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

The researchers found that colonies of bacteria form three-dimensionally into rough, crystal-like shapes.

Bacterial colonies often grow in streaks on Petri dishes in labs, but no one has figured out how colonies organize themselves in more realistic three-dimensional (3-D) environments, such as tissues and gels in the body human or soils and sediments in the environment. , so far. This knowledge could be important for advancing environmental and medical research.

A princeton university The team has now developed a method to observe bacteria in 3D environments. They discovered that when bacteria grow, their colonies consistently form fascinating rough shapes that resemble a branching head of broccoli, far more complex than what you see 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, assistant professor of chemical and biological engineering at Princeton. and lead author of the study. It was the result of practical limitations rather than a lack of curiosity. “If you try to watch the bacteria grow in the tissues or in the soils, those are opaque and you can’t really see what the colony is doing. That was really the challenge.

Researchers Sujit Datta, assistant professor of chemical and biological engineering, Alejandro Martinez-Calvo, postdoctoral researcher, and Anna Hancock, graduate student in chemical and biological engineering. Credit: David Kelly Crow for Princeton University

Datta’s research group discovered this behavior using a revolutionary experimental setup that allows them to make unprecedented observations of bacterial colonies in their natural three-dimensional state. Unexpectedly, 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 glass.

“These kinds of rough, branching shapes are ubiquitous in nature, but usually in the context of growing or clumping nonliving systems,” Datta said. “What we found is that the 3D growth of bacterial colonies exhibits a very similar process despite being collectives of living organisms.”

This new explanation of bacterial colony development in three dimensions has just been published in the journal Proceedings of the National Academy of Sciences. Datta and his colleagues hope their findings will contribute to a wide range of research on bacterial growth, from creating more effective antimicrobials to pharmaceutical, medical and environmental research, as well as procedures that exploit bacteria for industrial purposes. .

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

“At a fundamental level, we are thrilled 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 also, we think this new view of 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, Datta’s research team has been developing a system that allows them to analyze phenomena usually hidden in opaque environments, such as fluids flowing through floors. The team uses specially designed hydrogels, which are water-absorbing polymers similar to jelly and contact lenses, as matrices to support 3D bacterial growth. Unlike these common versions of hydrogels, Datta’s materials consist of extremely tiny hydrogel balls that are easily deformed by bacteria, allow the free passage of oxygen and nutrients that promote bacterial growth, and are transparent to the 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 soil or tissue structure. The hydrogel is strong enough to support the growth of the bacterial colony without having enough resistance to limit the growth.

“As the 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 plunging your arm into the ball pool. If you drag it, the balls rearrange around your arm.

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

“We changed the cell types, the nutritional conditions, the properties of the hydrogel,” Datta said. The researchers observed the same rough growth patterns in each case. “We have systematically changed all these parameters, but it seems to be a generic phenomenon.”

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

Second, the researchers observed that in three-dimensional growth, only bacteria close to the surface of the colony grew and divided. The bacteria crowded into the center of the colony seemed to fall into a dormant state. Because the bacteria inside did not grow and divide, the outer surface was not subjected to pressure that would cause it to expand evenly. Instead, its expansion is primarily driven by growth throughout the periphery 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 inside the colony and those on the periphery, it would be like filling a balloon,” said postdoctoral researcher Alejandro Martinez-Calvo. at Princeton and first author of the article. “Pressure from within would fill all disturbances on the periphery.”

To explain why this pressure was not present, the researchers added a fluorescent tag to proteins that become active in cells when bacteria grow. The fluorescent protein lights up when the bacteria are active and remains dark when they are not. Looking at the colonies, the researchers saw that the bacteria on 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 very differently,” Datta said.

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

“We think they are sleeping because they are hungry,” Datta said, although he cautioned that more research was needed to explore 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 surfaces of the colonies. The bumpy surface is the result of random variations in oxygen and nutrients in the environment, but the randomness tends to balance out within limits.

“Roughness has an upper limit on its size – the size of the floret if we compare it to broccoli,” he said. “We were able to predict this from the calculations, and it seems to be an unavoidable feature of large colonies growing in 3D.”

Because bacterial growth tended to follow a pattern similar to crystal growth and other well-studied phenomena of inanimate 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 coarse growth forms for colony functioning, and applying these lessons to other areas of science. interest.

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

Reference: “Morphological instability and roughness 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, New Jersey Health Foundation, National Institutes of Health, Eric and Wendy Schmidt Transformative Technology Fund, Pew Biomedical Scholars Fund, and Human Frontier Science Program.