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Timing the Checkpoints

March 28, 2022

Research in UT’s Mannik Lab follows up a quote from the renowned French geneticist François Jacob—a dream of every cell is to become two cells. Before a cell can realize that ambition, however, it has to go through a couple of crucial biological checkpoints. Jaan Mannik’s group has helped determine how the checkpoint related to division is timed relative to the checkpoint related to DNA replication. These data have been lacking in studies of bacteria until now. The more scientists learn about how these cells divide, the easier it is to translate that knowledge into advances like new antibiotics.

Bacterial Belt-Tightening

Most bacteria divide by building a wall in the center of the cell. This is where the cell constricts—like tightening a belt—before dividing in two. The onset of constriction is the critical checkpoint for cell division. The second critical cell cycle checkpoint is the onset of replication, when the cell starts to copy its genetic material. Once it finally splits into two daughter cells, both daughters need to have a copy of the mother cell’s DNA. Mannik and his colleagues wanted to figure out the timing of these two processes and how they are coordinated. Using Escherichia coli (E. coli, which possesses a single chromosome) as their model cells, they used genetic engineering to attach fluorescent labels to proteins that carry out replication and division. The labels comprised green fluorescent protein (GFP) type sequences, which show up as bright blue, green, or red when exposed to light. Then they took time-lapse images of the cells.

"From these images, we can then determine when the associated proteins start replication and division, respectively," Mannik, an associate professor, explained. "This timing information for both the replication and division was yet lacking for E. coli, or for any other bacteria for that matter."

For an idea of what these time-lapse movies look like, visit the Mannik Group website for movies of similar experiments.

His group found that the unreplicated chromosome blocks the start of constriction at mid-cell, and that the blockage lifts about the time replication is complete.

nucleoid block in e coli division

The unreplicated chromosome (nucleoid) blocks the onset of constriction in E. coli until the replication process terminates. (From "Coupling between DNA replication, segregation, and the onset of constriction in Escherichia coli" in Cell Reports.)

"To our understanding, this blockage is independent of the growth rate, but in faster growth rates this blockage is not typically holding back the division process," Mannik said.

The results are significant because they give scientists more information about how bacteria multiply and, when necessary (as when they cause illness), how to stop them.

"Division is an essential cellular process," Mannik said. "Many antibiotics target division. Once the division stops, the bacteria usually lose their viability."

Jaan Mannik designed the experiments with Jaana Mannik, a research scientist in the physics department, and Sriram Tiruvadi-Krishnan, a postdoctoral research associate who was lead author on the Cell Reports article describing the findings. They worked with Professor Ariel Amir’s group at Harvard University, a collaboration that evolved through conference and workshop attendance and a shared interest in the physics of living systems.

"In one of the meetings, we came together to the idea to address the topic of the current paper; our group coming from the experimental side and Ariel’s group from the modeling side," Jaan Mannik said.

The research builds on the Mannik group’s previous work on E. coli cell division. The group also uses lab-on-a-chip devices to get a clearer picture of bacterial behavior at the molecular level.

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