Deciphering Bacteria’s Defenses, One Gene at a Time

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.