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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.
By Scott Anderson
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."
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| Bacterial Club Med? Could this steaming
rainbow of heat-loving bacteria be the wellspring of life
-- and the source of some nasty ailments? |
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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
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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 |
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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.
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NASA high-power electron microscope
image of Martian meteorite. Does this look familiar?
Credit: NASA |
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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
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Thousands of nanobacteria
crowd the inside wall of a diseased artery.
Credit: Credit: Lynch, Kirkland
and Folk, University of Texas
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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?
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A virus prepares to infect
a normal bacteria; sausage-shaped nanobacteria are shown
for scale.
© 2004 by Scott
Anderson |
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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:
Scott_Anderson@ScienceForPeople.com
Here are some other suggested readings in nanobacteria:
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