The manned submersible Shinkai 6500 collected deep-sea mud from which researchers isolated microbes potentially key to the evolution of complex cells.

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Tentacled microbe could be missing link between simple cells and complex life

Patience proved the key ingredient to what researchers are saying may be an important discovery about how complex life evolved. After 12 years of trying, a team in Japan has grown an organism from mud on the seabed that they say could explain how simple microbes evolved into more sophisticated eukaryotes. Eukaryotes are the group that includes humans, other animals, plants, and many single-celled organisms. The microbe can produce branched appendages, which may have helped it corral and envelop bacteria that helped it—and, eventually, all eukaryotes—thrive in a world full of oxygen.

“This is the work that many people in the field have been waiting for,” says Thijs Ettema, an evolutionary microbiologist at Wageningen University in the Netherlands. The finding has not yet been published in a peer-reviewed journal, but on Twitter, other scientists reviewing a preprint on it have already hailed it as the “paper of the year” and the “moon landing for microbial ecology.”

The tree of life has three major branches—bacteria and archaea make up two, both of which are microbes that lack nuclei and mitochondria, distinct membrane-bound compartments to store DNA or generate energy, respectively. Those components, or organelles, characterize cells of the third branch, the eukaryotes. The prevailing thinking is that roughly 2 billion years ago, a microbe belonging to a group called the Asgard archaea absorbed a bacterium called an alphaproteobacterium, which settled inside and became mitochondria, producing power for its host by consuming oxygen as fuel. But isolating and growing Asgard archaea has proved a challenge, as they tend to live in inhospitable environments such as deep-sea mud. They also grow very slowly, so they are hard to detect. Most evidence of their existence so far has been fragments of DNA with distinctive sequences.

But Hiroyuki Imachi and Ken Takai, microbiologists from the Japan Agency for Marine-Earth Science and Technology in Yokosuka, and their colleagues have persisted in trying to grow such a microbe from a seabed core that a submersible brought up from a dive in 2006. For 2000 days, they kept mud from the 2500-meter-deep Omine Ridge off the coast of Japan in bioreactors fed continuously with methane, which is a gas common in deep-sea mud. The researchers then incubated small samples of the microbe-laden mud in glass tubes supplied with a wide variety of nutrients and other substances. A year later, they detected microbes in one of the tubes, which also contained four antibiotics to kill any contaminating bacteria.

DNA analyses of samples from the tube indicated it included an Asgard archaeon, the microbe they were hoping to grow. It took about 20 days for the numbers of this microbe to double—bacteria commonly double in less than an hour—but eventually, they got enough of the organism to study it. “It was really a gargantuan task,” says David Baum, an evolutionary biologist at the University of Wisconsin in Madison, who was not involved with the work.

The Japanese researchers, who could not be immediately reached for comment, named the microbe Prometheoarchaeum syntrophicum, after the Greek god Prometheus, who created humans out of mud. Experiments with this single-cell organism suggest it usually—if not always—grows in association with another microbe that makes methane, Imachi, Takai, and colleagues report today in a preprint on bioRxiv. The researchers further discovered that Prometheoarchaeum breaks down amino acids for food and releases hydrogen, which feeds its partner. That methanemaker in turn helps Prometheoarchaeum thrive by chewing through the hydrogen, the researchers say; a buildup of hydrogen could otherwise cause even slower growth of Prometheoarchaeum. The complex partnership is another reason why the Asgard arcahaea are so hard to grow in the lab.

The researchers sequenced all the microbe’s DNA, confirming that it does contain some genes that look like those found in eukaryotes. (When Ettema had pieced together an Asgardian genome from a broad sampling of DNA, he found the same, but skeptics wondered whether the genes were contaminants.) It’s as if Prometheoarchaeum “were primed to become eukaryotes,” Ettema says.

Having grown the microbe, the researchers used an electron microscope to image it, revealing multiple branched appendages. The team hypothesizes that, eons ago, an archaeon encircled the protomitochondrion and put it to work. The researchers propose that as the concentration of oxygen increased on early Earth, archaea like Prometheoarchaeum took in oxygen-using partners and did better than other microbes.

“This is exactly what we predicted,” Baum says. In 2014, he and a colleague published a similar “inside out” theory. Previously, most researchers had assumed that the mitochondria were pulled into their archaeal hosts—the “outside in” theory, with the nucleus and the cell’s internal membranes evolving from engulfed components. The newly cultured microbe’s appendages suggest otherwise. These appendages surrounded the protomitochondrion and their membranes gave rise to the internal ones.

Ettema cautions that the archaeal ancestor to eukaryotic cells that lived 2 billion years ago may not have looked and acted just like Prometheoarchaeum. Moreover, DNA studies indicate that other archaea are more closely related to eukaryotes than this one. He expects, however, that the 12 years the Japanese team devoted to culturing this microbe will help him and others isolate and grow related archaea in the lab: “I’m sure it will not take 12 years to get the next Asgard into culture.”

As impressive as the work is, the culturing of this Asgard—or others--doesn’t answer whether there are two kingdoms or three, says Patrick Forterre, a microbiogist at the Pasteur Institute in Paris. Based on his group’s extensive DNA studies of the microbes, Forterre argues that Asgard archaea are not close kin to eukaryotes and that eukaryotic-like genes were borrowed from the real eukaryotic ancestors, which evolved from a common ancestor to both archaea and eukaryotes. “They don’t look like [an] ‘intermediate’ cell between prokaryote and eukaryote but 100% as a classical (but very small) archaeon,” he wrote in an email.

But even if Asgard arcahea don’t prove to be the ancestor of eukaryotes, the new work “reveals all kinds of exciting biology,” says Willem van Schaik, a microbiologist at the University of Birmingham in the United Kingdom. “It feels like this will go into microbial textbooks immediately.”