Observation of living cells solves mystery of bacterial cell division

In the life sciences, bacteria are currently studied not only as pathogens or, conversely, as valuable symbionts of multicellular life, but they are also suitable as simple model organisms for investigating the fundamental principles of biology.

At Kiel University, Professor Marc Bramkamp's Microbial Biochemistry and Cell Biology research group is using the rod-shaped bacterium Bacillus subtilis to investigate, among other things, bacterial organisational and reproductive mechanisms.

In this way, the Kiel researchers are searching for biological patterns and universal principles that are of fundamental importance for the molecular organisation of life as a whole.

One such fundamental process is bacterial cell division, through which these single-celled organisms typically reproduce by copying their cellular contents. This enables bacteria to multiply very rapidly under suitable environmental conditions; a fundamental characteristic that contributes to their success in almost all habitats.

Duplicating the entire organism requires the precise separation and distribution of two identical copies of the bacterial genome. It is crucial that cell division is correctly localised and occurs only once per cell cycle. Central to this mechanism in bacterial cells is the so-called Min system, whose proteins regulate cell division spatially.

In the well-known rod-shaped bacterium Escherichia coli, this process has been studied in detail for years: here, the Min system ensures that the cells divide exactly in the middle and that no uncontrolled cell division takes place. In contrast, it has been known for some time that the bacterium B. subtilis lacks a specific component of this system, namely the MinE protein, which acts as an activator of the division process.

In a new study, Prof. Bramkamp and his team have now shown that this bacterial species does not, however, require a specific activation protein for the spatial organisation of the Min system. They discovered that the MinD protein in B. subtilis switches between cytosolic and membrane-bound states via its ATP-dependent cycle, whereby membrane binding alone is sufficient to activate the ATPase activity of MinD and thereby enable self-organised spatial patterning of the Min system. The Kiel-based scientists recently published their findings, which are based on a combination of biochemical experiments and single-molecule imaging in living cells, in the journal eLife.

Previously unknown regulation of cell division in B. subtilis

For decades, the cell division system of the extensively studied bacterium E. coli has been regarded in cell biology as the blueprint for the reproductive mechanism in rod-shaped bacteria. The Min system, consisting of several cell division proteins, ensures that the cell divides only once in the middle and not multiple times into many small, non-viable cells. An oscillating movement of the proteins between the cell poles also ensures that the division site forms precisely at the geometric centre of the cell.

"For this reason, it had previously been assumed that cell division in B. subtilis is organised in a similar way and that, due to the absence of the MinE protein, there must be another activator of the Min system," says Dr Helge Feddersen, a research fellow in Professor Bramkamp's group. In earlier research, Bramkamp and his team had already shown that the Min system in B. subtilis is highly dynamic and can rapidly relocate to active division sites - rather than being statically localised at the cell poles, as previously assumed. "We now wanted to investigate in detail how Min protein dynamics are regulated at the molecular level in B. subtilis and how cell division is initiated here," says Dr Feddersen, the first author of the current study.

Cell division mechanism functions differently than in other bacteria

The Kiel-based research team first used biochemical experiments to investigate how the Min system in B. subtilis behaves in the absence of the MinE protein. "Unlike in E. coli, there is no oscillation here, but rather a movement of the MinD protein from the cell pole towards the centre of the cell, where it binds to the cell membrane," emphasises Dr Feddersen. "The question was now what exactly happens at the cell membrane that regulates cell division in terms of both space and time," Feddersen continues.

Experimentally, the researchers were able to show that, in this case, the cell division protein MinD is activated solely by membrane binding. The ATP hydrolysis triggered by membrane binding provides the chemical energy required to sustain the continuous reaction cycle that underlies the spatial patterning within the bacterial cell.

"This intrinsic cycle is sufficient to transport the protein from anywhere in the cell to the membrane and initiate pattern formation; therefore, B. subtilis apparently does not require any other activator," says Dr Feddersen, summarising the new research findings, to which Charlotte Dyckmans also contributed as part of her bachelor's thesis in Professor Bramkamp's group - and thus very early on in her academic career.

The data suggest that Min systems are based on an evolutionarily conserved reaction-diffusion principle, which represents a fundamental physical organising principle of biological systems, whereas MinE in E. coli is a later adaptation that enables specific oscillatory dynamics of this basic mechanism.

Confirmation of the experimental results under the microscope

A significant milestone achieved by the research team was the direct confirmation of these new findings: over the past few years, Bramkamp's research group has developed the capability to validate experimental results through observation in living organisms, thanks to a unique combination of biochemical expertise and high-resolution microscopy. "Using single-molecule microscopy, we were able to look inside the cell and visually track how the MinD protein moves and behaves at the cell membrane," explains Prof. Bramkamp.

"Such observation of protein dynamics at the single-molecule level has only been possible for a few years using super-resolution microscopy. Here, it allowed us to track the binding to the membrane and subsequent detachment virtually in real time," Bramkamp continues. "Only jointly, experiment and observation provide the evidence that this molecular machinery in the bacterial cell actually functions as originally hypothesised."

Single-molecule imaging, alongside other innovative methods, thus holds the key to resolving numerous questions in cell biology that cannot be answered by biochemical methods alone. The Kiel University Light and Electron Microscopy Bioscience Facility (KLEM), which Bramkamp also heads, is a highly specialised facility that makes these and other imaging techniques available to other users in Kiel. This means that the university offers a wide range of innovative and state-of-the-art microscopy technologies, providing valuable support to researchers in numerous research fields within the framework of the Kiel University's priority research area Kiel Life Science (KLS).

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Source: University of Kiel