Skip to main content

Bacterial colonies or a branching head of broccoli?

In the lab, bacterial colonies usually grow in streaks in flat dishes, but until now no one has known how the colonies organize themselves in more realistic three-dimensional environments, such as tissues and gels in our bodies or soils and sediments in the environment. New research has unravelled the physical principles underlying bacterial growth in 3D, finding that as bacteria grow, their colonies consistently form fascinating rough shapes resembling a branching head of broccoli, much more intricate than forms typically seen in a flat dish. This knowledge could be critical for advancing medical and environmental research.

The morphogenesis of two-dimensional bacterial colonies has been well studied. However, little is known about the colony morphologies of bacteria growing in three dimensions, despite the prevalence of three-dimensional environments (e.g., soil, inside hosts) as natural bacterial habitats. 

Using experiments on bacteria in 3D granular hydrogel matrices, researchers found that dense multicellular colonies growing in three dimensions undergo a common morphological instability and roughen, adopting a characteristic broccoli-like morphology. Unexpectedly, the team found that wild colonies’ growth consistently resembles other natural phenomena like the growth of crystals or the spread of frost on a windowpane. Experiments and theory suggest that this behavior originates from an interplay of competition for nutrients with colony growth.

Snapshot of an E. coli colony displaying morphological instability and roughening after 37 hours of growth. The image shows maximum-intensity projections of the confocal optical slices taken at different depths in the medium
Snapshot of an E. coli colony displaying morphological instability and roughening after 37 hours of growth. The image shows maximum-intensity projections of the confocal optical slices taken at different depths in the medium.

 

Researchers said two factors seemed to cause the broccoli-shaped growth on a colony’s surface. First, bacteria with access to high levels of nutrients or oxygen will grow and reproduce faster than ones in a less nutritious environment. Even the most uniform environments have some uneven density of nutrients, and these variations as well as other inhomogeneities cause spots in the colony’s surface to surge ahead or fall behind. Repeated in three dimensions, this causes the bacterial colony to form bumps and nodules as some subgroups of bacteria grow more quickly than their neighbors.

Second, the researchers observed that in three-dimensional growth, only the bacteria close to the colony’s surface grew and divided. The bacteria crammed into the center of the colony seemed to lapse into a dormant state. Because the bacteria on the inside were not growing and dividing, the outer surface was not subjected to pressure that would cause it to expand evenly. Instead, its expansion is primarily driven by growth along the very edge of the colony. And the growth along the edge is subject to nutrient variations that eventually 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 Cross-Disciplinary HFSP postdoctoral fellow at Princeton University, and the paper’s first author. “The pressure from the inside would fill in any perturbations on the periphery.” To explain why this pressure was not present, the researchers added a fluorescent tag to particular proteins in the bacteria. Such proteins, called green fluorescent proteins, are sensitive to the local concentration of oxygen inside cells, thus lighting up when oxygen is available and bacteria are growing, and remaining dark when they are not. Observing the colonies, the researchers saw that bacteria on the colony’s edge were bright green, while the core remained dark.

Observing the colonies, the researchers saw that bacteria on the colony’s edge were bright green, while the core remained dark.
New research has unravelled the physical principles underlying bacterial growth in 3D, finding that as bacteria grow, their colonies consistently form fascinating rough shapes resembling a branching head of broccoli, much more intricate than forms typically seen in a flat dish. 

 

“The colony essentially self-organizes into a core and a shell that behave in very different ways,” Martinez-Calvo said. Researchers said the theory is that the bacteria on the colony’s edges scoop up most of the nutrients and oxygen, leaving little for the inside bacteria. “We think they are going dormant because they are starved,” Martinez-Calvo said, although he cautioned that further research was needed to explore this.

The experiments and mathematical models used by the researchers found that there was an upper limit to the bumps that formed on the colony surfaces. The bumpy surface is a result of random variations in the oxygen and nutrients in the environment, but the randomness tends to even out in certain limits. “The roughness has an upper limit of how large it can grow — the floret size, if we are comparing it to broccoli,” Martinez-Calvo said. “We were able to predict that from the math, and it seems to be an inevitable feature of large colonies growing in 3D.”

These results shed light on the fundamental biophysical principles underlying growth in three dimensions. “Ultimately, this work gives us more tools to understand, and eventually control, how bacteria grow in nature,” Martinez-Calvo said. Researchers hope the findings will assist with an array of research that deals with bacterial growth, from the development of more effective antimicrobials to pharmaceutical, medical and environmental research, as well as processes that harness bacteria for industrial use. Future research will likely focus on better understanding the mechanisms behind the growth, the implications of rough growth shapes for colony functioning, and applying these lessons to other areas of interest.

Reference

Alejandro Martínez-Calvo, Tapomoy Bhattacharjee, R. Konane Bay, Hao N. Luu, Anna M. Hancock, Ned S. Wingreen, & Sujit S. Datta (2022). Morphological instability and roughening of growing 3D bacterial colonies. Proceedings of the National Academy of Sciences119(43), e2208019119. doi: https://doi.org/10.1073/pnas.2208019119

Media contacts

Guntram Bauer
Director of Science Policy and Communications

Liliana Gonçalves
Science and Communications Officer

Rachael Bishop
Science Writer and Editor

Click here to show mail address

 

Reference

Alejandro Martínez-Calvo, Tapomoy Bhattacharjee, R. Konane Bay, Hao N. Luu, Anna M. Hancock, Ned S. Wingreen, & Sujit S. Datta (2022). Morphological instability and roughening of growing 3D bacterial colonies. Proceedings of the National Academy of Sciences119(43), e2208019119. doi: https://doi.org/10.1073/pnas.2208019119