Princeton scientists solve a bacterial mystery

Researchers found that bacterial colonies form in three dimensions in rough shapes that resemble crystals.

Bacterial colonies often grow in stripes on petri dishes in laboratories, but no one has understood how the colonies arrange themselves in more realistic three-dimensional (3D) environments, such as tissues and gels in human bodies or soils and sediments in the environment, until now. This knowledge can be important for advancing environmental and medical research.

A Princeton University team has now developed a method to observe bacteria in 3D environments. They found that as the bacteria grow, their colonies consistently form fascinating rough shapes resembling a branched head of broccoli, much more complex than what can be seen in a petri dish.

“Since bacteria were discovered more than 300 years ago, most laboratory research has studied them in test tubes or on petri dishes,” said Sujit Datta, an assistant professor of chemical and biological engineering at Princeton and the study’s senior author. This was due to practical limitations rather than a lack of curiosity. “If you try to see bacteria growing in tissues or soil, they’re opaque and you can’t really see what the colony is doing. That’s really been the challenge.”

Datta’s research group discovered this behavior using a groundbreaking experimental setup that allowed them to make previously unheard-of observations of bacterial colonies in their natural, three-dimensional state. Unexpectedly, the scientists discovered that the growth of the wild colonies consistently resembles other natural phenomena such as the growth of crystals or the spread of frost on a pane of glass.

“These kinds of rough, branching forms are ubiquitous in nature, but mostly in the context of growing or agglomerating nonliving systems,” Datta said. “What we found is that bacterial colonies growing in 3D show a very similar process, despite the fact that these are collectives of living organisms.”

This new explanation of how bacterial colonies develop in three dimensions was recently published in the journal Proceedings of the National Academy of Sciences. Datta and his colleagues hope their discoveries will aid a wide range of research into bacterial growth, from creating more effective antimicrobials to pharmaceutical, medical and environmental research, as well as procedures that harness bacteria for industrial use.

“On a fundamental level, we are pleased that this work reveals surprising connections between the development of form and function in biological systems and studies of inanimate growth processes in materials science and statistical physics. But we also think this new look at when and where cells grow in 3D will be of interest to anyone interested in bacterial growth, such as in environmental, industrial and biomedical applications,” Datta said.

For several years now, Datta’s research team has been developing a system that allows them to analyze phenomena that are usually hidden in opaque environments, such as fluid flowing through the soil. The team uses specially designed hydrogels, which are water-absorbing polymers similar to those in jello and contact lenses, as matrices to support bacterial growth in 3D. Unlike common versions of hydrogels, Datta’s materials consist of extremely small spheres of hydrogel that are easily deformed by the bacteria, allowing oxygen and nutrients that support bacterial growth to freely pass through, and they are transparent to light.

“It’s like a ball pit where each ball is an individual hydrogel. They’re microscopic, so you can’t really see them,” Datta said. The research team calibrated the makeup of the hydrogel to mimic the structure of soil or tissue. The hydrogel is strong enough to support the growing bacterial colony without providing enough resistance to limit growth.

“As the bacterial colonies grow in the hydrogel matrix, they can easily rearrange the balls around it so they don’t get stuck,” he said. “It’s like throwing your arm in the ball pit. As you drag it through, the balls rearrange around your arm.

The researchers conducted experiments with four different types of bacteria (including one that helps generate kombucha’s pungent taste) to see how they grew in three dimensions.

“We changed cell types, feeding conditions, hydrogel properties,” Datta said. The researchers saw the same, rough growth patterns over and over. “We have systematically changed all those parameters, but this appears to be a generic phenomenon.”

Datta said two factors seemed to cause the broccoli-shaped growth on a colony’s surface. First, bacteria with access to high levels of nutrients or oxygen will grow and reproduce faster than bacteria in a less abundant environment. Even the most uniform environments have uneven nutrient densities, and these variations cause patches in the colony’s surface to move forward or backward. Repeated in three dimensions, this causes the bacterial colony to form bumps and nodules as some subgroups of bacteria grow faster than their neighbors.

Second, the researchers noted that in three-dimensional growth, only the bacteria grew and divided close to the surface of the colony. The bacteria crammed into the center of the colony seemed to decay into a dormant state. Because the bacteria inside did not grow or divide, the outer surface was not subjected to pressure that would cause it to expand evenly. Instead, expansion is mainly driven by growth along the very edge of the colony. And growth along the edge is subject to nutrient variations that ultimately result in bumpy, uneven growth.

“If the growth was uniform and there was no difference between the bacteria in the colony and those on the periphery, it would be like filling a balloon,” said Alejandro Martinez-Calvo, a postdoctoral researcher at Princeton and the first author of the paper. article. “The pressure from within would fill any disturbances on the periphery.”

To explain why there was no such pressure, the researchers added a fluorescent label to proteins that become active in cells when the bacteria grow. The fluorescent protein lights up when bacteria are active and stays dark when they are inactive. When observing the colonies, the researchers noticed that bacteria on the periphery of the colony were bright green, while the core remained dark.

“The colony essentially organizes itself into a core and a shell that behave in very different ways,” Datta said.

Datta said the theory is that the bacteria on the edges of the colony scoop up most of the nutrients and oxygen, leaving little for the innermost bacteria.

“We think they’re inactive because they’re starving,” Datta said, though he cautioned that further research was needed to investigate this.

Datta said the experiments and mathematical models used by the researchers found there was an upper limit to the bumps that formed on the colony surfaces. The bumpy surface is a result of random variations in the oxygen and nutrients in the environment, but the randomness tends to equalize within limits.

“The roughness has an upper limit on how big it can get — the size of the rose if we compare it to broccoli,” he said. “We were able to predict that based on the math, and it seems to be an unavoidable feature of large colonies growing in 3D.”

Because the bacterial growth tended to follow a similar pattern to crystal growth and other well-studied phenomena of inanimate materials, Datta said the researchers could adapt standard mathematical models to represent bacterial growth. He said future research will likely focus on better understanding the mechanisms behind growth, the implications of raw growth forms for colony functioning, and applying these lessons to other areas of interest.

“Ultimately, this work gives us more tools to understand and ultimately control how bacteria grow in nature,” he said.

Reference: “Morphological Instability and Roughening of Growing 3D Bacterial Colonies” By Alejandro Martínez-Calvo, Tapomoy Bhattacharjee, R. Kōnane Bay, Hao Nghi Luu, Anna M. Hancock, Ned S. Wingreen, and Sujit S. Datta, October 18, 2022, Proceedings of the National Academy of Sciences.
DOI: 10.1073/pnas.2208019119

The study was funded by the National Science Foundation, the New Jersey Health Foundation, the National Institutes of Health, the Eric and Wendy Schmidt Transformative Technology Fund, the Pew Biomedical Scholars Fund and the Human Frontier Science Program.