Always a trailblazer, Susan Gottesman laid the foundation for two new fields in bacterial gene regulation.
As an undergraduate at Radcliffe College—Harvard's allgirl sister institution—in the 1960s, Susan Gottesman earned pocket money working as a technician in Jim Watson's Harvard lab. "I would hear stories of people going to mixers at Radcliffe and meeting this strange guy who said he was a professor," she laughs. "But in the lab he was perfectly well behaved." And he encouraged Gottesman to get some hands-on experience by helping a grad student with his experiments. "I didn't know enough science to understand everything that was going on," she says. "But I got to do what I wanted, which was playing in a lab and learning through osmosis."
And through the years, Gottesman has certainly built on everything she's absorbed. As an independent investigator at the National Institutes...
Early in her career, Gottesman dissected the functions of energy-dependent proteases that are the bacterial equivalents of the proteasome. "Before Susan, there wasn't much known about these proteins," says Thomas Silhavy, also at Princeton. "She figured out that their substrates are often regulatory proteins, and that proteolysis has a major impact on gene regulation. I think that work got her into the National Academy."
Gottesman also discovered dozens of small RNAs that direct E. coli's response to a variety of environmental assaults, from osmotic shock to iron deprivation. "Small RNAs are a very big deal, now," notes Botstein. "And Susan figured out they're major regulators in bacteria. Which makes her the reigning expert in that field."
"She had a first career in proteolysis," says former postdoc Eric Masse of the University of Sherbrooke in Quebec. "That career alone would have fulfilled most any researcher. But then she found these small RNAs and underwent a kind of renaissance. She launched this new field, all by herself."
Gottesman came of age, scientifically speaking, in the "dark ages" before recombinant DNA technology—that technological knight in shining armor—burst onto the scene. Back then, people relied on genetic manipulation to study their systems of interest. As a graduate student at Harvard in the late 1960s, Gottesman adapted an approach Jon Beckwith had developed for studying the lac operon to look at regulation of the E. coli arabinose operon. The technique involved getting lambda phage to do the dirty work of isolating one's favorite genes as it excised itself—along with the surrounding bits of chromosomal DNA. "It was a kind of cloning—but limited to the genes that lie next to the site where the phage was inserted," says Beckwith. "Susan was able to get a transducing phage for the arabinose genes. That was the first time we'd shown that this technique could work for something other than the lac operon."
The work gave her a taste for lambda, which is where she focused her attention as a postdoc at NIH in the laboratory of Max Gottesman (no relation). She was interested in studying how phages insert and remove themselves from the bacterial chromosome, and she began to work on setting up an in vitro integration/ excision reaction. "We were using crude extracts, then adding integrase, which does the integration, and Xis, which was needed for excision," she says. "And we did get it to work, but I'm not much of a biochemist, so I think it didn't go as quickly as it might have."
Part of the problem was the instability of Xis. The protein was degraded as soon as it was made, thus getting enough to run the reaction was a challenge. "So I started reading some papers that had been published about an ATP-dependent protease in E. coli called Lon," says Gottesman. "I wondered: Is this the protease that's eating up Xis? And if I mutate it, will I get more Xis so I can do the biochemistry better?"
Those mutants, however, were even more interesting than she'd expected. For one, they were sensitive to ultraviolet light: The protease is called Lon because mutants exposed to UV don't divide properly and instead become elongated. What's more, Lonless cells are a big, sticky mess. "The mutants overproduce this capsular polysaccharide, colanic acid, and they make these gooey, gooey colonies."
"Her bench would be piled high with Petri dishes," says Harvard's Nancy Kleckner, who was a postdoc in Botstein's Princeton lab when Gottesman worked there as a sort of post-postdoc in the mid-1970s before returning to NIH to start her own lab. "And these mutants produced slime. So not only did she have stacks of Petri dishes, but they were oozing slime all over the place."
Which made them especially intriguing. If both the lightsensitivity and the slime were caused by the absence of Lon, Gottesman says, "it suggested that this protease had a lot of regulatory effects that really weren't understood." First, she determined that the two effects were separate, by making mutations that would suppress one of the phenotypes, but not the other. That confirmed that Lon must have more than one target. "So we started making guesses, based on the genetics, and then seeing whether the proteins we identified were unstable in Lon-plus cells but stable in Lon-minus cells," she says. One such protein was a cell-division inhibitor called SulA. The protein is produced in response to DNA damage so that cells cease dividing while they make the necessary repairs. Of course, once the cell has recovered, it needs SulA to go away so it can resume reproducing. And in wild-type cells, Lon removes SulA—results Gottesman published in 1981 and 1983.
For the cell, generating proteins like SulA only to then destroy them "is an expensive way to do things," says Gottesman. "There are other ways to down-regulate protein activity." But that doesn't make it a less popular approach. That same sort of regulating proteolysis also controls progression through the cell cycle, even in eukaryotes, she says. "So it's become clear that all cells do this and they do it a lot."
Gottesman figured it out by making a mutation here, a mutation there, and seeing how things played out. "It's old-style and low-tech: You just do the right things with Petri dishes, pipettes, and bacteria," says Kleckner. "Just knowing what the molecules are doesn't help, you need to know what they do. The genetic approach is an art form—and Susan is a real artist."
"We used to spend more time discussing an experiment than we did actually doing it," adds Don Court, a colleague of Gottesman's at NIH. "We stood in the hallway at a chalkboard and everybody put in their two cents. With genetics, you're trying to make deductions with a sheet between you and the real world, so you can't see anything directly. So you'd reduce things down to the simplest experiment you could do to get the clearest results."
Which included doing all the necessary controls. "My students think I'm a bit of a 'control freak,'" laughs Janine Trempy of Oregon State University, Gottesman's former postdoc. "But Susan taught me the importance of both positive and negative controls. She wouldn't look at your data unless you'd done all the controls. Which really speaks to her scientific integrity."
That same sort of methodical, mutational approach helped Gottesman dissect the role that proteolysis plays in capsule synthesis. She discovered that RcsA, a protein that regulates capsular polysaccharide synthesis, is rapidly degraded by the Lon protease. Knock out lon or overexpress rcsA from a plasmid, and the cells go gooey. But Gottesman got to wondering: If Lon gets rid of RcsA when the cell no longer needs it, what triggers the protein's production in the first place? That's when they discovered DsrA: a small RNA, encoded near the rcsA gene, that boosts the protein's translation—as well as the translation of the sigma factor RpoS. RpoS, in turn, serves as a global regulator for a slew of other bacterial genes.
"Susan stuck with the question of capsular polysaccharide biosynthesis in a way I really admire," says Beckwith. "It's easy to start out with a problem and do the easy things, and then move on when things get harder. But sometimes when you stick with a system it starts generating all sorts of unexpected directions—like the small RNAs."
And DsrA was just the beginning. "She was not doing badly with that one small RNA," says Masse. "But then she realized that the RNA was highly conserved in bacteria, and that you could use bioinformatics to explore the whole genome for more." Which is how she and her NIH colleague Gigi Storz found 20 additional small RNAs, work published in Genes and Development in 2001. "To discover a whole set of genes like that is like, wow, you really hit the jackpot," says Masse, who still works on one of the small RNAs discovered in Gottesman's lab. "When she would upload a sequence and wait for the computer to give her a conservation rate, it was like she was in Las Vegas at a slot machine," he laughs. "But that's her style. She wants to have fun in lab."
And nothing is more fun than new data. "If she knew I was coming in to show her a result, she'd have this big smile, with her hands clutched together, like a 4-year-old about to receive a great gift," says Masse. "That kind of thing could keep you going forever."
As would a declaration that the result was "cute." "She has her own little slang," says former postdoc Cari Vanderpool of the University of Illinois at Urbana-Champaign. "Any time you could elicit an 'oh, that's cute' or get her to chuckle about your results was a good day, because you could surprise Susan and make her happy with what you'd found. She's just genuinely fascinated by the science."
These days Gottesman continues to chase down what these small RNAs are doing for the cell. "We're interested in the regulatory networks they're part of," she says. "What's making them, what's regulating their synthesis, and why are they regulating their targets? For each one, we'd really like to understand the physiology. So that's kept us occupied."
As does running the lab and playing with her grandchildren. "She's this well-known scientist and a member of the National Academy, but she's also family-oriented," says Vanderpool. Trempy agrees. "Twenty-four years ago she was the first woman scientist I'd met during my training—and the first woman who presented the entire package: a smart, successful scientist, well respected by her peers. But dedicated to her family."
"It never occurred to me that I couldn't do both," says Gottesman. And having a family gave her the balance she needed. "When your experiment isn't working, there's nothing better than going home to kids who couldn't care less."