For many bacteria, life is simple: grow, divide, and repeat. However, some bacteria take a more complicated route. This is true for the bacterium Caulobacter crescentus. Caulobacter crescentus is a non-pathogenic, gram-negative bacterium that lives in freshwater environments and is a model organism used to study bacterial cell cycle and protein degradation. The bacterium is also found to be a potential bacterial immunomodulator that could be used to treat tumors.
Figure 1. Crescent-shaped bacterium Caulobacter crescentus
The bacterium can also thrive in nutrient-deficient environments due to its ability to exhibit a dimorphic life cycle, by producing two functionally distinct daughter cells. This makes C. crescentus unique among other asymmetrically dividing bacteria that produce identical offspring. Each time the bacterium divides, one cell becomes a motile “ swarmer” and the other a “stalked” daughter cell.
Figure 3. The cell cycle of a bacterium
Every bacterium undergoes a cell cycle, a series of events a cell undergoes as it grows, replicates DNA, and produces two daughter cells. The more extended phase of the cell cycle is the interphase, where three stages occur. The first stage is the G1 phase, where the cell increases in size and produces the essential proteins before inducing DNA replication, which happens in the stationary (S) phase, and lastly, the G2 phase, which is a checkpoint to ensure that the cell is ready for mitosis.
Due to C. crescentus' unique regulated life cycle resulting in two functionally distinct daughter cells, the bacterium has been used as a model organism to deepen our understanding of bacterial development. These studies have laid the groundwork for how C. crescentus grows and divides, providing a deeper understanding of the bacterium's cell cycle.
Figure 2. The cell cycle of the bacterium Caulobacter crescentus. The swarmer cell contains a flagellum, which cannot undergo DNA replication until it transitions into a stalked cell. The stalked cell can undergo DNA replication (S phase), which ends in the early pre-divisional cell. Lastly, the cell prepares for cell division in the G2 phase.
Based on previous information, a research question was sparked on whether both daughter cells have the same growth rate, although they are distinctly different. A recent 2024 study titled Coupling of cell growth modulation to asymmetric division and cell cycle regulation in Caulobacter crescentus by Glenn et al. (2024) aimed to investigate this exact question. Glenn. et al tracked individual cells over time to determine the pattern of development between the life phases of C. crescentus in hopes of defining how bacteria can coordinate growth with function precisely, while also investigating the effects of the spoT mutation on cell growth.
SpoT is an enzyme that synthesizes and hydrolyzes (p)ppGpp, which is a class of alarmone nucleotides, guanosine pentaphosphate, that modulate stringent control, which is a stress response by bacterial cells that allows them to adapt to environmental stressors. Glenn. et al wanted to test whether the loss of the enzyme (∆spoT) would further delay the G1 phase in the daughter cells. Glenn. et al (2024) took isolated newborn cells (swarmer and stalked) to investigate the delay in cell growth between both daughter cells. They placed them onto low-agarose pads containing peptone-yeast extract medium and incubated at 30℃. From there, the researchers tracked both daughter cells through a time-lapse microscope from their birth on the pads. This method allowed them to visualize when the swarmer daughter cell was separated from their cell, swam for a minute, then reattached itself to the agarose pad (Movies S1 and S2).
These supplementary videos showed that the swarmer offspring moved away from the mother and transitioned from a swarmer to a stalked daughter cell, suggesting a lower average growth rate than its genetically identical sibling (stalked offspring). To investigate the differences in the slowdown of the swarmer and stalked offspring in their cell cycle, they extracted images of the offspring between birth and cell division. They calculated the area differences of each cell. This method showed a definite difference between the swarmer and stalked offspring growth rate (Figure.1E), as the swarmer growth rate slows down in the first few minutes of their cell cycle before increasing and matching the stalked offspring.
Figure 1. A GIF of Caulobacter crescentus swarmer offspring releasing its flagella and producing a stalk before continuing its cell cycle and maturing.
Glenn et al. identified that the slowdown period of the swarmer daughter cell cycle is associated with the G1 phase of the cell cycle. The researchers used the same low-agarose time-lapse microscopy method containing a DNA replicator marker (MipZ) that is fused to (eYFP), a yellow fluorescent protein variant, to track DNA replication in terms of cell growth. As the daughter cells' chromosomes would replicate, that area would fluoresce yellow under the microscope. Through this experiment, the researchers could define the time between the cell and DNA replication as the G1 phase. This suggested that the slowdown in the swarmer daughter cell cycle was not due to its identity but to whether the cell is experiencing DNA replication (Movie S3).
Upon discovering that the swarmer daughter cell experiences a slowdown in the G1 phase because it needs to transition from a swarmer to a stalked cell before DNA replication. Glenn et al. wanted to know if a stalked daughter cell could have a G1 phase in its cell cycle and experience a slow growth rate. Using a learning-based Omnipose/Supper Segger software and cell segmentation, they could identify that both the swarmer and daughter cells are subjected to a G1 phase slowdown. Still, the delay is longer in the swarmer cell than the stalked cell due to the flagellum-to-stalk transition (Figure 2D-E).
As shown in the results below, Figure 3 has different subsections that discuss the swarmer and stalked daughter cell cycle. (A) The white arrows show MipZ-eYFP, which indicates the segregation and duplication of chromosomes in the stalked cell compartment of the mother cell before the mother cell separates. (B) A simplified image representation is shown in (A), where cell birth happens near the cytokinesis completion in the mother cell rather than at the separation stage of the cells. The figure also shows that both daughter cells have a G1 phase, which is only delayed in the swarmer cell due to the flagella-to-stalked transition. (C) The swarmer cells display an extended DNA replication phase compared to the stalked daughter cell, which is not the case for the stalked daughter cell. (D-E) Both daughter cells experience a slowdown in the G1 phase regardless of their identity, but the swarmer daughter cell experiences a more prolonged slowdown.
Figure 3. (A) Offspring birth happens in the mother near cytokinesis completion. (B) Simplified representation of the image shown in (A) that both daughter cells are in a G1 phase, but the swarmer daughter cells are not. (C) The duration of the G1 phase of both the swarmer and stalked daughter cells. (D-E) There is a delay in the G1 phase for both daughter cells, which is shown by the dip at 0.2-0.3, and then starts to increase around 0.4, which means that the cells have shifted into the S phase.
Understanding that the growth delay happens in the G1 phase led to the idea of any possible molecular factor that could affect the G1 phase in both daughter cells. To test this speculation, they took daughter cells (swarmer and stalked) that had ∆spoT. Through analysis, it was suggested that the daughter cells' growth rate was slower in the ones that lacked spoT than the Wild-type cells, which means that (p)ppGpp hinders the growth rate in both daughter cells.
To conclude, the bacterium Caulobacter crescentus produces genetically identical daughter cells that are functionally distinct from each other. Both daughter cells experience a delay in their cell cycle during the G1 phase before they undergo DNA replication. Still, the slowdown is more prolonged in the swarmer daughter cells. The study raises the question of whether other plant symbionts and pathogens similar to C. crescentus experience a delayed growth rate between daughter cells.
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