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bioRxiv2018

Characterization of Diverse Homoserine Lactone Synthases in Escherichia coli

Quorum sensing networks have been identified in over one hundred bacterial species to date. A subset of these networks regulate group behaviors, such as bioluminescence, virulence, and biofilm formation, by sending and receiving small molecules called homoserine lactones (HSLs). Bioengineers have incorporated quorum sensing pathways into genetic circuits to connect logical operations. However, the development of higher-order genetic circuitry is inhibited by crosstalk, in which one quorum sensing network responds to HSLs produced by a different network. Here, we report the construction and characterization of a library of ten synthases including some that are expected to produce HSLs that are incompatible with the Lux pathway, and therefore show no crosstalk. We demonstrated their function in a common lab chassis, Escherichia coli BL21, and in two contexts, liquid and solid agar cultures, using decoupled Sender and Receiver pathways. We observed weak or strong stimulation of a Lux Receiver by longer-chain or shorter-chain HSL-generating Senders, respectively. We also considered the under-investigated risk of unintentional release of incompletely deactivated HSLs in biological waste. We found that HSL-enriched media treated with bleach is still bioactive, while autoclaving deactivates LuxR induction. This work represents the most extensive comparison of quorum sensing synthases to date and greatly expands the bacterial signaling toolkit while recommending practices for disposal based on empirical, quantitative evidence.

synthetic biology↗

bioRxiv2018

Genome-wide fitness assessment during diurnal growth reveals an expanded role of the cyanobacterial circadian clock protein KaiA

The recurrent pattern of light and darkness generated by Earths axial rotation has profoundly influenced the evolution of organisms, selecting for both biological mechanisms that respond acutely to environmental changes and circadian clocks that program physiology in anticipation of daily variations. The necessity to integrate environmental responsiveness and circadian programming is exemplified in photosynthetic organisms such as cyanobacteria, which depend on light-driven photochemical processes. The cyanobacterium Synechococcus elongatus PCC 7942 is an excellent model system for dissecting these entwined mechanisms. Its core circadian oscillator, consisting of three proteins KaiA, KaiB, and KaiC, transmits time-of-day signals to clock-output proteins, which reciprocally regulate global transcription. Research performed under constant light facilitates analysis of intrinsic cycles separately from direct environmental responses, but does not provide insight into how these regulatory systems are integrated during light-dark cycles. Thus, we sought to identify genes that are specifically necessary in a day-night environment. We screened a dense bar-coded transposon library in both continuous light and daily cycling conditions and compared the fitness consequences of loss of each nonessential gene in the genome. Although the clock itself is not essential for viability in light-dark cycles, the most detrimental mutations revealed by the screen were those that disrupt KaiA. The screen broadened our understanding of light-dark survival in photosynthetic organisms, identified unforeseen clock-protein interaction dynamics, and reinforced the role of the clock as a negative regulator of a night-time metabolic program that is essential for S. elongatus to survive in the dark.\n\nSignificanceUnderstanding how photosynthetic bacteria respond to and anticipate natural light-dark cycles is necessary for predictive modeling, bioengineering, and elucidating metabolic strategies for diurnal growth. Here, we identify the genetic components that are important specifically under light-dark cycling conditions and determine how a properly functioning circadian clock prepares metabolism for darkness, a starvation period for photoautotrophs. This study establishes that the core circadian clock protein KaiA is necessary to enable rhythmic de-repression of a night-time circadian program.

microbiology↗

bioRxiv2018

An unexpectedly effective Monte Carlo technique for the RNA inverse folding problem

Solving the RNA inverse folding problem, also known as the RNA design problem, is critical to advance several scientific fields like bioengineering, yet existing approaches have had limited success. The problem has several features that resist traditional computational techniques, such as its exponential complexity and the chaotic behavior of its cost function. Although some state-of-the-art AI approaches have reported promising results, all existing computational methods substantially underperform expert human designers. I combine a different technique, Nested Monte Carlo Search (NMCS), with domain-specific knowledge to create an algorithm that outperforms all prior published methods by wide margins and solves 95 of the 100 puzzles listed in a recently proposed RNA solving difficulty benchmark.

bioinformatics↗

bioRxiv2018

eQTL network analysis reveals that regulatory genes are evolutionarily older and bearing more types of PTM sites in Coprinopsis cinerea

Understanding the DNA variation in regulation of carbohydrate-active enzymes (CAZymes) is fundamental to the use of wood-decaying basidiomycetes in lignocellulose conversion into renewable energy. Our goal is to identify the regulators of lignocellulolytic enzymes in Coprinopsis cinerea, of which the genome harbors high number of Auxiliary Activities enzymes.\n\nThe DNA sequence of C. cinerea family including 46 single spore isolates (SSIs) from crosses of two homozygous strains are used to develop a panel of SNP markers. Then the RNA sequence were used to characterize the gene expression profiles. The RNA were extracted from cultures grown on softwood-enriched sawdust to induce lignocellulolytic enzymes and CCR de-repression genes. To assess the genetic contribution to enzyme expression variations among the 46 SSIs, associations between SNPs and gene expressions were examined genome-widely. 5148 local eQTLs and 7738 distant eQTLs were obtained. By analyzing these eQTLs, the potential regulatory factors of the CAZymes expression and the de-repression of Carbon Catabolism Repression (CCR) were identified.\n\nThe eQTL network is characterized in terms of hotspots, evolutionary age and post-translational modifications (PTMs). In the eQTL network of C. cinerea, the non-regulatory genes are younger than the regulatory genes. The proteins regulated by combinational multiple types of PTMs are more likely to function as super regulatory hotspots in protein-protein interactions. The evolutionary age analysis and the PTMome analysis could serve as alternative methods to identify master regulators from genomic data.\n\nThis work demonstrates a comprehensive bioinformatics approach to identify regulatory factors with next-generation sequencing data. The results provide candidate genes for bioengineering to increase the enzyme production, which will practically benefit the bioethanol production from lignocellulose.\n\nSignificanceThis eQTL analysis is designed to study the fungal CAZymes and carbon catabolism repression, especially during the mycelium stage.\n\nO_LIIn Coprinopsis cinerea, only the regions near two ends of the chromosomes have high recombination rate, and suitable for family based eQTL analysis.\nC_LIO_LIA sugar transporter is a hotspot controlling many CCR genes.\nC_LIO_LICAZymes are not regulated by a master regulator, but by individual regulators. This indicates that CAZymes are under specific regulatory pathways, so can response to specific conditions.\nC_LIO_LIIn the eQTL network, the rGenes are evolutionarily older, with more types of PTM sites than eGenes.\nC_LIO_LIIn the eQTL network, the proteins with more types of PTM sites are more likely associated with Information Storage and Processing, and act as super-hub in the network.\nC_LI

bioinformatics↗

bioRxiv2018

Dynamic load balancing enables large-scale flux variability analysis

Genome-scale metabolic models (GSMMs) of living organisms are used in a wide variety of applications pertaining to health and bioengineering. They are formulated as linear programs (LP) that are often under-determined. Flux Variability Analysis (FVA) characterizes the alternate optimal solution (AOS) space enabling thereby the assessment of the robustness of the solution. fastFVA (FFVA), the C implementation of MATLAB FVA, allowed to gain substantial speed up, although, the parallelism was managed through MATLAB. Here veryfastFVA (VFFVA) is presented, which is a pure C implementation of FVA, that relies on lower level management of parallelism through a hybrid MPI/OpenMP. The flexibility of VFFVA allowed to gain a threefold speedup factor and to decrease memory usage 14 fold in comparison to FFVA. Finally, VFFVA allows processing a higher number of GSMMs in faster times accelerating thereby biomedical modeling and simulation. VFFVA is available online at https://github.com/marouenbg/VFFVA.

bioinformatics↗

bioRxiv2018

Identification of a simple and novel cut-point based CSF and MRI signature for predicting Alzheimer disease progression that reinforces the 2018 NIA-AA research framework

The 2018 NIA-AA research framework proposes a classification system with beta-Amyloid deposition, pathologic Tau, and neurodegeneration (ATN) for the diagnosis and staging of Alzheimers Disease (AD). Data from the ADNI (AD neuroimaging initiative) database can be utilized to identify diagnostic signatures for predicting AD progression, and to determine the utility of this NIA-AA research framework. Profiles of 320 peptides from baseline cerebrospinal fluid (CSF) samples of 287 normal, mild cognitive impairment (MCI) and AD subjects followed over a 3-10 year period were measured via multiple reaction monitoring (MRM) mass spectrometry. CSF A{beta}42, total-Tau (tTau), phosphorylated-Tau (pTau-181) and hippocampal volume were also measured. From these candidate markers, optimal diagnostic signatures with decision thresholds to separate AD and normal subjects were first identified via unbiased regression and tree-based algorithms. The best performing signature determined via cross-validation was then tested in an independent group of MCI subjects to predict future progression. This multivariate analysis yielded a simple diagnostic signature comprising CSF pTau-181 to A{beta}42 ratio, MRI hippocampal volume and a novel PTPRN peptide, with a decision threshold on each marker. When applied to a separate MCI group at baseline, subjects meeting this signature criteria experience 4.3-fold faster progression to AD compared to a 2.2-fold faster progression using only conventional markers. This novel 4-marker signature represents an advance over the current diagnostics based on widely used marker, and is much easier to use in practice than recently published complex signatures. In addition, this signature reinforces the ATN construct from the 2018 NIA-AA research framework.\n\nDisclosuresViswanath Devanarayan is an employee of Charles River Laboratories, and as such owns equity in, receives salary and other compensation from Charles River Laboratories.\n\nData collection and sharing for this project was funded by the Alzheimers Disease Neuroimaging Initiative (ADNI) (National Institutes of Health Grant U01 AG024904) and DOD ADNI (Department of Defense award number W81XWH-12-2-0012). ADNI is funded by the National Institute on Aging, the National Institute of Biomedical Imaging and Bioengineering, and through generous contributions from the following: AbbVie, Alzheimers Association; Alzheimers Drug Discovery Foundation; Araclon Biotech; BioClinica, Inc.;Biogen; Bristol-Myers Squibb Company; CereSpir, Inc.; Eisai Inc.; Elan Pharmaceuticals, Inc.; Eli Lilly and Company; EuroImmun; F. Hoffmann-La Roche Ltd and its affiliated company Genentech, Inc.; Fujirebio; GE Healthcare; IXICO Ltd.; Janssen Alzheimer Immunotherapy Research & Development, LLC.; Johnson & Johnson Pharmaceutical Research & Development LLC.; Lumosity; Lundbeck; Merck & Co., Inc.; Meso Scale Diagnostics, LLC.; NeuroRx Research; Neurotrack Technologies; Novartis Pharmaceuticals Corporation; Pfizer Inc.; Piramal Imaging; Servier; Takeda Pharmaceutical Company; and Transition Therapeutics. The Canadian Institutes of Health Research is providing funds to support ADNI clinical sites in Canada. Private sector contributions are facilitated by the Foundation for the National Institutes of Health (www.fnih.org). The grantee organization is the Northern California Institute for Research and Education, and the study is coordinated by the Alzheimers Disease Cooperative Study at the University of California, San Diego. ADNI data are disseminated by the Laboratory for Neuro Imaging at the University of Southern California.

neuroscience↗

bioRxiv2018

Robotic platform for microinjection into single cells in intact tissue

Microinjection into single cells in intact tissue allows the delivery of membrane-impermeant molecules such as nucleic acids and proteins is a powerful technique to study and manipulate the behavior of these cells and, if applicable, their progeny. However, a high level of skill is required to perform such microinjection and is a low-throughput and low-yield process. The automation of microinjection into cells in intact tissue would empower an increasing number of researchers to perform these challenging experiments and could potentially open up new avenues of experimentation. We have developed the Autoinjector, a robot that utilizes images acquired from a microscope to guide a microinjection needle into tissue to deliver femtoliter volumes of liquids into single cells. The robotic operation enables microinjection of hundreds of cells within a single organotypic slice, resulting in an overall yield that is an order of magnitude greater than manual microinjection. We validated the performance of the Autoinjector by microinjecting both apical progenitors (APs) and newborn neurons in the embryonic mouse telencephalon, APs in the embryonic mouse hindbrain, and neurons in fetal human brain tissue. We demonstrate the capability of the Autoinjector to deliver exogenous mRNA into APs. Further, we used the Autoinjector to systematically study gap-junctional communication between neural progenitors in the embryonic mouse telencephalon and found that apical contact is a characteristic feature of the cells that are part of a gap junction-coupled cell cluster. The throughput and versatility of the Autoinjector will not only render microinjection a broadly accessible high-performance cell manipulation technique but will also provide a powerful new platform for bioengineering and biotechnology for performing single-cell analyses in intact tissue.

neuroscience↗

bioRxiv2019

A Genome-engineered Bioartificial Implant for Autoregulated Anti-Cytokine Drug Delivery

Biologic drug therapies are effective treatments for autoimmune diseases such as rheumatoid arthritis (RA) but may cause significant adverse effects, as they are administered continuously at high doses that can suppress the immune system. Using CRISPR-Cas9 genome editing, we engineered stem cells containing a synthetic gene circuit expressing biologic drugs to antagonize interleukin-1 (IL-1) or tumor necrosis factor (TNF) in an autoregulated, feedback-controlled manner in response to activation of the endogenous chemokine (C-C) motif ligand 2 (Ccl2) promoter. To test this approach in vivo, cells were tissue-engineered into a stable cartilaginous construct and implanted subcutaneously in mice with inflammatory arthritis. Bioengineered anti-cytokine implants mitigated arthritis severity as measured by joint pain, structural damage, and systemic and local inflammation. The coupling of synthetic biology with tissue engineering promises a range of potential applications for treating chronic diseases using custom-designed cells that express therapeutic transgenes in response to dynamically changing biological signals.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2019

DNA Nanostructures Coordinate Gene Silencing in Mature Plants

Plant bioengineering may generate high yielding and stress-resistant crops amidst a changing climate and a growing global population (1-3). However, delivery of biomolecules to plants relies on Agrobacterium infection (4) or biolistic particle delivery (5), the former of which is only amenable to DNA delivery. The difficulty in delivering functional biomolecules such as RNA to plant cells is due to the plant cell wall which is absent in mammalian cells and poses the dominant physical barrier to exogenous biomolecule delivery in plants. DNA nanostructure-mediated biomolecule delivery is an effective strategy to deliver cargoes across the lipid bilayer of mammalian cells, however, nanoparticle-mediated delivery remains unexplored for passive biomolecule delivery across the cell wall in plants. Herein, we report a systematic assessment of different DNA nanostructures for their ability to internalize into cells of mature plants, deliver small interfering RNAs (siRNAs), and effectively silence a constitutively-expressed gene in Nicotiana benthamiana leaves. We show that nanostructure internalization into plant cells and the corresponding gene silencing efficiency depends on the DNA nanostructure size, shape, compactness, stiffness, and location of the siRNA attachment locus on the nanostructure. We further confirm that the internalization efficiency of DNA nanostructures correlates with their respective gene silencing efficiencies, but that the endogenous gene silencing pathway depends on the siRNA attachment locus. Our work establishes the feasibility of biomolecule delivery to plants with DNA nanostructures, and details both the design parameters of importance for plant cell internalization, and also assesses the impact of DNA nanostructure geometry for gene silencing mechanisms.

plant biology↗

bioRxiv2019

GEMME: a simple and fast global epistatic model predicting mutational effects

AbstractsThe systematic and accurate description of protein mutational landscapes is a question of utmost importance in biology, bioengineering and medicine. Recent progress has been achieved by leveraging on the increasing wealth of genomic data and by modeling inter-site dependencies within biological sequences. However, state-of-the-art methods require numerous highly variable sequences and remain time consuming. Here, we present GEMME (www.lcqb.upmc.fr/GEMME), a method that overcomes these limitations by explicitly modeling the evolutionary history of natural sequences. This allows accounting for all positions in a sequence when estimating the effect of a given mutation. Assessed against 41 experimental high-throughput mutational scans, GEMME overall performs similarly or better than existing methods and runs faster by several orders of magnitude. It greatly improves predictions for viral sequences and, more generally, for very conserved families. It uses only a few biologically meaningful and interpretable parameters, while existing methods work with hundreds of thousands of parameters.

bioinformatics↗

bioRxiv2019

Data Driven Modelling of a Chemically Defined Growth Medium for Cupriavidus necator H16

Cupriavidus necator is a Gram-negative soil bacterium of major biotechnological interest. It is a producer of the bioplastic 3-polyhydroxybutyrate, has been exploited in bioremediation processes, and its lithoautotrophic capabilities suggest it may function as a microbial cell factory upgrading renewable resources to fuels and chemicals. It remains necessary however to develop appropriate experimental resources to permit controlled bioengineering and system optimisation of this microbial chassis. A key resource for physiological, biochemical and metabolic studies of any microorganism is a chemically defined growth medium. Here we use 1 mL micro-well cell cultures, automated liquid handling and a statistical engineering approach to develop a model that describes the effect of key media components and their interactions on C. necator culture cell density. The model is predictive and was experimentally validated against novel media compositions. Moreover, the model was further validated against larger culture volumes at 100 mL and 1 L volumes and found to correlate well. This approach provides valuable and quantifiable insights into the impact of media components on cell growth as well as providing predictions to guide culture scale-up.

microbiology↗

bioRxiv2019

Two resistance nodulation division-family efflux pumps in Chromobacterium species and their role in antibiotic resistance and tolerance

Very little is known of the antibiotic resistance mechanisms of members of the Chromobacterium genus. In previous studies of Chromobacterium subtsugae (formerly C. violaceum) strain CV017, we identified a resistance nodulation division (RND)-family efflux pump (CdeAB-OprM). Here, we show the cdeAB-oprM genes are widely distributed in members of the Chromobacterium genus. We use antimicrobial susceptibility testing with a CV017 cdeAB-oprM mutant to show the products of these genes confers resistance to a variety of antibiotics including ciprofloxacin, a clinically important antibiotic. We also identified a related RND-family pump, cseAB-oprN, in the genome of CV017 and other C. subtsugae species, that is not present in other members of the Chromobacterium genus. We demonstrate that CdeAB-OprM and CseAB-OprN are both transcriptionally induced in CV017 cells treated with sub-lethal antibiotic concentrations and they are important for induction of tolerance to different antibiotics. While CdeAB-OprM has a broad antibiotic specificity, the CseAB-OprN system is highly specific for a ribosome-targeting antibiotic produced by the saprophytic bacterium Burkholderia thailandensis, bactobolin. Finally, we use a previously developed B. thailandensis-C. subtsugae CV017 co-culture model to demonstrate that adding sub-lethal bactobolin at the beginning of co-culture growth increases the ability of CV017 to compete with B. thailandensis in a manner that is dependent on the CseAB-OprN system. Our results provide new information on the antibiotic resistance mechanisms of Chromobacterium species and highlight the importance of efflux pumps during competition with other bacterial species. IMPORTANCEThis study describes two closely related efflux pumps in members of the Chromobacterium genus, which includes opportunistic but often-fatal pathogens and species with highly versatile metabolic capabilities. Efflux pumps remove antibiotics from the cell and are important for antibiotic resistance. One of these pumps is broadly distributed in the Chromobacterium genus and increases resistance to clinically relevant antibiotics. The other efflux pump is present only in Chromobacterium subtsugae and is highly specific for bactobolin, an antibiotic produced by the soil saprophyte Burkholderia thailandensis. We demonstrate these pumps can be activated to increase resistance by their antibiotic substrates, and that this activation is important for C. subtsugae to survive in a laboratory competition experiment with B. thailandensis. These results have implications for managing antibiotic-resistant Chromobacterium infections, bioengineering of Chromobacterium species, and for understanding the evolution of efflux pumps.

microbiology↗

bioRxiv2019

Single-organelle quantification reveals the stoichiometric and structural variability of carboxysomes dependent on the environment

The carboxysome is a complex, proteinaceous organelle that plays essential roles in carbon assimilation in cyanobacteria and chemoautotrophs. It comprises hundreds of protein homologs that self-assemble in space to form an icosahedral structure. Despite its significance in enhancing CO2 fixation and potentials in bioengineering applications, the formation of carboxysomes and their structural composition, stoichiometry and adaptation to cope with environmental changes remain unclear. Here we use live-cell single-molecule fluorescence microscopy, coupled with confocal and electron microscopy, to decipher the absolute protein stoichiometry and organizational variability of single {beta}-carboxysomes in the model cyanobacterium Synechococcus elongatus PCC7942. We determine the physiological abundance of individual building blocks within the icosahedral carboxysome. We further find that the protein stoichiometry, diameter, localization and mobility patterns of carboxysomes in cells depend sensitively on the microenvironmental levels of CO2 and light intensity during cell growth, revealing cellular strategies of dynamic regulation. These findings, also applicable to other bacterial microcompartments and macromolecular self-assembling systems, advance our knowledge of the principles that mediate carboxysome formation and structural modulation. It will empower rational design and construction of entire functional metabolic factories in heterologous organisms, for example crop plants, to boost photosynthesis and agricultural productivity.\n\nOne Sentence SummaryDetermination of absolute protein stoichiometry reveals the organizational variability of carboxysomes in response to microenvironmental changes\n\nThe authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Author (www.plantcell.org) is: Lu-Ning Liu (luning.liu@liverpool.ac.uk).

biophysics↗

bioRxiv2019

UBR5 is a Novel E3 Ubiquitin Ligase involved in Skeletal Muscle Hypertrophy and Recovery from Atrophy

We aimed to investigate a novel and uncharacterised E3 ubiquitin ligase in skeletal muscle atrophy, recovery from atrophy/injury, anabolism and hypertrophy. We demonstrated an alternate gene expression profile for UBR5 versus well characterised E3-ligases, MuRF1/MAFbx, where after atrophy evoked by continuous-low-frequency electrical-stimulation in rats, MuRF1/MAFbx were both elevated yet UBR5 was unchanged. Furthermore, after recovery of muscle mass post tetrodotoxin (TTX) induced-atrophy in rats, UBR5 was hypomethylated and increased at the gene expression level, while a suppression of MuRF1/MAFbx was observed. At the protein level, we also demonstrated a significant increase in UBR5 after recovery of muscle mass from hindlimb unloading in both adult and aged rats, and after recovery from atrophy evoked by nerve crush injury in mice. During anabolism and hypertrophy, UBR5 gene expression increased following acute loading in three-dimensional bioengineered mouse muscle in-vitro, and after chronic electrical-stimulation-induced hypertrophy in rats in-vivo, without increases in MuRF1/MAFbx. Additionally, UBR5 protein abundance increased following functional overload-induced hypertrophy of the plantaris muscle in mice and during differentiation of primary human muscle cells. Finally, in humans, genetic association studies (>700,000 SNPs) demonstrated that the A alleles of rs10505025 and rs4734621 SNPs in the UBR5 gene were strongly associated with larger cross-sectional area of fast-twitch muscle fibres and favoured strength/power versus endurance/untrained phenotypes. Overall, we suggest that UBR5 is a novel E3 ubiquitin ligase that is inversely regulated to MuRF1/MAFbx, is epigenetically regulated, and is elevated at both the gene expression and protein level during recovery from skeletal muscle atrophy and hypertrophy.\n\nKey PointsO_LIWe have recently identified that a HECT domain E3 ubiquitin ligase, named UBR5, is altered epigenetically (via DNA methylation) after human skeletal muscle hypertrophy, where its gene expression is positively correlated with increasing lean leg mass after training and retraining.\nC_LIO_LIIn the present study we extensively investigate this novel and uncharacterised E3 ubiquitin ligase (UBR5) in skeletal muscle atrophy, recovery from atrophy and injury, anabolism and hypertrophy.\nC_LIO_LIWe demonstrated that UBR5 was epigenetically via altered DNA methylation during recovery from atrophy.\nC_LIO_LIWe also determined that UBR5 was alternatively regulated versus well characterised E3 ligases, MuRF1/MAFbx, at the gene expression level during atrophy, recovery from atrophy and hypertrophy.\nC_LIO_LIUBR5 also increased at the protein level during recovery from atrophy and injury, hypertrophy and during human muscle cell differentiation.\nC_LIO_LIFinally, in humans, genetic variations of the UBR5 gene were strongly associated with larger fast-twitch muscle fibres and strength/power performance versus endurance/untrained phenotypes.\nC_LI

physiology↗

bioRxiv2019

Tunable integrase-mediated differentiation facilitates improved output of burdensome functions in E. coli

Advances in synthetic biology, bioengineering, and computation allow us to rapidly and reliably program cells with increasingly complex and useful functions. However, because the functions we engineer cells to perform are typically unnecessary for cellular survival and burdensome to cell growth, they can be rapidly lost due to the processes of mutation and natural selection. To improve the evolutionary stability of engineered functions in a general manner, we developed an integrase-recombination-based differentiation gene circuit in Escherichia coli. In this system, differentiated cells uniquely carry out burdensome or toxic engineered functions but have limited capacity to grow (terminal differentiation), preventing the propagation of selectively advantageous loss of function mutations that inevitably arise. To experimentally implement terminal differentiation, we co-opted the R6K plasmid system, using differentiation to simultaneously activate T7 RNAP-driven expression of arbitrary engineered functions, and inactivate expression of {pi} protein (an essential factor for R6K plasmid replication), thereby allowing limitation of differentiated cell growth through antibiotic selection. We experimentally demonstrate terminal differentiation increases both duration and magnitude of high-burden T7 RNAP-driven expression, and that its evolutionary stability can be further improved with strategic redundancy. Using burdensome overexpression of a fluorescent protein as a model engineered function, our terminal differentiation circuit results in a ~2.8-fold (single-cassette) and ~4.2-fold (two-cassette) increase of total fluorescent protein produced compared to high-burden naive expression in which all cells inducibly express T7 RNAP. Finally, we demonstrate that differentiation can enable the expression of even toxic functions, a feat not achieved to our knowledge by any other strategy for addressing long-term evolutionary stability. Overall, this study provides an effective generalizable strategy for protecting engineered functions from evolutionary degradation.

synthetic biology↗

bioRxiv2019

Both clinical and environmental Caulobacter species act as opportunistic pathogens

The Caulobacter genus, including the widely-studied model organism Caulobacter crescentus, has been thought to be non-pathogenic and thus proposed as a bioengineering vector for various environmental remediation and medical purposes. However, Caulobacter species have been implicated as the causative agents of several hospital-acquired infections, raising the question of whether these clinical isolates represent an emerging pathogenic species or whether Caulobacters on whole possess previously-unappreciated virulence capability. Given the proposed environmental and medical applications for C. crescentus, understanding the potential pathogenicity and human health implications of this bacterium is crucial. Consequently, we sequenced a clinical Caulobacter isolate to determine if it has acquired novel virulence determinants. We found that the clinical isolate represents a new species, Caulobacter mirare. C. mirare phylogenetically resembles both C. crescentus and the related C. segnis, which was also thought to be non-pathogenic. The similarity to other Caulobacters and lack of obvious pathogenesis markers suggested that C. mirare is not unique amongst Caulobacters and that consequently other Caulobacters may also have the potential to be virulent. We tested this hypothesis by characterizing the ability of Caulobacters to infect the model animal host Galleria mellonella. In this context, two different lab strains of C. crescentus proved to be as pathogenic as C. mirare, while lab strains of E. coli were non-pathogenic. Further characterization showed that Caulobacter pathogenesis is mediated by a dose-dependent, cell-associated toxic factor that does not require active bacterial cells or host cellular innate immunity to elicit its toxic effects. Finally, we show that C. crescentus does not grow well in standard clinical culture conditions, suggesting that Caulobacter infections may be more common than generally appreciated but rarely cultured. Taken together, our findings redefine Caulobacters as opportunistic pathogens and highlight the importance of broadening our methods for identifying and characterizing pathogens.\n\nAUTHOR SUMMARYBacterial species have historically been classified as either capable of causing disease in an animal (pathogenic) or not. Caulobacter species represent a class of bacteria that were thought to be non-pathogenic. Caulobacters have been widely studied and proposed to be used for various industrial and medical applications due to their presumed safety. However, recent reports of human Caulobacter infections raised the question of whether disease-causing Caulobacters have acquired special factors that help them cause disease or whether the ability to infect is a more general feature of most Caulobacters. By combining genomic sequencing and animal infection studies we show that a clinical Caulobacter strain is similar to lab Caulobacters and that all Caulobacters studied can infect a model host. We explore the mechanism of this infectivity and show that it is due to a toxic factor that associates with Caulobacter cells. We also provide a possible explanation for why Caulobacters have not traditionally been isolated from human patients, owing to their inability to tolerate the salt levels used in most medical culturing systems.

microbiology↗

bioRxiv2019

Laboratory evolution of Escherichia coli enables life based on fluorinated amino acids

Organofluorine compounds are toxic to various living beings in different habitats. On the other hand, fluorine incorporation into single proteins via related amino acid analogues has become common practice in protein engineering. Thus, an essential question remains: can fluorinated amino acids generally be used as xeno-nutrients to build up biomass, or do large amounts of fluorine in the cells render them nonviable? To gain information about the effect of long-term exposure of a cellular proteome to fluorinated organic compounds, we constructed an experiment based on bacterial adaptation in artificial fluorinated habitats. We propagated Escherichia coli (E. coli) in the presence of either 4- or 5-fluoroindole as essential precursors for the in situ synthesis of tryptophan (Trp) analogues. We found that full adaptation requires astonishingly few genetic mutations but is accompanied by large rearrangements in regulatory networks, membrane integrity and quality control of protein folding. These findings highlight the cellular mechanisms of the evolutionary adaption process to unnatural amino acids and provide the molecular foundation for novel and innovative bioengineering of microbial strains with potential for biotechnological applications.\n\nOne Sentence SummaryLaboratory evolution enabled for the first time Escherichia coli to use fluorinated indoles as essential precursors for protein synthesis by introducing few genetic mutations but large rearrangements in regulatory networks, membrane integrity and quality control of protein folding.

synthetic biology↗

bioRxiv2019

A novel decellularization method to produce brain scaffolds

Scaffolds composed of extracellular matrix (ECM) can assist tissue remodeling and repair following injury. The ECM is a complex biomaterial composed of proteins, glycoproteins, proteoglycans, and glycosaminoglycans, secreted by cells. The ECM contains fundamental biological cues that modulate cell behavior and serves as a structural scaffold for cell adhesion and growth. For clinical applications, where immune rejection is a constraint, ECM can be processed using decellularization methods intended to remove cells and donor antigens from tissue or organs, while preserving native biological cues essential for cell growth and differentiation. Recent studies show bioengineered organs composed by a combination of a diversity of materials and stem cells as a possibility of new therapeutic strategies to treat diseases that affect different tissues and organs, including the central nervous system (CNS). Nevertheless, the methodologies currently described for brain decellularization involve the use of several chemical reagents with many steps that ultimately limit the process of organ or tissue recellularization. Here, we describe for the first time a fast and straightforward method for complete decellularization of mice brain by the combination of rapid freezing and thawing following the use of only one detergent (Sodium dodecyl sulfate (SDS)). Our data show that using the protocol we describe here the brain can be entirely decellularized, while still maintaining ECM components that are essential for cell survival and repopulation of the scaffold. Our results also show the repopulation of the decellularized brain matrix with Neuro2a cells, that were identified by immunohistochemistry in their undifferentiated form. We conclude that this novel and simple method for brain decellularization can be used as a biocompatible scaffold for cell repopulation.\n\nImpact StatementFor the first time we describe an easy, effective and low cost method for complete decellularization of murine brain by the use of only one detergent (SDS) combined with rapid freezing and thawing, that can be used as a 3D scaffold for cell culture of neuronal cells. The results show that the decellularized brains still maintain ECM components essential for cell survival and repopulation of the scaffold. Moreover, we found that the decellularized brain matrix can be repopulated with neural cells, showing its biocompatibility.\n\nGRAFICAL ABSTRACT\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC=\"FIGDIR/small/680702v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (26K):\norg.highwire.dtl.DTLVardef@15fd027org.highwire.dtl.DTLVardef@15acfeborg.highwire.dtl.DTLVardef@1087d2eorg.highwire.dtl.DTLVardef@529309_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗