Scientists from Princeton solved the bacterial mystery

Scientists from Princeton solved the bacterial mystery

Scientists from Princeton solved the bacterial mystery

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

The researchers discovered that bacterial colonies form in three dimensions in rough, crystal-like shapes.

Colonies of bacteria often grow in streaks on Petri dishes in laboratories, but no one understood how colonies organize 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 for the advancement of environmental and medical research.

A Princeton University the team has now developed a method to observe bacteria in 3-D environments. They found that when the bacteria grow, their colonies consistently form fascinating, rough shapes that resemble 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 senior 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 see what the colony is doing. It was really a challenge.”

Princeton Bacteria Researchers

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, the scientists found that the growth of wild colonies consistently resembled other natural phenomena such as the growth of crystals or the spreading of frost on a window pane.

“These types of coarse, branching forms are ubiquitous in nature, but typically in the context of growing or agglomerated non-living systems,” Datta said. “What we found is that bacterial colonies growing in 3-D show a very similar process despite the fact that they 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 help in a wide range of research on bacterial growth, from creating more effective antimicrobial agents to pharmaceutical, medical and environmental research, as well as procedures that use bacteria for industrial use.

Anna Hancock, Alejandro Martinez Calvo and Sujit Datta

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

“At a fundamental level, we are excited that this work reveals surprising connections between the evolution of form and function in biological systems and the study of non-living growth processes in materials science and statistical physics.” But also, we think this new look at when and where cells grow in 3D will be of interest to anyone interested in bacterial growth, such as 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 that are usually hidden in opaque environments, such as liquid flowing through soil. The team uses 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 the usual versions of hydrogels, Datta materials consist of extremely small hydrogel balls 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 really can’t 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 colony of bacteria without enough resistance to limit growth.

“As bacterial colonies grow in the hydrogel matrix, they can easily rearrange the beads around themselves so they don’t get trapped,” he said. “It’s like dipping your hand into a ball pit. If you pull it through, the balls will rearrange themselves around your hand.”

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

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

Datta says two factors caused the broccoli-like growth on the surface of the colony. First, bacteria with access to high levels of nutrients or oxygen will grow and reproduce faster than bacteria in an environment with less richness. Even the most uniform environments have some nonuniform nutrient density, and these variations cause spots on the surface of the colony to grow forward or backward. Repeating itself in three dimensions, this causes the colony of bacteria to form bumps and nodules as some subgroups 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 crammed into the center of the colony seemed to go into a state of dormancy. Since the bacteria inside were not growing and dividing, the outer surface was not subjected to pressure that would cause it to spread evenly. Instead, its spread is primarily 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 inside the colony and those on the periphery, it would be like filling a balloon,” said Alejandro Martinez-Calvo, a postdoctoral researcher at Princeton and first author of the paper. “The pressure from within would fill any perturbations 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 glows when the bacteria are active and stays dim when they are not. 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 a shell that behave in very different ways,” Datta said.

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

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

Datta said 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. A bumpy surface is the result of random variations in oxygen and nutrients in the environment, but the randomness tends to even out within certain limits.

“Roughness has an upper limit to how big it can grow – the size of a flower if you compare it to broccoli,” he said. “We could predict this mathematically, and it seems to be an inevitable feature of large colonies growing in 3D.”

Because bacterial growth tended to follow a similar pattern to the growth of crystals 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 is likely to focus on better understanding the mechanisms behind growth, the implications of gross growth patterns 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 roughness of growing 3D bacterial colonies” by Alejandro Martinez-Calvo, Tapomoy Bhattacharjee, R. Kōnane Bay, Hao Nghi Luu, Anna M. Hancock, Ned S. Wingreen, and Sujit S. Datta, 2028. 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 Technology, the Pew Biomedical Sciences Fund, and the Human Frontier Science Program.

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