Plasmid partitioning driven by collective migration of ParA between nucleoid lobes.

The ParABS system is crucial for the faithful segregation and inheritance of many bacterial chromosomes and low-copy-number plasmids. However, despite extensive research, the spatiotemporal dynamics of the ATPase ParA and its connection to the dynamics and positioning of the ParB-coated cargo have remained unclear. In this study, we utilize high-throughput imaging, quantitative data analysis, and computational modeling to explore the in vivo dynamics of ParA and its interaction with ParB-coated plasmids and the nucleoid. As previously observed, we find that F-plasmid ParA undergoes collective migrations ("flips") between cell halves multiple times per cell cycle. We reveal that a constricting nucleoid is required for these migrations and that they are triggered by a plasmid crossing into the cell half with greater ParA. Using simulations, we show that these dynamics can be explained by the combination of nucleoid constriction and cooperative ParA binding to the DNA, in line with the behavior of other ParA proteins. We further show that these ParA flips act to equally partition plasmids between the two lobes of the constricted nucleoid and are therefore important for plasmid stability, especially in fast growth conditions for which the nucleoid constricts early in the cell cycle. Overall, our work identifies a second mode of action of the ParABS system and deepens our understanding of how this important segregation system functions.

☠Track: Inferred counting and tracking of replicating DNA loci.

Fluorescent microscopy is the primary method to study DNA organization within cells. However, the variability and low signal/noise commonly associated with live-cell time-lapse imaging challenges quantitative measurements. In particular, obtaining quantitative or mechanistic insight often depends on the accurate tracking of fluorescent particles. Here, we present ★Track, an inference method that determines the most likely temporal tracking of replicating intracellular particles such DNA loci while accounting for missing, merged, and spurious detections. It allows the accurate prediction of particle copy numbers as well as the timing of replication events. We demonstrate ★Track's abilities and gain new insight into plasmid copy number control and the volume dependence of bacterial chromosome replication initiation. By enabling the accurate tracking of DNA loci, ★Track can help to uncover the mechanistic principles of chromosome organization and dynamics across a range of systems.

The quantitative basis for the redistribution of immobile bacterial lipoproteins to division septa.

The spatial localisation of proteins is critical for most cellular function. In bacteria, this is typically achieved through capture by established landmark proteins. However, this requires that the protein is diffusive on the appropriate timescale. It is therefore unknown how the localisation of effectively immobile proteins is achieved. Here, we investigate the localisation to the division site of the slowly diffusing lipoprotein Pal, which anchors the outer membrane to the cell wall of Gram-negative bacteria. While the proton motive force-linked TolQRAB system is known to be required for this repositioning, the underlying mechanism is unresolved, especially given the very low mobility of Pal. We present a quantitative, mathematical model for Pal relocalisation in which dissociation of TolB-Pal complexes, powered by the proton motive force across the inner membrane, leads to the net transport of Pal along the outer membrane and its deposition at the division septum. We fit the model to experimental measurements of protein mobility and successfully test its predictions experimentally against mutant phenotypes. Our model not only explains a key aspect of cell division in Gram-negative bacteria, but also presents a physical mechanism for the transport of low-mobility proteins that may be applicable to multi-membrane organelles, such as mitochondria and chloroplasts.

Rapid turnover of DnaA at replication origin regions contributes to initiation control of DNA replication.

DnaA is a conserved key regulator of replication initiation in bacteria, and is homologous to ORC proteins in archaea and in eukaryotic cells. The ATPase binds to several high affinity binding sites at the origin region and upon an unknown molecular trigger, spreads to several adjacent sites, inducing the formation of a helical super structure leading to initiation of replication. Using FRAP analysis of a functional YFP-DnaA allele in Bacillus subtilis, we show that DnaA is bound to oriC with a half-time of 2.5 seconds. DnaA shows similarly high turnover at the replication machinery, where DnaA is bound to DNA polymerase via YabA. The absence of YabA increases the half time binding of DnaA at oriC, showing that YabA plays a dual role in the regulation of DnaA, as a tether at the replication forks, and as a chaser at origin regions. Likewise, a deletion of soj (encoding a ParA protein) leads to an increase in residence time and to overinitiation, while a mutation in DnaA that leads to lowered initiation frequency, due to a reduced ATPase activity, shows a decreased residence time on binding sites. Finally, our single molecule tracking experiments show that DnaA rapidly moves between chromosomal binding sites, and does not arrest for more than few hundreds of milliseconds. In Escherichia coli, DnaA also shows low residence times in the range of 200 ms and oscillates between spatially opposite chromosome regions in a time frame of one to two seconds, independently of ongoing transcription. Thus, DnaA shows extremely rapid binding turnover on the chromosome including oriC regions in two bacterial species, which is influenced by Soj and YabA proteins in B. subtilis, and is crucial for balanced initiation control, likely preventing fatal premature multimerization and strand opening of DnaA at oriC.

Reconstitution and Coupling of DNA Replication and Segregation in a Biomimetic System.

A biomimetic system capable of replication and segregation of genetic material constitutes an essential component for the future design of a minimal synthetic cell. Here we have used the simple T7 bacteriophage system and the plasmid-derived ParMRC system to establish in vitro DNA replication and DNA segregation, respectively. These processes were incorporated into biomimetic compartments providing an enclosed reaction space. The functional lifetime of the encapsulated segregation system could be prolonged by equipping it with ATP-regenerating and oxygen-scavenging systems. Finally, we showed that DNA replication and segregation processes could be coupled in vitro by using condensed DNA nanoparticles resulting from DNA replication. ParM spindles extended over tens of micrometers and could thus be used for segregation in compartments that are significantly longer than bacterial cell size. Overall, this work demonstrates the successful bottom-up assembly and coupling of molecular machines that mediate replication and segregation, thus providing an important step towards the development of a fully functional minimal cell.

Nutritional Control of DNA Replication Initiation through the Proteolysis and Regulated Translation of DnaA.

Bacteria can arrest their own growth and proliferation upon nutrient depletion and under various stressful conditions to ensure their survival. However, the molecular mechanisms responsible for suppressing growth and arresting the cell cycle under such conditions remain incompletely understood. Here, we identify post-transcriptional mechanisms that help enforce a cell-cycle arrest in Caulobacter crescentus following nutrient limitation and during entry into stationary phase by limiting the accumulation of DnaA, the conserved replication initiator protein. DnaA is rapidly degraded by the Lon protease following nutrient limitation. However, the rate of DnaA degradation is not significantly altered by changes in nutrient availability. Instead, we demonstrate that decreased nutrient availability downregulates dnaA translation by a mechanism involving the 5' untranslated leader region of the dnaA transcript; Lon-dependent proteolysis of DnaA then outpaces synthesis, leading to the elimination of DnaA and the arrest of DNA replication. Our results demonstrate how regulated translation and constitutive degradation provide cells a means of precisely and rapidly modulating the concentration of key regulatory proteins in response to environmental inputs.

Computational and genetic reduction of a cell cycle to its simplest, primordial components.

What are the minimal requirements to sustain an asymmetric cell cycle? Here we use mathematical modelling and forward genetics to reduce an asymmetric cell cycle to its simplest, primordial components. In the Alphaproteobacterium Caulobacter crescentus, cell cycle progression is believed to be controlled by a cyclical genetic circuit comprising four essential master regulators. Unexpectedly, our in silico modelling predicted that one of these regulators, GcrA, is in fact dispensable. We confirmed this experimentally, finding that ΔgcrA cells are viable, but slow-growing and elongated, with the latter mostly due to an insufficiency of a key cell division protein. Furthermore, suppressor analysis showed that another cell cycle regulator, the methyltransferase CcrM, is similarly dispensable with simultaneous gcrA/ccrM disruption ameliorating the cytokinetic and growth defect of ΔgcrA cells. Within the Alphaproteobacteria, gcrA and ccrM are consistently present or absent together, rather than either gene being present alone, suggesting that gcrA/ccrM constitutes an independent, dispensable genetic module. Together our approaches unveil the essential elements of a primordial asymmetric cell cycle that should help illuminate more complex cell cycles.

Mid-cell migration of the chromosomal terminus is coupled to origin segregation in Escherichia coli.

Bacterial chromosomes are dynamically and spatially organised within cells. In slow-growing Escherichia coli, the chromosomal terminus is initially located at the new pole and must therefore migrate to midcell during replication to reproduce the same pattern in the daughter cells. Here, we use high-throughput time-lapse microscopy to quantify this transition, its timing and its relationship to chromosome segregation. We find that terminus centralisation is a rapid discrete event that occurs ~25 min after initial separation of duplicated origins and ~50 min before the onset of bulk nucleoid segregation but with substantial variation between cells. Despite this variation, its movement is tightly coincident with the completion of origin segregation, even in the absence of its linkage to the divisome, suggesting a coupling between these two events. Indeed, we find that terminus centralisation does not occur if origin segregation away from mid-cell is disrupted, which results in daughter cells having an inverted chromosome organisation. Overall, our study quantifies the choreography of origin-terminus positioning and identifies an unexplored connection between these loci, furthering our understanding of chromosome segregation in this bacterium.

Partition complex structure can arise from sliding and bridging of ParB dimers.

In many bacteria, chromosome segregation requires the association of ParB to the parS-containing centromeric region to form the partition complex. However, the structure and formation of this complex have been unclear. Recently, studies have revealed that CTP binding enables ParB dimers to slide along DNA and condense the centromeric region through the formation of DNA bridges. Using semi-flexible polymer simulations, we demonstrate that these properties can explain partition complex formation. Transient ParB bridges organize DNA into globular states or hairpins and helical structures, depending on bridge lifetime, while separate simulations show that ParB sliding reproduces the multi-peaked binding profile observed in Caulobacter crescentus. Combining sliding and bridging into a unified model, we find that short-lived ParB bridges do not impede sliding and can reproduce both the binding profile and condensation of the nucleoprotein complex. Overall, our model elucidates the mechanism of partition complex formation and predicts its fine structure.

High-throughput imaging and quantitative analysis uncovers the nature of plasmid positioning by ParABS.

The faithful segregation and inheritance of bacterial chromosomes and low-copy number plasmids requires dedicated partitioning systems. The most common of these, ParABS, consists of ParA, a DNA-binding ATPase and ParB, a protein that binds to centromeric-like parS sequences on the DNA cargo. The resulting nucleoprotein complexes are believed to move up a self-generated gradient of nucleoid-associated ParA. However, it remains unclear how this leads to the observed cargo positioning and dynamics. In particular, the evaluation of models of plasmid positioning has been hindered by the lack of quantitative measurements of plasmid dynamics. Here, we use high-throughput imaging, analysis and modelling to determine the dynamical nature of these systems. We find that F plasmid is actively brought to specific subcellular home positions within the cell with dynamics akin to an over-damped spring. We develop a unified stochastic model that quantitatively explains this behaviour and predicts that cells with the lowest plasmid concentration transition to oscillatory dynamics. We confirm this prediction for F plasmid as well as a distantly-related ParABS system. Our results indicate that ParABS regularly positions plasmids across the nucleoid but operates just below the threshold of an oscillatory instability, which according to our model, minimises ATP consumption. Our work also clarifies how various plasmid dynamics are achievable in a single unified stochastic model. Overall, this work uncovers the dynamical nature of plasmid positioning by ParABS and provides insights relevant for chromosome-based systems.

A Qualitative Exploration of Challenges and Opportunities for Dog Welfare in Ireland Post COVID-19, as Perceived by Dog Welfare Organisations.

This novel qualitative study identifies challenges and opportunities to improve dog welfare in Ireland, as perceived by dog welfare organisations (DWOs), a previously underutilised stakeholder. This study sought the views of this predominantly voluntary sector of the next steps for policy and action in dog welfare, in light of the effects of the "puppy pandemic", increased costs and COVID-19 restrictions. An integrated online focus group and interview design involving DWOs was analysed using inductive thematic analysis. Thematic analysis identified 2 key themes: (1) Key challenges and solutions in general dog welfare and (2) Challenges and opportunities in the welfare organisation sector. DWOs perceived poor public awareness of appropriate dog-husbandry, inadequate legislation enforcement, negative impact of puppy farms, and increased financial and volunteer burden. DWOs helped construct a best practice rehoming pathway, identified how overall standards could be improved and recommendations to enhance dog welfare. The DWOs perceived an increased numbers of households acquiring dogs, difficulties in rehoming, and financial challenges as threatening their viability as organisations and Irish dog welfare. Greater enforcement of legislation, greater communication between organisations and the government, and more media awareness were seen as helpful by the DWOs to improve dog welfare standards and their organisations.

Pattern selection in reaction diffusion systems.

Turing's theory of pattern formation has been used to describe the formation of self-organized periodic patterns in many biological, chemical, and physical systems. However, the use of such models is hindered by our inability to predict, in general, which pattern is obtained from a given set of model parameters. While much is known near the onset of the spatial instability, the mechanisms underlying pattern selection and dynamics away from onset are much less understood. Here, we provide physical insight into the dynamics of these systems. We find that peaks in a Turing pattern behave as point sinks, the dynamics of which is determined by the diffusive fluxes into them. As a result, peaks move toward a periodic steady-state configuration that minimizes the mass of the diffusive species. We also show that the preferred number of peaks at the final steady state is such that this mass is minimized. Our work presents mass minimization as a potential general principle for understanding pattern formation in reaction diffusion systems far from onset.

The lipoprotein Pal stabilises the bacterial outer membrane during constriction by a mobilisation-and-capture mechanism.

Coordination of outer membrane constriction with septation is critical to faithful division in Gram-negative bacteria and vital to the barrier function of the membrane. This coordination requires the recruitment of the peptidoglycan-binding outer-membrane lipoprotein Pal at division sites by the Tol system. Here, we show that Pal accumulation at Escherichia coli division sites is a consequence of three key functions of the Tol system. First, Tol mobilises Pal molecules in dividing cells, which otherwise diffuse very slowly due to their binding of the cell wall. Second, Tol actively captures mobilised Pal molecules and deposits them at the division septum. Third, the active capture mechanism is analogous to that used by the inner membrane protein TonB to dislodge the plug domains of outer membrane TonB-dependent nutrient transporters. We conclude that outer membrane constriction is coordinated with cell division by active mobilisation-and-capture of Pal at division septa by the Tol system.

Center Finding in E. coli and the Role of Mathematical Modeling: Past, Present and Future.

We review the key role played by mathematical modeling in elucidating two center-finding patterning systems in Escherichia coli: midcell division positioning by the MinCDE system and DNA partitioning by the ParABS system. We focus particularly on how, despite much experimental effort, these systems were simply too complex to unravel by experiments alone, and instead required key injections of quantitative, mathematical thinking. We conclude the review by analyzing the frequency of modeling approaches in microbiology over time. We find that while such methods are increasing in popularity, they are still probably heavily under-utilized for optimal progress on complex biological questions.

DNA Segregation in Natural and Synthetic Minimal Systems.

Faithful segregation of replicated genomes to dividing daughter cells is a major hallmark of cellular life and needs to be part of the future design of the robustly proliferating minimal cell. So far, the complexity of eukaryotic chromosome segregation machineries has limited their applicability to synthetic systems. Prokaryotic plasmid segregation machineries offer promising alternative tools for bottom-up synthetic biology, with the first three-component DNA segregation system being reconstituted a decade ago. In this review, the mechanisms underlying DNA segregation in prokaryotes, with a particular focus on segregation of plasmids and chromosomal replication origins are reviewed, along with a brief discussion of archaeal and eukaryotic systems. In addition, this review shows how in vitro reconstitution has allowed deeper insights into these processes and discusses possible applications of these machineries for a minimal synthetic segrosome as well as the challenge of its coupling to a minimal replisome.

Self-organised segregation of bacterial chromosomal origins.

The chromosomal replication origin region (ori) of characterised bacteria is dynamically positioned throughout the cell cycle. In slowly growing Escherichia coli, ori is maintained at mid-cell from birth until its replication, after which newly replicated sister oris move to opposite quarter positions. Here, we provide an explanation for ori positioning based on the self-organisation of the Structural Maintenance of Chromosomes complex, MukBEF, which forms dynamically positioned clusters on the chromosome. We propose that a non-trivial feedback between the self-organising gradient of MukBEF complexes and the oris leads to accurate ori positioning. We find excellent agreement with quantitative experimental measurements and confirm key predictions. Specifically, we show that oris exhibit biased motion towards MukBEF clusters, rather than mid-cell. Our findings suggest that MukBEF and oris act together as a self-organising system in chromosome organisation-segregation and introduces protein self-organisation as an important consideration for future studies of chromosome dynamics.

Functional Modules of Minimal Cell Division for Synthetic Biology.

Cellular reproduction is one of the fundamental hallmarks of life. Therefore, the development of a minimal division machinery capable of proper genome condensation and organization, mid-cell positioning and segregation in space and time, and the final septation process constitute a fundamental challenge for synthetic biology. It is therefore important to be able to engineer such modules for the production of artificial minimal cells. A bottom-up assembly of molecular machines from bulk biochemicals complemented by in vivo experiments as well as computational modelling helps to approach such key cellular processes. Here, minimal functional modules involved in genome segregation and the division machinery and their spatial organization and positioning are reviewed, setting into perspective the design of a minimal cell. Furthermore, the milestones of recent in vitro reconstitution experiments in the context of cell division are discussed and their role in shedding light on fundamental cellular mechanisms that constitute spatiotemporal order is described. Lastly, current challenges in the field of bottom-up synthetic biology as well as possible future developments toward the development of minimal biomimetic systems are discussed.

A System for Gene Expression Noise Control in Yeast.

Gene expression noise arises from stochastic variation in the synthesis and degradation of mRNA and protein molecules and creates differences in protein numbers across populations of genetically identical cells. Such variability can lead to imprecision and reduced performance of both native and synthetic networks. In principle, gene expression noise can be controlled through the rates of transcription, translation and degradation, such that different combinations of those rates lead to the same protein concentrations but at different noise levels. Here, we present a "noise tuner" which allows orthogonal control over the transcription and the mRNA degradation rates by two different inducer molecules. Combining experiments with theoretical analysis, we show that in this system the noise is largely determined by the transcription rate, whereas the mean expression is determined by both the transcription rate and mRNA stability and can thus be decoupled from the noise. This noise tuner enables 2-fold changes in gene expression noise over a 5-fold range of mean protein levels. We demonstrated the efficacy of the noise tuner in a complex regulatory network by varying gene expression noise in the mating pathway of Saccharomyces cerevisiae, which allowed us to control the output noise and the mutual information transduced through the pathway. The noise tuner thus represents an effective tool of gene expression noise control, both to interrogate noise sensitivity of natural networks and enhance performance of synthetic circuits.

A gated relaxation oscillator mediated by FrzX controls morphogenetic movements in Myxococcus xanthus.

Dynamic control of cell polarity is of critical importance for many aspects of cellular development and motility. In Myxococcus xanthus, MglA, a G protein, and MglB, its cognate GTPase-activating protein, establish a polarity axis that defines the direction of movement of the cell and that can be rapidly inverted by the Frz chemosensory system. Although vital for collective cell behaviours, how Frz triggers this switch has remained unknown. Here, we use genetics, imaging and mathematical modelling to show that Frz controls polarity reversals via a gated relaxation oscillator. FrzX, which we identify as a target of the Frz kinase, provides the gating and thus acts as the trigger for reversals. Slow relocalization of the polarity protein RomR then creates a refractory period during which another switch cannot be triggered. A secondary Frz output, FrzZ, decreases this delay, allowing rapid reversals when required. Thus, this architecture results in a highly tuneable switch that allows a wide range of reversal frequencies.

In vitro fermentation properties of selected fructooligosaccharide-containing vegetables and in vivo colonic microbial populations are affected by the diets of healthy human infants.

Salsify (Tragopogon porrifolius) and gobo (Arctium lappa) are root vegetables that contain high concentrations of naturally occurring fructooligosaccharides (FOS), nondigestible oligosaccharides that have prebiotic effects on the intestinal microflora. The objective of this study was to compare colonic microbial populations and the fermentation characteristics of gobo and salsify in breast-fed vs. formula-fed infants. Fecal inoculum from breast-fed and formula-fed infants consuming either no solid foods, cereal, or fruit and vegetable purees were fermented in vitro with sweet potato puree plus gobo, salsify puree or a control vegetable, carrot. Breast-fed and formula-fed infants had similar fecal bacteria concentrations, with the exception of Clostridium perfringens (P < 0.10). Introduction of solid foods into the diet of infants was associated with increased fecal concentrations of bifidobacteria (P < 0.10) and decreased concentrations of total aerobes (P < 0.01), C. perfringens (P < 0.001) and Escherichia coli (P < 0.10). Inoculum from feces of breast-fed infants resulted in greater acetate production, whereas inoculum from feces of formula-fed infants resulted in greater propionate and butyrate production (P < 0.05). Fermentation of FOS-containing vegetables by infant fecal inoculum did not differ significantly from fermentation of carrots as assessed by total short-chain fatty acid (SCFA) production. The addition of solids to the diet of infants was associated with increased production of acetate and total SCFA (P < 0.05). It appears that both the composition and fermentative activity of the colonic microflora of human infants is affected by breast-feeding and solid food consumption, but not by short-term exposure to low concentrations of FOS-containing substrates.