Bacterial Colonies’ Clumpy Growth

300 years of research: Princeton scientists solve a bacterial mystery

The researchers were able to observe the clumpy growth of the bacterial colonies in three dimensions. Credit: Neil Adelantar/Princeton University

The researchers found that bacterial colonies form in three dimensions in rough shapes similar to crystals.

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

A team from Princeton University has now developed a method for observing bacteria in 3D environments. They discovered that as the bacteria grew, their colonies repeatedly formed intriguing rough shapes that resembled a branched head of broccoli, far more complex than what’s seen in a Petri dish.

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

Princeton bacterial researcher

Researcher Sujit Datta, Assistant Professor of Chemical and Bioengineering, Alejandro Martinez-Calvo, Postdoctoral Researcher, and Anna Hancock, PhD student in Chemical and Bioengineering. Photo credit: David Kelly Crow for Princeton University

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

“These types of rough, branching shapes are ubiquitous in nature, but typically associated with growing or agglomerating non-living systems,” Datta said. “What we found is that bacterial colonies growing in 3-D show a very similar process even though they are collectives of living organisms.”

This new explanation for 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 help a wide range of bacterial growth research, from creating more effective antimicrobials, to pharmaceutical, medical and environmental research, to processes that harness bacteria for industrial use.

Anna Hancock, Alejandro Martinez Calvo and Sujit Datta

Princeton researchers in the lab. Photo credit: David Kelly Crow for Princeton University

“In general, we are excited that this work reveals surprising connections between the evolution of form and function in biological systems and studies of inanimate growth processes in materials science and statistical physics. But we also believe that this new view of when and where cells grow in 3D will be of interest to anyone interested in bacterial growth, such as for environmental, industrial and biomedical applications,” said Datta.

For several years, Datta’s research team has been developing a system that allows them to analyze phenomena normally hidden in opaque environments, such as B. Liquids flowing through floors. The team uses specially engineered hydrogels, which are water-absorbing polymers similar to those found in jelly and contact lenses, as matrices to support bacterial growth in 3-D. Unlike common versions of hydrogels, Datta’s materials are made up of extremely small hydrogel beads that are easily deformed by bacteria, allow the free passage of oxygen and nutrients that support bacterial growth, and are translucent.

“It’s like a ball pool where each ball is an individual hydrogel. They’re microscopic, so you can’t really see them,” Datta said. The research team calibrated the composition 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 restrict growth.

“As the bacterial colonies grow in the hydrogel matrix, they can easily arrange the beads around them so they don’t get trapped,” he said. “It’s like dipping your arm in a ball pit. If you pull it off, the bullets will rearrange around your arm.”

The researchers ran experiments on four different types of bacteria (including one that helps create kombucha’s tart taste) to see how they grew in three dimensions.

“We changed cell types, nutrient conditions, and hydrogel properties,” Datta said. The researchers each saw the same, edgy growth patterns. “We systematically changed all of these parameters, but this seems to be a generic phenomenon.”

Datta said two factors appeared to cause the broccoli-shaped growth on the surface of a colony. First, bacteria with access to high levels of nutrients or oxygen will grow and multiply faster than those in a less-abundant environment. Even the most even environments have uneven nutrient densities, and these fluctuations cause patches on the colony’s surface to shoot forward or fall back. Repeated in three dimensions, this causes the bacterial colony to form bumps and nodules as some subsets of bacteria grow faster than their neighbors.

Second, the researchers observed that with three-dimensional growth, only the bacteria near the colony surface grew and divided. The bacteria crammed into the center of the colony seemed to go dormant. Because the bacteria inside did not grow or divide, the outer surface was not subjected to any pressure that would cause it to expand evenly. Instead, its expansion is driven primarily by growth at the very fringes of the colony. And growth along the edge is subject to nutrient fluctuations that eventually lead to bumpy, uneven growth.

“If growth were 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 fellow at Princeton and first author of the paper. “The pressure from within would fill up any disturbances on the periphery.”

To explain why this pressure wasn’t there, the researchers added a fluorescent label to proteins that become active in cells as the bacteria grow. The fluorescent protein glows when bacteria are active and stays dark when they are not. When observing the colonies, the researchers noticed that the bacteria at the edge of the colony were light green, while the core remained dark.

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

Datta said the theory is that the bacteria at the edges of the colony take up most of the nutrients and oxygen, leaving little for the bacteria inside.

“We believe they are dormant because they are starving,” Datta said, although he cautioned that more research is needed to investigate this.

Datta said the experiments and mathematical models used by the researchers revealed that there was an upper limit to the bumps that formed on the colony’s surfaces. The bumpy surface is the result of random fluctuations in the oxygen and nutrients in the environment, but the randomness tends to even out within certain limits.

“Roughness has an upper limit on how big it can get — the bud size if we compare it to broccoli,” he said. “We were able to predict this from math, and it seems to be an inevitable feature of large colonies growing in 3D.”

Because bacterial growth tends to follow a similar pattern to crystal growth and other well-studied phenomena of inanimate materials, Datta says the researchers were able to adapt standard mathematical models to reflect bacterial growth. He said future research will likely focus on better understanding the mechanisms behind growth, the impact of gross growth patterns on colony functioning, and applying these findings to other areas of interest.

“Ultimately, this work gives us more tools to understand and eventually 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.

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