Princeton scientists solve bacterial mystery

The researchers found that the bacterial colonies formed in three dimensions in rough, crystal-like shapes.

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

A Princeton University the team has already developed a method for observing bacteria in 3-D environments. They found that as the bacteria grew, their colonies consistently formed fascinating, gross shapes that resembled a branching head of broccoli, far more complex than what is seen in a petri dish.

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

Datta’s research group discovered this behavior using an innovative 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 wild colonies consistently resembled other natural phenomena such as the growth of crystals or the spread of frost on window glass.

“These types of coarse, branching forms are ubiquitous in nature, but usually in the context of growing or agglomerating nonliving systems,” Datta said. “What we found is that growing in 3-D, bacterial colonies 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 findings will inform a wide range of research on bacterial growth, from creating more effective antimicrobials to pharmaceutical, medical and environmental research, as well as procedures that harness the bacteria for industrial use.

“At a fundamental level, 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 think that this new insight into when and where cells grow in 3D will be of interest to anyone interested in bacterial growth, such as environmental, industrial and biomedical applications,” Datta said.

For several years, Datta’s research team has been developing a system that allows them to analyze phenomena that are usually obscured in opaque settings, such as liquid flowing through soils. The team used specially designed 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 these common versions of hydrogels, Datta’s materials are composed 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 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 composition of the hydrogel to mimic the structure of soil or tissue. The hydrogel is strong enough to support a growing bacterial colony without providing enough resistance to limit growth.

“As bacterial colonies grow in the hydrogel matrix, they can easily rearrange the balls around them so they don’t get trapped,” he said. “It’s like putting your hand in the ball pit. If you swipe it, the balls rearrange around your hand.

The researchers performed experiments with four different types of bacteria (including one that helps generate the tart taste of kombucha) to see how they grow in three dimensions.

“We changed cell types, nutrient conditions, hydrogel properties,” Datta said. The researchers see the same, rough growth patterns in each case. “We systematically varied all these parameters, but this seems to be a general phenomenon.”

Datta said two factors appear to cause the broccoli-shaped growth on the surface of the colony. First, bacteria with access to high levels of nutrients or oxygen will grow and reproduce faster than those in less abundant environments. Even the most uniform medium has some uneven nutrient density, and these variations cause patches on the surface of the colony to advance or lag behind. Repeated in three dimensions, this causes the bacterial colony to form bumps and knots, as some subsets of bacteria grow faster than their neighbors.

Second, the researchers observed that in three-dimensional growth, only bacteria near the surface of the colony grew and divided. The bacteria crowded into the center of the colony seemed to go dormant. Since the bacteria inside do not grow or divide, the outer surface is not subjected to pressure to cause it to expand uniformly. Instead, its expansion is primarily driven by growth at the very edge of the colony. And edge growth is subject to nutrient variations, ultimately resulting in patchy, uneven growth.

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

To explain why this pressure was not present, the researchers added a fluorescent marker to proteins that become active in the cells when the bacteria grow. The fluorescent protein glows when the bacteria are active and stays dark when they are not. By observing the colonies, the researchers saw that the bacteria at the edge of the colony were bright green, while the core remained dark.

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

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

“We think they’re latent because they’re hungry,” Datta said, though he cautioned that more research is needed to investigate this.

Datta said that the experiments and mathematical models used by the researchers found that there is an upper limit to the bumps that form on the surfaces of the colonies. The uneven surface results from random variations in oxygen and nutrients in the environment, but the randomness tends to level out within certain limits.

“Roughness has an upper limit to how big it can grow – the size of a floret, if we compare it to broccoli,” he said. “We were able to predict this from the mathematics, and it seems to be an inevitable feature of large colonies that grow in 3D.”

Because bacterial growth tends to follow a similar pattern to crystal growth and other well-studied phenomena of nonliving materials, Datta said 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 implications of gross 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 Coarsening of Growing 3D Bacterial Colonies” by Alejandro Martinez-Calvo, Tapomoi Bhattacharjee, R. Konane Bei, Hao Ngi 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 Health Foundation of New Jersey, the National Institutes of Health, the Eric and Wendy Schmidt Fund for Transformative Technologies, the Pew Foundation for Biomedical Scientists, and the Human Frontiers Science Program.