Studies in bacterial genome engineering and its applications

textMany different approaches exist for engineering bacterial genomes. The most common current methods include transposons for random mutagenesis, recombineering for specific modifications in Escherichia coli, and targetrons for targeted knock-outs. Site-specific recombinases, which can catalyze a variety of large modifications at high efficiency, have been relatively underutilized in bacteria. Employing these technologies in combination could significantly expand and empower the toolkit available for modifying bacteria. Targetrons can be adapted to carry functional genetic elements to defined genomic loci. For instance, we re-engineered targetrons to deliver lox sites, the recognition target of the site-specific recombinase, Cre. We used this system on the E. coli genome to delete over 100 kilobases, invert over 1 megabase, insert a 12-kilobase polyketide-synthase operon, and translocate a 100 kilobase section to another site over 1 megabase away. We further used it to delete a 15-kilobase pathogenicity island from Staphylococcus aureus, catalyze an inversion of over 1 megabase in Bacillus subtilis, and simultaneously deliver nine lox sites to the genome of Shewanella oneidensis. This represents a powerful, versatile, and broad-host-range solution for bacterial genome engineering. We also placed lox sites on mariner transposons, which we leveraged to create libraries of millions of strains harboring rearranged genomes. The resulting data represents the most thorough search of the space of potential genomic rearrangements to date. While simple insertions were often most adaptive, the most successful modification found was an inversion that significantly improved fitness in minimal media. This approach could be pushed further to examine swapping or cutting and pasting regions of the genome, as well. As potential applications, we present work towards implementing and optimizing extracellular electron transfer in E. coli, as well as mathematical models of bacteria engineered to adhere to the principles of the economic concept of comparative advantage, which indicate that the approach is feasible, and furthermore indicate that economic cooperation is favored under more adverse conditions. Extracellular electron transfer has applications in bioenergy and biomechanical interfaces, while synthetic microbial economics has applications in designing consortia-based industrial bioprocesses. The genomic engineering methods presented above could be used to implement and optimize these systems.Cellular and Molecular Biolog

The transcription factors ActR and SoxR differentially affect the phenazine tolerance of Agrobacterium tumefaciens

Bacteria in soils encounter redox‐active compounds, such as phenazines, that can generate oxidative stress, but the mechanisms by which different species tolerate these compounds are not fully understood. Here, we identify two transcription factors, ActR and SoxR, that play contrasting yet complementary roles in the tolerance of the soil bacterium Agrobacterium tumefaciens to phenazines. We show that ActR promotes phenazine tolerance by proactively driving expression of a more energy‐efficient terminal oxidase at the expense of a less efficient alternative, which may affect the rate at which phenazines abstract electrons from the electron transport chain (ETC) and thereby generate reactive oxygen species. SoxR, on the other hand, responds to phenazines by inducing expression of several efflux pumps and redox‐related genes, including one of three copies of superoxide dismutase and five novel members of its regulon that could not be computationally predicted. Notably, loss of ActR is far more detrimental than loss of SoxR at low concentrations of phenazines, and also increases dependence on the otherwise functionally redundant SoxR‐regulated superoxide dismutase. Our results thus raise the intriguing possibility that the composition of an organism's ETC may be the driving factor in determining sensitivity or tolerance to redox‐active compounds

Genes required for sediment fitness in Desulfovibrio desulfuricans G20.

97 genes required for sediment fitness of G20 were identified and the identification of chemotaxis genes validated our STM screening method since it would be expected to enhance sediment fitness. The growth of these sediment fitness mutants was monitored in laboratory growth media to determine whether growth rate influenced sediment fitness. Homology with other delta- proteobacteria was determined and the putative sediment functions of many of these genes were described. Amino acid biosynthesis mutants, hydrogenase mutants, and DNA repair mutants were characterized to prove the importance of specific functions for sediment fitness.A modified version of the signature-tagged mutagenesis (STM) procedure was developed, which incorporated microarray technology to streamline the entire screening process. A mini-Tn10 transposon that is capable of randomly mutagenizing our model sulfate-reducing bacterium was also identified. This system can therefore be applied to other sulfate-reducing bacteria to answer questions of ecological or economical significance. This is the first study using STM to study bacterial environmental fitness.Preliminary experiments were conducted on Desulfovibrio desulfuricans G20 to obtain some basic information of this bacterium. Strain G20 has a single flagellum and a doubling time of 3.2 hours at 37° C in the lactate-sulfate medium, compared to the doubling time of 7.3 and 5.0 hours for strain Essex6 and strain ASR, respectively. G20 is resistant to several antibiotics including nalidixic acid, spectinomycin, and streptomycin.The proteomes of the regulatory mutants were analyzed and compared to the proteome of the parent strain. The reproducible and reliable data produced by the accurate mass and time tag approach allow us to compare protein production in the mutants. The results showed that these regulators regulated different sets of genes which may be required for sediment survival, although the regulatory pathways remain to be elucidated. It is evident that the laboratory-adapted strain is different from sediment-adapted strain at the protein level

In Vivo Gene Essentiality and Metabolism in Bordetella pertussis

Bordetella pertussis is the causative agent of whooping cough, a serious respiratory illness affecting children and adults, associated with prolonged cough and potential mortality. Whooping cough has reemerged in recent years, emphasizing a need for increased knowledge of basic mechanisms of B. pertussis growth and pathogenicity. While previous studies have provided insight into in vitro gene essentiality of this organism, very little is known about in vivo gene essentiality, a critical gap in knowledge, since B. pertussis has no previously identified environmental reservoir and is isolated from human respiratory tract samples. We hypothesize that the metabolic capabilities of B. pertussis are especially tailored to the respiratory tract and that many of the genes involved in B. pertussis metabolism would be required to establish infection in vivo. In this study, we generated a diverse library of transposon mutants and then used it to probe gene essentiality in vivo in a murine model of infection. Using the CON-ARTIST pipeline, 117 genes were identified as conditionally essential at 1 day postinfection, and 169 genes were identified as conditionally essential at 3 days postinfection. Most of the identified genes were associated with metabolism, and we utilized two existing genome-scale metabolic network reconstructions to probe the effects of individual essential genes on biomass synthesis. This analysis suggested a critical role for glucose metabolism and lipooligosaccharide biosynthesis in vivo. This is the first genome-wide evaluation of in vivo gene essentiality in B. pertussis and provides tools for future exploration. IMPORTANCE Our study describes the first in vivo transposon sequencing (Tn-seq) analysis of B. pertussis and identifies genes predicted to be essential for in vivo growth in a murine model of intranasal infection, generating key resources for future investigations into B. pertussis pathogenesis and vaccine design

Synthetic biology approach towards engineering of Shewanella oneidensis MR-1 for microbial fuel cell technologies

In the past decade the emerging field of microbial electrochemical technologies (METs) has gained increased attention due to its potential for bioenergy production and bioremediation. By utilizing pollutants or waste as carbon sources electroactive bacteria (EAB) can convert chemical energy into electricity, thereby conceivably closing the waste disposal energy generation loop. These EABs can generate current anaerobically by forming an electroactive biofilm on conductive electrode materials via extracellular electron transfer (EET). The genetically tractable EAB model organism Shewanella oneidensis MR-1 (SOMR-1) already possesses several EET routes and a large respiration versatility. These traits make it feasible as a synthetic biology chassis to increase predictability, stability and novel functionalities of MET applications. However, as synthetic gene circuits become more elaborate in size and complexity and only relatively few well-characterized biological parts have been described for this organism, precise genetic engineering increasingly presents a bottleneck for this new technology. Here, the synthetic biology toolbox for SOMR-1 was expanded by establishing the Standardised European Vector Architecture (SEVA) plasmid platform providing characterisation of plasmid maintenance with a large range of replication origins, quantification of plasmid copy numbers and their compatibility as multi-plasmid bearing systems in SOMR-1. Further, establishment of transcriptional regulation using oxygen independent inducible promoters was realised. In this work the novel cyclohexanone inducible promoter PChnB/ChnR was introduced among others and characterised using oxygen independent reporter assays. A synthetic flavin gene operon under the control of PChnB/ChnR was used to show enhancement of SOMR-1 EET in small-scale MFCs using screen-printed electrode technology. Additional screening methods are presented which were aimed to identify novel EET capabilities in SOMR-1 using a colorimetric tungsten trioxide (WO3) assay

Energy recovery from organic wastewater via microbial fuel cell technology: a novel approach

One promising technology that offers a potential breakthrough in terms of energy recovery from wastewater efforts is Microbial Fuel Cells (MFCs). This technology exploits the ability of microorganisms, usually bacteria, to oxidise organic matter contained in wastewater and harness the produced electrons to generate electricity. However, despite its promise, current bottlenecks such as long start-up time, low power output and limited understanding of microbial communities central to the process, prevent this technology to achieve its maximum potential. Through this DPhil project, we have expanded our understanding of the underlying science and mechanisms behind Microbial Fuel Cell technology, as well as pushing towards its applications as a simultaneous solution for wastewater treatment and energy crisis problems in industrial scale. In this project, two main Extracellular Electron Transfer (EET) mechanisms for S. oneidensis MR-1 – mediated electron transfer (MET) and direct electron transfer (DET) were evaluated and analysed for their contributions. The results confirm that electron transfer via mediator contributed 70% of power output, and genetic engineering of cells to include additional flavin-production gene from B. subtilis increased the power output by over two-fold. In addition, the in-situ transfer of flavin-overexpression genes into the bacterial cell using ultrasound in an MFC setup was achieved for the first time. This study has also demonstrated a significant scale-up to ultrasound gene transfer technology – with working volume of 300 mL, providing ~150X scale-up than those previously reported elsewhere. Furthermore, the ability of S. oneidensis MR-1 to utilise acetate as sole carbon and energy source in an MFC setup was demonstrated. A voltage of 0.032 0.011 V was generated across 1kΩ resistor with 20 mM sodium acetate as the sole carbon source, with maximum power output that reached 1.2 0.1 mW/m2. The acetate utilisation by S. oneidensis was also demonstrated when using anaerobic digester liquor as MFC substrates – with 16.2 4.1 mg/L of acetate content consumed within 5 days, resulting in ~11% coulombic efficiency. This is a novel finding – as there are no previous literatures that report successful utilization of pure culture S. oneidensis to degrade acetate from real AD liquor for electricity generation. Furthermore, this further supports claims that have been made by other researchers – that acetate utilization by MR-1 is not limited only under aerobic condition. Finally, the viable application of magnetic nanoparticles (MNPs) in MFC setup was demonstrated. The deployment of silica-coated Iron-Oxide Nanoparticles (ION) prevented oxidative nature of the iron core, while maintaining the magnetic property of the nanoparticles. The combined result of these characteristics enabled the use of nanoparticles to form engineered biofilm on the electrode surface without compromising its electricity production. A voltage of ~40 mV was achieved using E. coli – S. oneidensis MR-1 consortium to degrade glucose, with maximum power production of 39.8 ± 2.4 mW/m2. The biofilm composition was found to have shifted towards a community predominated by the favoured electrigen which is MR-1 strains, reaching 38.3 ± 7.0% of total cell population – around 5-fold higher compared to 7.4 ± 4.2% of that the control where the nanoparticles were not present. To the best of our knowledge, this is the first reported study of MNP application in MFC as coating agent for bacterial cell – for the purpose of selective biofilm formation of electrigen-enriched electrode. Future research trends should be focused on the advancement of electrode materials towards cheaper, more biocompatible, and higher effective surface area which promote better biofilm attachment, and further understanding of the biology within bacteria consortium that is often very complex – coupled with genetic engineering and modifications to implement capabilities across bacterial species for complex substrate degradation and enhanced electricity generation capabilities. This study has contributed majorly to some of these aspects through its novel result and findings, although further studies, sensitivity analyses and development are still required to reach our target end-state. In the future, we believe that the application of MFC should not be limited to wastewater treatment, but also form part of important integrations with other technologies such as biosensory systems, anaerobic digester as well as energy storage and chemical productions. All these milestones should be achieved as we advance our understanding of the science and underlying mechanisms behind the technology

Systems analysis of minimal metabolic networks In prokaryotes

PhD Thesis in Chemical and Biological EngineeringThe complexity of living cells is staggering, as a result of billions of years of evolution through natural selection in constantly changing environments. Systems biology emerges as the preferred approach to the disentangling of this complexity by looking at living cells and their responses to environments in a holistic manner. Complete annotated sequences of genomes are now available for thousands of species of the simplest unicellular life forms known, the prokaryotes. Together with other large-scale datasets as proteomes and phenotypic screenings and a careful analysis of the literature, genome annotations allow for the reconstruction of large constraint-based models of cellular metabolism. Here, genome-scale metabolic models (GSMs) of prokaryotes are used together with other disparate large-scale datasets and literature assessments to study and predict essential components in minimal metabolic networks. A conceptual clarification is presented in a review of systems biology perspectives on minimal and simpler cells. An assessment of the biomass compositions in 71 GSMs of prokaryotes was then performed, revealing heterogeneity that impacted predictions of reaction essentiality. The integration of 33 large-scale essentiality assays with other data and literature revealed universally and conditionally essential cofactors for prokaryotes. These were used to revise predictions of essential genes and in the prediction of one biosynthetic pathway in the GSM of M. tuberculosis. Additionally, a large-scale assessment of essentiality of different metabolic subsystems was performed with 15 comparable GSMs. The results were validated with 36 large-scale experimental assays of gene essentiality. The ancestry of metabolic genes and subsystems was estimated by blasting representative genomes of all the phyla in the prokaryotic tree of life. Ancestry was correlated with essentiality in general but not with non-essentiality. Finally, a method was devised to generate minimal viable metabolic networks based on a curated and diverse universe of prokaryotic metabolic reactions. Different growth media were tested and shown to generate different networks regarding size, cofactor requirements and maximum biomass production. The results of this work are expected to contribute for fundamental investigations of core and ancestral prokaryotic metabolism and the design of modularized and controllable chassis cells.A complexidade das células vivas é surpreendente, como resultado de milhares de milhões de anos de evolução através de seleção natural em ambientes em constante mudança. A Biologia de sistemas surge como a abordagem preferencial para analisar esta complexidade por examinar as células e as suas respostas ao meio de uma forma holística. Estão hoje disponíveis sequências completas e anotadas de genomas para milhares de espécies das formas de vida unicelulares mais simples conhecidas, os procariotas. Juntamente com outros conjuntos de dados de larga escala como proteomas e triagens fenotípicas e uma análise cuidadosa da literatura, os genomas anotados permitem a reconstrução de grandes modelos do metabolismo celular baseados em restrições. Neste trabalho utilizam-se modelos metabólicos à escala genómica (GSMs) de procariotas em conjunto com outros grandes conjuntos de dados díspares e avaliações da literatura para estudar e prever componentes essenciais em redes metabólicas mínimas. Um esclarecimento conceptual é apresentado numa revisão de perspectivas da biologia de sistemas sobre células mínimas e mais simples. Segue-se uma avaliação das composições de biomassa em 71 GSMs de procariotas, revelando a heterogeneidade que afecta as previsões de essencialidade de reações. Com a integração de 33 ensaios em grande escala de essencialidade com outros dados e literatura, revelam-se cofactores essenciais universais e condicionais em procariotas. Estes foram utilizados na revisão de previsões de genes essenciais e na previsão de uma via biossintética no GSM de M. tuberculosis. Adicionalmente, foi realizada uma avaliação em larga escala de essencialidade de diferentes subsistemas metabólicos com 15 GSMs comparáveis. Os resultados foram validados com 36 ensaios experimentais de essencialidade em larga escala. A ancestralidade de genes metabólicos e subsistemas foi estimada por blast a genomas representativos de todos os filos na árvore da vida procariota. A ancestralidade revelou-se correlacionada com a essencialidade em geral, mas não com a não essencialidade. Finalmente, concebeu-se um método para gerar redes metabólicas mínimas viáveis com base num universo curado e diversificado de reações metabólicas procariotas. Diferentes meios de crescimento foram testados, mostrando-se a geração de diferentes redes em relação ao tamanho, os requisitos de cofactores e a produção de biomassa máxima. Espera-se que os resultados deste trabalho contribuam para investigações fundamentais dos metabolismos essencial e ancestral de procariotas e para o desenho de células chassis modulares e controláveis.This work was funded by FCT, the Portuguese Foundation for Science and Technology, with the grant SFRH/BD/81626/201

Implementation of nano-liquid chromatography hyphenated to tandem mass spectrometry for protein identification in gel and non-gel based proteomics

This thesis is divided into three parts. Part I deals with the current status of large scale strategies for the analysis of proteins in biological systems. In Chapter I, genomics and the need to shift towards proteomic approaches are outlined. An overview of key technologies used in functional and structural proteomics is provided in Chapter II, whereas mass spectrometry and the strategies for profiling of proteins are discussed in detail in the final chapter of this part (Chapter III). Part II describes the analysis of cytosolic proteins by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. First, the techniques are highlighted that were used to identify gel separated proteins, i.e. the implementation of nano-liquid chromatography and mass spectrometry (Chapter IV). The next two chapters are applications of the developed methodology in two case studies: the protein composition of the dissimilatory iron-reducing bacterium Shewanella oneidensis MR-1 grown on ferric oxide (Chapter V) and the effect of a short-term heat shock on the plant barley (Chapter VI). Part III deals with the development of alternative strategies for the analysis of membrane proteins. A special electrophoretic technique, i.e. blue-native polyacrylamide gel electrophoresis, in combination with mass spectrometry, was used for profiling the different subunits of the oxidative phosphorylation system (Chapter VII), while multi-dimensional liquid chromatography coupled to MALDI tandem mass spectrometry was used for the profiling of the proteins present in the murine myelin sheath (Chapter VIII)