A Home for the Microbiome

 Biologists Identify How Beneficial Bacteria Reside and Thrive in Gastrointestinal Tract

A section of mouse colon is shown with gut bacteria (outlined in yellow) residing within the crypt channel. (Credit: Caltech / Mazmanian Lab)

The human body is full of tiny microorganisms -- hundreds to thousands of species of bacteria collectively called the microbiome, which are believed to contribute to a healthy existence. The gastrointestinal (GI) tract -- and the colon in particular -- is home to the largest concentration and highest diversity of bacterial species. But how do these organisms persist and thrive in a system that is constantly in flux due to foods and fluids moving through it? A team led by California Institute of Technology (Caltech) biologist Sarkis Mazmanian believes it has found the answer, at least in one common group of bacteria: a set of genes that promotes stable microbial colonization of the gut.

A study describing the researchers' findings was published as an advance online publication of the journal Natureon August 18.

"By understanding how these microbes colonize, we may someday be able to devise ways to correct for abnormal changes in bacterial communities -- changes that are thought to be connected to disorders like obesity, inflammatory bowel disease and autism," says Mazmanian, a professor of biology at Caltech whose work explores the link between human gut bacteria and health.

The researchers began their study by running a series of experiments to introduce a genus of microbes called Bacteriodesto sterile, or germ-free, mice. Bacteriodes, a group of bacteria that has several dozen species, was chosen because it is one of the most abundant genuses in the human microbiome, can be cultured in the lab (unlike most gut bacteria), and can be genetically modified to introduce specific mutations.

"Bacteriodes are the only genus in the microbiome that fit these three criteria," Mazmanian says.

Lead author S. Melanie Lee (PhD '13), who was an MD/PhD student in Mazmanian's lab at the time of the research, first added a few different species of the bacteria to one mouse to see if they would compete with each other to colonize the gut. They appeared to peacefully coexist. Then, Lee colonized a mouse with one particular species, Bacteroides fragilis, and inoculated the mouse with the same exact species, to see if they would co-colonize the same host. To the researchers' surprise, the newly introduced bacteria could not maintain residence in the mouse's gut, despite the fact that the animal was already populated by the identical species.

"We know that this environment can house hundreds of species, so why the competition within the same species?" Lee says. "There certainly isn't a lack of space or nutrients, but this was an extremely robust and consistent finding when we tried to essentially 'super-colonize' the mice with one species."

To explain the results, Lee and the team developed what they called the "saturable niche hypothesis." The idea is that by saturating a specific habitat, the organism will effectively exclude others of the same species from occupying that niche. It will not, however, prevent other closely related species from colonizing the gut, because they have their own particular niches. A genetic screen revealed a set of previously uncharacterized genes -- a system that the researchers dubbed commensal colonization factors (CCF) -- that were both required and sufficient for species-specific colonization by B. fragilis.

But what exactly is the saturable niche? The colon, after all, is filled with a flowing mass of food, fecal matter and bacteria, which doesn't offer much for organisms to grab onto and occupy.

"Melanie hypothesized that this saturable niche was part of the host tissue" -- that is, of the gut itself -- Mazmanian says. "When she postulated this three to four years ago, it was absolute heresy, because other researchers in the field believed that all bacteria in our intestines lived in the lumen -- the center of the gut -- and made zero contact with the host…our bodies. The rationale behind this thinking was if bacteria did make contact, it would cause some sort of immune response."

Nonetheless, when the researchers used advanced imaging approaches to survey colonic tissue in mice colonized with B. fragilis, they found a small population of microbes living in miniscule pockets -- or crypts -- in the colon. Nestled within the crypts, the bacteria are protected from the constant flow of material that passes through the GI tract. To test whether or not the CCF system regulated bacterial colonization within the crypts, the team injected mutant bacteria -- without the CCF system -- into the colons of sterile mice. Those bacteria were unable to colonize the crypts.

"There is something in that crypt -- and we don't know what it is yet -- that normal B. fragilis can use to get a foothold via the CCF system," Mazmanian explains. "Finding the crypts is a huge advance in the field because it shows that bacteria do physically contact the host. And during all of the experiments that Melanie did, homeostasis, or a steady state, was maintained. So, contrary to popular belief, there was no evidence of inflammation as a result of the bacteria contacting the host. In fact, we believe these crypts are the permanent home ofBacteroides, and perhaps other classes of microbes."

He says that by pinpointing the CCF system as a mechanism for bacterial colonization and resilience, in addition to the discovery of crypts in the colon that are species specific, the current paper has solved longstanding mysteries in the field about how microbes establish and maintain long-term colonization.

"We've studied only a handful of organisms, and though they are numerically abundant, they are clearly not representative of all the organisms in the gut," Lee says. "A lot of those other bacteria don't have CCF genes, so the question now is: Do those organisms somehow rely on interactions with Bacteroides for their own colonization, or their replication rates, or their localization?"

Suspecting that Bacteroides are keystone species -- a necessary factor for building the gut ecosystem -- the researchers next plan to investigate whether or not functional abnormalities, such as the inability to adhere to crypts, could affect the entire microbiome and potentially lead to a diseased state in the body.

"This research highlights the notion that we are not alone. We knew that bacteria are in our gut, but this study shows that specific microbes are very intimately associated with our bodies," Mazmanian says. "They are living in very close proximity to our tissues, and we can't ignore microbial contributions to our biology or our health. They are a part of us."

Funding for the research outlined in the Nature paper, titled "Bacterial colonization factors control specificity and stability of the gut microbiota," was provided by the National Institutes of Health and the Crohn's and Colitis Foundation of America. Additional coauthors were Gregory Donaldson and Silva Boyajian from Caltech and Zbigniew Mikulski and Klaus Ley from the La Jolla Institute for Allergy and Immunology in La Jolla, California.

Source : California Institute of Technology.

By Science and Universe

Social Amoebae Travel With a Posse: Tiny Single-Celled Organisms Have Amazingly Complicated Social Lives

The social amoeba called Dictyostelium discoideum, picks up edible bacteria, carries them to new locations and harvests them like crops. (Credit: Joan Strassmann)

In 2011, Nature announced that scientists had discovered a single-celled organism that is a primitive farmer. The organism, a social amoeba called Dictyostelium discoideum, picks up edible bacteria, carries them to new locations and harvests them like crops.

D. discoideum enjoyed a brief spell in the media spotlight, billed as the world's smallest farmer.

Now a collaboration of scientists at Washington University in St. Louis and Harvard University has taken a closer look at one lineage, or clone, of a D. discoideum farmer.

This farmer carries not one but two strains of bacteria. One strain is the "seed corn" for a crop of edible bacteria, and the other strain is a weapon that produces defensive chemicals.

The edible bacteria, the scientists found, evolved from the toxic one. The two strains differ by many mutations but a single key mutation, which hit an important controller in the genome of the nonfood strain, alters expression of 10 percent of its genome. This alteration increases the expression of some genes and decreases the expression of others.

A mutation that affects this much of a genome could be lethal, but in this case it had the surprising effect of making the bacterium edible by changing its chemical profile.

The discovery is reported in the July 29 issue of the Proceedings of the National Academy of Sciences.

The first farmer

The first farmers were found by Debra Brock, then a graduate student in the laboratory run by David Queller and Joan Strassmann at Rice University in Houston, Texas. (All three scientists have since moved to Washington University in St. Louis, where Queller and Strassmann are professors of biology and Brock is a research scientist.)

Brock, who had worked for years with the standard axenic (pure, or uncontaminated) lab clone, noticed something strange about the D. discoideum in the Queller/Strassmann lab, which had been collected from the wild.

When she looked at wild D. discoideum clones under a microscope, she saw bacteria in the sori of some clones. Oddly it was always the same clones that carried bacteria. The bacteria caught her attention because she had never seen anything like this in the lab clone.

"As I tell the students, it's all about the details," Brock says.

A fancy farmer

Whenever she found a D. discoideum clone carrying bacteria, Brock tried to isolate the bacteria. This was a bit hit or miss, she explains, because many organisms that live in the soil cannot be grown in the lab.

Eventually she found a champion D. discoideum: a farmer clone from which she was able to isolate two strains of bacteria. At least the strains looked different when they were cultured in a dish.

She sent the two bacteria out to be identified genetically and both came back as Pseudomonas fluorescens: the same species, even though they were morphologically so different.

"It was a bit of a puzzle," Brock said. On top of that one of the two morphs was edible and the other was not, and the edible one was the first edible strain she had isolated that wasn't a lab feedstock.

"So, I now had two bacteria that seemed the same and one was a food and the other wasn't," Brock said. "That was really odd.."

Toting guns and butter

When the farmer paper appeared in Nature, Jon Clardy of the Harvard Medical School in Boston noticed a passing reference to the D. discoideum farmer with two hitchhikers in the supplement section of the paper. Clardy, who studies the chemistry of mutualism, contacted the Queller/Strassmann lab to suggest the two labs collaborate to unravel the interactions among the newly discovered threesome.

Brock sent the bacteria to Harvard, where Pierre Stallforth, a postdoctoral associate in the Clardy lab, grew them in liquid media. He sent extracts from the media back to Brock, who tested them on D. discoideum to see if they were active.

"Ultimately Pierre figured out that the nonfood strain was producing two chemicals: chromene and pyrrolnitrin. And excitingly, chromene is a new compound," Strassmann said.

"We determined chromene increases spore production in the farmer strain and suppresses spore formation in the nonfarmer strain," she explained. "We saw the same increases in the farmer and decreases in the non-farmer with pyrrolnitrin. A known antibiotic and antifungal, pyrrolnitrin probably also suppresses other organisms in the soil that might compete with the farmer strain."

Assays showed that it was not merely the absence of chromene and pyrrolnitrin that made the food bacterium edible. Something else is going on as well.

Why become butter?

Stallforth next sequenced the entire genome of the two bacterial strains to look for mutations that might explain the differences between them.

The genes responsible for producing pyrrolnitrin were intact in both strains. So he looked at the genes for a two-part global activator that regulates the pyrrolnitrin pathway, among many other genes.

Sure enough, there was a mutation in one of the controller genes of the food bacterium that turned it off and broke the controller. As shown by others in a previous study, breaking the controller changed the expression of 10 percent of the bacteria's genome.

"That's pretty cool, but then you still don't really know for sure if that mutation is the one that matters," Strassmann said.

To check, Stallforth artificially broke the controller -- and only the controller -- in the nonedible P. fluorescens bacterium. The knockout strain he created had the same chemical profile as the food bacterium and it, too, was edible.

Had a similar mutation in the evolutionary past created the edible strain? To answer that question, the scientists constructed a family tree of P. fluorescens clones in the Strassmann/Queller lab by comparing 20 genes.

"It turns out that of all the bacteria strains we've ever isolated, the two we collected from the D. discoideum farmer clone Brock discovered are the most closely related, Queller said.

"The tree also tells us that edibility is a derived trait. These guys used to be inedible and became edible. That's just a weird thing to evolve: to be able to eaten," Queller said.

It makes sense only because it benefits kin, more of whom will be carried to new locations by the well-fed farmer D. discoideumclone, the scientists said.

It's altruism, ultimately. Altruism in miniature
.

Source : Washington University in St. Louis.

By Science and Universe