Nanobacteria, extremely tiny “microorganisms” that have sparked controversy and may cause disease, have been declared dead. Again.

Some say the nanobacteria were never really alive. Once touted as the world’s smallest living organisms, and even an entirely new form of life, the entities are actually nothing more than sub-microscopic balls of minerals and proteins, independent teams of scientists in Taiwan and France report.

Surviving the nanobacteria is their “father,” Robert Folk, a geologist from the University of Texas at Austin, who discovered them in deposits from Italian hot springs in the early 1990s. Folk and his colleagues have since found the objects, which are a thousandth the size of common bacteria such as E. coli, in sedimentary mineral deposits ranging from limestone to iron oxides to silicates. The microbes may be major players in eroding rocks to make soil, Folk believes.

Initial evidence for nanobacteria sparked excitement among many microbiologists, geologists and other researchers. Some experts were skeptical that the ingredients to make a living cell could be crammed into such a tiny package. DNA is 2 nanometers wide, and some proteins are as large as the proposed size of nanobacteria, 80 to 500 nanometers. But nanobacteria have been cultured in laboratories and seen to grow in number, albeit slowly.

Finnish researchers found nanobacteria in human and cow blood about the time that Folk discovered the organisms in rocks. Since then, nanobacteria have been linked to kidney stone and gallstone formation (SN: 8/1/98, p. 75), polycystic kidney diseases, rheumatoid arthritis, some cancers, co-infection with HIV, Alzheimer’s disease and chronic prostatitis. They have also been implicated in hardening of the arteries and aging.

The discovery of what appeared to be evidence of nanobacteria in a Martian meteorite (SN: 8/10/96 p. 84) sparked extreme reactions from people who were excited about a new form of life (especially one that might have lived on Mars) and people who doubted the existence of nanoscale living organisms.

“It had the optimists turning cartwheels and the naysayers enraged, and it’s stayed the same ever since,” Folk says.

John Ding-E Young of Chang Gung University in Taiwan believed in nanobacteria so much that he and graduate student Jan Martel embarked on a series of experiments to prove that the microorganisms couldn’t be just mineral deposits.

“I really wanted these people to be right,” Young says. Instead, the team found just the opposite.

Calcium minerals (found in seashells, eggshells and marble) and calcium phosphate (the stuff that bones are made of) are thought to compose nanobacteria’s crusty exterior. When the researchers made calcium carbonate in a solution used to nourish cells, the mineral formed round particles that looked like the nanobacteria found in blood and rocks. Some of the particles even resembled bacteria in the act of reproducing.

Then the researchers made the calcium carbonate particles grow by seeding the solution with human blood serum—a common ingredient in solutions used to grow cells—and incubating the solution with carbon dioxide. More tiny calcium particles formed over the next few days, similar to the slow reproduction time of nanobacteria.

A protein called albumin in the blood serum seems to seed nanoparticles but keeps the calcium carbonate from forming crystals, Young and Martel reported in the April 8 Proceedings of the National Academy of Sciences.

Working independently, a group of researchers in France also showed that nanobacteria aren’t living organisms.

“These things can be replicated, but we quickly found that they aren’t bacteria at all,” says Didier Raoult, a microbiologist at the National Center for Scientific Research in Marseille, France.

In a study published February 15 in the online journal PLoS Pathogens, Raoult and his colleagues described nanobacteria as complexes of minerals and a protein called fetuin. Fetuin prevents minerals from forming crystals but may aid in clumping calcium molecules into the hollow spheres characteristic of nanobacteria.

But the particles can “infect” other solutions, producing more nanobacteria. “There is something transmissible,” Raoult says. “I don’t know why, and I don’t know what it is. It’s not living.” He theorizes that nanobacteria, which he dubbed nanons, may work like infectious proteins called prions, which cause mad cow disease, scrapie in sheep and several human brain diseases.

This is not the first time the bell has tolled for nanobacteria. A report in 2000 showed that the entities lack DNA. “In fact, there’s no nucleic acid at all,” Raoult says.

Folk concedes that the two groups “did a good job” of showing that the particular nanobacteria they studied are nonliving. But the organisms are so diverse that “one explanation is obviously not going to fit all.” If the groups had investigated some of the nanobacteria that he has collected, they would find a different story, he says.

“Some are shaped like kidney beans. These are not shapes that minerals take. It’s clearly biological,” Folk says.

But even researchers who believe nanobacteria may cause disease had already started edging away from the idea that they are minuscule bacteria, even before the latest publications.

“For me it’s not important if they are living or not,” says Andrei Sommer of the University of Ulm in Germany. “It’s more important to focus on their clear link to disease and how to eliminate them.” Wrangling over whether nanobacteria are alive or dead is “only blocking the brain and blocking action,” Sommer says.

Sommer and his colleague Dan Zhu isolated nanobacteria from fluid found in the joints of people with arthritis. The researchers described their technique in an article that appeared online April 2 in Environmental Science & Technology.

Those who study nanobacteria are used to controversy. “We hear, ‘You’re studying something that doesn’t exist,’” says Virginia Miller, a physiologist at the Mayo Clinic Medical School in Rochester, Minn., who studies “self-replicating calcifying nanoparticles” and their link to disease. Others have called the particles living nanovesicles or nanobiota.

“We’re just beginning to understand how particles at the nanoscale interact with cells,” Miller says. “It would be a very sad thing if these two papers capped that avenue of research. We’re just on the brink of understanding how these particles participate in disease.”

Young isn’t ready to bury nanobacteria yet, either. “My thinking is that these structures are not only real, but they are important,” he says.

But if the particles aren’t itty-bitty bacteria, scientists have to decide what to call them. “It’s nonsense to use the name nanobacteria,” Sommer says. “At the same time, it’s unfair not to use it because it would be cutting off a whole history of research.”

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