Richard Losick and Jonathan Dworkin In 1918, German embryologist Hans Spemann took an early newt embryo and removed some cells that were destined to become skin. He transplanted these cells to a region destined to develop into nervous tissue and watched as the prospective skin cells adopted the fate of their neighbors. When he did the same experiment using embryos at a later stage of development, the prospective skin cells stuck to their guns and grew into skin cells. These results, he surmised, meant that at some point during development, cells become committed to a particular destiny–and there’s no turning back.
This type of commitment is not peculiar to newts. Similar decision points direct the growth and development of many organisms, even simple bacteria. Now, studying Bacillus subtilis, a microbe that can form spores when times are tough, Richard Losick of Harvard University and Jonathan Dworkin, now at Columbia University, have identified the genes necessary for commitment to sporulation. Their studies appear in the May 6 issue of Cell.
In the wild, B. subtilis reacts to extended periods of starvation by forming a tough little spore that can withstand elevated temperatures, high pressure, UV irradiation, and other environmental assaults. These spores, which Losick describes as “among the most macho spores on the planet,” can lie dormant for years–millennia, some think–and then germinate when conditions become more favorable.
In times of plenty, bacteria reproduce by splitting in half–forming two daughter cells of equal size. When nutrients are scarce, however, the bacterium divides asymmetrically, generating a large “mother” cell with a small “forespore” at one end. The forespore gets engulfed by the mother and its cell wall toughens, producing the long-lasting macho spore; to release the spore, the mother cell will self-destruct—”as mothers often do in giving rise to their mature offspring,” jokes David Dubnau of the Public Health Research Institute in Newark, NJ.
At some point in this process, the bacterium reaches a point of no return: even if nutrients become available, the mother dies and the forespore enters sporehood. But what is it that locks the mother and her forespore into their individual fates? “We originally assumed that it had to do with formation of the septum”–the wall that separates the mother from the forespore–says Losick. But that’s not the whole story. It turns out that in each of the progeny–the mother and the forespore–certain genes get switched on to keep them headed down their chosen developmental paths. Using a combination of genetic and microscopic techniques, Dworkin and Losick have found these commitment genes.
“This is the first paper that identifies a specific gene involved in the commitment process,” says Linc Sonensheinof Tufts University School of Medicine.
“It’s a substantial achievement,” adds Patrick Piggot of Temple University School of Medicine.
The team’s search for the genes that dictate commitment began with a pair of proteins called sigma F and sigma E. Shortly after the septum forms, each progeny cell activates its own special regulatory protein: sigma F in the forespore and sigma E in the mother. Each of these master regulators then switches on its own specific suite of genes.
Dworkin and Losick suspected that sigma E and sigma F were involved in commitment because when they eliminated these genes, the progeny cells were able to shirk their destinies. Mothers lacking sigma E, when presented with nutrients, abandon their suicidal tendencies and begin to divide normally, producing daughter cells of equal size. And forespores lacking sigma F foreswear their sporish fate and do the same. Thus, the authors reasoned, sigma E and sigma F must turn on genes that keep this type of outgrowth in check–in other words, genes that control commitment.
To figure out which genes they were, Dworkin and Losick knocked out every gene that’s controlled by sigma F and looked for cells that could resume growth when plopped into rich medium. After a bit of trial and error, the researchers determined that a pair of genes–called spoIIP and spoIIQ–contribute to commitment in the forespore. And that spoIIP on its own seals the mother cell’s irreversible fate.
“The next big challenge,” says Losick, “is to figure out exactly what P and Q are doing.” Work from his lab and others suggests that proteins encoded by these genes might be involved in synthesizing the material that forms the septum.
These sorts of studies are possible now, says Piggot, because the B. subtilis genome has been completely sequenced and microarray analyses have allowed researchers to track the activities of large numbers of genes. So Losick and Dworkin knew, for example, which genes were controlled by sigma F–which gave them a place to start their search.
Also, microscopic techniques have gotten better: the researchers were able to use green fluorescent proteins, produced inside the bacterial cells, to help them watch the forespores and their mothers divide. “For a long time the dogma was that you couldn’t do this kind of microscopy with bacteria: they were just too damned small,” says Dubnau. “Rich was one of the pioneers in showing that you could use sophisticated microscopic techniques to do really meaningful work in bacteria. This paper is another example of how this pioneering approach has paid off.”
Interestingly, Losick finds that B. subtilis’s “evil cousin” Bacillus anthracis–the bug that causes anthrax–has two genes that resemble spoIIP: one that gets switched on by sigma F, and another that’s under the control of sigma E. Whether the pathogen uses these proteins in forming its spores remains to be seen. Losick doesn’t plan to pursue that particular problem because working with B. anthracis requires high-level containment facilities that he doesn’t have access to.
Happily for Losick, not all bacillus are life-threatening. Some are even considered a delicacy. Natto, a traditional Japanese dish, is made from soybeans fermented by a strain of B. subtilis. “I’ve tried it,” says Losick. “It’s completely disgusting.” Such gustatory investigations are surely above and beyond the call of duty. It’s hard to imagine that Hans Spemann ever ate a newt.