Mastering the Microbe

UM lab engages tiny organisms to study aging, multicellularity

By Cary Shimek

Professor Frank Rosenzweig admits his interests are many and diverse. For the past decade, researchers in his UM lab have studied everything from creating super yeast for possible use in ethanol biorefineries to how pathogens evolve in a cystic fibrosis lung.

“I’ve been accused of being interested in everything, which I suppose is true,” he says in his slight, precise Tennessee drawl. “But the unifying thread from all
our projects is that we use microorganisms to ask fundamental questions about the evolution of life on Earth.”

Two of the many questions that excite him most of late: What can yeast teach us about cellular aging? And how might a single-celled microbe produce offspring that start cooperating — even to the point of making the jump to true multicellularity?

.   .   .   .   .   .   .   .   .   .

Useful for making beer, wine and bread, yeast also are a powerful tool for probing the basic biology of mammalian cells. One of the great mysteries about that biology is how and why cells age.

Yeast lifespan can be measured in two ways: the number of times a cell divides before it dies or the number of days a yeast cell can live without dividing at all. Because some of the most important cell types in human bodies, such as nerves, rarely divide, Rosenzweig became interested in this second kind of yeast aging, called chronological aging. In order to stop yeast from dividing, researchers usually just starve cells and see how long they live.

“But nerve cells in a healthy human are not starving — far from it!” Rosenzweig says. “They are among the most metabolically active cell types in the human body.”

So he set out to find a way to uncouple yeast-cell division from metabolism to study how yeast age when there is plenty of food. Rosenzweig’s lab has toyed with a “cute little trick,” which seems greatly to extend yeast chronological lifespan.

“It’s called an immobilization, and it’s been around for at least 30 years,” he says. “You take yeast cells and put them into a sort of gelatin-type matrix. You grow yeast in one container, stir up a batch of something like sodium alginate in another, mix the two together then pump the mixture into a container containing calcium chloride, which causes alginate to cross-link and harden.”

The end result is a soft, tapioca-sized bead. These beads, each impregnated with yeast, then are packed into a bioreactor and continuously fed a liquid medium containing the vitamins, minerals and food they need to grow happily.

“Over the first couple of days, yeast undergo four or five rounds of cell division, and then they stop,” Rosenzweig says. “You can then supply a pint-size reactor of such yeast with several pounds of sugar per week. The cells don’t divide, but they do eat voraciously, producing huge amounts of carbon dioxide and ethanol, which are both removed from the reactor.”

The cells live on and on — far past the lifespans they would have if they were not in this altered state. Immobilized yeast reactors efficiently produce ethanol because they don’t use any of the carbon in the sugar to make more babies; all of it goes into ethanol and routine cell maintenance. Rosenzweig says it’s an amazing process, but it has proved devilishly complicated for chemical engineers to scale up for use in biofuel production.

While immobilized yeast have remained something of a biotechnology toy for researchers interested in ethanol production, Rosenzweig believes their extended longevity will provide a useful model to study aging.

Ultimately, the length of your life is determined by the lifespan of your cells. Yeast and humans seemingly don’t have much in common — people have about 35,000 genes and yeast have about 6,200 — but the core genes that control cell metabolism and reproduction are similar.

Yeast is among the most-studied organisms in science, and Rosenzweig says yeast collections exist where, one-by-one, almost every gene has been selectively deleted or amplified. These collections consist of thousands of strains that are genetically identical except for the one gene that has been knocked out or increased in copy number.

“What we want to do is take the whole collection and immobilize it in bioreactors and ask these questions: Which are the first dropout mutants to drop out? Which are the last ones standing?” he says. “The first men down and the last ones standing will give you a lot of insight into the genes that shorten or extend cell lifespan.”

At the end of the exercise, Rosenzweig’s lab hopes to generate a list of genes that when knocked out cause lifespan to be extremely brief, even under conditions where it should be long. Conversely, there should be a list of genes that when increased in copy number cause lifespan to extend.

When asked whether this research could someday extend human lifespans, he laughs and says, “That’s a long ways off, but we think we have figured out a way to model certain long-lived cell types like human muscle and nerve cells — not the whole organism. We’ll definitely probe for genes that cause foreshortened or extended cell lifespans.”

.   .   .   .   .   .   .   .   .   .

On the topic of single cells starting to cooperate and possibly move towards multicellularity — one of the biggest leaps forward in the history of life on Earth — Rosenzweig says his interest sparked while he was doing postdoctoral work at the University of Michigan.

Two of his mentors, Julian Adams and Robert Helling, years ago made an interesting observation: If they took a single clone of a common E. coli bacterium, fed it only glucose and let it reproduce over multiple generations — the simplest of experimental conditions — they wound up with multiple E. coli strains that lived happily together.

At the time this observation flew in the face of both theory and prior experiments, which suggested that they should have wound up with a succession of mutant clones each more fit than its predecessor. This didn’t happen. One mutant became best at eating glucose, but it churned out byproducts that other genotypes became adept at consuming. From a single E. coli came many that worked together. Apparently, cooperative behavior could evolve in a test tube.

“My postdoctoral work showed it was cross-feeding interactions that drove the evolution of diversity,” Rosenzweig says. “Biodiversity builds upon itself. We created the simplest experimental conditions possible, put in one cell type but life found a way to produce multiple cell types.”

The researchers learned that a single E. coli could evolve into a community in about 100 days, which encompasses about 500 bacterial generations. The first distinct strains arose in as little as 20 to 40 days.

“The great thing about working with microbes in a laboratory is that you can conduct evolution experiments at time scales of weeks or months that would require many thousands of years in long-lived species like humans or elephants,” he says.

Rosenzweig and one of his postdoctoral associates, Margie Kinnersley, intend to revisit this work as a system to study the evolution of cooperation, since the bacteria work together to extract the maximum amount of energy from their limited resources. They will study at the genetic level what it is about certain genotypes, or certain environments, that favor the evolution of these kinds of cross-feeding communities.

The UM lab plans to tweak the original experiment. Rosenzweig says they learned the strain he studied in Michigan had an unknown mutation that may have helped the E. coli develop biodiversity. Also, evolving them on glucose, a relatively complex carbon source, may have boosted the founder E. coli chances to evolve complexity, as its metabolism produces multiple “edible” byproducts such as acetate and glycerol.

“We have some serious questions we would like to see answered,” Rosenzweig says. “Are there certain genotypes that make evolution of biodiversity more or less likely? Are there certain types of limiting resources that make the evolution of biodiversity more or less likely?”

The researchers also will test whether more diverse bacterial communities can withstand stress (such as rising temperatures) more readily than bacteria in a simple system, and whether diverse, cooperating bacterial communities are more resistant to invasion by “foreign” E. coli than non-cooperative systems.

Another of Rosenzweig’s lab associates, Matthew Heron, recently landed a NASA postdoctoral fellowship to study whether he can induce a single-celled organism to become multicellular in the laboratory. The space agency wants more knowledge about the ecological conditions that promote development of multicellular or quasi-multicellular structures.

Within one closely related group of algae, the volvocines, scientists recognize both truly multicellular species such as Volvox and truly single-celled species like Chlamydomonas. Herron will use the power of experimental evolution to see if he can induce, via one or another type of selection, single-celled critters to group together and to do so in a way that is stably heritable. In other words, Herron and Rosenzweig want to identify a genetic switch that enables a unicellular life form to become multicellular. They will be aided in this work by undergraduate researcher Jacob Boswell.

“Starting with an organism, Chlamydomonas, which is unicellular, Matt and Jacob will see if they can produce cultures consisting of cells that uniformly give rise to clusters of four, eight or more cells that work together,” Rosenzweig says. “It’s a long shot, but NASA thinks it’s a good enough bet to invest some money. We hope to gain insight into the genetic basis of one of the key innovations in the history of life — one that almost certainly would have to occur for intelligent life to arise elsewhere in the universe. Also, on the practical side of things, it did not escape NASA’s attention that this photosynthetic alga, Chlamydomonas, has become a workhorse in the bioenergy field.”

Studying how cells age to how they become multicellular only scratches the surface of all the work going on in the Rosenzweig lab, which engages swarms of microorganisms to understand some of the most basic questions in biology. Rosenzweig hopes the diverse projects under way in his UM lab will lead to greater understanding of evolution and origins of life itself.

For more information, email frank.rosenzweig@mso.umt.edu.

Next >