From the identification of the pathogens causing major human diseases, through the understanding of the most basic forms of life, and in the struggle to develop new antibiotics to counter the increasing bacterial resistance to these widely used substances, research on microbial growth and survival has rightfully seen a booming interest across the last two centuries. The guanosine tetraphosphate (ppGpp) signaling system is of particular significance for these fields of research. It is a bacterial response to stress and starvation which allows these organisms to activate the necessary genes to survive in these conditions. It also plays a key role in modulating the abundance of various machineries necessary for growth in order to maximize the rate at which bacteria are growing in different nutrient conditions. In poorer nutrient conditions requiring more enzymes to import and digest these nutrients into the same amount of essential building blocks for growth, production of ppGpp is triggered in response to the lack of these building blocks. Higher ppGpp concentrations lead to downregulation of the abundance of ribosomes, the machineries operating growth, allowing larger abundance of enzymes producing the building blocks from nutrients. Recent advances, by genetically modifying bacteria, allowed to finely tune ppGpp concentrations independently of growth conditions. Using this approach, we investigate the scope of the regulation operated by ppGpp: which proteins does it influence in changing growth conditions and to what extent? We present novel quantifications of different proteins and other relevant biomolecules in E. coli strains artificially modified to have higher or lower ppGpp concentration than naturally found. These results allow us to identify which proteins are regulated by ppGpp and why the right concentration of ppGpp is necessary for bacteria to achieve optimal growth. We confirm the role of ppGpp in upregulating the proteins related to the synthesis of new proteins: translation. We also show that, apart from translation-related proteins, ppGpp is not responsible for consistently regulating other groups of proteins, including some that do vary with growth rate variations caused by change in nutrient source, which were suspected to be under ppGpp’s control. With a simplified mathematical model of the ppGpp regulation mathematical model, we attempt to understand why slowing down bacterial growth by artificially increasing ppGpp requires a lot more ppGpp than naturally found. By doing so, we identify characteristics that a model seeking to explain ppGpp perturbations should have. These conditions strengthened our understanding of the ppGpp system by refuting some of our intuitions and laying a foundation for future models elucidating the missing pieces. We also present another story regarding how yeast can survive one of the harshest stress: being completely desiccated. With these different studies, we extend our knowledge of two different systems relevant to growth and survival of microorganisms, which leads to potential new directions for those who seek to investigate such systems and answer some of the questions that our findings raise.

Open questions are whether life can be enabled in uninhabitable environments, and whether there is a limit to howmuch one can tune the speed of proliferation. Answering such questions has broad implications. It may reveal whether we can live in unforeseen habitats, whether we can slow down aging, and whether there are limits to lifespan. In this dissertation, we explore such questions for the budding yeast by changing the temperature. We will use temperature, a physical parameter, as a knob to tune the speed of cellular life. Temperature affects all organisms and habitats, and is of contemporary interest in light of climate change. Indeed, cells of microbes, plants and cold-blooded animals often endure temperatures that can be considered extreme. For reference, budding yeast lives comfortably at 30 ◦C and has a doubling time of roughly 1.5 hours – the time a cell needs to grow and divide into two cells. During our studies, we will elucidate the common principles that govern the life of yeast at extreme temperatures – how a cell survives, grows, replicates, ages, and dies. We combine models, experiments and measurements of single cells and at amolecular level, and integrate these into a systems-level view of the life of yeast at extreme temperatures..

Tales of a Fountain of Youth and the invention of medicine illustrate our age-long obsession with two themes: life and death. What it takes to stay alive, and not to be dead, is a basic question in science that is easy to state, and yet difficult to address at a profound level. One striking feature of many living organisms is the ability of individuals to behave in unison by communicating with each other. At life’s microscopic level, living cells can also send and receive chemical signals to communicate with each other in their habitat but for a population of many thousands of cells it remains enigmatic who is communicating with whom, what are the signals, and how the signals work over space and time. We used quantitative experiments and mathematical modelling to systematically explore how mouse Embryonic Stem (ES) cells might cooperate by communicating when differentiating into the first two lineages. We discovered that differentiating mouse ES cells scattered across many centimeters on a dish form one macroscopic entity that either survives or dies in unison if and only if its population-density is above a threshold value. This switch-like behavior is determined by cells that secrete and sense FGF4 that diffuses over many millimeters to activate YAP1-induced survival mechanisms. Our work shows that living cells (in vitro) can rely on macroscopic cooperation to stay alive.

Microorganisms can cooperate with each other, meaning that individual cells work together to pursue a common interest. Cooperation can be crucial for microorganisms to survive stressful environmental conditions. In this work, we report on the cooperative behaviour by the yeast S. cerevisiae and the bacterium E. coli, which stimulates survival and growth at high temperatures. Our lab had already discovered that genetically identical yeast cells cooperate with each other via the secretion of a public good (the antioxidant glutathione), to extend habitability of high temperatures. However, microbes naturally live in communities, where they coexist with other strains and species. These microbial communities are crucial to the health of an ecosystem, but their habitats are warming up due to climate change. So, understanding their response to a rising temperature may be crucial to keep ecosystems healthy. We take it one step further and study the cooperation between different yeast strains at high temperatures. We found that not only a population of genetically identical cells, but also different strains cooperate with each other, to collectively survive under high temperature conditions. We performed so-called co-existence experiments at high temperatures, where we combined two different yeast strains, one genetically fitter than the other. Depending on the initial composition of the mixed population, we obtained two outcomes. In the first outcome, the genetically fitter strain helps the less fit strain to grow. In the second outcome, vice versa, the less fit strain helps the fitter strain to grow. A mathematical model that reproduces the experimental data, predicts an additional outcome, where both strains would help each other to grow. Additionally, we discovered a novel cooperative behaviour at high temperatures in the bacterium E. coli. We found that E. coli cells show similar growth behaviour compared to yeast and we hypothesize that a comparable cooperative behaviour lies at the basis, also regulated by the secretion of a public good. These results suggest that cooperation at high temperatures could be conserved amongst different microbial species.

This thesis is about a little molecule called guanosine tetraphosphate. ppGpp. Consider it the bacterial brain, at the core of the coordination and regulation of bacterial growth. For over half a century, it has haunted microbiologists as it appears involved in every aspect of microbial physiology, yet incredibly difficult to study due to its fast dynamics, chemical instability and pleiotropic effects. Like the human brain, it cannot simply be removed to show its true nature. In contrast to the pronunciation of its name, ppGpp is a rather simple molecule, and built from two of the most abundant substrates in the bacterial cell (ATP and GTP). The enzymes that make or break ppGpp are highly efficient, such that at any moment, the bacteria can decide to instantly 100-fold increase ppGpp concentrations, or virtually remove all of it. Thanks to this intelligent system, E. coli can decide to arrest growth, protecting itself against any threats, or to rapidly feast upon the sparse nutrients it may be tossed, within the order of minutes.

Metal nanoparticles have promising potential for use in medical applications, such as imaging or targeted drug delivery. Current methods of production of nanoparticles are expensive and make use of harsh reagents. Because of these limitations, nanoparticles are not yet widely available, limiting the use in medical applications. To commercialize nanoparticle production, it is necessary to develop a nanoparticle synthesis process that is cheap, safe and environmentally friendly. Earlier research has shown that biosynthesis of nanoparticles is possible in many different organisms using certain reducing peptide sequences. Biosynthesis of metallic nanoparticles is cheaper, safer and occurs under physiological conditions, making it an ideal process for the up scaling of nanoparticle production. However, shape and size of the nanoparticle are hard to control and yields are low when biosynthesis is used, limiting the use of biosynthesis. To overcome the problem of shape and size control, it is proposed to use viral capsids as protein cages to restrict the size of synthesized nanoparticles to the inside of the capsid. To implement this, metal ion reducing peptides were fused to the monomers of self-assembling capsids in a way that they were displayed on the inside of the capsid. It was shown that with the adjusted monomers, the capsids still self-assemble. Nanoparticle formation was attempted, but results were inconclusive.