Towards Synthetic Life: Establishing a Minimal Segrosome for the Rational Design of Biomimetic Systems

DNA segregation is a fundamental life process, crucial for renewal, reproduction and propagation of all forms of life. Hence, a dedicated segregation machinery, a segrosome, must function reliably also in the context of a minimal cell. Conceptionally, the development of such a minimal cell follows a minimalistic approach, aiming at engineering a synthetic entity only consisting of the essential key elements necessary for a cell to survive. In this thesis, various prokaryotic segregation systems were explored as possible candidates for a minimal segrosome. Such a minimal segrosome could be applied for the rational design of biomimetic systems including, but not limited to, a minimal cell. DNA segregation systems of type I (ParABS) and type II (ParMRC) were compared for ensuring genetic stabilities in vivo using vectors derived from the natural secondary chromosome of Vibrio cholerae. The type II segregation system R1-ParMRC was chosen as the most promising candidate for a minimal segrosome, and it was characterized and reconstituted in vitro. This segregation system was encapsulated into biomimetic micro-compartments and its lifetime prolonged by coupling to ATP-regenerating as well as oxygen-scavenging systems. The segregation process was coupled to in vitro DNA replication using DNA nanoparticles as a mimic of the condensed state of chromosomes. Furthermore, another type II segregation system originating from the pLS20 plasmid from Bacillus subtilis (Alp7ARC) was reconstituted in vitro as a secondary orthogonal segrosome. Finally, a chimeric RNA segregation system was engineered that could be applied for an RNA-based protocell. Overall, this work demonstrates successful bottom-up assemblies of functional molecular machines that could find applications in biomimetic systems and lead to a deeper understanding of living systems

Microbial Active Matter: A Topological Framework

Topology transcends boundaries that conventionally delineate physical, biological and engineering sciences. Our ability to mathematically describe topology, combined with our access to precision tracking and manipulation approaches, has triggered a fresh appreciation of topological ramifications, specifically in mediating key functions in biological systems spanning orders of magnitude in length and time scales. Microbial ecosystems, a frequently encountered example of living matter, offer a rich test bed where the role of topological defects and their mechanics can be explored in the context of microbial composition, structure and functions. Emergent processes, triggered by anisotropy and activity characteristic of such structured, out-of-equilibrium systems, underpin fundamental properties in microbial systems. An inevitable consequence of anisotropy is the long-range orientational (or positional) correlations, which give rise to topological defects nucleating due to spontaneous symmetry breaking. The scene stealer of this emerging cross-disciplinary field is the topological defects: singularities embedded within the material field that elicit novel, if not unexpected, dynamics that are at the heart of active processes underpinning soft and living matter systems. In this short review, I have put together a summary of the key recent advances that highlight the interface of liquid crystal physics and the physical ecology of microbes; and combined it with original experimental data on sessile species as a case to demonstrate how this interface offers a biophysical framework that could help to decode and harness active microbial processes in 'true' ecological settings. Topology and its functional manifestations - a crucial and well-timed topic - offer a rich opportunity for both experimentalists and theoreticians willing to take up an exciting journey across scales and disciplines.Comment: 9 pages, 5 figure

Swarming and complex pattern formation in Paenibacillus vortex studied by imaging and tracking cells

<p>Abstract</p> <p>Background</p> <p>Swarming motility allows microorganisms to move rapidly over surfaces. The Gram-positive bacterium <it>Paenibacillus vortex </it>exhibits advanced cooperative motility on agar plates resulting in intricate colonial patterns with geometries that are highly sensitive to the environment. The cellular mechanisms that underpin the complex multicellular organization of such a simple organism are not well understood.</p> <p>Results</p> <p>Swarming by <it>P. vortex </it>was studied by real-time light microscopy, by <it>in situ </it>scanning electron microscopy and by tracking the spread of antibiotic-resistant cells within antibiotic-sensitive colonies. When swarming, <it>P. vortex </it>was found to be peritrichously flagellated. Swarming by the curved cells of <it>P. vortex </it>occurred on an extremely wide range of media and agar concentrations (0.3 to 2.2% w/v). At high agar concentrations (> 1% w/v) rotating colonies formed that could be detached from the main mass of cells by withdrawal of cells into the latter. On lower percentage agars, cells moved in an extended network composed of interconnected "snakes" with short-term collision avoidance and sensitivity to extracts from swarming cells. <it>P. vortex </it>formed single Petri dish-wide "supercolonies" with a colony-wide exchange of motile cells. Swarming cells were coupled by rapidly forming, reversible and non-rigid connections to form a loose raft, apparently connected <it>via </it>flagella. Inhibitors of swarming (<it>p</it>-Nitrophenylglycerol and Congo Red) were identified. Mitomycin C was used to trigger filamentation without inhibiting growth or swarming; this facilitated dissection of the detail of swarming. Mitomycin C treatment resulted in malcoordinated swarming and abortive side branch formation and a strong tendency by a subpopulation of the cells to form minimal rotating aggregates of only a few cells.</p> <p>Conclusion</p> <p><it>P. vortex </it>creates complex macroscopic colonies within which there is considerable reflux and movement and interaction of cells. Cell shape, flagellation, the aversion of cell masses to fuse and temporary connections between proximate cells to form rafts were all features of the swarming and rotation of cell aggregates. Vigorous vortex formation was social, i.e. required > 1 cell. This is the first detailed examination of the swarming behaviour of this bacterium at the cellular level.</p

Reviewing agent-based modelling of socio-ecosystems: a methodology for the analysis of climate change adaptation and sustainability

The integrated - environmental, economic and social - analysis of climate change calls for a paradigm shift as it is fundamentally a problem of complex, bottom-up and multi-agent human behaviour. There is a growing awareness that global environmental change dynamics and the related socio-economic implications involve a degree of complexity that requires an innovative modelling of combined social and ecological systems. Climate change policy can no longer be addressed separately from a broader context of adaptation and sustainability strategies. A vast body of literature on agent-based modelling (ABM) shows its potential to couple social and environmental models, to incorporate the influence of micro-level decision making in the system dynamics and to study the emergence of collective responses to policies. However, there are few publications which concretely apply this methodology to the study of climate change related issues. The analysis of the state of the art reported in this paper supports the idea that today ABM is an appropriate methodology for the bottom-up exploration of climate policies, especially because it can take into account adaptive behaviour and heterogeneity of the system's components.Review, Agent-Based Modelling, Socio-Ecosystems, Climate Change, Adaptation, Complexity.

Possible origins of macroscopic left-right asymmetry in organisms

I consider the microscopic mechanisms by which a particular left-right (L/R) asymmetry is generated at the organism level from the microscopic handedness of cytoskeletal molecules. In light of a fundamental symmetry principle, the typical pattern-formation mechanisms of diffusion plus regulation cannot implement the "right-hand rule"; at the microscopic level, the cell's cytoskeleton of chiral filaments seems always to be involved, usually in collective states driven by polymerization forces or molecular motors. It seems particularly easy for handedness to emerge in a shear or rotation in the background of an effectively two-dimensional system, such as the cell membrane or a layer of cells, as this requires no pre-existing axis apart from the layer normal. I detail a scenario involving actin/myosin layers in snails and in C. elegans, and also one about the microtubule layer in plant cells. I also survey the other examples that I am aware of, such as the emergence of handedness such as the emergence of handedness in neurons, in eukaryote cell motility, and in non-flagellated bacteria.Comment: 42 pages, 6 figures, resubmitted to J. Stat. Phys. special issue. Major rewrite, rearranged sections/subsections, new Fig 3 + 6, new physics in Sec 2.4 and 3.4.1, added Sec 5 and subsections of Sec

Stigmergy co-ordinates multicellular collective behaviours during Myxococcus xanthus surface migration

Surface translocation by the soil bacterium Myxococcus xanthus is a complex multicellular phenomenon that entails two motility systems. However, the mechanisms by which the activities of individual cells are coordinated to manifest this collective behaviour are currently unclear. Here we have developed a novel assay that enables detailed microscopic examination of M. xanthus motility at the interstitial interface between solidified nutrient medium and a glass coverslip. Under these conditions, M. xanthus motility is characterised by extensive micro-morphological patterning that is considerably more elaborate than occurs at an air-surface interface. We have found that during motility on solidified nutrient medium, M. xanthus forges an interconnected furrow network that is lined with an extracellular matrix comprised of exopolysaccharides, extracellular lipids, membrane vesicles and an unidentified slime. Our observations have revealed that M. xanthus motility on solidified nutrient medium is a stigmergic phenomenon in which multi-cellular collective behaviours are co-ordinated through trail-following that is guided by physical furrows and extracellular matrix materials

Meso-scale turbulence in living fluids

Turbulence is ubiquitous, from oceanic currents to small-scale biological and quantum systems. Self-sustained turbulent motion in microbial suspensions presents an intriguing example of collective dynamical behavior amongst the simplest forms of life, and is important for fluid mixing and molecular transport on the microscale. The mathematical characterization of turbulence phenomena in active non-equilibrium fluids proves even more difficult than for conventional liquids or gases. It is not known which features of turbulent phases in living matter are universal or system-specific, or which generalizations of the Navier-Stokes equations are able to describe them adequately. Here, we combine experiments, particle simulations, and continuum theory to identify the statistical properties of self-sustained meso-scale turbulence in active systems. To study how dimensionality and boundary conditions affect collective bacterial dynamics, we measured energy spectra and structure functions in dense Bacillus subtilis suspensions in quasi-2D and 3D geometries. Our experimental results for the bacterial flow statistics agree well with predictions from a minimal model for self-propelled rods, suggesting that at high concentrations the collective motion of the bacteria is dominated by short-range interactions. To provide a basis for future theoretical studies, we propose a minimal continuum model for incompressible bacterial flow. A detailed numerical analysis of the 2D case shows that this theory can reproduce many of the experimentally observed features of self-sustained active turbulence.Comment: accepted PNAS version, 6 pages, click doi for Supplementary Informatio

Unicellular but not asocial. Life in community of a bacterium

All living organisms have acquired the outstanding ability to overcome the limitations imposed by changeable environments through the gain of genetic traits over years of evolution and the tendency of individuals to associate in communities. The complementation of a singular weakness, the deployment of reinforcement for the good of the community, the better use of resources, or effective defense against external aggression are advantages gained by this communal behavior. Communication has been the cohesive element prompting the global responses that promote efficiency in two features of any community: specialization in differentiated labor and the spatio-temporal organization of the environment. These principles illustrate that what we call human ecology also applies to the cellular world and is exemplified in eukaryotic organisms, where sophisticated cell-to-cell communication networks coordinate cell differentiation and the specialization of multiple tissues consisting of numerous cells embedded in a multifunctional extracellular matrix. This sophisticated molecular machinery appears, however, to be invented by the &ldquo;simple&rdquo; but still fascinating bacteria. What I will try to expand in the following sections are notions of how &ldquo;single prokaryotic cells&rdquo; organize a multicellular community. [Int Microbiol 19(2):81-90 (2016)]Keywords: evolution &middot; molecular machinery &middot; multicellular community &middot; prokaryotic cells &middot; global response