Scientists from Michigan State University’s Department of Microbiology and Molecular Genetics  (MMG) in the College of Natural Science connected remarkable bacterial research across decades, from Minnesota to Michigan.

Their results were published in the most recent issue of the Proceedings of the National Academy of Sciences.

The V. cholerae research team (top left to bottom): Yann Dufour, MMG assistant professor, Chris Waters, MMG professor, Nico Fernandez, postdoctoral researcher, and microbiologist Arthur Henrici (1889-1943). Credit: Chris Waters and Sarah Zwickle

In 1928, Arthur Henrici, a pioneer of microbiology at the University of Minnesota, drew with pen on paper his observations of straight Vibrio cholerae, the bacteria that causes cholera. But his observations remained unexplored, and for more than a century, the scientific community accepted that the preferred shape of V. cholerae was curved.

Fast forward almost 100 years to the lab of Chris Waters, professor of microbial genetics at MSU, where MMG graduate student Nicolas Fernandez was investigating differences in cellular behavior between genetically modified V. cholerae bacteria with a lot of the bacterial signaling chemical known as cyclic di-GMP, or c-di-GMP, versus those with just a little.

“Out of the blue, I decided to look at the V. cholerae cells under the microscope, and cells with a lot of the signal appeared straighter than cells with less,” Fernandez explained.

Thanks to a tip from Gary Dunny, Waters’ Ph.D. advisor at the University of Minnesota, Fernandez realized he was seeing the same straight shape Henrici saw decades earlier. When other lab members confirmed his unexpected observation, Fernandez knew he had to explore the morphological phenomenon further.

The astounding results of Fernandez’s experiments, along with additional research by collaborators Yann DuFour, MMG assistant professor, and Nhu Nguyen, a graduate student in MMG and co-author on the paper, were 100-year-old corroborations of Henrici’s conclusion that V. cholerae changes shape and that these changes are not random.

In fact, using advanced techniques unimaginable to Henrici, the MSU scientists have provided genetic and mechanistic support for his hypothesis.

The virulent form of V. cholerae claims hundreds of thousands of human lives each year, but it also confronts numerous challenges to its own survival. The Waters Lab aims to understand the mechanisms that allow it to overcome these challenges, and shape-shifting may be one of them.

Of upmost importance is V. cholerae’s “decision” to stick together as a biofilm, bacterial colonies that attach to surfaces and encase themselves in an extracellular polymer that shields it from environmental stresses and antibiotics, or to swim independently. C-di-GMP, the chemical Fernandez was investigating when he observed the straight V. cholerae, is the primary signal driving that choice.

Henrici’s drawings of straight V. cholerae overlayed on a micrograph of a wild-type V. cholerae taken in the Dufour Lab. Henrici’s observations of the straight form of the bacteria would remain unexplored, but Fernandez would bring them back into the scientific community’s eye. Credit: Nhu Nguyen and Arthur Henrici.

“In Vibrios, an accumulation of proteins initiated by c-di-GMP turns on genes to make a biofilm matrix and turns off genes that control making the flagella, the apparatus used to swim in liquid media,” said Fernandez, now a postdoctoral researcher at the University of Michigan. “This fit exactly with what Henrici saw 100 years ago—they are straighter in conditions where they want to form a biofilm and curved when they want to swim.”

“C-di-GMP is this common intracellular currency that integrates dozens of environmental signals that feed into that decision, and when the signal is increased, it drives V. cholerae to form a biofilm, and when it is decreased, it drives them to swim” Waters added. “We knew this for a while, but we didn’t know changing cell shape was one of the things that was being controlled by the signal along with biofilm.”

Fernandez’s discovery that the bacteria’s shape could be changed not just through genetic mutations, but also through signaling, led him to Dufour, whose lab uses a combination of mathematical modeling, single-cell microscopy and tracking to precisely calculate the parameters and swimming capabilities of V. cholerae cells.

“Our lab was also interested in V. cholerae, but from a different angle, and this is where our research collided,” Dufour said. “We wanted to know if the shape of V. cholerae affected how they swim.”

The morphologically induced changes discovered by Fernandez, combined with the behavioral studies of Nguyen, resulted in novel insights about possible reasons for curved bacteria.

“The curved cells swam faster than straight ones, giving them the advantage of increased efficiency for the same amount of energy,” explained Dufour, who filmed the Vibrios swimming in his lab. “This is important because in the sea, where they originate, food is scarce, but curvature costs them. Curved Vibrios do not grow as quickly.”

Nhu Nguyen, graduate student in the Dufour Lab, conducted behavioral studies of V. cholerae that led to novel conclusions about the objectives of morphological change Henrici had hypothesized almost 100 years earlier. Credit: Yann Dufour

But why had Henrici’s observations of both straight and curved V. cholerae remained hidden for so many years? Part of the answer is embedded in modern scientific labs where standardization is crucial for reducing complexity to compare and control results.

“But when you control experiments, you are restricting the parameters you look at for the bacteria to begin with, and most conditions in the lab don’t show changes in morphology,” Dufour said. “Only when we get away from the beaten path and grow bacteria under different conditions, do we stumble on old results from a time when they would get diverse bacteria from the wild.”

The paper exemplifies how MMG’s atmosphere of collaboration and advanced scientific techniques can open both new and 100-year-old collaborative doors into the purpose and mechanisms behind bacteria’s morphological objectives.

And the scientific connections go even deeper. Waters spent his graduate years at the University of Minnesota surrounded by Henrici’s images of single cells changing shape.

“The departmental library at Minnesota, where I did my Ph.D. work and had many meetings, was named the Henrici library and had images of his work on how bacteria can change cell shape!” Waters said. “I had no idea at the time that thanks to Nico, my lab would be studying this exact process.”  

Story via College of Natural Science 

Banner image: A scanning electron micrograph of Vibrio cholerae showing the classical curved morphology. The long tail is a flagella that it uses to swim. Credit: Geoff Severin