From Jones and Bartlett, a book on Stem Cells from Dr. Ann A. Kiessling and Scott C. Anderson:

Selected Articles:

June 13, 2004

The Attack of the Killer Nanobacteria

Tiny creatures so small that they've been overlooked by scientists may be slinking around your body right now - and they're probably up to no good.

So, naturalists observe, a flea
Has smaller fleas that on him prey;
And these have smaller still to bite 'em;
And so proceed ad infinitum.

-- Jonathan Swift

In the 1970s, while doing field work in his beloved Italy, a geologist named Dr. Robert Folk from the University of Texas discovered that bacteria seemed to be precipitating - excreting, really - an unusual type of limestone. Known as travertine, it's been used for thousands of years in statuary and buildings all around the world, from the Coliseum in Rome to the Lincoln Center in New York. Why study travertine? As Dr. Folk puts it, "I was simply looking for a good excuse to continue doing field work in Italy because I loved the food and lifestyle, and hit upon the idea of working on the travertines of Rome."

Bacterial Club Med? Could this steaming rainbow of heat-loving bacteria be the wellspring of life -- and the source of some nasty ailments?

Dr. Folk wasn't the first to notice that travertines are related to bacteria. These rocks are formed in steaming sulfurous cauldrons like those in Yellowstone, and it has been known for some time that so-called thermophilic bacteria actually thrive in the boiling, mineral-laden water. They create mats that geologists have always assumed would trap minerals and, over time, become fossilized. But Dr. Folk saw it differently. Rather than idle bystanders simply covered by sediment, he realized that the bacteria were actively creating calcium carbonate causing it to grow at rates far faster than seemed possible with ordinary chemistry.

A decade later, Dr. Folk and his colleagues, Leo Lynch and Brenda Kirkland, got access to a high-power electron microscope and had a chance to review some of his samples closer up. What they saw made them squint in disbelief. Organic-looking clumps of nodules were scattered all over the samples, far smaller than ordinary bacteria but with many similar shapes. When he asked around, he found that these had been noted before by other researchers, but they were assumed to be artifacts, perhaps caused by the preparation of the sample.

But Folk had seen bacteria in rocks before, so he was less inclined to shrug off these tiny spheres as mere artifacts. And the more rocks he looked at, the more of the little spheres he found. After some research in the unfamiliar turf of bacteriology, he found a few references to ultramicrobacteria, and felt he had found the answer. He was ready to tell the world.

The Sound of One Hand Clapping

"The important role of nannobacteria in the mineralogical world was
discovered through dumb luck, idle curiosity and random reading."

-- Dr. Robert L. Folk

Chains of nanobacteria grow across a sample from the Bagnaccio hot springs in Viterbo, Italy. Inorganic processes rarely lead to chains, but bacteria do it all the time.
Credit: Lynch, Kirkland and Folk, University of Texas

The smallest living thing, as you undoubtedly learned in school, is the virus. It is so small that it doesn't even have enough genes to reproduce itself (making its claim to life rather tenuous). Instead, it depends on the kindness of strangers, mostly bacteria and higher-level cells, to do the copying for them.

So in 1992, when the feisty Dr. Folk announced that several kinds of rock seemed to be created by what looked like tiny bacteria not much bigger than a virus, he was greeted with a stony silence. He was ridiculed as a kook both out of his field and out of his depth.

Folk was claiming much more than a new Guinness Record for tiniest creature: he was proposing that these hitherto invisible life forms actually constituted the bulk of the world's biomass. As well as creating rocks, Dr. Folk said they were also responsible for breaking them down, creating dirt in the process. For good measure, Dr. Folk even blamed them for much of the metal corrosion in the world. These little creatures were just a few dozen nanometers (billionths of a meter) in size, so Dr. Folk dubbed them nanobacteria. Actually he used the prefix nanno, a variant favored by geologists, but most researchers have since adopted the shorter form.

Not everyone at the presentation laughed at Dr. Folk. Chris Romanek, a NASA scientist, thought about an interesting rock he had in his possession: a little chunk of Mars, in the form of a meteorite that had landed in Antarctica. When he looked at the meteorite under the same magnification Dr. Folk was using, he was amazed to see what looked like tiny 50-nanometer bacteria. They were connected in chains much like ordinary bacteria. When he checked for certain specific hydrocarbons associated with fossilized bacteria, he found a familiar signature. In addition, there were tiny magnetic particles found in the meteorite that were similar to the ones found inside many terrestrial bacteria. In 1996, Dave McKay of NASA went public with Romanek's work, suggesting that the Martian meteorite known as ALH84001 may show evidence of Martian life. The news captured headlines around the world.

NASA high-power electron microscope image of Martian meteorite. Does this look familiar?
Credit: NASA

Many skeptics remained to be convinced. NASA asked a panel of experts to render an opinion on the minimum size of life. Scientists at the 1999 NAS Workshop on Size Limits of Very Small Microorganisms agreed that a minimal living nanobe would require enough DNA for a core set of 250 proteins and at least one ribosome. A ribosome is a structure possessed by all known life forms; it acts like a workbench where proteins are hammered out according to the specs contained in the DNA. No ribosome, no proteins. All the DNA in the world won't help if you can't translate it into working proteins. As far as size is concerned, the ribosome is the kicker. It's at least 25 nanometers wide, all by itself. If you wadded up the core DNA along with the required ribosome, the tightest ball you can get would be about 200 nanometers in diameter. This might explain the largest of the nanobacteria, but it seemed to rule out the smaller ones, some of which are smaller than the ribosome alone.

That dampened the enthusiasm for nanobacteria somewhat. But hidden in that same workshop report was an another, more speculative, viewpoint: perhaps life could be smaller if it forsook DNA and used RNA instead.

A decade earlier, Sidney Altman and Thomas Cech had won the Nobel prize for demonstrating that RNA can have enzyme activity, meaning it can do some of the jobs previously considered the sole province of proteins. Cech even showed that RNA can reproduce itself without the aid of any other proteins. This amazing result led to speculation that life might have started out with a simple self-replicating polymer predating proteins by billions of years. This theory also seemed to imply that such RNA-based life must have been exterminated by Darwinian pressure with the advent of the "superior" DNA / protein scheme. After all, we haven't found any life forms based on self-replicating RNA, so they must have been wiped out, right? But what if that ancient life still exists in the world -- and still has the upper hand?

Meanwhile, Over in Finland.

At around the same time, at the University of Kuopio in Finland, Drs. Neva Ciftcioglu and Olavi Kajander were having a hell of a time with their cell cultures. Biologists the world round curse the lottery of the serum. It is not a minor complaint: a goodly percent of experiments with mammalian cells start out just fine only to succumb to some kind of infection along the way. Whatever it is, it kills the cells and ruins the experiment. There is nothing else to do but start over again, and hope that your serum isn't somehow compromised.

The serum we're talking about is FBS, Fetal Bovine Serum, which is derived from cow fetuses. It is blood that has been passed through a filter with tiny pores typically either 220 or 450 nanometers wide. These pores filter out cells and bacteria, creating a golden liquid with a complex mix of growth hormones and nutrients that help keep a mammalian cell culture alive.

Kajander and Ciftcioglu used 100-nanometer pores to produce a more pure serum, but still they found that their cell cultures were dying. They gamma-radiated the serum enough to kill any remnant bacteria, but again the serum was deadly to their cultures. Something had to be making it through the filter, but when they looked at the serum and tried to cultivate bacterial colonies, they invariably came up empty. Whatever was in the serum, it wasn't an ordinary bug. They were stumped, and went on to other research.

However, someone accidentally forgot a serum sample in the incubator, where it was left to slow cook for four months. When it was finally noticed, there was a slimy scum over the serum. Intrigued, they put the scum through some tests. They couldn't see anything interesting through the optical scope. But when they looked at it with a high-powered electron microscope, they were perplexed. It seemed that tiny bacteria had created a biofilm, a thin mat buttressed with deposits of the mineral apatite (calcium phosphate). But they were far too tiny, at least a hundredth of the size of normal bacteria. Whatever they were looking at had never been described in any of the microbiology literature.

When scientists see something that defies the current wisdom, they need to be extremely circumspect. This is especially true in biology, where there are so many variables to control for that it's hard to rule out contamination. They ran dozens of experiments, dosing the tiny bacteria with antibiotics, disinfectants and radiation. They discovered that for such a tiny thing, it was pretty tough. One wag dubbed it Conan the Bacteria. Whatever it was, it was slow-growing. While bacteria might double their population in minutes, it seemed to take days for these new bugs to double. That would explain why they hadn't been able to culture anything on such a short time scale.

But time and again, when they let the serum incubate for a sufficient period of time, it would turn cloudy, indicating a slow but steady growth. Except for the size and the slow growth rate, these creatures seemed a lot like bacteria. Apparently unaware of Dr. Folk's work, in a rare instance of parallel naming, they dubbed them nanobacteria.

The more they looked, the more they saw nanobacteria. Even human blood seemed to be infected with nanobacteria. But if nanobacteria were building calcium phosphate shells while cruising through the bloodstream, that couldn't possibly be good. Kajander and Ciftcioglu looked at arterial plaque and kidney stones and found nanobacteria in both places. Perhaps heart disease and kidney stones both had a nanobacterial origin. In 1997, they decided to publish.

You don't announce that you've discovered a new form of life without stirring up a little excitement in the biological community. Most biologists were incredulous, but at least one scientist decided to replicate the work. Dr. John Cisar, a researcher at the NIH, undertook a study to find these tiny spheres in serum and saliva. Very quickly, he did - but he came to a conclusion completely at odds with the Finnish team. To Dr. Cisar, the nodules were simply crystalline aggregates, something that can happen with ordinary chemistry. In 2000, Dr. Cisar published his report, claiming that bacterial contamination was responsible for the organic aspects of the phenomena and that self-propagating apatite was responsible for the apparent growth of the nodules.

Not everyone agreed. In fact, self- propagating apatite might be just as amazing as nanobacteria. In science, if you want to debunk something outrageous, it's best not to be outrageous yourself. Nevertheless, Dr. Cisar's results convinced many scientists that Kajander and Ciftcioglu had jumped the gun and that nanobacteria were simply an artifact.

Putting further strain on their credibility, Drs. Kajander and Ciftcioglu formed a company based on their research. It promised to test for and treat nanobacterial infections in people. Put yourself in their shoes: if you believed you could create a cure for a deadly disease, wouldn't you proceed to do so? As good as their intentions may be, however, some scientists suspect their objectivity might be colored by their business interests. The case for nanobacteria was tantalizing, they felt, but not sufficiently proven.

Don't Forget Texas

Thousands of nanobacteria crowd the inside wall of a diseased artery.
Credit: Credit: Lynch, Kirkland and Folk, University of Texas

Science is so big and diversified today that it's difficult to keep up with your own field, let alone an unrelated one. So when NASA announced the discovery of nanobacteria in the Martian meteorite, they had no idea that biologists in Finland were working on something similar. But an enterprising reporter for an Austin newspaper did a deep search for nanobacteria and came across the Finnish research. As Dr. Folk puts it, "This was great news both for me and for NASA -- independent confirmation by medical researchers of what ignorant geologists had thought they 'discovered.'" Soon after, NASA began cooperative work with some of the Finnish group.

In the meantime, Dr. Folk wasn't resting on his laurels. Supposedly retired, he continued to look under every rock for nanobacteria using the scanning electron microscope at the University of Texas. He was often successful. Along with Drs. Leo Lynch and Brenda Kirkland, he took hundreds of pictures (many of them have since been cataloged in a beautiful Photo Gallery by Dr. Lynch at Mississippi State University). One day, Dr. Kirkland brought in a sample of arterial plaque to put under the scope. They were surprised to see what looked like piles of nanobacteria coating the damaged artery. In 1997, they published the first photo of arterial plaque at 100,000 magnification.

By chance, they managed to hook up with the Mayo clinic. There were more than a few raised eyebrows at the prospect of these three geologists working on arterial plaque, but anyone who saw the pictures of plaque side-by-side with the pictures of travertine quickly got the connection. The biologists and the rock hounds complemented each other perfectly, and their research sped ahead.

Their hard work paid off in May of 2004 when the Mayo clinic announced that they had successfully repeated the Finnish experiments and added new data. Drs. John Lieske, Virginia Miller and their team ground up diseased human arteries and then filtered out everything smaller than 200 nanometers. That got rid of human cells and any ordinary bacteria. After a few weeks of incubation, they found increasing cloudiness in the serum. When it was examined with a high-power electron microscope, they saw small nodules, 20-200 nanometers in diameter, just as described by Kajander and Ciftcioglu. Interestingly, they found that the nodules absorbed uridine, one of the constituents of RNA.

They showed that particles filtered from genetically-caused aneurysms didn't show DNA activity, but particles from calcified aneurysms did. That implies that there is a living, replicating agent, under 200 nanometers in size, that is associated with a major cause of heart disease. Suddenly, nanobacteria were hot again.

The evidence is not indisputable. For instance, ordinary apatite crystals can soak up a little uridine all by themselves. And contaminants are always hard to rule out. But the Mayo team study helps to buttress the case for nanobacteria. Could it be that a slow-moving relic of the original primordial soup is still slinking around?

The First Earthlings?

A virus prepares to infect a normal bacteria; sausage-shaped nanobacteria are shown for scale.
© 2004 by Scott Anderson

A possible scenario starts to emerge: Billions of years ago, fed by the hot mineral springs that bubbled all over the early earth, different forms of pseudo-life started to emerge. Consisting of self-replicating RNA and simple proteins, these precursors to life were subject to the whims of their particular pond. With no cells walls, each genetic strand would depend on naturally-occurring amino acids to create proteins that might be shared by other bits of RNA or DNA. In a sense, the whole pond was the living creature. With much of the energy coming from thermal and chemical sources, there really weren't too many requirements for exotic metabolic proteins. It might be possible to create a replicating soup with just a few dozen genes.

Proteins are typically made of a small number of basic building blocks, like sheets and twisted ribbons. Great power comes from combining these small units into bigger complexes. Large-scale structures, like membranes and ribosomes, can be built simply by the continued addition of these basic bits. These larger objects typically require proteins to guide their construction. There might, however, be another way.

In a hot spring, currents set up when heat from below causes a plume of water to rise. When it hits the surface, it cools and then falls down the sides of the pool where it gets heated again.

Anything carried along with these currents would experience a regular cycle of heating and cooling. As it turns out, that's how scientists create working amounts of DNA in the lab: they heat a tiny sample, causing the paired strands of DNA to split apart. Then they let it cool, and as it does, chemicals in the mix reassemble a mirror image on each strand. The heating/cooling cycle is repeated, and for each temperature cycle the amount of DNA is doubled. After ten doublings, you have about 1,000 times more DNA than when you started. A hot spring might make an ideal incubator, delivering hot food to all parts of the pool and cycling through temperatures changes to encourage protein growth and genetic replication.

At some point, sheets large enough to serve as membranes may have formed. This would allow the formation of cells that could regulate their own affairs and separate their inner, cellular, chemistry from the rest of the pool. Separation had a big upside: if a pool changed in some fundamental way, the  blob of chemicals inside a membrane might outlive the unprotected life forms. But the membrane also brings problems: in order to really offer protection, it needs a bouncer at the door. The bouncer protein will let in the chosen molecules and turn away the rest. To create bouncers, though, you need extra genes, bringing the total up to sixty or so. These cells might look like mycoplasmas, tiny bacteria only 200 nanometers wide with a flimsy membrane. Still, with so much nutrition being cycled by the hot spring, the genetic needs of this creature would be small.

If one of these little creatures were somehow able to incorporate the abundance of dissolved calcium and phosphate into a tough shell, they might have an extra evolutionary advantage. They might even be able to survive the worst fate of such a pool - drying out. Such a bug could qualify as an ancestor of modern-day nanobacteria.

Jump ahead a few billion years to modern animals. With their hearts continuously delivering warm nutrients, including calcium and phosphate, a nanobacteria might find itself quite at home. As Kajander and Ciftcioglu put it, "The modern-day primordial soup is blood."

With the animal host doing most of the work, the nanobacteria would need just a minimal gene kit, keeping it very small and reducing its impact. Its slow growth rate wouldn't cause problems for the host for at least thirty or forty years - longer than the typical life span, and thus sparing it from Darwinian pressure.

Although this scenario is plausible, it is not the only one. Some scientists think nanobacteria have only recently taken up residence in human blood. Dr. Kajander points out that atherosclerotic plaque is a relatively new and growing cause of heart disease. As recently as 100 years ago, it was quite rare. On the other hand, people didn't live as long or eat as many French-fries back then either, so they may have had less of an opportunity to clog their arteries.

But whether nanobacteria are recent visitors or long-term residents, they seem to be up to no good. Their nastiness is twofold: they make hard shells and they cause human cells to die. The first wouldn't be so bad if it was limited to the bones, but hardness is not a desirable trait for a blood vessel. As to killing human cells, it may be excreted proteins that do the deed, but it is definitely bad manners for a guest. The possible ailments caused by nanobacteria include heart disease, kidney stones and cataracts. If Kajander and Ciftcioglu are correct, these maladies may yield to novel anti-nanobacterial medicines.

With so much at stake, the story of nanobacteria is not going to fade away. For the next few years we can expect to see many more studies like the one at Mayo. Hopefully, one of them will be able to sequence the DNA (or RNA). Only then will most scientists concede the existence of nanobacteria. Until then, it's bound to be an interesting journey.

Dr. Robert Folk responds:

Buongiorno, Scotto! Just finished your article -- congrats, a great job... But I wish you would credit Lynch and Kirkland as they are an integral part of the "Texas wing" of the connection between nannobacteria and heart disease -- geologists made a contribution as did medical researchers...

-- Ciao, RLFolk

A sign of a great scientist is eagerness to credit others. I've added more info about Drs. Lynch and Kirkland, both now at Mississippi State University.

-- Scott

Dr. Virginia Miller responds:

Dear Scott, Just finished reading your article and will add my congratulations to those of Dr. Folk. I think I reflect the collective enthusiasm of our group at Mayo by saying that working collaboratively on this project with the "Texas team" has been one of the most interesting and exciting projects of our careers. The results of this joint project are accepted for publication in the prestigious American Journal of Physiology: Heart and Circulatory Physiology, a peer reviewed journal published by the American Physiological Society.

-- Sincerely yours, Virginia Virginia M. Miller, PhD Professor, Surgery and Physiology

Here's a link to the article by Drs. Miller and Lieske (the abstract is free, the article requires a subscription or payment):

Mayo Clinic Study in the American Journal of Physiology

More links:

Mississippi State Nanobacteria Photo Gallery

Nanobac Life Sciences

The Calcium Bomb: The Nanobacteria Link to Heart Disease and Cancer

Copyright © 2000-2014 by Scott Anderson
For reprint rights, email the author:

Here are some other suggested readings in nanobacteria: