, and which promote the growth of Tregs in the gut. Rather than taking the kitchen-sink approach of fecal transplants, they would try to deliver a surgically precise germ.

They took stool samples from a healthy Japanese volunteer and doused them with chloroform, so that once more they only had to contend with spore-forming species. Then they inoculated germ-free mice with different combinations of the surviving bacteria. After letting the bacteria grow in the mice, they then inspected the animals to see if any of them had high levels of Tregs as a result. They did find some of those combinations, and they went on narrowing down the suspects until  they were left with just seventeen species, along belong to a type of bacteria called Clostridia.

One particularly interesting result of the experiment was that they couldn’t get these good results from any fewer than those seventeen species. On its own, each of the species was unable to foster the immune cells. It may be a combination of signals from all seventeen species that promotes the Tregs.

The researchers wondered if these seventeen species played a special role in autoimmune diseases in humans. To find out, they looked at the microbiomes from health people and people with colitis. The seventeen species tended to be rarer in the sick people.  Perhaps, the researchers reasoned, losing this network of microbes weakens the signals that keep Treg levels normal. The immune system spins out of control, leading to colitis.

If that were true, then giving someone a pill with all seventeen species might be an effective way to fight colitis. But Honda and his colleagues weren’t ready to start inoculating people with Clostridia. Clostridia is a huge group of species that includes some very nasty characters that cause diseases like tetanus and botulism. They would have to do some preliminary work first.

All seventeen species were new to science (something that’s pretty typical for microbiome research), so the scientists sequenced their genomes. None of the seventeen species carried genes for toxins or other disease-causing proteins. From an inspection of their DNA, at least, the microbes looked safe.

Next, the scientists tested the bacteria out on mice. They gave the microbes to animals either suffering from colitis or from allergy-triggered diarrhea. In both cases, the bacteria raised the level of Tregs dramatically in the guts of the mice. The mice also partly recovered from their diseases. The mice with colitis had less inflammation, and the mice with diarrhea had healthier stool.

From here, the Vedanta researchers eventually hope to get to clinical trials on humans. As I wrote in April, turning bugs into drugs is a big challenge on many levels. For one thing, the FDA doesn’t have a long tradition of approving such research. And while Honda and his colleagues have certainly gone a long way to pinpointing how microbes foster Tregs, they have yet to work out the precise balance of signals that really matters to the immune system.

Nevertheless, a seventeen-bug cocktail would be appealing in many ways. For one thing, the microbes are regular residents of the human gut, dwelling there for people’s entire lifetime. And they only live in the gut, and not the heart or the liver or some other organ. Both these facts suggest that such a cocktail would be unlikely to cause harmful side effects. What’s more, the microbes would be able to deliver a steady, long-running dose of the chemicals necessary to keep Treg levels healthy.

The new study also points to a way to systematically search the microbiome for treatments for other diseases. It’s possible that small teams of other species handle other jobs in the body. They may nurture other types of immune cells, for example. Or they may send signals into the body that regulate body weight. By winnowing down the microbiome, scientists may be able to deploy those elite units to fight other diseases.

[Update: Nature paper link fixed]