An evaluation of fusion partner proteins for paratransgenesis in Asaia bogorensis.

Mosquitoes transmit many pathogens responsible for human diseases, such as malaria which is caused by parasites in the genus Plasmodium. Current strategies to control vector-transmitted diseases are increasingly undermined by mosquito and pathogen resistance, so additional methods of control are required. Paratransgenesis is a method whereby symbiotic bacteria are genetically modified to affect the mosquito's phenotype by engineering them to deliver effector molecules into the midgut to kill parasites. One paratransgenesis candidate is Asaia bogorensis, a Gram-negative bacterium colonizing the midgut, ovaries, and salivary glands of Anopheles sp. mosquitoes. Previously, engineered Asaia strains using native signals to drive the release of the antimicrobial peptide, scorpine, fused to alkaline phosphatase were successful in significantly suppressing the number of oocysts formed after a blood meal containing P. berghei. However, these strains saw high fitness costs associated with the production of the recombinant protein. Here, we report evaluation of five different partner proteins fused to scorpine that were evaluated for effects on the growth and fitness of the transgenic bacteria. Three of the new partner proteins resulted in significant levels of protein released from the Asaia bacterium while also significantly reducing the prevalence of mosquitoes infected with P. berghei. Two partners performed as well as the previously tested Asaia strain that used alkaline phosphatase in the fitness analyses, but neither exceeded it. It may be that there is a maximum level of fitness and parasite inhibition that can be achieved with scorpine being driven constitutively, and that use of a Plasmodium specific effector molecule in place of scorpine would help to mitigate the stress on the symbionts.

Progress towards an inducible, replication-proficient transposon delivery vector for Chlamydia trachomatis.

Background Genetic systems have been developed for Chlamydia but the extremely low transformation frequency remains a significant bottleneck.  Our goal is to develop a self-replicating transposon delivery vector for C. trachomatis which can be expanded prior to transposase induction. Methods We made E. coli/ C. trachomatis shuttle vectors bearing the Himar1 C9  transposase under control of the tet promoter and a novel rearrangement of the Himar1 transposon with the ß-lactamase gene.  Activity of the transposase was monitored by immunoblot and by DNA sequencing. Results We constructed pSW2-mCh-C9, a C. trachomatis plasmid designed to act as a self-replicating vector carrying both the Himar1 C9  transposase under tet promoter control and its transposon.  However, we were unable to recover this plasmid in C. trachomatis following multiple attempts at transformation. Therefore, we assembled two new deletion plasmids pSW2-mCh-C9-ΔTpon carrying only the Himar1 C9  transposase (under tet promoter control) and a sister vector (same sequence backbone) pSW2-mCh-C9-ΔTpase carrying its cognate transposon.  We demonstrated that the biological components that make up both pSW2-mCh-C9-ΔTpon and pSW2-mCh-C9-ΔTpase are active in E. coli.  Both these plasmids could be independently recovered in C. trachomatis. We attempted to perform lateral gene transfer by transformation and mixed infection with C. trachomatis strains bearing pSW2-mCh-C9-ΔTpon and pSW2-RSGFP-Tpon (a green fluorescent version of pSW2-mCh-C9-ΔTpase).  Despite success in achieving mixed infections, it was not possible to recover progeny bearing both versions of these plasmids. Conclusions We have designed a self-replicating plasmid vector pSW2-mCh-C9 for C. trachomatis carrying the Himar1 C9  transposase under tet promoter control.  Whilst this can be transformed into E. coli it cannot be recovered in C. trachomatis.  Based on selected deletions and phenotypic analyses we conclude that low level expression from the tet inducible promoter is responsible for premature transposition and hence plasmid loss early on in the transformation process.

An inducible transposon mutagenesis approach for the intracellular human pathogen Chlamydia trachomatis.

Background: Chlamydia trachomatis is a prolific human pathogen that can cause serious long-term conditions if left untreated. Recent developments in Chlamydia genetics have opened the door to conducting targeted and random mutagenesis experiments to identify gene function. In the present study, an inducible transposon mutagenesis approach was developed for C. trachomatis using a self-replicating vector to deliver the transposon-transposase cassette - a significant step towards our ultimate aim of achieving saturation mutagenesis of the Chlamydia genome. Methods: The low transformation efficiency of C. trachomatis necessitated the design of a self-replicating vector carrying the transposon mutagenesis cassette (i.e. the Himar-1 transposon containing the beta lactamase gene as well as a hyperactive transposase gene under inducible control of the tet promoter system with the addition of a riboswitch). Chlamydia transformed with this vector (pSW2-RiboA-C9Q) were induced at 24 hours post-infection. Through dual control of transcription and translation, basal expression of transposase was tightly regulated to stabilise the plasmid prior to transposition. Results: Here we present the preliminary sequencing results of transposon mutant pools of both C. trachomatis biovars, using two plasmid-free representatives: urogenital strain   C. trachomatis SWFP- and the lymphogranuloma venereum isolate L2(25667R). DNA sequencing libraries were generated and analysed using Oxford Nanopore Technologies' MinION technology. This enabled 'proof of concept' for the methods as an initial low-throughput screen of mutant libraries; the next step is to employ high throughput sequencing to assess saturation mutagenesis. Conclusions: This significant advance provides an efficient method for assaying C. trachomatis gene function and will enable the identification of the essential gene set of C. trachomatis. In the long-term, the methods described herein will add to the growing knowledge of chlamydial infection biology leading to the discovery of novel drug or vaccine targets.

Blood meal-induced inhibition of vector-borne disease by transgenic microbiota.

Vector-borne diseases are a substantial portion of the global disease burden; one of the deadliest of these is malaria. Vector control strategies have been hindered by mosquito and pathogen resistances, and population alteration approaches using transgenic mosquitos still have many hurdles to overcome before they can be implemented in the field. Here we report a paratransgenic control strategy in which the microbiota of Anopheles stephensi was engineered to produce an antiplasmodial effector causing the mosquito to become refractory to Plasmodium berghei. The midgut symbiont Asaia was used to conditionally express the antiplasmodial protein scorpine only when a blood meal was present. These blood meal inducible Asaia strains significantly inhibit pathogen infection, and display improved fitness compared to strains that constitutively express the antiplasmodial effector. This strategy may allow the antiplasmodial bacterial strains to survive and be transmitted through mosquito populations, creating an easily implemented and enduring vector control strategy.

Charles Darwin Synthetic Interview: A 19th Century Scientist Speaks in the 21st Century.

Charles Darwin is largely unknown and poorly understood as a historical figure. Similarly the fundamental principles of evolution are often misstated, misunderstood, or entirely rejected by large numbers of Americans. Simply trying to communicate more facts about Darwin, or facts supporting the principles of evaluation, is inadequate; neither students nor members of the public will care or retain the information. On the contrary, building facts into a one-on-one conversational narrative creates a memorable opportunity to learn. Here we create a digital media, self-guided question and answer 'synthetic interview' with Charles Darwin. Questions are derived from a survey of nearly 1,000 people. Answers spoken by an actor portraying Darwin are derived from Darwin's own writings. Questions on modern topics are answered by scientists, theologians, and lawyers. First produced as a museum exhibit and then later reproduced as an app (iOS/Android), the Darwin Synthetic Interview has been evaluated with more than 3,000 surveyed users, of which 69% indicated that they learned and more than 75% would recommend the experience. Students who interacted with the synthetic interview in a classroom setting found answers were unexpected and clarifying. Using a format of personal narrative, the Darwin Synthetic Interview creates a new way to engage students and the public in a process of self-directed discovery of a topic that is often considered difficult to teach.

Inhibition of Plasmodium berghei Development in Mosquitoes by Effector Proteins Secreted from Asaia sp. Bacteria Using a Novel Native Secretion Signal.

Novel interventions are needed to prevent the transmission of the Plasmodium parasites that cause malaria. One possible method is to supply mosquitoes with antiplasmodial effector proteins from bacteria by paratransgenesis. Mosquitoes have a diverse complement of midgut microbiota including the Gram-negative bacteria Asaia bogorensis. This study presents the first use of Asaia sp. bacteria for paratransgenesis against P. berghei. We identified putative secreted proteins from A. bogorensis by a genetic screen using alkaline phosphatase gene fusions. Two were secreted efficiently: a siderophore receptor protein and a YVTN beta-propeller repeat protein. The siderophore receptor gene was fused with antiplasmodial effector genes including the scorpine antimicrobial peptide and an anti-Pbs21 scFv-Shiva1 immunotoxin. Asaia SF2.1 secreting these fusion proteins were fed to mosquitoes and challenged with Plasmodium berghei-infected blood. With each of these effector constructs, significant inhibition of parasite development was observed. These results provide a novel and promising intervention against malaria transmission.

Current perspectives on unconventional shale gas extraction in the Appalachian Basin.

The Appalachian Basin is home to three major shales, the Upper Devonian, Marcellus, and Utica. Together, they contain significant quantities of tight oil, gas, and mixed hydrocarbons. The Marcellus alone is estimated to contain upwards of 500 trillion cubic feet of natural gas. The extraction of these deposits is facilitated by a combination of horizontal drilling and slick water stimulation (e.g., hydraulic fracturing) or "fracking." The process of fracking requires large volumes of water, proppant, and chemicals as well as a large well pad (3-7 acres) and an extensive network of gathering and transmission pipelines. Drilling can generate about 1,000 tons of drill cuttings depending on the depth of the formation and the length of the horizontal bore. The flowback and produced waters that return to the surface during production are high in total dissolved solids (TDS, 60,000-350,000 mg L(-1)) and contain halides (e.g., chloride, bromide, fluoride), strontium, barium, and often naturally occurring radioactive materials (NORMs) as well as organics. The condensate tanks used to store these fluids can off gas a plethora of volatile organic compounds. The waste water, with its high TDS may be recycled, treated, or disposed of through deep well injection. Where allowed, open impoundments used for recycling are a source of air borne contamination as they are often aerated. The gas may be "dry" (mostly methane) or "wet," the latter containing a mixture of light hydrocarbons and liquids that need to be separated from the methane. Although the wells can produce significant quantities of natural gas, from 2-7 bcf, their initial decline rates are significant (50-75%) and may cease to be economic within a few years. This review presents an overview of unconventional gas extraction highlighting the environmental impacts and challenges.

Draft Genome Sequence of Asaia sp. Strain SF2.1, an Important Member of the Microbiome of Anopheles Mosquitoes.

Asaia spp. are abundant members of the microbiota of Anopheles mosquitoes, the principle vectors of malaria. Here, we report the draft genome sequence of Asaia sp. strain SF2.1. This strain is under development as a platform to deliver antimalarial peptides and proteins to adult female Anopheles mosquitoes.

Fighting malaria with engineered symbiotic bacteria from vector mosquitoes.

The most vulnerable stages of Plasmodium development occur in the lumen of the mosquito midgut, a compartment shared with symbiotic bacteria. Here, we describe a strategy that uses symbiotic bacteria to deliver antimalaria effector molecules to the midgut lumen, thus rendering host mosquitoes refractory to malaria infection. The Escherichia coli hemolysin A secretion system was used to promote the secretion of a variety of anti-Plasmodium effector proteins by Pantoea agglomerans, a common mosquito symbiotic bacterium. These engineered P. agglomerans strains inhibited development of the human malaria parasite Plasmodium falciparum and rodent malaria parasite Plasmodium berghei by up to 98%. Significantly, the proportion of mosquitoes carrying parasites (prevalence) decreased by up to 84% for two of the effector molecules, scorpine, a potent antiplasmodial peptide and (EPIP)(4), four copies of Plasmodium enolase-plasminogen interaction peptide that prevents plasminogen binding to the ookinete surface. We demonstrate the use of an engineered symbiotic bacterium to interfere with the development of P. falciparum in the mosquito. These findings provide the foundation for the use of genetically modified symbiotic bacteria as a powerful tool to combat malaria.

Ribosome display of combinatorial antibody libraries derived from mice immunized with heat-killed Xylella fastidiosa and the selection of MopB-specific single-chain antibodies.

Pierce's disease is a devastating lethal disease of Vitus vinifera grapevines caused by the bacterium Xylella fastidiosa. There is no cure for Pierce's disease, and control is achieved predominantly by suppressing transmission of the glassy-winged sharpshooter insect vector. We present a simple robust approach for the generation of panels of recombinant single-chain antibodies against the surface-exposed elements of X. fastidiosa that may have potential use in diagnosis and/or disease transmission blocking studies. In vitro combinatorial antibody ribosome display libraries were assembled from immunoglobulin transcripts rescued from the spleens of mice immunized with heat-killed X. fastidiosa. The libraries were used in a single round of selection against an outer membrane protein, MopB, resulting in the isolation of a panel of recombinant antibodies. The potential use of selected anti-MopB antibodies was demonstrated by the successful application of the 4XfMopB3 antibody in an enzyme-linked immunosorbent assay (ELISA), a Western blot assay, and an immunofluorescence assay (IFA). These immortalized in vitro recombinant single-chain antibody libraries generated against heat-killed X. fastidiosa are a resource for the Pierce's disease research community that may be readily accessed for the isolation of antibodies against a plethora of X. fastidiosa surface-exposed antigenic molecules.

Secretion of anti-Plasmodium effector proteins from a natural Pantoea agglomerans isolate by using PelB and HlyA secretion signals.

The insect-vectored disease malaria is a major world health problem. New control strategies are needed to supplement the current use of insecticides and medications. A genetic approach can be used to inhibit development of malaria parasites (Plasmodium spp.) in the mosquito host. We hypothesized that Pantoea agglomerans, a bacterial symbiont of Anopheles mosquitoes, could be engineered to express and secrete anti-Plasmodium effector proteins, a strategy termed paratransgenesis. To this end, plasmids that include the pelB or hlyA secretion signals from the genes of related species (pectate lyase from Erwinia carotovora and hemolysin A from Escherichia coli, respectively) were created and tested for their efficacy in secreting known anti-Plasmodium effector proteins (SM1, anti-Pbs21, and PLA2) in P. agglomerans and E. coli. P. agglomerans successfully secreted HlyA fusions of anti-Pbs21 and PLA2, and these strains are under evaluation for anti-Plasmodium activity in infected mosquitoes. Varied expression and/or secretion of the effector proteins was observed, suggesting that the individual characteristics of a particular effector may require empirical testing of several secretion signals. Importantly, those strains that secreted efficiently grew as well as wild-type strains under laboratory conditions and, thus, may be expected to be competitive with the native microbiota in the environment of the mosquito midgut.

Bacterial genetic methods to explore the biology of mariner transposons.

Mariners are small DNA mediated transposons of eukaryotes that fortuitously function in bacteria. Using bacterial genetics, it is possible to study a variety of properties of mariners, including transpositional ability, dominant-negative regulation, overexpresson inhibition, and the function of cis-acting sequences like the inverted terminal repeats. In conjunction with biochemical techniques, the structure of the transposase can be elucidated and the activity of the elements can be improved for genetic tool use. Finally, it is possible to uncover functional transposase genes directly from genomes given a suitable bacterial genetic screen.

Technological advances to enhance agricultural pest management.

Biotechnology offers new solutions to existing and future pest problems in agriculture including, for the first time, possible tools to use against insect transmitted pathogens causing plant diseases. Here, we describe the strategy first described as Autocidal Biological Control applied for the development of conditional lethal pink bollworm strains. When these strains are mass-reared, the lethal gene expression is suppressed by a tetracycline repressor element, which is activated by the presence of chlorotetracycline, a normal component of the mass-rearing diet. Once removed from the tetracycline diet, the lethal genes are passed on to offspring when ordinary lab-reared pink bollworms mate with special lethal strains. Lethality is dominant (one copy sufficient for lethality), expressed in the egg stage and affects all eggs (100% lethal expression). The initial investment by the California Cotton Pest Control Board is an outstanding example of research partnerships between agriculture industry, the USDA and land grant universities.

Hyperactive Himar1 transposase mediates transposition in cell culture and enhances gene expression in vivo.

The use of nonviral delivery systems results in transient gene expression, in part because of the low efficiency of DNA integration. Previously, vectors based on transposon systems such as Sleeping Beauty have been shown to be able to increase stable transfection efficiencies in cell culture and in animal models. Himar1, a reconstructed active transposon belonging to the Tc1/mariner superfamily, also has been used as a vector for stable gene delivery, but the rate of transposition after transfection is low. In this paper, we evaluate the potential of the hyperactive Himar1 transposase C9, in combination with the Himar1 inverted repeat transposon, as a gene delivery vector. The C9 transposase is a hyperactive mutant of Himar1 with two amino acid substitutions, Q131R and E137K, that result in an increase in activity relative to wild type. Here we demonstrate that cotransfection of the C9 transposase with a Himar1-based vector increases the frequency of stable gene expression in human cells in a transposase concentration-dependent manner. In addition, we establish that C9 transposase mediates integration of the transgene in mammalian cells at a frequency similar to that of Sleeping Beauty under some of the conditions tested. Last, we show significantly higher levels of reporter gene expression in vivo in mouse liver and in synovium of rabbit knee joints after injection of the transposon plasmid expressing the transgene and the C9 transposase. These data suggest that vectors based on the Himar1 transposable element, in conjunction with the hyperactive mutant transposase C9, may be suitable vectors for gene therapy applications.

The N-terminus of Himar1 mariner transposase mediates multiple activities during transposition.

Mariner family transposons are perhaps the most widespread transposable elements of eukaryotes. While we are beginning to understand the precise mechanism of transposition of these elements, the structure of their transposases are still poorly understood. We undertook an extensive mutagenesis of the N-terminal third of the transposase of the Himar1 mariner transposon to begin the process of determining the structure and evolution of mariner transposases. N and C-terminal deletion analyses localized the DNA binding domain of Himar1 transposase to the first 115 amino acids. Alanine scanning of 23 selected sites within this region uncovered mutations that not only affected DNA binding but DNA cleavage as well. The behavior of other mutations strongly suggested that the N-terminus is also involved in multimerization of the transposase on a single inverted terminal repeat and in paired ends complex formation which brings together the two ends of the transposon. Finally, two hyperactive mutations at conserved sites suggest that mariner transposases are under a pattern of stabilizing selection in nature with regard to how efficiently they mediate transposition, resulting in a population of "average" transposons.

Early intermediates of mariner transposition: catalysis without synapsis of the transposon ends suggests a novel architecture of the synaptic complex.

The mariner family is probably the most widely distributed family of transposons in nature. Although these transposons are related to the well-studied bacterial insertion elements, there is evidence for major differences in their reaction mechanisms. We report the identification and characterization of complexes that contain the Himar1 transposase bound to a single transposon end. Titrations and mixing experiments with the native transposase and transposase fusions suggested that they contain different numbers of transposase monomers. However, the DNA protection footprints of the two most abundant single-end complexes are identical. This indicates that some transposase monomers may be bound to the transposon end solely by protein-protein interactions. This would mean that the Himar1 transposase can dimerize independently of the second transposon end and that the architecture of the synaptic complex has more in common with V(D)J recombination than with bacterial insertion elements. Like V(D)J recombination and in contrast to the case for bacterial elements, Himar1 catalysis does not appear to depend on synapsis of the transposon ends, and the single-end complexes are active for nicking and probably for cleavage. We discuss the role of this single-end activity in generating the mutations that inactivate the vast majority of mariner elements in eukaryotes.

A bacterial genetic screen identifies functional coding sequences of the insect mariner transposable element Famar1 amplified from the genome of the earwig, Forficula auricularia.

Transposons of the mariner family are widespread in animal genomes and have apparently infected them by horizontal transfer. Most species carry only old defective copies of particular mariner transposons that have diverged greatly from their active horizontally transferred ancestor, while a few contain young, very similar, and active copies. We report here the use of a whole-genome screen in bacteria to isolate somewhat diverged Famar1 copies from the European earwig, Forficula auricularia, that encode functional transposases. Functional and nonfunctional coding sequences of Famar1 and nonfunctional copies of Ammar1 from the European honey bee, Apis mellifera, were sequenced to examine their molecular evolution. No selection for sequence conservation was detected in any clade of a tree derived from these sequences, not even on branches leading to functional copies. This agrees with the current model for mariner transposon evolution that expects neutral evolution within particular hosts, with selection for function occurring only upon horizontal transfer to a new host. Our results further suggest that mariners are not finely tuned genetic entities and that a greater amount of sequence diversification than had previously been appreciated can occur in functional copies in a single host lineage. Finally, this method of isolating active copies can be used to isolate other novel active transposons without resorting to reconstruction of ancestral sequences.

Recent horizontal transfer of mellifera subfamily mariner transposons into insect lineages representing four different orders shows that selection acts only during horizontal transfer.

We report the isolation and sequencing of genomic copies of mariner transposons involved in recent horizontal transfers into the genomes of the European earwig, Forficula auricularia; the European honey bee, Apis mellifera; the Mediterranean fruit fly, Ceratitis capitata; and a blister beetle, Epicauta funebris, insects from four different orders. These elements are in the mellifera subfamily and are the second documented example of full-length mariner elements involved in this kind of phenomenon. We applied maximum likelihood methods to the coding sequences and determined that the copies in each genome were evolving neutrally, whereas reconstructed ancestral coding sequences appeared to be under selection, which strengthens our previous hypothesis that the primary selective constraint on mariner sequence evolution is the act of horizontal transfer between genomes.