eTopics

eTopic 3.1 Richard Losick: The Thrill of Discovery in Molecular Microbiology: An Interview

Richard Losick has taught at Harvard for more than three decades, and his creative teaching was honored when he was named a Howard Hughes Medical Institute (HHMI) Professor. A member of the National Academy of Sciences, Losick has authored more than 200 research articles on bacterial genetics and cell biology. He discovered how Bacillus sporulation is controlled by alternative sigma factors that regulate transcription. To dissect sporulation and the cell division cycle, he uses fluorescent proteins to visualize individual subcellular parts of bacteria.

Why did you decide to make a career in microbiology? Did you begin research as an undergraduate?

When I was in grade school, I used to read science books and do experiments on my own. I thought that school was boring, and I didn’t realize that science was a subject that I would learn about later in school!

When I was an undergraduate at Princeton, I joined the laboratory of a newly arrived professor, Charles Gilvarg, who studied amino acid biosynthesis in bacteria. I fell in love with bacteria from that experience, and I have remained fascinated by bacteria ever since.

Why do you study Bacillus subtilis? How is your work relevant to the insecticide B. thuringiensis and the human pathogen B. anthracis?

Bacillus subtilis is extraordinarily rich in its biology. Its ability to metamorphose into a spore is endlessly fascinating. B. subtilis has the capacity to differentiate into specialized cells for DNA uptake (competence), to swim, to swarm on surfaces, to cannibalize sibling cells, and to form architecturally elaborate, multicellular communities. At the same time, its property of genetic competence (gene transfer between cells) makes it exceptionally facile for molecular genetic manipulation.

B. thuringiensis and B. anthracis are less favorable than B. subtilis for genetic manipulation, but the main features of spore formation are very similar in all three organisms. The crystal toxin of B. thuringiensis that has useful insecticidal properties is produced under sporulation control. The remarkable environmental resistance properties of B. subtilis spores are shared with its “evil cousin” B. anthracis and help us understand the challenge posed by the bioterrorism agent.

Your career has spanned a generation of revolutionary developments in genetics and molecular biology, such as the discovery of the sigma factors that regulate transcription of many genes. Would you describe how one of these developments came about?

After winning my PhD from MIT, I came to Harvard to work on a project involving the effect of a virus on bacterial cell membranes. The sigma subunit of E. coli RNA polymerase had just been discovered (by Dick Burgess and Andrew Travers at Harvard and by John Dunn and Ekkehard Bautz at Rutgers), and people were excited about the possible existence of alternative sigma factors (proteins that regulate large groups of genes). Meanwhile, A. L. Sonenshein was studying how certain bacterial viruses (bacteriophages) get trapped during spore formation by Bacillus subtilis. The phage is virulent but is shut off transcriptionally upon entering a sporulating cell. The phages are unable to grow in cells that have started to sporulate and instead become trapped in what will become the spore. So the unfortunate spore, after waiting for long periods of time to germinate, gets killed upon germination by the enemy within.

Putting two and two together, we reasoned that maybe changes in the RNA polymerase during entry into sporulation could help explain the shutdown in viral gene expression. We got so excited about this idea that we began to work on RNA polymerase full time. This eventually led to the discovery that sporulation is governed by a cascade of bacterial sigma factors.

Meanwhile, in collaboration with Jan Pero, we found that phage SP01 encodes and produces two alternative sigma factors upon infecting growing cells. These regulatory proteins, the first examples of alternative sigmas, drive the phage program of gene transcription. Since then, alternative sigmas have emerged as a widespread theme in the control of gene sets in many different kinds of bacteria.

What is the significance of sporulation as a developmental process?

Sporulation is an extremely attractive system for understanding cellular differentiation because it is both complicated enough to mimic features of development in higher organisms and accessible enough to be subject to genetic, biochemical, and cytological manipulation. Sporulating cells undergo a true process of cellular differentiation, in which the two cellular compartments of the sporangium communicate with each other in a back-and-forth manner by intricate signaling systems.

How was the technology developed to visualize chromosome dynamics in Bacillus?

In the old days, bacteria were viewed as an amorphous vessel with enzymes floating around inside. Now we understand that bacteria are highly organized, that proteins often have subcellular addresses in bacteria; thus, cytology has become an important aspect of bacterial research.

One of the keys to this revolution was the introduction of green fluorescent protein (GFP) as a tool for visualizing the subcellular localization of proteins in cells. Andrew Murray and Andrew Belmont figured out that GFP could also be harnessed to visualize the location of sites on DNA by attaching the fluorescent protein to a protein that binds to specific sequences in DNA. Murray and Belmont used this trick to visualize centromeres in yeast. Andrew Wright and I extended this methodology to visualizing sites on chromosomes in bacteria. Traditional thinking was that origins tended to remain in cell middles, but we found that replication origins moved toward the cell poles during chromosome segregation, and with GFP we could visualize this in living cells.

How do students get involved with your research? Do you work with undergraduates as well as graduate students?

Yes, I always have undergraduates as well as graduate students in my lab, and both have contributed to some of our most significant findings. One special undergraduate was Aurelio Teleman, who was both a brilliant student and a gifted investigator. Working with a graduate student, Teleman helped create and exploit the system for visualizing the movement of a specific site in the bacterial chromosome that had been tagged with tandem lactose operon operators. He is now at the European Molecular Biology Laboratory.

As an educator, what innovations have you developed?

I enjoy the challenge of explaining complicated concepts and of making large-class instruction lively and interactive. I run a program for placing freshmen from disadvantaged backgrounds in laboratories of faculty for long-term research mentoring. Such students are often at greatest risk for dropping out of science, but a long-term relationship with a laboratory that begins early during the college years can keep these students (indeed all students) excited about science throughout college and beyond.

I believe that a positive experience in experimental work can represent one of the most memorable aspects of undergraduate education. And its benefit is not limited to future scientists. A science major with an associated experience in discovery research is excellent preparation for a wide variety of careers, including medicine, business, law, public policy, journalism, and education.

What advice do you have for today’s students?

Join a lab! In science you can learn something that nobody else on Earth ever knew before. There’s a special thrill about learning how living things function—the inner workings of living things. If you learn some new aspect of it, even if it’s a tiny part, you’ve advanced knowledge for the whole of humankind.

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Richard Losick, Harvard College Professor. Losick has made major discoveries on the molecular and cellular biology of Bacillus subtilis. Photo courtesy of Richard Losick.

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Sporulating cells of Bacillus subtilis with molecular parts labeled by fluorophores. Red fluorescence (FM4-64) labels the cell membrane; blue (DAPI) the DNA; and green (green fluorescent protein) a DNA pump that translocates a chromosome across the division septum from the mother cell (the big cell) into the forespore (the small cell on the upper right). (Cell length, 2–3 μm.) Photo courtesy of Richard Losick.

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Losick teaching undergraduates at Harvard. Harvard Gazette staff photo by Kris Snibbe.