Steps and Tools for PCR-Based Technique Design

The identity and clonal differences within bacterial populations have been broadly explored through PCR-based techniques. Thus, bacterial identification and elucidation of DNA fingerprinting have provided insights regarding their phenotypic and genotypic variations. Indeed, some diversity of rates may reflect changes among subpopulations that have their own ecological dynamic and individual traits on coexisting genotypes. Therefore, identification of polymorphic regions from nucleic acid sequences is based on the identification of both conserved and variable regions. Advantages of PCR-based methods are high sensitivity, specificity, speed, cost-effectiveness, and the opportunity for simultaneous detection of many microbial agents or variants. Fingerprint information might allow the tracking of certain outbreaks globally in several reference databases containing valuable genotyping information. In this chapter, we will review applications from Web resources and computational tools online for the designing of PCR-based methods to identify bacterial species. We will also focus on lab applications and key conditions for technique standardization

Bacterial Degradation of Isoprene in the Terrestrial Environment

Isoprene is a climate active gas emitted from natural and anthropogenic sources in quantities equivalent to the global methane flux to the atmosphere. 90 % of the emitted isoprene is produced enzymatically in the chloroplast of terrestrial plants from dimethylallyl pyrophosphate via the methylerythritol pathway. The main role of isoprene emission by plants is to reduce the damage caused by heat stress through stabilizing cellular membranes. Isoprene emission from microbes, animals, and humans has also been reported, albeit less understood than isoprene emission from plants. Despite large emissions, isoprene is present at low concentrations in the atmosphere due to its rapid reactions with other atmospheric components, such as hydroxyl radicals. Isoprene can extend the lifetime of potent greenhouse gases, influence the tropospheric concentrations of ozone, and induce the formation of secondary organic aerosols. While substantial knowledge exists about isoprene production and atmospheric chemistry, our knowledge of isoprene sinks is limited. Soils consume isoprene at a high rate and contain numerous isoprene-utilizing bacteria. However, Rhodococcus sp. AD45 is the only terrestrial isoprene-degrading bacterium characterized in any detail. A pathway for isoprene degradation involving a putative soluble monooxygenase has been proposed. In this study, we report the isolation of two novel isoprene-degrading bacteria and characterization of the isoprene gene clusters in their draft genomes. Using marker exchange mutagenesis, transcription assays and proteomics analyses, we provide conclusive evidence that isoprene is metabolized in Rhodococcus sp. AD45 through the induced activity of soluble isoprene monooxygenase, a close relative to well known soluble diiron center monooxygenase enzymes. Metabolic gene PCR assays based on a key component of isoprene monooxygenase were also developed to detect isoprene degraders in the environment. The diversity of active isoprene degraders in the terrestrial environment was investigated using DNA-stable isotope probing experiments combined with 454 pyrosequencing

Analysis of the interaction between chromosomal replication and transposition mediated by sliding clamps

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 20 de mayo de 2016Insertion sequences (ISs) are small mobile genetic elements widely distributed in prokaryotes. They often encode only one enzyme, the transposase, required for their own transposition. ISs are promiscuous elements that can proliferate within genomes, where they play a key role in genome evolution by promoting chromosomal rearrangements and genetic flow. Furthermore, ISs have the ability to cross species barriers and transpose actively in new hosts, which also makes ISs essential players in the process of horizontal gene transfer. Although highly autonomous, ISs activity is linked to and can be regulated by various host processes, especially chromosomal replication; however no general mechanism had been proposed connecting replication with transposition. In this thesis we investigated the interplay between transposases and host replication factors. First, we performed a survey of orientation patterns of IS in fully-sequenced bacterial chromosomes. We found that a significant fraction of IS families present a consistent and family-specific orientation bias with respect of the movement of the replication fork, especially in Firmicutes. Then, we found that the transposases of up to ten different IS families with different transposition pathways interact with E. coli β sliding clamp, an essential replication factor. Additionally, we demonstrated that purified transposase of Tn5 also interact with β sliding clamp. Moreover, we studied to what extent the interaction limits or favors the ability of ISs to colonize a chromosome from a phylogenetically-distant organism. We describe the proliferation of a member of the IS1634 family in a long-term culture of Acidiphilium sp. We found that the Acidiphilium IS1634 transposase binds to β sliding clamp of Acidiphilium, Leptospirillum and E. coli. Further, we also demonstrated that Acidiphilium IS1634 transposase binds to the archaeal sliding clamp (PCNA) from Methanosarcina, and that the transposase encoded by Methanosarcina IS1634 binds Acidiphilium β. Finally, we demonstrated that strengthening the interaction between β and the transposase results in an increased transposition rate in vivo. Our results strongly suggest that transposase interaction with sliding clamps is a widespread mechanism that allows ISs integration with host chromosomal replication. Interaction with β and asymmetries in β distribution in the replication fork could explain the observed strong orientation bias found in some IS families in Firmicutes. Sliding clamps may represent a universal and highly conserved platform for ISs dispersal between species. The strength of the interaction could determine the potential of ISs to be mobilized in bacterial populations and also their ability to proliferate within chromosome.Las secuencias de inserción (SI) son pequeños elementos genéticos móviles ampliamente distribuidos en procariotas. Habitualmente codifican para una solo enzima, la transposasa, requerida para su propia transposición. Las SI son elementos promiscuos que puede proliferar en los cromosomas, donde juegan un papel clave en la evolución de los genomas promoviendo la reorganización cromosómica y el flujo genético. Además, las SI tienen la habilidad para cruzar la barrera inter-especie y transponerse activamente en nuevos huéspedes, lo que también las convierte en actores esenciales en procesos de transferencia génica horizontal. Aunque son altamente autónomas, la actividad de las SI está ligada y puede ser regulada por varios procesos del hospedador, especialmente la replicación del cromosoma; sin embargo no se ha propuesto ningún mecanismo general conectando la replicación con la transposición. En esta tesis investigamos la interacción entre transposasas y factores de replicación del hospedador. Inicialmente, realizamos un estudio de los patrones de orientación de las SI en cromosomas bacterianos completamente secuenciados. Encontramos que una fracción significativa de familias de SI presentan un sesgo de orientación consistente y específico de la familia, con respecto al movimiento de la horquilla de replicación, especialmente en Firmicutes. Además hallamos que la transposasa de hasta 10 familias distintas de SI con diferentes mecanismos de transposición, interaccionan con β sliding clamp de E. coli, un factor esencial de la replicación. Asimismo, demostramos que la transposasa purificada de Tn5 también interacciona con β sliding clamp. Además estudiamos hasta qué punto esta interacción limita o favorece la habilidad de las SI para colonizar cromosomas de organismos distantes filogenéticamente. Describimos la proliferación de un miembro de la familia IS1634 en un cultivo de larga duración de Acidiphilium sp. y demostramos que la transposasa de IS1634 de Acidiphilium, interacciona con β sliding clamp de Acidiphilium, Leptospirillum y E. coli. Más aún, demostramos que la transposasa de IS1634 de Acidiphilium también interacciona con el sliding clamp de la arquea (PCNA) Methanosarcina, y que la transposasa codificada por IS1634 de Methanosarcina interacciona con β de Acidiphilium. Finalmente, demostramos que fortaleciendo la interacción entre β y la transposasa resulta en un incremento en la tasa de transposición in vivo. Nuestros resultados sugieren consistentemente que la interacción entre transposasa y sliding clamp es un mecanismo ampliamente distribuido que permite a las SI integrarse con la replicación de los cromosomas hospedadores. La interacción con β y las asimetrías en la distribución de β en la horquilla de replicación, podrían explicar los fuertes sesgos en la orientación de ciertas familias de SI en Firmicutes. Los sliding clamps pueden representar una plataforma universal y altamente conservada para la dispersión de las SI entre especies. La afinidad de la interacción puede determinar el potencial de las SI para movilizarse en poblaciones bacterianas y también su habilidad para proliferar dentro de los propios cromosomas

Characterization and design of C2H2 zinc finger proteins as custom DNA binding domains

As the storage medium for the source code of life, DNA is fundamentally linked to all cellular processes. Nature employs hundreds of sequence-specific DNA binding proteins as transcription factors and repressors to regulate the flow of genetic expression and replication. By adapting these DNA-binding domains to target desired genome locations, they can be harnessed to treat diseases by regulating genes and repairing diseased gene sequences. The C2H2 zinc finger motif is perhaps the most promising and versatile DNA binding framework. Each C2H2 zinc finger domain (module) is capable of recognizing approximately three adjacent nucleotide bases in standard B form DNA. Through directed mutagenesis, novel zinc finger modules (ZFMs) can be selected for most of the 64 possible DNA triplets. By assembling multiple ZFMs with the appropriate linkers, zinc finger proteins (ZFPs) can be generated to specifically bind extended DNA sequence motifs. Several methods of varying complexity are currently available for ZFP engineering. ZFPs generated from the relatively simple modular design method often fail to function in vivo. Those generated using the most reliable module subsets, those recognizing triplets with a 5\u27 guanine (GNN), only function successfully only an estimated 50% of the time, while modularly assembled ZFPs comprising primarily non-GNN modules rarely function in vivo. These low success rates are extremely problematic for applications requiring multiple ZFPs that target adjacent sequence motifs. More complex ZFP engineering approaches provide enhanced success rates, as compared to modular design, with the drawback that they are also more labor intensive and require additional biological expertise. In this research we developed and engineered novel ZFPs, analyzed characteristics of functional custom zinc finger proteins and their targets, formulated algorithms predictive of ZFP success for both modular assembly and OPEN (Oligomerized Pool Engineering) selection methods, and generated a web-based server and software tools to aid others in the successful application of this technology

Discovery and evolution of novel Cre-type tyrosine site-specific recombinases for advanced genome engineering

Tyrosine site-specific recombinases (Y-SSRs) are DNA editing enzymes that play a valuable role for the manipulation of genomes, due to their precision and versatility. They have been widely used in biotechnology and molecular biology for various applications, and are slowly finding their spot in gene therapy in recent years. However, the limited number of available Y-SSR systems and their often narrow target specificity have hindered the full potential of these enzymes for advanced genome engineering. In this PhD thesis, I conducted a comprehensive investigation of novel Y-SSRs and their potential for advancing genome engineering. This PhD thesis aims to address the current limitations in the genetic toolbox by identifying and characterizing novel Cre-type recombinases and demonstrating their impact on the directed evolution of designer recombinases for precise genome surgery. To achieve these aims, I developed in a collaboration a comprehensive prediction pipeline, combining a rational bioinformatical approach with knowledge of the biological functions of recombinases, to enable high success rate and high-throughput identification of novel tyrosine site-specific recombinase (Y-SSR) systems. Eight putative candidates were molecularly characterized in-depth to ensure their successful integration into future genome engineering applications. I assessed their activity in prokaryotes (E. coli) and eukaryotes (human cell lines), and determined their specificity in the sequence space of all known Cre- type target sites. The potential cytotoxicity associated with cryptic genomic recombination sites was also explored in the context of recombinase applicability. This approach allowed the identification of novel Y-SSRs with distinct target sites, enabling simultaneous use of multiple Y-SSR systems, and provided knowledge that will facilitate the assignment of novel and known recombinases to specific uses or organisms, ensuring their safe and effective implementation. The introduction of these novel Y-SSRs into the genome engineering toolbox opens up new possibilities for precise genome manipulation in various applications. The broader targetability offered by these enzymes could accelerate the development of novel gene therapies, as well as advance the understanding of gene function and regulation. Moreover, these recombinases could be used to design custom genetic circuits for synthetic biology, allowing researchers to create more complex and sophisticated cellular systems. Finally, I introduced the novel Y-SSRs into efforts aimed at developing designer recombinases for precise genome surgery, demonstrating their impact on accelerating the directed evolution process. Therapeutically relevant recombinases with altered DNA specificity have been developed for excision or inversion of specific DNA sequences. However, the potential for evolving recombinases capable of integrating large DNA cargos into naturally occurring lox-like sites in the human genome remained untapped so far. Thus, I embarked on evolving the Vika recombinase to mediate the integration of DNA cargo into a native human sequence. I discovered that Vika could integrate DNA into the voxH9 site in the human genome, and then, I enhanced the process through directed evolution. The evolved variants of Vika displayed a marked improvement in integration efficiency in bacterial systems. However, the translation of these results into mammalian systems has not yet been entirely successful. Despite this, the study laid the groundwork for future research to optimize the efficiency and applicability of Y-SSRs for genomic integration. In summary, this thesis made significant strides in the identification, characterization, and development of novel Y-SSRs for advanced genome engineering. The comprehensive prediction pipeline, combined with in-depth molecular characterization, has expanded the genetic toolbox to meet the growing demand for better genome editing tools. By exploring efficiency, cross-specificity, and potential cytotoxicity, this research lays the foundation for the safe and effective application of novel Y-SSRs in various therapeutic settings. Furthermore, by demonstrating the potential of these recombinases to improve efforts in creating designer recombinases through directed evolution, this research has opened new avenues for precise genome surgery. The successful development and implementation of these novel recombinases have the potential to revolutionize gene therapy, synthetic biology, and our understanding of gene function and regulation

From prediction to function: Polyamine biosynthesis and formate metabolism in the α- and ε-Proteobacteria

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

On Computable Protein Functions

Proteins are biological machines that perform the majority of functions necessary for life. Nature has evolved many different proteins, each of which perform a subset of an organism’s functional repertoire. One aim of biology is to solve the sparse high dimensional problem of annotating all proteins with their true functions. Experimental characterisation remains the gold standard for assigning function, but is a major bottleneck due to resource scarcity. In this thesis, we develop a variety of computational methods to predict protein function, reduce the functional search space for proteins, and guide the design of experimental studies. Our methods take two distinct approaches: protein-centric methods that predict the functions of a given protein, and function-centric methods that predict which proteins perform a given function. We applied our methods to help solve a number of open problems in biology. First, we identified new proteins involved in the progression of Alzheimer’s disease using proteomics data of brains from a fly model of the disease. Second, we predicted novel plastic hydrolase enzymes in a large data set of 1.1 billion protein sequences from metagenomes. Finally, we optimised a neural network method that extracts a small number of informative features from protein networks, which we used to predict functions of fission yeast proteins

Decoding Microbial Genomes: Novel User-Friendly Tools Applied to Fermented Foods

Over the past two decades, the cost of DNA sequencing per base has significantly outpaced Moore's law. Many organizations and research groups have exploited this trend and generated large amounts of genomic data and made it possible to tackle new research questions. This growth also brings challenges, including the need for faster algorithms, more efficient ways to visualize and explore data, more automatized data processing, and systematic data management. For more than a century, Agroscope collects lactic acid bacteria (LAB) extracted from the Swiss dairy environment. Today, the collection comprises more than 10’000 strains and so far, for about 15% of the strains the genome was sequenced. The over-arching goal of this thesis is to find new ways of exploiting this genetic potential to design new fermented food products including potential additional health benefits, and to understand the underlying mechanisms. One compound with potential health benefits is indole. Previous experiments have shown that indole compounds modulate the gut immune system via the aryl hydrocarbon receptor (AhR). Our objective was to create a yoghurt enriched in indole metabolites through fermentation, and then to examine whether maternal consumption of this yoghurt would enhance gut immune system maturation in germ-free mice. To reduce the number of strains to test, I developed comparative genomics tools to pre-select strains from the strain collection. This led to the successful development of a yoghurt with significantly increased AhR activation activity. In germ-free mice, we could show the expected effect. Based on these comparative genomics tools, I developed the software OpenGenomeBrowser to enable biologists, who know their organisms of interest in great detail, to efficiently explore the genomic data by themselves, without bioinformatics skills or the need for a middleman bioinformatician. The foundation of OpenGenomeBrowser is a simple system for transparent data management of microbial genomes which makes the automation of common bioinformatics workflows possible. In addition, I built a user-friendly website based on modern web technologies to facilitate common bioinformatics workflows. Because of OpenGenomeBrowser's solid foundation, it is the first software of its kind that can be self-hosted and is dataset-independent, making it potentially useful for many similar genome datasets. During the project, we measured thousands of metabolites in yoghurts made using different strains. However, we experienced that no existing tools could adequately connect such a high-dimensional phenotypic dataset to the genomic information, i.e., presence-absence of orthogenes. Finding high-confidence causative links between these datasets is challenging because of the properties of microbial genomes. For instance, clonal reproduction leads to genome-wide linkage disequilibrium, which prohibits the use of techniques developed for human genome-wide association studies (hGWAS). To this end, I developed Scoary2, a complete rewrite and extension of the original microbial GWAS (mGWAS) software Scoary. The key improvements include an implementation of the core algorithm that is orders of magnitude faster and an interactive web-app that enables efficient data exploration of the output, which is crucial given the size of the dataset. With this software, we discovered two previously uncharacterized genes involved in the carnitine metabolism

DEVELOPMENT AND APPLICATION OF MASS SPECTROMETRY-BASED PROTEOMICS TO GENERATE AND NAVIGATE THE PROTEOMES OF THE GENUS POPULUS

Historically, there has been tremendous synergy between biology and analytical technology, such that one drives the development of the other. Over the past two decades, their interrelatedness has catalyzed entirely new experimental approaches and unlocked new types of biological questions, as exemplified by the advancements of the field of mass spectrometry (MS)-based proteomics. MS-based proteomics, which provides a more complete measurement of all the proteins in a cell, has revolutionized a variety of scientific fields, ranging from characterizing proteins expressed by a microorganism to tracking cancer-related biomarkers. Though MS technology has advanced significantly, the analysis of complicated proteomes, such as plants or humans, remains challenging because of the incongruity between the complexity of the biological samples and the analytical techniques available. In this dissertation, analytical methods utilizing state-of-the-art MS instrumentation have been developed to address challenges associated with both qualitative and quantitative characterization of eukaryotic organisms. In particular, these efforts focus on characterizing Populus, a model organism and potential feedstock for bioenergy. The effectiveness of pre-existing MS techniques, initially developed to identify proteins reliably in microbial proteomes, were tested to define the boundaries and characterize the landscape of functional genome expression in Populus. Although these approaches were generally successful, achieving maximal proteome coverage was still limited by a number of factors, including genome complexity, the dynamic range of protein identification, and the abundance of protein variants. To overcome these challenges, improvements were needed in sample preparation, MS instrumentation, and bioinformatics. Optimization of experimental procedures and implementation of current state-of-the-art instrumentation afforded the most detailed look into the predicted proteome space of Populus, offering varying proteome perspectives: 1) network-wide, 2) pathway-specific, and 3) protein-level viewpoints. In addition, we implemented two bioinformatic approaches that were capable of decoding the plasticity of the Populus proteome, facilitating the identification of single amino acid polymorphisms and generating a more accurate profile of protein expression. Though the methods and results presented in this dissertation have direct implications in the study of bioenergy research, more broadly this dissertation focuses on developing techniques to contend with the notorious challenges associated with protein characterization in all eukaryotic organisms