Symbiotic Burkholderia Species Show Diverse Arrangements of nif/fix and nod Genes and Lack Typical High-Affinity Cytochrome cbb3 Oxidase Genes.

Genome analysis of fourteen mimosoid and four papilionoid beta-rhizobia together with fourteen reference alpha-rhizobia for both nodulation (nod) and nitrogen-fixing (nif/fix) genes has shown phylogenetic congruence between 16S rRNA/MLSA (combined 16S rRNA gene sequencing and multilocus sequence analysis) and nif/fix genes, indicating a free-living diazotrophic ancestry of the beta-rhizobia. However, deeper genomic analysis revealed a complex symbiosis acquisition history in the beta-rhizobia that clearly separates the mimosoid and papilionoid nodulating groups. Mimosoid-nodulating beta-rhizobia have nod genes tightly clustered in the nodBCIJHASU operon, whereas papilionoid-nodulating Burkholderia have nodUSDABC and nodIJ genes, although their arrangement is not canonical because the nod genes are subdivided by the insertion of nif and other genes. Furthermore, the papilionoid Burkholderia spp. contain duplications of several nod and nif genes. The Burkholderia nifHDKEN and fixABC genes are very closely related to those found in free-living diazotrophs. In contrast, nifA is highly divergent between both groups, but the papilionoid species nifA is more similar to alpha-rhizobia nifA than to other groups. Surprisingly, for all Burkholderia, the fixNOQP and fixGHIS genes required for cbb3 cytochrome oxidase production and assembly are missing. In contrast, symbiotic Cupriavidus strains have fixNOQPGHIS genes, revealing a divergence in the evolution of two distinct electron transport chains required for nitrogen fixation within the beta-rhizobia.

Plant-associated symbiotic Burkholderia species lack hallmark strategies required in mammalian pathogenesis.

Burkholderia is a diverse and dynamic genus, containing pathogenic species as well as species that form complex interactions with plants. Pathogenic strains, such as B. pseudomallei and B. mallei, can cause serious disease in mammals, while other Burkholderia strains are opportunistic pathogens, infecting humans or animals with a compromised immune system. Although some of the opportunistic Burkholderia pathogens are known to promote plant growth and even fix nitrogen, the risk of infection to infants, the elderly, and people who are immunocompromised has not only resulted in a restriction on their use, but has also limited the application of non-pathogenic, symbiotic species, several of which nodulate legume roots or have positive effects on plant growth. However, recent phylogenetic analyses have demonstrated that Burkholderia species separate into distinct lineages, suggesting the possibility for safe use of certain symbiotic species in agricultural contexts. A number of environmental strains that promote plant growth or degrade xenobiotics are also included in the symbiotic lineage. Many of these species have the potential to enhance agriculture in areas where fertilizers are not readily available and may serve in the future as inocula for crops growing in soils impacted by climate change. Here we address the pathogenic potential of several of the symbiotic Burkholderia strains using bioinformatics and functional tests. A series of infection experiments using Caenorhabditis elegans and HeLa cells, as well as genomic characterization of pathogenic loci, show that the risk of opportunistic infection by symbiotic strains such as B. tuberum is extremely low.

Designing synthetic biology.

Synthetic biology is frequently defined as the application of engineering design principles to biology. Such principles are intended to streamline the practice of biological engineering, to shorten the time required to design, build, and test synthetic gene networks. This streamlining of iterative design cycles can facilitate the future construction of biological systems for a range of applications in the production of fuels, foods, materials, and medicines. The promise of these potential applications as well as the emphasis on design has prompted critical reflection on synthetic biology from design theorists and practicing designers from many fields, who can bring valuable perspectives to the discipline. While interdisciplinary connections between biologists and engineers have built synthetic biology via the science and the technology of biology, interdisciplinary collaboration with artists, designers, and social theorists can provide insight on the connections between technology and society. Such collaborations can open up new avenues and new principles for research and design, as well as shed new light on the challenging context-dependence-both biological and social-that face living technologies at many scales. This review is inspired by the session titled "Design and Synthetic Biology: Connecting People and Technology" at Synthetic Biology 6.0 and covers a range of literature on design practice in synthetic biology and beyond. Critical engagement with how design is used to shape the discipline opens up new possibilities for how we might design the future of synthetic biology.

A survey of the microbial community in the rhizosphere of two dominant shrubs of the Negev Desert highlands, Zygophyllum dumosum (Zygophyllaceae) and Atriplex halimus (Amaranthaceae), using cultivation-dependent and cultivation-independent methods.

PREMISE OF THE STUDY: Plant roots comprise more than 50% of the plant's biomass. Part of that biomass includes the root microbiome, the assemblage of bacteria and fungi living in the 1-3 mm region adjacent to the external surface of the root, the rhizosphere. We hypothesized that the microorganisms living in the rhizosphere and in bulk soils of the harsh environment of the Negev Desert of Israel had potential for use as plant-growth-promoting bacteria (PGPB) to improve plant productivity in nutrient-poor, arid soils that are likely to become more common as the climate changes. • METHODS: We used cultivation-dependent methods including trap experiments with legumes to find nitrogen-fixing rhizobia, specialized culture media to determine iron chelation via siderophores and phosphate-solubilizing and cellulase activities; cultivation-independent methods, namely 16S rDNA cloning and sequencing; and also community-level physiological profiling to discover soil microbes associated with the Negev desert perennials Zygophyllum dumosum and Atriplex halimus during the years 2009-2010. • KEY RESULTS: We identified a number of PGPB, both epiphytes and endophytes, which fix nitrogen, chelate iron, solubilize phosphate, and secrete cellulase, as well as many other bacteria and some fungi, thereby providing a profile of the microbiomes that support the growth of two desert perennials. • CONCLUSION: We generated a snapshot of the microbial communities in the Negev Desert, giving us an insight in its natural state. This desert, like many arid environments, is vulnerable to exploitation for other purposes, including solar energy production and dry land farming.

Smelling in multiple dimensions.

Smell is perhaps the most subjective of the human senses, making odors difficult to measure and define. In everyday language, in the philosophy of aesthetics, and in the lab, this low opinion of odors means that smells are often characterized simply along an axis of good or bad. Odors and the ways they are perceived, however, are varied and incredibly complex, requiring an understanding of chemistry, neuroscience, aesthetics, and social science. Science and art that engage the sense of smell have the potential to expand our understanding of how biology and chemistry interact.

A BioBrick compatible strategy for genetic modification of plants.

BACKGROUND: Plant biotechnology can be leveraged to produce food, fuel, medicine, and materials. Standardized methods advocated by the synthetic biology community can accelerate the plant design cycle, ultimately making plant engineering more widely accessible to bioengineers who can contribute diverse creative input to the design process. RESULTS: This paper presents work done largely by undergraduate students participating in the 2010 International Genetically Engineered Machines (iGEM) competition. Described here is a framework for engineering the model plant Arabidopsis thaliana with standardized, BioBrick compatible vectors and parts available through the Registry of Standard Biological Parts (http://www.partsregistry.org). This system was used to engineer a proof-of-concept plant that exogenously expresses the taste-inverting protein miraculin. CONCLUSIONS: Our work is intended to encourage future iGEM teams and other synthetic biologists to use plants as a genetic chassis. Our workflow simplifies the use of standardized parts in plant systems, allowing the construction and expression of heterologous genes in plants within the timeframe allotted for typical iGEM projects.

Natural strategies for the spatial optimization of metabolism in synthetic biology.

Metabolism is a highly interconnected web of chemical reactions that power life. Though the stoichiometry of metabolism is well understood, the multidimensional aspects of metabolic regulation in time and space remain difficult to define, model and engineer. Complex metabolic conversions can be performed by multiple species working cooperatively and exchanging metabolites via structured networks of organisms and resources. Within cells, metabolism is spatially regulated via sequestration in subcellular compartments and through the assembly of multienzyme complexes. Metabolic engineering and synthetic biology have had success in engineering metabolism in the first and second dimensions, designing linear metabolic pathways and channeling metabolic flux. More recently, engineering of the third dimension has improved output of engineered pathways through isolation and organization of multicell and multienzyme complexes. This review highlights natural and synthetic examples of three-dimensional metabolism both inter- and intracellularly, offering tools and perspectives for biological design.

A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism.

BACKGROUND: FeFe-hydrogenases are the most active class of H2-producing enzymes known in nature and may have important applications in clean H2 energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by O2. RESULTS: We have developed a synthetic metabolic pathway in E. coli that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate O2 levels eliminate growth. This pathway forms the basis for a genetic selection for O2 tolerance. Genetically selected hydrogenases did not show improved stability in O2 and in many cases had lost H2 production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer. CONCLUSIONS: Rational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient in vivo assays to aid in the engineering of synthetic H2 metabolism. Our results also indicate a H2-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of H2-activating catalysts.

Towards a synthetic chloroplast.

BACKGROUND: The evolution of eukaryotic cells is widely agreed to have proceeded through a series of endosymbiotic events between larger cells and proteobacteria or cyanobacteria, leading to the formation of mitochondria or chloroplasts, respectively. Engineered endosymbiotic relationships between different species of cells are a valuable tool for synthetic biology, where engineered pathways based on two species could take advantage of the unique abilities of each mutualistic partner. RESULTS: We explored the possibility of using the photosynthetic bacterium Synechococcus elongatus PCC 7942 as a platform for studying evolutionary dynamics and for designing two-species synthetic biological systems. We observed that the cyanobacteria were relatively harmless to eukaryotic host cells compared to Escherichia coli when injected into the embryos of zebrafish, Danio rerio, or taken up by mammalian macrophages. In addition, when engineered with invasin from Yersinia pestis and listeriolysin O from Listeria monocytogenes, S. elongatus was able to invade cultured mammalian cells and divide inside macrophages. CONCLUSION: Our results show that it is possible to engineer photosynthetic bacteria to invade the cytoplasm of mammalian cells for further engineering and applications in synthetic biology. Engineered invasive but non-pathogenic or immunogenic photosynthetic bacteria have great potential as synthetic biological devices.

Insulation of a synthetic hydrogen metabolism circuit in bacteria.

BACKGROUND: The engineering of metabolism holds tremendous promise for the production of desirable metabolites, particularly alternative fuels and other highly reduced molecules. Engineering approaches must redirect the transfer of chemical reducing equivalents, preventing these electrons from being lost to general cellular metabolism. This is especially the case for high energy electrons stored in iron-sulfur clusters within proteins, which are readily transferred when two such clusters are brought in close proximity. Iron sulfur proteins therefore require mechanisms to ensure interaction between proper partners, analogous to many signal transduction proteins. While there has been progress in the isolation of engineered metabolic pathways in recent years, the design of insulated electron metabolism circuits in vivo has not been pursued. RESULTS: Here we show that a synthetic hydrogen-producing electron transfer circuit in Escherichia coli can be insulated from existing cellular metabolism via multiple approaches, in many cases improving the function of the pathway. Our circuit is composed of heterologously expressed [Fe-Fe]-hydrogenase, ferredoxin, and pyruvate-ferredoxin oxidoreductase (PFOR), allowing the production of hydrogen gas to be coupled to the breakdown of glucose. We show that this synthetic pathway can be insulated through the deletion of competing reactions, rational engineering of protein interaction surfaces, direct protein fusion of interacting partners, and co-localization of pathway components on heterologous protein scaffolds. CONCLUSIONS: Through the construction and characterization of a synthetic metabolic circuit in vivo, we demonstrate a novel system that allows for predictable engineering of an insulated electron transfer pathway. The development of this system demonstrates working principles for the optimization of engineered pathways for alternative energy production, as well as for understanding how electron transfer between proteins is controlled.

Modular electron transfer circuits for synthetic biology: insulation of an engineered biohydrogen pathway.

Electron transfer is central to a wide range of essential metabolic pathways, from photosynthesis to fermentation. The evolutionary diversity and conservation of proteins that transfer electrons makes these pathways a valuable platform for engineered metabolic circuits in synthetic biology. Rational engineering of electron transfer pathways containing hydrogenases has the potential to lead to industrial scale production of hydrogen as an alternative source of clean fuel and experimental assays for understanding the complex interactions of multiple electron transfer proteins in vivo. We designed and implemented a synthetic hydrogen metabolism circuit in Escherichia coli that creates an electron transfer pathway both orthogonal to and integrated within existing metabolism. The design of such modular electron transfer circuits allows for facile characterization of in vivo system parameters with applications toward further engineering for alternative energy production.

Synthetic biology: exploring and exploiting genetic modularity through the design of novel biological networks.

Synthetic biology has been used to describe many biological endeavors over the past thirty years--from designing enzymes and in vitro systems, to manipulating existing metabolisms and gene expression, to creating entirely synthetic replicating life forms. What separates the current incarnation of synthetic biology from the recombinant DNA technology or metabolic engineering of the past is an emphasis on principles from engineering such as modularity, standardization, and rigorously predictive models. As such, synthetic biology represents a new paradigm for learning about and using biological molecules and data, with applications in basic science, biotechnology, and medicine. This review covers the canonical examples as well as some recent advances in synthetic biology in terms of what we know and what we can learn about the networks underlying biology, and how this endeavor may shape our understanding of living systems.

Myriaporone 3/4 structure--activity relationship studies define a pharmacophore targeting eukaryotic protein synthesis.

Myriaporones are naturally occurring compounds which structurally resemble the southern hemisphere of the tedanolide family of macrolide antitumor agents. Despite the fact that myriaporone 3/4 represents only a portion of tedanolide, it nonetheless retains much of its biological activity. We show here that like tedanolide, myriaporone 3/4 inhibits protein synthesis and proliferation of mammalian cells with low nanomolar potencies but displays no prokaryotic growth inhibitory effect. Moreover, myriaporone 3/4 displays a very rapid, reversible and p21-independent activity to block S phase progression in mammalian cells. Structure-activity relationship studies revealed that the C18-C19 epoxide and the C14 hydroxymethyl group (tedanolide numbering) of myriaporone 3/4 are required for cell cycle inhibition. These constitute previously unidentified and/or novel pharmacophores for myriaporone 3/4. Our results show that the important biological activities associated with the structurally complex tedanolides are present and can be harnessed in the chemically much simpler myriaporones. This greatly increases the value of the latter as investigative tools for biochemical research as well as for development of potential therapeutics.

High-density haplotype structure and association testing of the insulin-degrading enzyme (IDE) gene with type 2 diabetes in 4,206 people.

The insulin-degrading enzyme is responsible for the intracellular proteolysis of insulin. Its gene IDE is located on chromosome 10, in an area with suggestive linkage to type 2 diabetes and related phenotypes. Due to the impact of genetic variants of this gene in rodents and the function of its protein product, it has been proposed as a candidate gene for type 2 diabetes. Various groups have explored the role of the common genetic variation of IDE on insulin resistance and reported associations of various single nucleotide polymorphisms (SNPs) and haplotypes on both type 2 diabetes and glycemic traits. We sought to characterize the haplotype structure of IDE in detail and replicate the association of common variants with type 2 diabetes, fasting insulin, fasting glucose, and insulin resistance. We assessed linkage disequilibrium, selected single-marker and multimarker tags, and genotyped these markers in several case-control and family-based samples totalling 4,206 Caucasian individuals. We observed no statistically significant evidence of association between single-marker or multimarker tests in IDE and type 2 diabetes. Nominally significant differences in quantitative traits are consistent with statistical noise. We conclude that common genetic variation at IDE is unlikely to confer clinically significant risk of type 2 diabetes in Caucasians.

Association testing of the protein tyrosine phosphatase 1B gene (PTPN1) with type 2 diabetes in 7,883 people.

Protein tyrosine phosphatase (PTP)-1B, encoded by the PTPN1 gene, inactivates the insulin signal transduction cascade by dephosphorylating phosphotyrosine residues in insulin signaling molecules. Due to its chromosomal location under a chromosome 20 linkage peak and the metabolic effects of its absence in knockout mice, it is a candidate gene for type 2 diabetes. Recent studies have associated common sequence variants in PTPN1 with type 2 diabetes and diabetes-related phenotypes. We sought to replicate the association of common single nucleotide polymorphisms (SNPs) and haplotypes in PTPN1 with type 2 diabetes, fasting plasma glucose, and insulin sensitivity in a large collection of subjects. We assessed linkage disequilibrium, selected tag SNPs, and typed these markers in 3,347 cases of type 2 diabetes and 3,347 control subjects as well as 1,189 siblings discordant for type 2 diabetes. Despite power estimated at >95% to replicate the previously reported associations, no statistically significant evidence of association was observed between PTPN1 SNPs or common haplotypes with type 2 diabetes or with diabetic phenotypes.

Identification of a molecular recognition role for the activation loop phosphotyrosine of the SRC tyrosine kinase.

A human cDNA phage display library screen, using a phosphopeptide designed to mimic the activation loop phosphotyrosine of the Src tyrosine kinase, has identified the N-terminal SH2 domain of the p85 regulatory subunit of phosphatidyl inositol-3 kinase (PI3K) as an interacting recognition domain. Activation loop phosphorylation is known to play a conformational role in kinase activation, but is largely not thought to play a role in protein/protein recognition. Affinity chromatography and biochemical evaluation in mouse fibroblast cells has confirmed the dependence of this interaction on both the Src activation loop phosphotyrosine and the N-terminal SH2 domain of PI3K.