Cells use lipid membranes to organize and define their chemical environments. All cell membranes are based on a common structure: bilayers composed of phospholipids with two hydrocarbon chains. How did biology converge on this particular solution for cellular encapsulation? The first cell membranes are proposed to have assembled from simple, single-chain lipids, such as fatty acids and their derivatives, which would have been available in the prebiotic environment. Here we argue that the physical properties of fatty acid membranes would have made them well suited for a role as primitive cell membranes and predisposed their evolution to modern, phospholipid-based membranes. We first considered models for primitive membrane self-assembly, which faces significant concentration barriers due to the entropic cost of aggregation and the solubility of single-chain lipids. We therefore identified two physical mechanisms by which fatty acid membrane assembly can proceed from dilute solutions. Thermal diffusion columns, a proposed prebiotic concentration method, drive the formation of fatty acid vesicles by concentrating an initially isotropic solution past the critical concentration necessary for aggregation. Alternatively, mixtures of fatty acids with varying chain lengths, the expected products of abiotic lipid synthesis, intrinsically reduce the concentration barrier to aggregation through their polydispersity. These results motivated us to better understand the phase behavior of fatty acids in solutions. We found that the composition of fatty acid aggregates, whether vesicles or micelles, is also determined by concentration. Fatty acid vesicles feature significant amounts of coexisting micelles, whose abundance is enriched in low concentration solutions. We utilized this micelle-vesicle equilibrium to drive the growth of pre-existing fatty acid vesicles by changing amphiphile concentration. We next considered the evolution of phospholipid membranes, which was a critical and necessary step for the early evolution of cells. We found that the incorporation of even small amounts of phospholipids drives the growth of fatty acid vesicles by competition for monomers with neighboring vesicles lacking phospholipids. This competitive growth would have provided a strong selective advantage for primitive cells to evolve the catalytic machinery needed to synthesize phospholipids from their single-chain precursors. Growth is caused by any relative difference in phospholipid content, suggesting an evolutionary arms race among primitive cells for increasingly phospholipid membranes. What would have been the consequences for early cells of such a transition in membrane composition? We found that increasing phospholipid content inhibits the permeability of fatty acid membranes through changes in bilayer fluidity. For early heterotrophic cells, the emergence of increasingly phospholipid membranes would have therefore imposed new selective pressures for the evolution of membrane transport machinery and metabolism. Our model for early membrane evolution led us to develop prebiotic models for phospholipid chemistry. The assembly of phospholipids from single-chain substrates requires a single reaction: the acyltransfer of an activated fatty acid onto a glycerol monoester or lysophospholipid. We developed a synthetic model for this reaction that incorporates a copper-catalyzed azide-alkyne cycloaddition and showed that it drives de novo vesicle assembly.

The evolution of a large, diverse group of intracellular bacteria was previously very difficult to study. Recent advancements in both metagenomic methods and bioinformatics has made it possible. This thesis investigates the evolution of the order Legionellales. The study concentrates on a group of proteins essential for pathogenesis and host manipulation in the order, called effector proteins. The role of effectors in host adaptation, evolutionary history and the diversification of the order were investigated using a multitude of bioinformatics methods. First, the abundance and distribution of the known effector proteins in the orderwas found to cover newly discovered clades. There was a clear distinction between the proteins present in Legionellales and the outgoup, indicating the important role of the effectors in the order. Further, the effectors with known functions found in the new clades, particularly in Berkiella, revealed potential modes of host manipulation of this group. Secondly, the evolution of the effector gene content in the order shed light on theevolution of the order, as well as on the potential evolutionary differences between Legionellaceae and Coxiellaceae. In general, most of the effectors were gained early in the last common ancestor of Legionellales and Legionellaceae, as further indication of their role in the diversification of the order. New effector genes were acquired in the Legionellaceae even up to recent speciation events, whereas Coxiellacea have lost more protein coding genes with time. These differences may be due to horizontal gene transfer in the case of gene gains in Legionellaceae and loss of selection in the case of gene losses in Coxiellaceae. Third, the early evolution of core gained effector proteins for the order was studied.Two of the eight investigated core effectors seem to have a connection to eukaryotes, the rest to other bacteria, indicating both inter-domain and within bacteria horizontal gene transfer. In particular, one effector protein with eukaryotic motif gained at the last common ancestor of Legionellales, was found in all the clades and is therefore an important evolutionary link that may have allowed Legionellales to utilize eukaryotic hosts.