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Bacillus subtilis going into lockdown mode. This spore-forming bacteria is a close cousin of anthrax.
[Credit: Patrick Eichenberger]

Both deadly and benign spore-forming bacteria’s genes allow them to eke it out in extreme conditions.

By Adam T. Hadhazy, SciencelineNYU – The bacteria in the test tube are harmless, but boy are they stinky—a general swampiness with hints of manure, leavened with a moldy food funk. Lab workers retrieve vials of these foul-smelling, soil-dwelling microbes and then set about starving them.

But this cruelty on a microscopic scale has a purpose, and besides, Bacillus subtilis is a tough customer. The bacteria have a special method of survival—when under duress, they encapsulate themselves in protective shells called spores. By studying the genetic underpinnings of B. subtilis’ fortress-making abilities, scientists hope to be better armed against its dangerous bacterial cousin anthrax. In addition, analyzing the dynamics of spore formation may reveal more about the cellular mechanics of microorganisms in general.

Back in the lab, B. subtilis is out of food and begins to show off its bunker mentality by entering a process called sporulation. This undertaking produces a durable, spherical spore that allows B. subtilis to withstand severe dehydration, boiling, freezing and even high levels of radiation. The microbe can remain in this stasis indefinitely, waiting it out until conditions improve. Bacterial spores are so rugged there is concern they could survive on spacecraft and end up on places like Mars.

“They are the most resistant of all [known] living cells,” says Patrick Eichenberger, a professor of biology at New York University whose laboratory is dedicated to the study of B. subtilis.

This invulnerability makes spore-forming bacteria like B. subtilis and its relative Bacillus anthracis, better known as anthrax, notoriously difficult to wipe out. Take the case of the 2001 attacks on the Hart Senate Office Building: After the arrival of a couple of envelopes containing anthrax spores, it took months to thoroughly decontaminate the premises at a cost of about $27 million, according to the Government Accountability Office. Five people died and 17 were injured as a result of this still-unsolved biological assault. Cleansing of a nearby mail facility cost an additional $130 million and took over two years.

Any insight into how stubborn microbes remain safe behind their defensive shields would be useful in rooting out a future anthrax infestation. In fact, a 2002 grant from the Department of Defense initially funded the lab that Eichenberger now runs. “If we know more about what makes subtilis resistant, then we know more about what makes anthracis resistant,” he explains.

Finding Answers in the Genes

Eichenberger intends to uncover the network of genes involved in B. subtilis’ spore-making ability. Besides aiding efforts to thwart bioterrorism, this research may help curtail more common infections from other spore-formers, such as Clostridium difficile. The scourge of hospitals, this bacterium afflicts one in five in-patients with diarrhea and can even cause death in elderly victims.

Though B. subtilis is among the most studied organisms on the planet, researchers understand relatively little about how the single-celled bacterium accomplishes the feat of sporulation. Once researchers have obtained a complete “assembly map” for spore-forming, drugs and chemicals could be manufactured to either halt the transformation or go after chinks in the bacteria’s armor, according to Eichenberger.

While scientists have unveiled the genome, or entire DNA sequence, of hundreds of species (including our own), this is not the same thing as knowing how that sequence is actually translated into action. DNA is segmented into a number of genes, which are the instructions that cells use to make proteins. In turn, these proteins perform specific tasks in the cell or around a creature’s body.

But even with the genome of the single-celled B. subtilis organism in hand, microbiologists still do not know when certain genes are turned on and off, and what they all do once activated. Overall, gene regulation is a byzantine process of organic molecules having far-flung, miniscule effects amid a myriad of redundancies.

“It’s comparable to the economy of, say, Thailand,” offers Rich Bonneau, a professor of biology and computer science at New York University who is collaborating with Eichenberger. “We can make general predictions and observations, and we can tell to some extent what disrupting one trucking line will do, or if a port is shut down, for example.” But he and his colleagues cannot really extrapolate how each “truck,” or protein, influences the entire organism’s overall economy.

And until scientists know how each part contributes to the whole, they cannot bridge the gap between a genetic blueprint and a living bacterium, whether it is innocuous B. subtilis or deadly B. anthracis. “You haven’t solved a system unless you can predict results,” says Eichenberger.

Even the smallest genomes are awfully large chunks of information for researchers to organize into a sensible system. The size of B. subtilis’ genome, at least when compared to the human genome’s 3 billion base pairs, is a slightly more manageable 4 million base pairs. These are segregated into a little over 4,000 genes. It is quite difficult to fathom how a fully functioning, odoriferous creature springs forth from these sparse genetic instructions. But by isolating the 400 to 500 genes responsible for sporulation, or roughly 10 percent of the bacterium’s genome, researchers hope to start small and then work their way up.

“We have only reduced the complexity of our problem by a factor of 10,” says Eichenberger, but he says this is still a big step in finding out what each gene and the proteins it codes for are doing.

Tracking the Proteins Wherever They Go

Meanwhile, the experiment in Eichenberger’s laboratory continues—the famished B. subtilis bugs have given up all hope of sustenance and are hunkering down for the interim. This transition from vulnerable wet specks to ultra-hardy spores will take about eight hours. Eichenberger wants to find out how the proteins in this process assemble into regular patterns and form the outermost perimeter of the spore, called the spore coat. He has already identified 24 novel proteins involved in setting up these fortifications.

“We have certain tricks we can use,” he says. The students in his lab have tagged individual proteins in the fasting B. subtilis with green or red fluorescent markers so they can catalog when the protein is created and where it ends up in the spore-making process.

Another technique is to “knock out” individual genes that alter the bacterium’s formation of the spore coat. If the coat develops improperly, the researchers can infer which genes are needed to yield the spore coat and what role each particular gene plays. But this is a time-consuming process, and many times there will be no discernible damage to the formed spore coat, if it develops at all. Instead there may just be a bunch of dead microbes, which doesn’t reveal a whole lot.

“It’s like taking pliers to your DVD player and randomly popping something out. Then you try turning it back on to figure out how the whole thing works,” says Bonneau.

To further complicate things, sporulation itself is not a straightforward, sequential process. While the genes do turn on in a step-wise, biblical “X begets Y begets Z” manner, the effect that the genes have is not that simple. A study (pdf) published in the Journal of Bacteriology last year showed how a series of consecutively activated genes modify the forming spore coat in opposing ways. It is similar to putting more than enough icing on the first layer in a cake, only to then wipe some of the icing off to achieve the ideal, intended coating before moving on to the next layer.

“We were surprised by this level of fine-tuning in the sporulation process,” says Lee Kroos, an author of the paper and a professor of biochemistry and molecular biology at Michigan State University in East Lansing. But despite this observed specificity, the paper also showed that B. subtilis can tolerate suboptimal situations—even if some of the genes in the sequence are “skipped” or de-emphasized, the spore coat can still turn out just fine. “It’s a remarkably robust system,” adds Kroos.

Enlightening Evidence

The lab work at NYU carries on: After centrifuging the spores in a beaker to obtain a nice, sample-ready clump of B. subtilis, Eichenberger or a team member prepares a slide and then heads to the microscopy room. By applying special illuminators, researchers can make the fluorescent dyes in the microbe’s proteins light up like glow sticks at a jam-band concert. A mini-camera then snaps some shots of the glowing germs, and with that, the two-day experiment is over.

Often, particular strains are preserved for future lab inquiry in a bureau-like freezer that has drawers full of mutated B. subtilis. Nearby, a growing chamber agitates more microbes in a nutrient solution for the next round of tests. Life is good right now for the feasting little guys, and in accordance, they emit a powerful reek.

“The smell gets pretty nasty sometimes,” Eichenberger admits, still not quite accustomed after years of manipulating the microorganisms.

This mild annoyance, however, isn’t all that bad when compared to the life-threatening traits of anthrax, B. subtilis’ close relative. As Eichenberger continues to decipher sporulation, and eventually the entire life cycle of B. subtilis gene-by-gene, perhaps he will unveil the origin of this milder form of nastiness as well.