Light controlled motility of Escherichia coli. Characterization and applications

Characterization of wild type E. coli motility in response to light stimuli. Gene editing of bacteria to implement specifc functions (e.g. photokinesis). The engineered strain has been used to demonstrate that density modulation of photokinetic bacteria can be obtained by projecting spatially structured light on the sample. Additionally these bacteria have been also used as propelling units in microfabricated structures

Mechanistic modeling of a rewritable recombinase addressable data module

Many of the most important applications predicted to arise from Synthetic Biology will require engineered cellular memory with the capability to store data in a rewritable and reversible manner upon induction by transient stimuli. DNA recombination provides an ideal platform for cellular data storage and has allowed the development of a rewritable recombinase addressable data (RAD) module, capable of efficient data storage within a chromosome. Here, we develop the first detailed mechanistic model of DNA recombination, and validate it against a new set of in vitro data on recombination efficiencies across a range of different concentrations of integrase and gp3. Investigation of in vivo recombination dynamics using our model reveals the importance of fully accounting for all mechanistic features of DNA recombination in order to accurately predict the effect of different switching strategies on RAD module performance, and highlights its usefulness as a design tool for building future synthetic circuitry

Light-regulated Gene Expression in Bacteria : Fundamentals, Advances, and Perspectives

Numerous photoreceptors and genetic circuits emerged over the past two decades and now enable the light-dependent i.e., optogenetic, regulation of gene expression in bacteria. Prompted by light cues in the near-ultraviolet to near-infrared region of the electromagnetic spectrum, gene expression can be up- or downregulated stringently, reversibly, non-invasively, and with precision in space and time. Here, we survey the underlying principles, available options, and prominent examples of optogenetically regulated gene expression in bacteria. While transcription initiation and elongation remain most important for optogenetic intervention, other processes e.g., translation and downstream events, were also rendered light-dependent. The optogenetic control of bacterial expression predominantly employs but three fundamental strategies: light-sensitive two-component systems, oligomerization reactions, and second-messenger signaling. Certain optogenetic circuits moved beyond the proof-of-principle and stood the test of practice. They enable unprecedented applications in three major areas. First, light-dependent expression underpins novel concepts and strategies for enhanced yields in microbial production processes. Second, light-responsive bacteria can be optogenetically stimulated while residing within the bodies of animals, thus prompting the secretion of compounds that grant health benefits to the animal host. Third, optogenetics allows the generation of precisely structured, novel biomaterials. These applications jointly testify to the maturity of the optogenetic approach and serve as blueprints bound to inspire and template innovative use cases of light-regulated gene expression in bacteria. Researchers pursuing these lines can choose from an ever-growing, versatile, and efficient toolkit of optogenetic circuits

EXPANDING MOLECULAR TOOLS FOR THE METABOLIC ENGINEERING OF RALSTONIA EUTROPHA H16

Ralstonia eutropha H16 (also known as Cupriavidus necator H16) is a non-pathogenic chemolithoautotrophic soil bacterium. It has increasingly gained biotechnological interest for its use as a microbial cell factory for the production of several valuable bio-based chemicals. However the absence of a large repertoire of molecular tools to engineer this organism remains a critical limiting factor to exploiting its full biotechnological potential. Also, adopting established molecular tools applicable to the more notable microbial hosts such as E. coli and Saccharomyces cerevesiae is severely hampered by chassis-incompatibility and functional variability of essential biological parts. The work detailed in this thesis focuses on the development of key molecular tools crucial to improving the biosynthesis of malonyl-CoA - a precursor metabolite required for the biosynthesis of fatty acids and potentially several valuable bio-products in Ralstonia eutropha H16. All molecular tools developed were based on the broad host range (BHR) plasmid vector backbone of pBBR1MCS1 – a R. eutropha H16-compatible vector. Firstly, to facilitate heterologous pathway optimization, a combination of pre-existing and novel methods of genetic modifications were applied to engineer a collection of 42 promoters. Promoter strengths were characterized using a fluorescence-based assay and benchmarked to the dose-dependent activity of an L-arabinose-inducible PBAD promoter. Next, to detect intracellular accumulation of malonyl-CoA, transcriptional factor-based malonyl-CoA-sensing genetic circuits were developed via careful selection from the promoter collection. Thirdly, BHR L-arabinose-inducible λ-Red plasmid vectors were developed for mediating λ-Red-based genome editing. These were first tested in E. coli BW25113 to confirm their functionality and then subsequently tested in R. eutropha H16. Overall, the collection of engineered promoters yielded a 137-fold range of promoter activity and the malonyl-CoA biosensors responded to changing malonyl-CoA concentrations. The BHR λ-Red plasmids showed high recombination efficiency in E. coli BW25113. The molecular tools developed from this work will further facilitate rapid control and regulation of gene expression in R. eutropha, particularly for malonyl-CoA engineering

E. coli Nissle 1917 as a potential chassis for osmolality biosensors in the gut

The human gut is a heterogeneous environment. Diseases like inflammatory bowel disease (IBD) modify the physical properties of the gut, such as osmolality. These changes are thus desirable biomarkers for personalized diagnosis and treatment. Current diagnostic tools are invasive and insufficient to precisely detect changes of the physical environment. Likewise, disease heterogeneity hinders final diagnosis, showing the importance of creating personalized and sensitive diagnostic tools. For these reasons, its necessary to develop clinically relevant technologies that can safely and accurately report on these physical changes across different regions of the human gut. Extensive research on the human microbiome has revealed that microbes are able to sense shifts in the gut’s physical properties, making them strong candidates to report on these novel biomarkers. Furthermore, advances in synthetic biology have allowed the creation of microbial whole-cell biosensors that robustly report disease biomarkers in the human gut. Therefore, this thesis shows the potential of the probiotic strain Escherichia coli Nissle 1917 (EcN) as a chassis for gut osmolality biosensors. Here, RNA-seq and differential gene expression analysis were used to filter and compare genes that strongly and exclusively respond to different osmolality levels relative to general stress conditions. It was found that five promoters met the conditions, nevertheless, there was cross-reactivity within levels of osmolality and with other general stress conditions. Interestingly, some of the selected promoters had not been shown to react to elevated osmolality conditions, hinting at differences between EcN and other E. coli strains. Based on this, further experimentation is necessary to validate the activity of these promoters in the conditions of interest. Taken together, this work provides a starting point for gut osmolality biosensors using the probiotic strain EcN, providing more options for building biosensors of gut biomarkers

EFFECTS OF NON-LETHAL CONCENTRATIONS OF BIOACTIVE COMPOUNDS ON PLANT-RELATED BIOFILMS

It has been estimated that at least 99 % of the world\u2019s microbial biomass exists in form of biofilm, a complex differentiated surface-associated community embedded in a self-produced polymeric matrix enabling microorganisms to develop coordinated and efficient survival strategies. Biofilm formation is a dynamic and cyclical process involving attachment, maturation and a final dispersal phase, and these steps are initiated by a variety of signals. Despite their positive effects in some cases, biofilms can be detrimental in different environmental domains since microorganisms are able to colonize almost all types of surfaces both abiotic and biotic, leading to consequences in terms of social and economic impact. These include human tissues, implantable medical devices, natural aquatic systems, plants, food and industrial lines. Once biofilm is formed, its eradication becomes difficult because its resilience to environmental stresses, disinfectants, and antimicrobial treatments. Plants support a diverse array of microorganisms that exist in form of biofilms. Even if in some cases the association with plants leads to beneficial interactions promoting plant growth, inducing plant defense mechanisms and preventing the deleterious effects of pathogenic microorganisms, in other cases they have a significant negative impact. For instance, in agriculture, plant colonization of fungi and bacteria in form of biofilm is a cause of plant diseases, affecting crop quality and productivity. Indeed, despite the planktonic growth, biofilm lifestyle improves microbial resistance to antimicrobials up to several orders of magnitude, often reducing the possibility of treating biofilm effectively. In addition, due to the worrisome consequences related to the use of these substances on human health and on their persistence in the environment, increasingly regulations are arising to limit antimicrobial application. Furthermore, in addition to the principles of integrated pest management (IPM) embraced by the worldwide legislation aims to recommend alternative approaches to the application of pesticides, an innovative approach could be the use of biocide-free bioactive compounds characterized by novel targets, unique modes of action and properties that are separate from those currently highlighted in the use of antimicrobials. Indeed, the application of non-lethal doses of bio-inspired molecules able to interfere with specific key-steps involved in the biofilm formation process has been suggested as a complementary/alternative strategy to hinder biofilm formation. In addition, this approach also lead to deprive microorganisms of their virulence factors without affecting their viability and decreasing the selection pressure for biocides resistance. In this PhD thesis, the in vitro effects of non-lethal concentrations of several bioactive compounds were evaluated on the biofilm formation of different plant-associated microorganisms. Specifically, the aim of this work was to provide new effective preventive or integrated solutions against bacterial and fungal biofilm formation. In chapter III, the methanol extracts obtained by different plant portions of three seagrass species collected in Vietnam and in India (Enhalus acoroides, Halophila ovalis and Halodule pinifolia) were investigated for their effects in mediating non-lethal interactions on sessile Escherichia coli and Candida albicans cultures taken as models of bacterial and fungal biofilms respectively. The study was focused on anti-biofilm activities of seagrass extracts, without killing cells. Seagrass extracts appeared to be more effective in deterring microbial adhesion on hydrophobic surfaces than on hydrophilic. Results revealed that E. acoroides leaf extract proved to be the most promising extract among those tested. Indeed, the selected non-lethal concentrations of E. acoroides leaf extract were found to exert an anti-biofilm effect on C. albicans and E. coli biofilm in the first phase of biofilm genesis, opening up the possibility of developing preventive strategies to hinder the adhesion of microbial cells to surfaces. The leaf extract also affected the dispersion and maturation steps in C. albicans and E. coli respectively, suggesting an important role in cell signaling processes. Methanolic extracts were characterized and major phenolic compounds were identified by MS/MS analysis, showing the unique profile of the E. acoroides leaf extract. In chapter IV, two essential oils (PK and PK-IK) derived from two cultivars of Perilla frutescens, an annual short day plant widely used in therapeutics in the traditional medicine as well as in food preparations in Asian countries. Essential oils were extracted from the leaves and were characterized. Subsequeltly, their ability to affect biofilm formation of the phytopathogenic model fungi Colletotrichum musae, Fusarium dimerum and F. oxysporum have been studied. PK and PK-IK neither inhibited fungal growth nor were they utilized as a carbon energy source. In addition, PK and PK-IK essential oils showed excellent anti-biofilm performances inhibiting conidia germination and reducing conidia adhesion. Furthermore, they revealed a magnificent anti-biofilm effect even during biofilm maturation, affecting biofilm structural development, with a reduction of dried weight, extracellular polysaccharides and proteins. In all cases PK-IK displayed better activity than PK. Thus, the anti-biofilm effects were exploited with a non-lethal mechanism. This research supported the spreading of PK and PK-IK essential oils as biocide-free agents suitable for a preventive or integrative approach for sustainable crop protection. Lastly, in chapter V, a non-lethal concentration of N-Acetylcysteine (NAC) was evaluated on the biofilm formation of Xylella fastidiosa, a phytopathogen bacterium that causes a range of economically important plant diseases worldwide and that has been recently found in Italy in olive plants, where it causes the olive quick decline syndrome (OQSD). NAC is a naturally occurring compound found in several vegetables (including garlic, onion, peppers and asparagus) and it is mostly known in clinical area, in which it is employed at lethal concentrations in the treatment of human diseases due to its ability to reduce bacterial adhesion, inhibit the production of extracellular polysaccharides and promote the dispersion of pre-formed mature biofilms. In this study, N-Acetylcysteine (NAC) was tested for its ability to affect biofilm response of X. fastidiosa CoDiRO strain, mimicking a preventive, a curative and a combination of both approaches. The not-lethal dose 0.08 mg/ml was chosen as representative of plant concentration after its application. NAC did not alter planktonic bacterial growth but promoted biofilm formation in terms of biofilm biomass (above 62 %) and matrix polysaccharides (above 53%) through a ROS-mediated mechanism. Additionally, NAC was not able to destroy X. fastidiosa biofilm when already established on the surface but rather, it was suitable to contain the biofilm infection limiting biofilm dispersal. On the contrary, a combination of both preventive and curative approach has been found promising in biofilm dissolving making it more vulnerable

The compatible solutes ectoine and 5-hydroxyectoine: Catabolism and regulatory mechanisms

To cope with osmotic stress many microorganisms make use of short, osmotically active, organic compounds, the so-called compatible solutes. Examples for especially effective members of this type of molecules are the tetrahydropyrimidines ectoine and 5-hydroxyectoine. Both molecules are produced by a large number of microorganisms, not only to fend-off osmotic stress, but also for example low and high temperature challenges. The biosynthetic pathway used by these organisms to synthesize ectoines has already been studied intensively and the enzymes used therein are characterized quite well, both biochemically as well as structurally. However, synthesis of ectoines is only half the story. Inevitably, ectoines are frequently released from the producer cells in different environmental settings. Especially in highly competitive habitats like the upper ocean layers some bacteria specialized on a niche like this. The model organism used in this work is such a species. It is the marine bacterium Ruegeria pomeroyi DSS-3 which belongs to the Roseobacter-clade. Roseobacter species are heterotrophic Proteobacteria which can live in symbiosis with phytoplankton as well as turning against them in a bacterial warfare fashion to scavenge valuable nutrients. Ectoines can be imported by R. pomeroyi DSS-3 in a high-affinity fashion and be used as energy as well as carbon- and nitrogen-sources. To achieve this, both ectoines rings are degraded by the hydrolase EutD and deacetylated by the deacetylase EutE. The first hydrolysis products α-ADABA (from ectoine) and hydroxy-α-ADABA (from hydroxyectoine) are deacetylated to DABA and hydroxy-DABA which are in additional biochemical reactions transformed to aspartate to fuel the cell’s central metabolism. The role and functioning of the EutDE enzymes which work in a concerted fashion are a central aspect of this work. Both enzymes could be biochemically and structurally characterized, and the architecture of the metabolic pathway could be illuminated. α-ADABA and hydroxy-α-ADABA are not only central to ectoine catabolism, but also to the regulatory mechanisms associated with it. Both molecules serve as inducers of the central regulatory protein of this pathway, the MocR-/GabR-type regulator protein EnuR. In the framework of this dissertation molecular details could be clarified which enable the EnuR repressor molecule to sense both molecules with high affinity to subsequently derepress the genes for the import and catabolism of ectoines

Diatom milking? A review and new approaches

The rise of human populations and the growth of cities contribute to the depletion of natural resources, increase their cost, and create potential climatic changes. To overcome difficulties in supplying populations and reducing the resource cost, a search for alternative pharmaceutical, nanotechnology, and energy sources has begun. Among the alternative sources, microalgae are the most promising because they use carbon dioxide (CO2) to produce biomass and/or valuable compounds. Once produced, the biomass is ordinarily harvested and processed (downstream program). Drying, grinding, and extraction steps are destructive to the microalgal biomass that then needs to be renewed. The extraction and purification processes generate organic wastes and require substantial energy inputs. Altogether, it is urgent to develop alternative downstream processes. Among the possibilities, milking invokes the concept that the extraction should not kill the algal cells. Therefore, it does not require growing the algae anew. In this review, we discuss research on milking of diatoms. The main themes are (a) development of alternative methods to extract and harvest high added value compounds; (b) design of photobioreactors; (c) biodiversity and (d) stress physiology, illustrated with original results dealing with oleaginous diatoms