The researchers were able to observe the lumpy growth of the bacterial colonies in three dimensions. Credit: Neil Forward/Princeton University
The researchers found that bacterial colonies form in three dimensions in crude crystal-like shapes.
Bacterial colonies often grow in streaks in petri dishes in laboratories, but no one has understood how colonies are organized 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 medical and environmental research.
A Princeton University The team has now developed a method to observe bacteria in three-dimensional environments. They found that when the bacteria grow, their colonies consistently form fascinating rough shapes resembling a branching head of broccoli, far more complex than those seen in a petri dish.
“Since bacteria were discovered more than 300 years ago, most laboratory research has studied them in test tubes or petri dishes,” said Sujit Datta, an assistant professor of chemical and biological engineering at Princeton and lead author. of the study. This was the result of practical limits rather than a lack of curiosity. “If you try to look at how the bacteria grow in the tissues or in the soil, they’re opaque and you can’t really see what the colony is doing. That’s really been 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 an innovative experimental setup that allows them to make never-before-seen observations of bacterial colonies in their natural three-dimensional state. Unexpectedly, the scientists discovered that the growth of wild colonies consistently resembles other natural phenomena such as the growth of crystals or the spread of frost on a window pane.
“These kinds of rough, branching forms are ubiquitous in nature, but usually in the context of growing or agglomerated non-living systems,” Datta said. “What we found is that when growing in 3D, bacterial colonies exhibit 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 that his discoveries will help with 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 bacteria for industrial use.
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 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 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 normally hidden in opaque settings, such as fluids flowing through soils. The team uses specially designed hydrogels, which are water-absorbent polymers similar to those in gelatin and contact lenses, as matrices to support bacterial growth in 3-D. Unlike common versions of hydrogels, Datta materials are made of extremely small hydrogel balls that are easily deformed by bacteria, allow free passage of oxygen and nutrients that support bacterial growth, and are transparent to light.
“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 presenting enough resistance to restrict 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 putting your arm in the ball pit. If you drag it, the balls rearrange themselves around your arm.”
The researchers conducted experiments with four different species of bacteria (including one that helps generate the sour taste of kombucha) to see how they grew in three dimensions.
“We changed the cell types, the nutrient conditions, the properties of the hydrogel,” Datta said. The researchers observed the same irregular growth patterns in each case. “We systematically change all of those parameters, but this seems to be a generic phenomenon.”
Datta said two factors appeared to cause broccoli-shaped 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 densities, and these variations cause spots on the colony’s surface to come early or late. Repeated in three dimensions, this causes the bacterial colony to form bumps and nodules, as some subgroups of bacteria grow more rapidly 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 huddled together in the center of the colony seemed to fall into a dormant state. Because the bacteria inside did not grow or divide, the outer surface was not subjected to the pressure that would cause it to expand evenly. Instead, their expansion is primarily driven by growth along the colony boundary. And growth along the edge is subject to nutrient variations that eventually result in patchy, uneven growth.
“If the growth were 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 Martínez-Calvo, a postdoctoral researcher at Princeton and first author of the paper. “Pressure from within would fill any disturbance on the periphery.”
To explain why this pressure was not present, the researchers added a fluorescent tag to proteins that are activated 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 at the edge of the colony were bright green, while the center remained dark.
“The colony essentially self-organizes into a nucleus and a shell that behave in very different ways,” Datta said.
Datta said the theory is that bacteria at the edges of the colony pick up most of the nutrients and oxygen, leaving little for the bacteria inside.
“We think they are inactive because they are hungry,” Datta said, though he cautioned that more research is needed to explore this.
Datta said that the experiments and mathematical models used by the researchers found that there was an upper limit to the bumps that formed on the surfaces of the colonies. The uneven 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 much it can grow: the size of the floret compared to broccoli,” he said. “We were able to predict that from the math, 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 that future research will likely focus on a better understanding of the mechanisms behind growth, the implications of approximate growth forms for colony functioning, and the application of these lessons 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 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 of 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.
