杏吧原创

PHILIPPA UWINS wasn鈥檛 looking for the smallest bacteria known to science when she aimed her scanning electron microscope at a chunk of rock extracted from thousands of metres below the Australian seabed. An oil company had hired her to search for illite, a type of clay that can damage precious oil reservoirs 鈥� but what Uwins saw was far more perplexing: tiny filaments that looked suspiciously like bacterial or fungal colonies growing on the Triassic and Jurassic sandstone.

Even so, Uwins, a geologist from the University of Queensland in St Lucia, wasn鈥檛 jumping to any conclusions. It鈥檚 not that it鈥檚 unheard of for bacteria to live far below the Earth鈥檚 surface, but Uwins鈥檚 organisms seemed to adapt surprisingly easily to totally different conditions in an above-ground lab. What鈥檚 more, each organism was minuscule 鈥� some no more than 20 nanometres (20 billionths of a metre) in diameter. That was a problem, because creatures that small aren鈥檛 meant to exist 鈥� at least, not according to conventional microbiology. Viruses can be that tiny because they rely on host cells to reproduce, but any free-living organism needs to have a diameter of at least 100 to 250 nanometres (depending on whose calculations you trust) to cram in the machinery necessary for life.

Now Uwins is convinced that she has found an organism 鈥� she calls it a nanobe 鈥� that breaks that rule. In the past two years, she claims to have grown numerous nanobe colonies in her lab. And, with her University of Queensland colleagues, microbiologists Anthony Taylor and Richard Webb, Uwins says she has used three different stains for detecting DNA, with positive results, and made ultrathin sections of the organisms to reveal what appear to be cell walls 鈥� findings that are consistent with these being living creatures, not an inorganic process masquerading as bacterial colonies.

Uwins is not alone. Researchers in Finland and the US also claim to have discovered organisms that smash the generally accepted lower size limit for free-living life. What鈥檚 more, they suspect that their bugs might be making people sick. And just to add spice to the story, there鈥檚 even wild speculation that some of these living 鈥渘anobacteria鈥� could have arrived on Earth aboard meteorites that hurtled here from the far reaches of space.

If you thought such ruminations would turn off mainstream microbiologists, you鈥檇 be wrong. True, most try to distance themselves from the more bizarre claims of the champions of nanobacteria. But it鈥檚 also true that many microbiologists find thinking about creatures that challenge the conventions of their science a compelling exercise. What鈥檚 more, bacteria still have the ability to surprise us. In April, for example, biologists reported that they had discovered bacteria with diameters of up to 0.75 millimetres 鈥� large enough to be seen with the naked eye (Science, vol 284, p 493). More than three times as large by volume as the previous record holder and between 100 and 200 times longer than the average bacterium, Thiomargarita namibiensis is larger than this full stop. Nanobacteria researchers now hope to set records for tininess.

The nanobacteria controversy reached fever pitch on 7 August 1996, when NASA scientists stepped up to microphones and declared to the world that they had discovered 3.6-billion-year-old fossilised mini-bacteria in a Martian meteorite. Most of the egg-shaped and tubular globs were only 100 nanometres long, one thousandth the diameter of a human hair. This is tiny when compared with what most microbiologists accept as the smallest free-living bacteria 鈥� the spherical mycoplasma at 300 nanometres across. Still, NASA officials put the best spin on things they could, announcing that the fossils 鈥渁re strikingly similar to microscopic fossils of the tiniest bacteria found on Earth鈥�.

They were referring to the work of geologist Robert Folk at the University of Texas in Austin. In 1992, Folk claimed to have found fossils of dwarf bacteria in the white limestone around hot springs in Viterbo, Italy. He calls them nannobacteria 鈥� and that鈥檚 with two 鈥渘鈥漵, not the more conventional one. Folk acknowledges that inorganic processes can produce similar shapes ( Geology, vol 27, p 347), but says that an expert can distinguish between the two. Most microbiologists had simply ignored Folk鈥檚 claims 鈥� Folk himself cheerfully admits that 鈥�99 per cent of biologists think I鈥檓 crazy鈥� 鈥� but they could no longer turn a blind eye after the NASA meteorite appeared on the scene, followed all too soon by the first claims of living nanobacteria.

How low can you go?

So why has it been so difficult to accept the existence of mini-bacteria? The answer comes from some back-of-an-envelope calculations based on what an organism needs to sustain life. As long ago as the 1950s, Harold Morowitz at Yale University in New Haven, Connecticut, began working out the minimum number of genes a cell needs to live. Quite recently, researchers using new molecular biology tools completed that project by deleting genes from a species of mycoplasma, M. genitalium. This bacteria normally gets by with only 468 genes, but researchers now think it can survive with as few as 250 (New 杏吧原创, 16 August 1997, p 30).

If you took that minimum number of genes, added the requisite amount of RNA and assumed that the bacteria had a slow metabolism 鈥� with each gene having just one or two protein-generating ribosomes 鈥� you couldn鈥檛 cram it into anything less than 250 nanometres in diameter, says microbiologist Mary Jane Osborn of the University of Connecticut in Farmington. Michael Adams of the University of Georgia in Athens, who studies heat-loving bacteria, comes up with slightly different numbers. He calculates that the smallest diameter for a spherical cell would be about 180 nanometres. Some 10 per cent of that volume would be DNA, 10 per cent would be 65 ribosomes, 20 per cent would be proteins, 50 per cent would be water and the remaining 10 per cent sundries such as lipids. 鈥淚t would be very slow-growing and would have a major problem getting the nutrients it needed,鈥� he says. 鈥淵ou鈥檇 hardly find it on some stark meteorite. You might find it in blood.鈥�

Specs for the smallest theoretical bacterium are taken to even more minuscule limits by Jack Maniloff at the University of Rochester in New York, who studies the tiny mycoplasmas. His 鈥渞ather austere cell鈥� would be roughly 75 per cent water, need two to three copies of about 100 different protein plus the necessary DNA, and only one ribosome. Even then, says Maniloff, 鈥渕y quick-and-dirty calculation is that such a cell would have a volume equivalent to a 100-nanometre sphere鈥�. In light of that estimate, he adds, the nano-enthusiasts 鈥渉ave not seemed to appreciate how exceptional their claim is鈥�.

Alive and kicking

Uwins is well aware that people have a hard time believing in a free-living organism that is only 20 nanometres across 鈥� after all, a ribosome is roughly 20 nanometres long. 鈥淔inding out how the very small nanobes `live鈥� will be something we will be following up,鈥� she says. And although Uwins feels certain that her samples have not been contaminated with outside DNA as some critics have suggested, her team is now analysing the organism鈥檚 genes in the hope of demonstrating that it has a unique genome. Such genetic scrutiny should also reveal where the organisms are perched on the tree of life. 鈥淲e suspect that they are thermophilic organisms belonging to Archaea,鈥� says Uwins, referring to the kingdom of ancient microbial life forms that have free-floating DNA like bacteria and genetic similarities to nucleated cells.

Olavi Kajander and Neva Ciftcioglu at the University of Kuopio in Finland, on the other hand, are certain that they have found mini-bacteria. In July last year, they announced in the Proceedings of the National Academy of Sciences (vol 95, p 8274) that they had found nanobacteria alive and kicking in kidney stones. Kajander was searching with an electron microscope for a contaminant that was stopping laboratory cell cultures growing well when he found what appeared to be a white film of tiny bacteria. Culturing the strange microorganisms was difficult, not least because they had a remarkably slow metabolism 鈥� about 10 000 times slower than normal 鈥� which meant that the population took a leisurely one to five days to double in size. And although most of the spherical bacteria ranged in diameter from between 200 and 500 nanometres, 鈥渉uge numbers鈥� of them were between 50 and 80 nanometres.

That slow metabolism helps to explain how the bacteria can scrape by with so little space inside for the bare necessities of life, says Kajander. But he also suggests a far more radical explanation for their minuscule size. Each of the smaller forms could be a single piece of a whole nanobacterium 鈥� a part of its genome would be stored in one 鈥渃ell鈥�, a ribosome or two in another. These fragments could then link up, using molecules on their surfaces to create a complete organism that would be seen under the microscope as one of the larger cells in the population.

That suggestion, plus Kajander鈥檚 claims that the nanobacteria have a 鈥渄ifferent鈥� sort of DNA, are difficult for most microbiologists to digest. 鈥淭he bit about `viruses that get together and create their own cell鈥� is bizarre,鈥� says Maniloff. He suspects that Kajander and other nanobacteria researchers are mistaking bits of debris for life. Forty years ago, he notes, researchers thought that mycoplasmas went through a stage in their life cycle when they were just 100 nanometres across 鈥� they turned out to be pieces of lifeless detritus. 鈥淭hose who do not know history are indeed condemned to repeat it,鈥� says Maniloff. 鈥溞影稍�创s studying `nanobacteria鈥� in the 1990s have done an excellent job of repeating the errors of scientists studying mycoplasmas in the 1950s and early 1960s. In the words of that great philosopher Yogi Berra 鈥� a tough reference for your British readers 鈥� it鈥檚 d茅j脿 vu all over again.鈥�

Still, Kajander鈥檚 bigger nanobacteria are deemed perfectly acceptable, as a cell 500 nanometres in diameter is larger than mycoplasmas. 鈥淭hese guys definitely have something. Precisely how small they are is not really clear. If [Kajander] would get off this nanobusiness, I think it wouldn鈥檛 get people鈥檚 hair up so much,鈥� says Adams. Dennis Carson at the University of California in San Diego, who wrote an editorial to accompany Kajander鈥檚 PNAS report, believes that comparisons between Kajander鈥檚 nanobacteria and the ones in the Martian meteorite have also harmed his case. 鈥淭hose people are looking at fossils and writing philosophy. This is medicine,鈥� he says.

That medicine revolves around the nanobacteria鈥檚 ability to build protective 鈥渃astles鈥� around themselves, apparently by precipitating carbonate apatite, the same substance that makes up most kidney stones. These castles, says Kajander, could act in the kidneys like grains of sand in a pearl-producing oyster, triggering the growth of stones. Using transmission electron microscopy, Kajander and Ciftcioglu have identified nanobacteria in kidney stones from 72 patients. Antibody staining confirmed that result, but the pi猫ce de r茅sistance was culturing living nanobacteria from the stones. And when they injected nanobacteria into the blood of rabbits, they later appeared in the kidneys and damaged the part of the organ where stones typically form.

To kidney specialists the idea that a bacterium 鈥� albeit a minuscule one 鈥� causes kidney stones doesn鈥檛 seem so bizarre. Although the cause of most kidney stones has been a mystery, bacterial infections are known to cause a rarer kind of stone 鈥� the bacterium in question makes urine more acidic, encouraging mineral precipitation.

Small but deadly

Another kind of kidney disorder, polycystic kidney disease, may also be linked to nanobacteria, according to findings presented in June by Marcia Miller-Hjelle and J. Thomas Hjelle from the University of Illinois in Peoria at a Chicago meeting of the American Society for Microbiology. Worldwide, over 12 million people suffer from this inherited, incurable disorder in which the kidneys gradually swell up with cysts until they no longer function. Studies in animals suggest that the disease is part genetic, part infectious agent 鈥� mice that have been genetically engineered to get the disease remain healthy as long as they are kept in a germ-free environment. What鈥檚 more, people with polycystic kidney diseases are far more likely to develop kidney stones than other people, so it would seem possible that the same agent causes both diseases.

Miller-Hjelle and Hjelle, working with Kajander, have cultured nanobacteria from 10 out of 12 kidneys from patients with polycystic kidney disease, and detected the nanobacteria in all the cystic kidneys they have examined with electron microscopy. What鈥檚 more, Miller-Hjelle and Hjelle claim that the nanobacteria make a toxin that they have identified in the cyst fluids, and that they have found nanobacteria in the blood and urine of a 23-year-old patient with rapidly enlarging cysts in his kidneys. Even so, they admit that they have yet to complete comparable studies in people without kidney disease, and at this stage it鈥檚 impossible to say whether the nanobacteria are causing the disease or are innocent bystanders.

鈥淭his opens a new door if it鈥檚 true,鈥� says David Chan, a urologist at Johns Hopkins Medical Institutions in Baltimore. But, he cautions, 鈥渘o one has repudiated it or confirmed it. We have to be excited, but also careful.鈥�

And what about those claims that nanobacteria may have come from outer space? Most people are familiar with the hypothesis that life on Earth is descended from extraterrestrial microbes that reached our planet on a comet or asteroid (New 杏吧原创, 12 September 1998, p 24). Kajander says his nanobacteria would be ideal for such a trip. Ensconced in their 鈥渃astles鈥� 鈥� a mineral coat similar to the heat shields on the space shuttle, he says 鈥� nanobacteria could withstand the extremes of heat and pressure, as well as the radiation exposure, that such a journey would entail. Uwins says her nanobes are equally robust. What鈥檚 to say, muses Kajander, that these microbes that defy the rules of life on Earth didn鈥檛 arrive here from Mars?

It鈥檚 reflections like this that perhaps best illustrate why the nano-enthusiasts stay fired up in the face of widespread scepticism and not a little ridicule. They know that if what they suspect turns out to be true, it would not only guarantee them scientific fame and fortune, but also challenge everything we think we know about life on Earth.