Implementation of Fluorescent-Protein-Based Quantification Analysis in L-Form Bacteria.

Cell-wall-less (L-form) bacteria exhibit morphological complexity and heterogeneity, complicating quantitative analysis of them under internal and external stimuli. Stable and efficient labeling is needed for the fluorescence-based quantitative cell analysis of L-forms during growth and proliferation. Here, we evaluated the expression of multiple fluorescent proteins (FPs) under different promoters in the Bacillus subtilis L-form strain LR2 using confocal microscopy and imaging flow cytometry. Among others, Pylb-derived NBP3510 showed a superior performance for inducing several FPs including EGFP and mKO2 in both the wild-type and L-form strains. Moreover, NBP3510 was also active in Escherichia coli and its L-form strain NC-7. Employing these established FP-labeled strains, we demonstrated distinct morphologies in the L-form bacteria in a quantitative manner. Given cell-wall-deficient bacteria are considered protocell and synthetic cell models, the generated cell lines in our work could be valuable for L-form-based research.

Comparative genomics hints at dispensability of multiple essential genes in two Escherichia coli L-form strains.

Despite the critical role of bacterial cell walls in maintaining cell shapes, certain environmental stressors can induce the transition of many bacterial species into a wall-deficient state called L-form. Long-term induced Escherichia coli L-forms lose their rod shape and usually hold significant mutations that affect cell division and growth. Besides this, the genetic background of L-form bacteria is still poorly understood. In the present study, the genomes of two stable L-form strains of E. coli (NC-7 and LWF+) were sequenced and their gene mutation status was determined and compared with their parental strains. Comparative genomic analysis between two L-forms reveals both unique adaptions and common mutated genes, many of which belong to essential gene categories not involved in cell wall biosynthesis, indicating that L-form genetic adaptation impacts crucial metabolic pathways. Missense variants from L-forms and Lenski's long-term evolution experiment (LTEE) were analyzed in parallel using an optimized DeepSequence pipeline to investigate predicted mutation effects (α) on protein functions. We report that the two L-form strains analyzed display a frequency of 6-10% (0% for LTEE) in mutated essential genes where the missense variants have substantial impact on protein functions (α<0.5). This indicates the emergence of different survival strategies in L-forms through changes in essential genes during adaptions to cell wall deficiency. Collectively, our results shed light on the detailed genetic background of two E. coli L-forms and pave the way for further investigations of the gene functions in L-form bacterial models.

Quantification of Giant Unilamellar Vesicle Fusion Products by High-Throughput Image Analysis.

Artificial cells are based on dynamic compartmentalized systems. Thus, remodeling of membrane-bound systems, such as giant unilamellar vesicles, is finding applications beyond biological studies, to engineer cell-mimicking structures. Giant unilamellar vesicle fusion is rapidly becoming an essential experimental step as artificial cells gain prominence in synthetic biology. Several techniques have been developed to accomplish this step, with varying efficiency and selectivity. To date, characterization of vesicle fusion has relied on small samples of giant vesicles, examined either manually or by fluorometric assays on suspensions of small and large unilamellar vesicles. Automation of the detection and characterization of fusion products is now necessary for the screening and optimization of these fusion protocols. To this end, we implemented a fusion assay based on fluorophore colocalization on the membranes and in the lumen of vesicles. Fluorescence colocalization was evaluated within single compartments by image segmentation with minimal user input, allowing the application of the technique to high-throughput screenings. After detection, statistical information on vesicle fluorescence and morphological properties can be summarized and visualized, assessing lipid and content transfer for each object by the correlation coefficient of different fluorescence channels. Using this tool, we report and characterize the unexpected fusogenic activity of sodium chloride on phosphatidylcholine giant vesicles. Lipid transfer in most of the vesicles could be detected after 20 h of incubation, while content exchange only occurred with additional stimuli in around 8% of vesicles.

Lipid Exchange Promotes Fusion of Model Protocells.

Vesicle fusion is an important process underlying cell division, transport, and membrane trafficking. In phospholipid systems, a range of fusogens including divalent cations and depletants have been shown to induce adhesion, hemifusion, and then full content fusion between vesicles. This work shows that these fusogens do not perform the same function for fatty acid vesicles, which are used as model protocells (primitive cells). Even when fatty acid vesicles appear adhered or hemifused to each other, the intervening barriers between vesicles do not rupture. This difference is likely because fatty acids have a single aliphatic tail, and are more dynamic than their phospholipid counterparts. To address this, it is postulated that fusion could instead occur under conditions, such as lipid exchange, that disrupt lipid packing. Using both experiments and molecular dynamics simulations, it is verified that fusion in fatty acid systems can indeed be induced by lipid exchange. These results begin to probe how membrane biophysics could constrain the evolutionary dynamics of protocells.

Construction of a mini-RNA replicon in Escherichia coli.

How the ribonucleic acid (RNA) world transited to the deoxyribonucleic acid (DNA) world has remained controversial in evolutionary biology. At a certain time point in the transition from the RNA world to the DNA world, 'RNA replicons', in which RNAs produce proteins to replicate their coding RNA, and 'DNA replicons', in which DNAs produce RNA to synthesize proteins that replicate their coding DNA, can be assumed to coexist. The coexistent state of RNA replicons and DNA replicons is desired for experimental approaches to determine how the DNA world overtook the RNA world. We constructed a mini-RNA replicon in Escherichia coli. This mini-RNA replicon encoded the ß subunit, one of the subunits of the Qß replicase derived from the positive-sense single-stranded Qß RNA phage and is replicated by the replicase in E. coli. To maintain the mini-RNA replicon persistently in E. coli cells, we employed a system of α complementation of LacZ that was dependent on the Qß replicase, allowing the cells carrying the RNA replicon to grow in the lactose minimal medium selectively. The coexistent state of the mini-RNA replicon and DNA replicon (E. coli genome) was successively synthesized. The coexistent state can be used as a starting system to experimentally demonstrate the transition from the RNA-protein world to the DNA world, which will contribute to progress in the research field of the origin of life.

Cell Sorting-Directed Selection of Bacterial Cells in Bigger Sizes Analyzed by Imaging Flow Cytometry during Experimental Evolution.

Cell morphology is an essential and phenotypic trait that can be easily tracked during adaptation and evolution to environmental changes. Thanks to the rapid development of quantitative analytical techniques for large populations of cells based on their optical properties, morphology can be easily determined and tracked during experimental evolution. Furthermore, the directed evolution of new culturable morphological phenotypes can find use in synthetic biology to refine fermentation processes. It remains unknown whether and how fast we can obtain a stable mutant with distinct morphologies using fluorescence-activated cell sorting (FACS)-directed experimental evolution. Taking advantage of FACS and imaging flow cytometry (IFC), we direct the experimental evolution of the E. coli population undergoing continuous passage of sorted cells with specific optical properties. After ten rounds of sorting and culturing, a lineage with large cells resulting from incomplete closure of the division ring was obtained. Genome sequencing highlighted a stop-gain mutation in amiC, leading to a dysfunctional AmiC division protein. The combination of FACS-based selection with IFC analysis to track the evolution of the bacteria population in real-time holds promise to rapidly select and culture new morphologies and association tendencies with many potential applications.

Optimization and compartmentalization of a cell-free mixture of DNA amplification and protein translation.

Recent studies have shown that the reconstituted cell-free DNA replisome and in vitro transcription and translation systems from Escherichia coli are highly important in applied and synthetic biology. To date, no attempt has been made to combine those two systems. Here, we study the performance of the mixed two separately exploited systems commercially available as RCR and PURE systems. Regarding the genetic information flow from DNA to proteins, mixtures with various ratios of RCR/PURE gave low protein expression, possibly due to the well-known conflict between replication and transcription or inappropriate buffer conditions. To further increase the compatibility of the two systems, rationally designed reaction buffers with a lower concentration of nucleoside triphosphates in 50 mM HEPES (pH7.6) were evaluated, showing increased performance from RCR/PURE (85%/15%) in a time-dependent manner. The compatibility was also validated in compartmentalized cell-sized droplets encapsulating the same RCR/PURE soup. Our findings can help to better fine-tune the reaction conditions of RCR-PURE systems and provide new avenues for rewiring the central dogma of molecular biology as self-sustaining systems in synthetic cell models. KEY POINTS: • Commercial reconstituted DNA amplification (RCR) and transcription and translation (PURE) systems hamper each other upon mixing. • A newly optimized buffer with a low bias for PURE was formulated in the RCR-PURE mixture. • The performance and dynamics of RCR-PURE were investigated in either bulk or compartmentalized droplets.

Photoinducible Azobenzene trimethylammonium bromide (AzoTAB)-mediated giant vesicle fusion compatible with synthetic protein translation reactions.

Lipid giant vesicles represent a versatile minimal model system to study the physicochemical basis of lipid membrane fusion. Membrane fusion processes are also of interest in synthetic cell research, where cell-mimicking behavior often requires dynamically interacting compartments. For these applications, triggered fusion compatible with transcription-translation systems is key in achieving complexity. Recently, a photosensitive surfactant, azobenzene trimethylammonium bromide (AzoTAB), has been reported to induce membrane fusion by a photoinduced conformational change. Using imaging flow cytometer (IFC) and confocal microscopy we quantitatively investigated photoinduced AzoTAB-mediated fusion of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine vesicles. The IFC analysis result showed that the fusion rate could reach about 40% following AzoTAB addition and UV irradiation in optimized conditions. We confirmed the compatibility between AzoTAB-induced vesicle fusion and a synthetic cell-free protein translation system using green fluorescent protein as reporter. With the techniques presented, cell-sized vesicle fusion can be quantitatively analyzed and optimized, paving the way to controllable synthetic cells with fundamental biological functions like the ability to express proteins from encapsulated plasmids.

Primordial mimicry induces morphological change in Escherichia coli.

The morphology of primitive cells has been the subject of extensive research. A spherical form was commonly presumed in prebiotic studies but lacked experimental evidence in living cells. Whether and how the shape of living cells changed are unclear. Here we exposed the rod-shaped bacterium Escherichia coli to a resource utilization regime mimicking a primordial environment. Oleate was given as an easy-to-use model prebiotic nutrient, as fatty acid vesicles were likely present on the prebiotic Earth and might have been used as an energy resource. Six evolutionary lineages were generated under glucose-free but oleic acid vesicle (OAV)-rich conditions. Intriguingly, fitness increase was commonly associated with the morphological change from rod to sphere and the decreases in both the size and the area-to-volume ratio of the cell. The changed cell shape was conserved in either OAVs or glucose, regardless of the trade-offs in carbon utilization and protein abundance. Highly differentiated mutations present in the genome revealed two distinct strategies of adaption to OAV-rich conditions, i.e., either directly targeting the cell wall or not. The change in cell morphology of Escherichia coli for adapting to fatty acid availability supports the assumption of the primitive spherical form.

The requirement of cellularity for abiogenesis.

The history of modern biochemistry started with the cellular theory of life. By putting aside the holistic protoplasmic theory, scientists of the XX century were able to advance the functional classification of cellular components significantly. The cell became the unit of the living. Current theories on the abiogenesis of life must account for a moment in evolution (chemical or biological) when this was not the case. Investigating the role of compartments and membranes along chemical and biotic evolution can lead a more generalised idea of living organisms that is fundamental to advance our efforts in astrobiology, origin of life and artificial life studies. Furthermore, it may provide insights in unexplained evolutionary features such as the lipid divide between Archaea and Eubacteria. By surveying our current understanding of the involvement of compartments in abiogenesis and evolution, the idea of cells as atomistic units of a general theory of biology will be discussed. The aim is not to undermine the validity of the cellular theory of life, but rather to elucidate possible biases with regards to cellularity and the origin of life. An open discussion in these regards could show the inherent limitations of non-cellular compartmentalization that may lead to the necessity of cellular structures to support complex life.

Giant Vesicles Produced with Phosphatidylcholines (PCs) and Phosphatidylethanolamines (PEs) by Water-in-Oil Inverted Emulsions.

(1) Background: giant vesicles (GVs) are widely employed as models for studying physicochemical properties of bio-membranes and artificial cell construction due to their similarities to natural cell membranes. Considering the critical roles of GVs, various methods have been developed to prepare them. Notably, the water-in-oil (w/o) inverted emulsion-transfer method is reported to be the most promising, owning to the relatively higher productivity and better encapsulation efficiency of biomolecules. Previously, we successfully established an improved approach to acquire detailed information of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)-derived GVs with imaging flow cytometry (IFC); (2) Methods: we prepared GVs with different lipid compositions, including phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), and PC/PE mixtures by w/o inverted emulsion methods. We comprehensively compared the yield, purity, size, and encapsulation efficiency of the resulting vesicles; (3) Results: the relatively higher productivities of GVs could be obtained from POPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), DOPC: DLPE (7:3), and POPC: DLPE (6:4) pools. Furthermore, we also demonstrate that these GVs are stable during long term preservation in 4 °C. (4) Conclusions: our results will be useful for the analytical study of GVs and GV-based applications.

Clustering of Bacterial Growth Dynamics in Response to Growth Media by Dynamic Time Warping.

Bacterial growth curves, representing population dynamics, are still poorly understood. The growth curves are commonly analyzed by model-based theoretical fitting, which is limited to typical S-shape fittings and does not elucidate the dynamics in their entirety. Thus, whether a certain growth condition results in any particular pattern of growth curve remains unclear. To address this question, up-to-date data mining techniques were applied to bacterial growth analysis for the first time. Dynamic time warping (DTW) and derivative DTW (DDTW) were used to compare the similarity among 1015 growth curves of 28 Escherichia coli strains growing in three different media. In the similarity evaluation, agglomerative hierarchical clustering, assessed with four statistic benchmarks, successfully categorized the growth curves into three clusters, roughly corresponding to the three media. Furthermore, a simple benchmark was newly proposed, providing a highly improved accuracy (~99%) in clustering the growth curves corresponding to the growth media. The biologically reasonable categorization of growth curves suggested that DTW and DDTW are applicable for bacterial growth analysis. The bottom-up clustering results indicate that the growth media determine some specific patterns of population dynamics, regardless of genomic variation, and thus have a higher priority of shaping the growth curves than the genomes do.

A protocell with fusion and division.

A protocell is a synthetic form of cellular life that is constructed from phospholipid vesicles and used to understand the emergence of life from a nonliving chemical network. To be considered 'living', a protocell should be capable of self-proliferation, which includes successive growth and division processes. The growth of protocells can be achieved via vesicle fusion approaches. In this review, we provide a brief overview of recent research on the formation of a protocell, fusion and division processes of the protocell, and encapsulation of a defined chemical network such as the genetic material. We also provide some perspectives on the challenges and future developments of synthetic protocell research.

Using Imaging Flow Cytometry to Quantify and Optimize Giant Vesicle Production by Water-in-oil Emulsion Transfer Methods.

Many biologists, biochemists, and biophysicists study giant vesicles, which have a diameter of >1 µm, owing to their ease of characterization using standard optical methods. More recently, there has been interest in using giant vesicles as model systems for living cells and for the construction of artificial cells. In fact, there have been a number of reports about functionalizing giant vesicles using membrane-bound pore proteins and encapsulating biochemical reactions. Among the various methods for preparing giant vesicles, the water-in-oil emulsion transfer method is particularly well established. However, the giant vesicles prepared by this method have complex and heterogeneous properties, such as particle size and membrane structure. Here, we demonstrate the characterization of giant vesicles by imaging flow cytometry to provide quantitative and qualitative information about the vesicle products prepared by the water-in-oil emulsion transfer method. Through image-based analyses, several kinds of protocol byproducts, such as oil droplets and vesicles encapsulating no target molecules, were identified and successfully quantified. Further, the optimal agitation conditions for the water-in-oil emulsion transfer method were found from detailed analysis of imaging flow cytometry data. Our results indicate that a sonication-based water-in-oil emulsion transfer method exhibited a higher efficiency in producing giant vesicles, about 10 times or higher than that of vortex and rumble strip-based methods. It is anticipated that these approaches will be useful for fine-tuning giant vesicle production and subsequent applications.

Development of an Automated UV Irradiation Device for Microbial Cell Culture.

Ultraviolet (UV) mutagenesis is a widely used technique to increase bacterial mutation rates in laboratory experiments. UV mutagenesis requires fine regulation of UV dose, because the number of dead cells increases exponentially as the dose increases. Ignoring this hazard can cause extinction of UV-exposed populations. Therefore, an automated system that cooperatively conducts both growth measurement and UV irradiation is needed for efficient UV mutagenesis experiments. To address this task, we constructed an automated UV irradiation device for microbial cell culture. This device can measure cell density and irradiate the bacterial cells with UV light automatically according to the state of cell growth. We demonstrated that this growth feedback control avoided extinction and enabled accumulation of mutations in bacterial genomes at a rapid rate for a long period. Whole-genome sequencing revealed the high accumulation rate, neutrality, and spectrum of UV-induced mutations. These characteristics were all consistent with those obtained by manual UV irradiation. These results indicate that our automated device is useful in accelerating mutation accumulation over a long duration.

A decay effect of the growth rate associated with genome reduction in Escherichia coli.

BACKGROUND: Bacterial growth is an important topic in microbiology and of crucial importance to better understand living cells. Bacterial growth dynamics are quantitatively examined using various methods to determine the physical, chemical or biological features of growing populations. Due to methodological differences, the exponential growth rate, which is a parameter that is representative of growth dynamics, should be differentiated. Ignoring such differentiation in the growth analysis might overlook somehow slight but significant changes in cellular features of the growing population. Both experimental and theoretical investigations are required to address these issues. RESULTS: This study experimentally verified the differentiation in growth rates attributed to different methodologies, and demonstrated that the most popular method, optical turbidity, led to the determination of a lower growth rate in comparison to the methods based on colony formation and cellular adenosine triphosphate, due to a decay effect of reading OD600 during a population increase. Accordingly, the logistic model, which is commonly applied to the high-throughput growth data reading the OD600, was revised by introducing a new parameter: the decay rate, to compensate for the lowered estimation in growth rates. An improved goodness of fit in comparison to the original model was acquired due to this revision. Applying the modified logistic model to hundreds of growth data acquired from an assortment of Escherichia coli strains carrying the reduced genomes led to an intriguing finding of a correlation between the decay rate and the genome size. The decay effect seemed to be partially attributed to the decrease in cell size accompanied by a population increase and was medium dependent. CONCLUSIONS: The present study provides not only an improved theoretical tool for the high-throughput studies on bacterial growth dynamics linking with optical turbidity to biological meaning, but also a novel insight of the genome reduction correlated decay effect, which potentially reflects the changing cellular features during population increase. It is valuable for understanding the genome evolution and the fitness increase in microbial life.

Self-replication of circular DNA by a self-encoded DNA polymerase through rolling-circle replication and recombination.

A major challenge in constructing artificial cells is the establishment of a recursive genome replication system coupled with gene expression from the genome itself. One of the simplest schemes of recursive DNA replication is the rolling-circle replication of a circular DNA coupled with recombination. In this study, we attempted to develop a replication system based on this scheme using self-encoded phi29 DNA polymerase and externally supplied Cre recombinase. We first identified that DNA polymerization is significantly inhibited by Cre recombinase. To overcome this problem, we performed in vitro evolution and obtained an evolved circular DNA that can replicate efficiently in the presence of the recombinase. We also showed evidence that during replication of the evolved DNA, the circular DNA was reproduced through recombination by Cre recombinase. These results demonstrate that the evolved circular DNA can reproduce itself through gene expression of a self-encoded polymerase. This study provides a step forward in developing a simple recursive DNA replication system for use in an artificial cell.

Automated in vitro evolution of a translation-coupled RNA replication system in a droplet flow reactor.

Automation is a useful strategy to make laborious evolutionary experiments faster and easier. To date, several types of continuous flow reactors have been developed for the automated evolutionary experiments of viruses and bacteria. However, the development of a flow reactor applicable to compartmentalized in vitro self-replication systems is still a challenge. In this study, we demonstrate automated in vitro evolution of a translation-coupled RNA system in a droplet flow reactor for the first time. This reactor contains approximately 1010 micro-scale droplets (average diameter is approximately 0.8 µm), which continuously fuse and divide among each other at a controllable rate. In the droplets, an RNA (artificial genomic RNA) replicate through the translation of self-encoded RNA replicase with spontaneously appearing parasitic RNA. We performed two automated replication experiments for more than 400 hours with different mixing intensities. We found that several mutations displayed increased frequencies in the genomic RNA populations and the dominant RNA mutants acquired the ability to replicate faster or acquired resistance to the parasitic RNA, demonstrating that Darwinian evolution occurred during the long-term replication. The droplet flow reactor we developed can be a useful tool to perform in vitro evolutionary experiments of translation-coupled systems.

Influence of adaptive mutations, from thermal adaptation experiments, on the infection cycle of RNA bacteriophage Qß.

A population's growth rate is determined by multiple 'life history traits'. To quantitatively determine which life history traits should be improved to allow a living organism to adapt to an inhibitory environment is an important issue. Previously, we conducted thermal adaptation experiments on the RNA bacteriophage Qß using three independent replicates and reported that all three end-point populations could grow at a temperature (43.6°C) that inhibited the growth of the ancestral strain. Even though the fitness values of the endpoint populations were almost the same, their genome sequence was not, indicating that the three thermally adapted populations may have different life history traits. In this study, we introduced each mutation observed in these three end-point populations into the cDNA of the Qß genome and prepared three different mutants. Quantitative analysis showed that they tended to increase their fitness by increasing the adsorption rate to their host, shortening their latent period (i.e., the duration between phage infection and progeny release), and increasing the burst size (i.e., the number of progeny phages per infected cell), but all three mutants decreased their thermal stability. However, the degree to which these traits changed differed. The mutant with the least mutations showed a smaller decrease in thermal stability, the largest adsorption rate to the host, and the shortest latent period. These results indicated that several different adaptive routes exist by which Qß can adapt to higher temperatures, even though Qß is a simple RNA bacteriophage with a small genome size, encoding only four genes.

De novo design and synthesis of a 30-cistron translation-factor module.

Two of the many goals of synthetic biology are synthesizing large biochemical systems and simplifying their assembly. While several genes have been assembled together by modular idempotent cloning, it is unclear if such simplified strategies scale to very large constructs for expression and purification of whole pathways. Here we synthesize from oligodeoxyribonucleotides a completely de-novo-designed, 58-kb multigene DNA. This BioBrick plasmid insert encodes 30 of the 31 translation factors of the PURE translation system, each His-tagged and in separate transcription cistrons. Dividing the insert between three high-copy expression plasmids enables the bulk purification of the aminoacyl-tRNA synthetases and translation factors necessary for affordable, scalable reconstitution of an in vitro transcription and translation system, PURE 3.0.