Takuro Shimaya (Department of Physics, 3rd year of Doctoral program)

Kazumasa Takeuchi, Associate Professor, Department of Physics

Key Points of the Presentation

• We constructed a new device for highly controlling the environment of a bacterial population and observed how the E. coli population responds to nutrient starvation.
• We found that although bacterial cell size fluctuates dramatically with starvation, the shape of the cell size distribution is essentially unchanged during the rapid starvation process.
• We found that bacterial populations may adopt different response strategies depending on the rate of environmental fluctuations caused by nutritional starvation or drug administration.

Publication Summary

Cells vary in size from individual to individual, even if they are of the same type. Various studies have been conducted to understand how the cell size distribution follows the law in a steady-state environment. However, cells generally live in fluctuating environments, and the laws of size distribution in such environments have not been well understood due to the technical difficulties involved in observation. To address this issue, a joint research team led by Takuro Shimaya, Graduate Student, and Kazumasa Takeuchi, Associate Professor at the Graduate School of Science, The University of Tokyo, Reiko Okura, Project Associate Researcher at the Graduate School of Arts and Sciences, and Yuichi Wakamoto, Associate Professor at the Graduate School of Arts and Sciences/ Institute of Engineering Innovation, has constructed a new device that highly controls the environment of a cell population to observe the response of E. coli populations to sudden nutritional starvation. They observed the response of E. coli populations to sudden nutrient starvation (Fig. 1). They found that while starvation reduced the overall size of the cells, the shape of the size distribution remained unchanged and continued to satisfy the statistical property of scale invariance ^{(Note 1)}. In conjunction with simulations, they showed that the "speed" of the starvation process determines whether scale invariance is valid or not. These results indicate that bacterial populations recognize the speed of environmental change at the population level and may lead to the development of control methods for biofilms ^{(Note 2} ).

Publication details

Background of Research
When we look at cells of microorganisms and other organisms under a microscope, we can see that there is variation in the size of each individual cell. Individual microorganisms grow and divide, grow and divide, and repeat the cycle of growth and proliferation. Since the speed of growth and the timing of division in this process are determined stochastically for each individual, the cell size in a population forms a fluctuating distribution.

In recent years, it has been argued that there may be a universal property of cell size distribution across species. For example, when cell size distributions are measured for each species of unicellular organisms such as paramecium and green lacewings in a steady-state culture environment, the shape of the distribution also differs because the typical cell size differs for each species. However, when the distribution of "relative volume," which is the individual cell volume divided by the average volume of each species, is examined, it is reported to be common across many species. This concept is called scale invariance and implies that there are universal aspects to the growth mechanisms of many species.

Although many such studies have been conducted "in a steady-state environment," it is not known how robust the above scale invariance is in a "fluctuating environment" where microorganisms generally live. The root cause of this problem is the technical difficulty of controlling spatially uniform and large-scale fluctuating environments for microbial populations.

Details of the Study
In this study, we constructed a device called "wide-area microperfusion system ^{(Note 3)} " that can switch the environment surrounding a microbial population uniformly in space, on a large scale, and at high speed, and investigated how the cell size distribution of the E. coli population responds to a fluctuating environment (Figure 1).

Figure 1: The wide-area microperfusion system developed in this study (left) and the starvation response of the E. coli population observed using the system (right).

The device traps the cell population in a flat space through a double porous membrane while continuously feeding the culture medium from the top to constantly control the environment. In this study, we focused on the known fact that bacteria such as Escherichia coli typically decrease their cell size under nutrient starvation, and measured the variation in cell size distribution during the nutrient starvation process. When nutrients were rapidly removed from the culture environment by a wide-area microperfusion system, we observed a large fluctuation in the size distribution as the cells were dwarfed (Figure 2a). However, when we examined the distribution of "relative volume," which is the individual cell volume divided by the average volume at each time point, we found that scale invariance holds true during the starvation process and the shape of the distribution remains constant. On the other hand, we also found that the shape of this distribution widens (i.e., the inter-individual fluctuation of cell size increases) as the growth environment prior to starvation becomes more favorable.

Furthermore, by simulating the growth cycle of bacteria, it was shown that the nature of the cell size distribution changes significantly depending on the rate of nutrient starvation. In the case of rapid starvation, the cell size distribution continues to satisfy scale invariance, i.e., its shape remains constant, while in the process of slow starvation, the cell size distribution narrows, i.e., size fluctuations are suppressed (Figure 2b). In the simulations, we investigated the size distribution fluctuations by changing the time scales at which the DNA replication process of each cell stops and the cell growth stops due to starvation. lower left region), while the distribution narrows when each process does not stop easily due to slow starvation (Figure 2b, upper right region). A possible mechanism for this behavior is that the intracellular state (chromosome number, etc.) in the pre-starvation environment is maintained up to a certain time after starvation, and that the shape of the cell size distribution is also maintained as growth stops while retaining the memory of this state during the rapid starvation process.

Figure 2: (a) Scale invariance of the cell size distribution. The distribution of cell volume fluctuates significantly after nutrients are removed from the culture environment (left), while the distribution of "relative volume" divided by the average volume at each time point remains constant (right). (b) Results of a simulation of the bacterial growth cycle during starvation, showing the variation in the shape of the distribution. Scale invariance holds for "distribution constant" as in the result of a. For "distribution narrowing," the distribution of relative volume becomes narrower. The "volume increase" indicates that, unlike the experimental results, starvation has increased the volume.

Future Prospects
The results of this study suggest that bacterial populations may be able to discriminate the speed of nutrient starvation through the shape of the cell size distribution. Bacteria such as E. coli often form biofilms, which are densely aggregated populations, and this variation in cell size distribution may lead to structural changes in such biofilms. Further research is expected to be conducted to determine how the speed of environmental change affects structural changes in biofilms and how this may lead to defense strategies of the bacterial population. Although this study focused on the effects of nutrient starvation, future research may also focus on the importance of the speed of environmental change in the effects of antibiotics and other drug administration on biofilms.

Journals

Journal name	Communications Physics
Title of paper	Scale invariance of cell size fluctuations in starving bacteria
Authors	Takuro Shimaya*, Reiko Okura, Yuichi Wakamoto, and Kazumasa A. Takeuchi*
DOI Number	10.1038/s42005-021-00739-5

Terminology

Note 1 Scale invariance

The fact that the intrinsic property of a phenomenon does not change when the size scale of the phenomenon changes. In the case of this study, it means that the shape of the distribution does not change as an intrinsic property even if the overall cell size changes. ↑up

Note 2 Biofilm

A high-density aggregate formed by a population of bacteria, such as E. coli, adhering to some surface. Biofilms form in a wide range of environments, from the intestines and oral cavity of living creatures to industrial and medical products, and are known to cause a variety of problems. ↑up

Note 3 Wide-area micro perfusion system

A device whose shape can be designed with micrometer (μ m) precision and which can continuously supply fresh culture medium through a porous membrane while trapping cells in a wide two-dimensional space. The culture environment can be manipulated in real time by switching the fluid supply while observing the cell population. ↑up