|Olavi Kajander suggests that nanobacteria attach to each other to form chains.
Credit: Doctors Health Supply
"Nanobacteria." The name sounds obvious enough. They’re small. They’re bacteria. You might assume they resemble the smallest archaean, Nanoarchaeum, or the smallest bacterium, Mycoplasma, in size. You might assume they resemble ordinary bacteria in nature.
But instead of taking the prize as the smallest organism, nanobacteria-a tenth the size of ordinary bacteria and half as large as Nanoarchaeum and Mycoplasma-have continued to cause controversy since their description in the early 1990s by a Finnish research team led by Olavi Kajander. The team avoided overstating their claim – "These autonomously replicating particles are tentatively named nanobacteria" – but the name stuck, and many of the team’s publications state that nanobacteria are alive. The spheres, covered with a hard calcium phosphate coat, were found in various fluids used to grow cells in the laboratory, such as cow serum.
Medical microbiologist Neva Ciftcioglu, now at NASA’s Johnson Space Center in Houston, her colleague Kajander and collaborators in the United States, Canada, the UK, Japan and Russia continue to research nanobacteria. But scientists in some labs say that, while they can produce the spheres and can see them with an electron microscope, they have been unable to confirm any sign of life in them.
Other scientists raise a theoretical objection, saying that at 200 nanometers, nanobacteria are just too small to contain the machinery of life. "Generally," Ciftcioglu says, "we say that a microorganism should not be any smaller than 200 nanometers. So nanobacteria is within this range, however, there are some forms we detected that they are even 80 or 50 nanometers." These smaller forms, she says, may not be complete cells.
"So the difficulty came with two reasons," Ciftcioglu says, "number one was naming; we called them bacteria before we characterized them and proved that they are bacteria. And the second difficulty was the size discussion."
|Click here for larger image. The process of DNA amplification by Polymerase Chain Reaction (PCR). This sensitive method is also used to detect nucleic acids in hopes of discovering life.
The debate heated up in 1998, when Kajander and Ciftcioglu published a paper in the July Proceedings of the National Academies of Science (PNAS) linking nanobacteria with diseases involving calcification, such as kidney stones and hardening of the arteries. They didn’t mince words, calling nanobacteria "the smallest cell-walled bacteria" and even showing where nanobacteria fit in the tree of life. Ciftcioglu says, "First of all I convinced myself that these things are living."
National Institutes of Health research microbiologist John O. Cisar was one of many scientists intrigued by the 1998 PNAS paper. "We were quite convinced from Kajander’s paper that nanobacteria may be real," Cisar says.
Cisar and colleagues isolated the particles from several sources, including cow serum and both human saliva and dental plaque. They saw the particles increase in number very slowly, the same results Ciftcioglu reported. "We were very excited when we were able to reproduce their critical findings," Cisar says.
Cisar’s team did find evidence of nucleic acid. Kajander and Ciftcioglu described a particular RNA sequence in the ’98 PNAS paper and in another paper published in ’98. To detect nucleic acids, researchers use an extremely sensitive method called the polymerase chain reaction, or PCR. PCR makes millions of copies of short stretches of nucleic acid, copying over and over again even a single original molecule.
On further examination of the RNA sequence found by both research teams, Cisar determined that it was identical to an RNA sequence belonging to a well-known bacterial contaminant often found in PCR experiments.
"We are very confident of our interpretation of the PCR data in terms of pointing out where a mistake was made in the original report," Cisar says. "If you discount the PCR results from the original description of nanobacteria, you take away all molecular evidence for a new type of living organism."
"It eventually became clear to us that we were chasing an artifact," Cisar concludes. He has not pursued nanobacteria further. But, he says, "If somebody would provide molecular evidence showing that nanobacteria are alive, we’d be happy to jump back into the problem."
|Harold Morowitz, a Mycoplasma veteran, is doubtful of nanobacteria being a self-replicating cell.
Credit: George Mason University
Ciftcioglu says techniques to grow nanobacteria in the lab are very exacting. "Unfortunately, [Cisar and colleagues] did not seek nanobacteria control cultures or the best available nanobacteria-specific reagents for use in their experiments." While the correct methods remain unpublished, Ciftcioglu says, she is more than willing to communicate with researchers about lab techniques. "I would be very happy to help them through e-mail to say treat your sample this or that way, that might be the reason you don’t get results."
Harold Morowitz, of George Mason University, in Fairfax Virginia, is a Mycoplasma veteran. "The first question is, do I theoretically think that something [that small] could be a self replicating cell? And the answer is, I’d be extremely doubtful."
Back in the 1950s, Morowitz found colleagues charging that Mycoplasma was too small to be a cell, so he and his colleagues had to accumulate proof. "In the 1950s, we were growing Mycoplasma gallisepticum, and there were all sorts of things in the literature as to whether they were organisms," he says. The team responded by isolating cell structures such as ribosomes, finding functional enzymes and experimentally showing that Mycoplasma has a cell membrane.
"I think the burden is on the people who say that they’re alive to prove that [nanobacteria] fit the normal criteria of cellular biology: they are surrounded by a membrane, they have a genome (at least an RNA genome), and they have ribosomes and other structures that are associated with all life that we know," Morowitz says.
Current research may be nibbling away at the theoretical size objections, Ciftcioglu says. Canadian researchers have published a description of bacteria in healthy human blood that measure in at a mere 200 nanometers in diameter (as measured in an electron micrograph), about the size of the larger nanobacteria.
|"The results are convincing that these things are living and have metabolic activity and nucleic acid." -Neva Ciftcioglu
Credit: Kuopion Yliopisto
Ciftcioglu thinks the evidence is piling up. "The results are convincing that these things are living and they have metabolic activity, and they have nucleic acid," she says. But because of the controversy, she wants to dot every "i" and cross every "t" before publishing. "If you are working on controversial research, people are not looking with [a] loupe, they are looking at your research with [an] electron microscope and you have to be absolutely sure what you are talking about is scientifically correct and can be repeated." she says.
"First you have to convince yourself what you are working with is not an artifact. Nobody wants to work full time with an artifact. You first want to understand if there is nucleic acid, this is number one. And number two, if they have membrane structure. They should have some metabolic activity. So we concentrated mostly on those three issues. And our biggest question was if they have unique protein structure. So far, we have pilot research for all of them."
These days, Ciftcioglu works at JSC with David McKay. "We are working on biomarkers, what biomarkers we [should search for] on meteorites for being able to say there is life or not. And what is the definition of life. And what is the best way to analyze mineralized samples," Ciftcioglu says. Research into how to analyze mineralized meteorite samples will translate easily, she says, into work on nanobacteria. McKay and colleagues think nanobacteria research could aid understanding of the earliest cells on Earth and any possible cells on Mars.
"Additionally, my colleagues in Mayo Clinic have some data on nanobacteria nucleic acids, so the collaboration is going on."
Researchers interested in nanobacteria have met twice in international conferences. Ciftcioglu says plans are in the works for another conference. "In 2003, our plan is to come together and bring our so-far results because we are very sensitive to publish any results before we confirm each other." With results from several labs on the table, she hopes, clear evidence that nanobacteria are alive can finally be published.