Bacterial cell cycle dynamics: size regulation during exponential growth and role of polyphosphate during starvation response

Bacteria are the most diverse and abundant kingdom of life and have adapted to survive and thrive in habitats around the globe. When provided with ample nutrients they grow and divide at staggering rates, increasing their population exponentially. Upon nutrient depletion on the other hand, they quickly adapt by drastically altering their metabolism, halting growth to survive for a very long time. Since bacteria are tiny -about a few micrometers-, visualizing these processes requires microscopy. To measure the dynamics of their shape and inner structures precisely, one needs to choose a technique that balances spatial resolution, temporal resolution and photo-toxicity. In this thesis, I present two projects using advanced dynamic microscopy, first to study cell size regulation during exponential growth in the abundance of nutrients and then to elucidate the role and positioning of polyphosphate granules during cell cycle exit in response to nutrient starvation. During exponential growth, bacteria balance growth and division to regulate their size, resulting in a narrow size distribution, referred to as cell size homeostasis. Recent work tried to uncover what cells sense to decide to divide in order to achieve size homeostasis: time, size, growth or a combination of those. Control of cell division is often equated to control of constriction onset; however, the constriction period still accounts for up to 40% of cell growth and could thus contribute significantly to cell size regulation. We used SIM microscopy to measure constriction kinetics and their impact on cell size regulation in Caulobacter crescentus. We found that constriction rate regulation can determine cell size. Moreover, constriction rate modulation compensates for variability in elongation before constriction, allowing a higher fidelity cell size homeostasis. We suggest a parsimonious model where excess cell wall precursors accumulate proportionally to elongation before constriction and set the rate of constriction. This is the first direct demonstration that constriction rate can contribute to cell size control and homeostasis in bacteria. Upon nutrient starvation, bacteria exit their cell cycle to preserve energy and nutrients. In many bacteria, such as Pseudomonas aeruginosa, this is associated with the accumulation of polyphosphate (polyP) in intracellular granules. PolyP is created by polyphosphate kinases (ppkâs), which are required for successful cell cycle exit and survival of and recovery from long-term starvation. Interestingly, these polyP granules are regularly spaced within the nucleoid. To date, it is not known during which stage polyP is required for cell cycle exit, and what causes the spacing of the granules. Here, we use fluorescence microscopy to probe the cell cycle stage of Îppk cells arrested during nutrient starvation as well as the localization and dynamics of ppkâs. We show that a majority of Îppk cells are arrested with open replication forks. Furthermore, we find that ppkâs localize in distinct patterns, already prior to starvation and polyP granule production, which could be responsible for the positioning of polyP granules. To this end, we developed a background subtraction algorithm to remove cytoplasmic fluorescence, improving accuracy of spot detection and localization.

Time-lapse high-resolution microscopy to study the morphogenesis of microorganisms

Mélanie Thérèse Marie Hannebelle

Mycobacterium tuberculosis, the etiological agent for the tuberculosis disease, is a bacterial pathogen thought to infect about a quarter of the global human population. It is the first cause of death among infectious diseases, and is most prevalent in low-income populations. Much remains unknown about how these bacteria grow, divide, and manage to persist in the host even during month-long antibiotic treatments. Bacteria are typically at the edge of the spatial resolution that conventional optical microscopy techniques can offer, and so new tools are required to study bacterial substructures or surface morphologies with nanometer resolution. We developed a platform for automated optical microscopy and atomic force microscopy (AFM) in order to study the morphogenesis of mycobacteria. What is the growth dynamics of mycobacteria? Opposite models coexist in the literature for the pole growth dynamics of mycobacteria: the unipolar model and the bipolar model. We show that the growth dynamics of mycobacteria follows a new end take off NETO model. There is a surprising similarity between the NETO growth dynamics of mycobacteria and of fission yeast, which hints at shared mechanisms for pole growth in evolutionarily distant pole growing organisms. How do pole-growing organisms maintain their shape through insertion of new cell wall material at the tip? To explore further pole growth morphogenesis, we developed a method for imaging with AFM the pole of a growing rod-shaped microorganism. We observed the formation of a stiff fibril mesh on the pole of fission yeast cells, and showed that this mesh is stretched as the cell grows and insert new cell wall material at the tip. How do mycobacteria divide into two sibling cells? Another essential aspect of morphogenesis of rod-shaped cell is the division of a mother cell into two sibling cells. Using AFM, we measured the relative contributions of mechanical forces and molecular mechanisms driving the division process in mycobacteria. We showed that there is a concentration of mechanical stress at the future division site, using a combination of COMSOL finite element modeling and AFM measurements while controlling the cell turgor pressure. The accumulation of mechanical stress, in combination of enzymatic activity, ultimately leads to mechanical fracture and separation of the sibling bacteria. Conventional optical microscopy techniques tend to have low resolution and high speed, while conventional AFMs have comparatively high resolution and low speed. We designed and built a 3D-printed structured illumination (SIM) module, the openSIM, as an add-on for fluorescence microscopes. It provides structured illumination to obtain SIM images with higher resolution. With the aim of improving the imaging speed of AFM, we implemented photothermal actuation in a tip-scanning AFM head, and quantified the design constraints for high-speed photothermal off-resonance tapping imaging. AFM is most often used to scan and acquire high-resolution images of a sample. Its nanometer precision make it a unique tool for other applications. We combined an AFM head with a volcano-shaped probe for electrogram measurements, and demonstrated simultaneous recording of extracellular field potentials and contraction of the cell. This new tool opens the way for future studies exploring the intriguing links between mechanobiology and electrophysiology, with single-cell resolution.

EPFL2020

Single-cell dynamics of the chromosome replication and cell division cycles in mycobacteria

Djenet Bousbaine, Neeraj Dhar, John McKinney, Isabella Santi

During the bacterial cell cycle, chromosome replication and cell division must be coordinated with overall cell growth in order to maintain the correct ploidy and cell size. The spatial and temporal coordination of these processes in mycobacteria is not understood. Here we use microfluidics and time-lapse fluorescence microscopy to measure the dynamics of cell growth, division and chromosome replication in single cells of Mycobacterium smegmatis. We find that single-cell growth is size-dependent (large cells grow faster than small cells), which implicates a size-control mechanism in cell-size homoeostasis. Asymmetric division of mother cells gives rise to unequally sized sibling cells that grow at different velocities but show no differential sensitivity to antibiotics. Individual cells are restricted to one round of chromosome replication per cell division cycle, although replication usually initiates in the mother cell before cytokinesis and terminates in the daughter cells after cytokinesis. These studies reveal important differences between cell cycle organization in mycobacteria compared with better-studied model organisms.

Nature Publishing Group2013

Mechanical and Functional Study of Cell Physiology at the Nanoscale

Pascal Damian Odermatt

By live-cell imaging of biological samples dynamic cellular processes can be resolved. Fluorescence microscopy (FM) and atomic force microscopy (AFM) are both capable of imaging live cells. By combining these techniques structural as well as functional information of the same biological samples are measured. While the dynamics of specific proteins can be imaged by FM, the AFM provides the structural context. To study how intracellular processes affect the mechanics, structure and kinetics of cells at the nanoscale we combined a high-speed AFM (HS-AFM) and an inverted optical microscope (OM) into one instrument. With this system cell physiological processes at different time scales are imaged. Mammalian cell migration happening at minute time scales is studied by correlated super-resolution microscopy and HS-AFM. Bacterial cell separation happening within 10s of milliseconds is characterized as well as bacterial growth over multiple generations. Additionally to imaging, the capability of the AFM to quantitatively measure mechanical properties of tissues is deployed in combination with FM on mouse corneal tissues. Different to most other rod-shaped bacteria where division happens through constriction of the cell wall at midcell, mycobacteria keep the shape of their outer cell envelope unchanged during septation. The septal wall is formed by peptidoglycan branching off from the outer cell envelop eventually splitting the cell into two sister compartments. Nanomechanical measurements show that from the initiation of septation the stiffness at the septum increases significantly. High-temporal resolution imaging of the following bacterial separation process indicated that the sibling cells separate within tens of milliseconds. The connection of the outer cell envelop at the former septum is broken apart suggesting a mechanical break. The timing of the cell separation is regulated by the tensile stress at the septum and the ultimate tensile strength of the cell wall material. The positions where these separations occur are closely associated with morphological features on the undulating cell surface. These features are formed at the poles and migrate towards midcell as the cell grows. Interestingly, sibling cell separation is restricted to the center-most wave-trough making these features the first characteristic associated with division site placement. Experiments with mutant strains show that even asymmetric divisions occur within wave-troughs emphasizing their role in division site placement. Chronic inflammation induces an increase in the stiffness of the corneal tissue and is associated with dramatic changes in the composition of the tissue. Stem cells migrating along this tissue layer of increased stiffness experience increased activation of mechanotransduction pathways. This eventually leads to induction of a skin-like rather than corneal-like character of these regenerating stem cells. It demonstrates that the stiffness of the tissue and thus pathways involved in mechanotransduction decisively influence the stem cell fate.