While life is present everywhere on earth, each individual species can only grow and proliferate in a specific set of conditions. Moreover due to inherent fluctuation of the environment, individual organisms are often periodically confronted to stressful conditions that prohibit growth, reproduction and increase mortality. One of the most common strategy to cope with these harmful conditions is to shield and quietly wait for the storm to finish. Indeed upon change of the environment, many organisms enter "dormancy", whereby they differentiate into a distinct resting form with additional protection, storage and with greatly reduced internal activity (dormant state). Although relying on widely different molecular basis, specialized dormant stage are found in virtually every group of living organisms, including endospores of bacteria, spores of fungi, seeds of plants, cysts of protists and diapaused eggs of animals. Even after sometimes years of apparent inactivity, dormant organisms can resume growth and reproduction upon sudden improvement of environmental conditions. How such organisms manage to keep the potential to resume growth while having a nearly ceased activity? Over the last 20 years, scientists have uncovered various physiological and molecular mechanisms that control entry and exit of dormancy. However a basic understanding of what exactly happens during dormancy, i.e how to survive while stopping nearly all internal activity, is still missing. Two reasons are likely responsible for that knowledge gap. A first conceptual obstacle is a general view that since by definition nothingmuch happens during dormancy, nothing important happens. A second technical obstacle is the lack of sensitive enough instruments to quantify the "nearly ceased" activity during dormancy. As a result, very few studies have established the concrete link between a vanishing internal activity and the ability to survive during dormancy in a single experimental system. In this thesiswe propose to focus on dormant Saccharomyces cerevisiae yeast spores to specifically investigate the links between gene expression and the ability to survive during week-long dormancy.@en

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.

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.

For millennia, humans have used microbes to produce industrial products of social and economical value through fermentation processes. In recent years, the application of engineering principles to microbiology have dramatically expanded our ability to modify and optimize microbes for the production of a wide variety of commercial products from renewable feedstocks: food and commodity chemicals, to biofuels and fine chemicals such as pharmaceuticals, fragrances, cosmetics or dyes. The use of microbial bioprocesses for the production of natural products represents an attractive and sustainable alternative to current industrial production methods, which mainly rely on chemical synthesis and/ or extraction from native producers. Advanced biomanufacturing technologies would not only provide sustainable economic benefits (by reducing the monetary cost of production of useful chemicals), but also offer social and environmental benefits. Synthetic biology has allowed engineering the production of many industrial compounds within microbes that do not naturally produce them – this is called “heterologous microbial biosynthesis”. In addition to replacing current manufacturing processes, heterologous microbial biosynthesis likely offers the only viable platform to produce certain natural products at industrial scales. Indeed, many relevant compounds cannot be viably manufactured through chemical synthesis, and/or are produced at undetectable/insufficient levels in native organisms. However, many heterologous bioprocesses remain in their infancy to fully enable an economically viable delivery of relevant natural products to the market. In order to build and sustain the promise of a bioeconomy for the 21st century, metabolic engineering is under pressure to continue to provide largescale, sustainable and cost-competitive bioprocesses that meet global needs. In this thesis, we focus on the development of microbial strains to accelerate the microbial production of 2 different families of high-value compounds of prominent biotechnological relevance within the established microbial chassis Escherichia coli: antibiotics and isoprenoids. The fight against antimicrobial resistance is considered one of the greatest public health challenges of the 21st century. Recent technologies have uncovered new antibiotics that, if harnessed, might help alleviate this crisis. However, most of these new antibiotic compounds are far too complex for economical chemical synthesis, and are naturally produced by unculturable and/or genetically intractable microbes. Developing new heterologous microbial platforms for antibiotic production may be an efficient solution for harnessing the clinical potential of these molecules and their commercialization. Isoprenoids represent one of the largest families of natural compounds (over 50,000 molecules) with an incredible number of practical uses, and of great commercial value: from high-value compounds such as many pharmaceuticals, fragrances and flavors, to commodity chemicals such as solvents, rubber or advanced biofuels. We focus in particular on relevant obstacles associated with the development of proof-of-principle strains for the laboratory-scale production of these high-value chemicals. @en

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. @en

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.. @en

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.@en

Bacteria have been an integral part of human life since the ancient times either as cooperative tenants living in and around us or as constant threats to our health and wellbeing. Since prehistoric times we unknowingly used them as tools of fermentation and fought against them with haphazardly discovered natural remedies. Today, after more than 3 centuries since they were first observed with a microscope, our understanding of their functions has increased immensely along with our ability to alter it. We have discovered on a molecular level how life stores and transfers information, how this information is used to build biochemical machines with a myriad of functions, namely proteins, and how these proteins undertake their functions. Along with a better understanding came the ability to alter the biological information within DNA and to create new proteins that does not occur in nature.@en

Implant-associated infections by antibiotic-resistant biofilm-forming pathogenic bacteria such as Staphylococcus aureus have become a growing concern as they are difficult to treat and lead to implant revision surgery. Implant coatings consisting of superparamagnetic iron oxide nanoparticles may prevent the attachment to and infection of implants by bacteria. However, these nanoparticles release iron, which is also a nutrient necessary for bacterial growth, and may inadvertently contribute to infection. In this thesis the uptake of iron from iron nanoparticles by S. aureus is investigated.  To achieve this goal, nanoparticles were characterized, separation methods were developed and growth curves were constructed to determine the influence of nanoparticles on the growth of S. aureus. The release and uptake of iron from nanoparticles was investigated through the irradiation of iron nanoparticles and the measurement of the remaining 59Fe after nanoparticle-supplemented growth. It was found that only 3.7 ± 2.4% of nanoparticle iron was taken up by S.aureus after 24 hours, while 53 ± 12% of free iron was taken up. The percentage of iron that was taken up was found to be greater than the weight percentage of iron that was released from the nanoparticles. While this demonstrates that iron remains with S. aureus after separation. It is not yet known whether this iron is truly internalized, or simply attached to the membrane. It is also not conclusively known whether nanoparticle iron contributes to determine bacterial growth. Therefore, future experiments should be conducted to determine the internalization and the subcellular distribution of radioactive nanoparticle iron and its effects on bacterial metabolism.