Functional genomics, analysis of adaptation in and applications of models to the metabolism of engineered Escherichia coli

In order to examine the metabolism of bacteria in the genus Enterobacteriaceae tools for gene complement comparison and stoichiometric model building have been developed to take advantage of both the number of complete bacterial genome sequences currently available and the relationship between genes and metabolism. A functional genomic approach to improving knowledge of the metabolism of Escherichia coli CFT073 (a uropathogen) has been undertaken taking into account not only its genome sequence, but its close relationship to E. coli MG1655. A fresh comparison of E. coli CFT073 has been done with E. coli MG1655 to identify all those genes in CFT073 that are not present in MG1655 and may have metabolic characteristics. These genes have further been bioinformatically assessed to determine whether they might encode enzymes for the metabolism of chemicals commonly found in human urine, and one set of such genes has been experimentally confirmed to encode an L-sorbose utilisation pathway. Little experimental work has been done as yet to elucidate how bacteria adaptively respond to the introduction of heterologous metabolic genes. To investigate how bacteria respond to such DNA, genes encoding the L-sorbose utilisation and uptake operon from CFT073 have been cloned and transformed into DH5 and a selective pressure (minimal medium with L-sorbose as sole carbon source) has been applied over 100 generations of growth of this strain in serial passage to investigate the change in its behaviour. The availability of large numbers of completely sequenced genomes, along with the development of a stoichiometric metabolic model with very high coverage of E. coli metabolism (iAF1260 [1]) have made possible the analysis of the core metabolism of large numbers of bacteria to investigate gene essentiality in these bacteria. A novel way of assessing gene complement has been developed using BLAST and DiagHunter to improve reliability of gene synteny comparisons with contextual information about the genes and to extend work by others to cover all E. coli and Shigella genome sequences with available sequences on GanBank (as of 1st June 2009) in order to bioinformatically investigate essential genes in these bacteria and the heterogeneity of their metabolic networks. Further to this a metabolic model has been constructed for DH5 with an added L-sorbose pathway and for CFT073 and these models have been used to investigate behavioural changes during adaptation of bacteria to novel heterologous genes

Bioprospecting of Trichococcus species

Since 1928 with the discovery of penicillin, the value of microbes in our society significantly was reconsidered. Nowadays, 60% of commercial drugs and products mimic or derive from microbialmetabolites. After almost a century, can we find new compounds and where? For addressing thisquestion, we need a large-scale screening of the microbial capabilities. Trichococcus species have multiple genes for producing 1,3-propanediol (1,3-PDO), which synthesizes the partially biodegradable plastic PTT. Based on this, we developed a strategy for analyzing 90,000 bacterial genomes that eventually generated information for every microbial characteristic. The outstanding factor is that all this information is stored in a database that can be easily mined for everything. This collective andunbiased strategy resulted in identifying the key genes for efficient production of 1,3-PDO. We discovered 187 novel candidates that can produce 1,3-PDO and some were in the lab confirmed. Another result of the screening was about Trichococcus patagoniensis. This bacterium grows in minus 5 degrees without oxygen and was discovered by NASA scientists to simulate life in other planets. When it is cold and without oxygen, T. patagoniensis “extra-terrestrial” properties allow it to create its own ”blanket” by producing exopolymer saccharides. We characterized this cryoprotectant compound as inulin, which prevents crystallization of water and many plants use it for preserving their roots in subzero temperatures. Furthermore, inulin is a commercial prebiotic and is connected with gut health. Considering the bacterial kingdom, there are limited members producing inulin and none of them wereidentified as prychrotolerant species. T. patagoniensisis produces plenty of inulin and due to its robustness, easily can be the next biofactory for the compound.The applied methods in this PhD thesis is a platform for mining every bacterial or metabolic information. All the knowledge is there and we need to dive into it. Every finding will be revolutionary and expand our perspective for microbes. Big data mining is like Zenos Dichotomy paradox, we will always know half and never everything.</p

Environmental modulation of microbial ecosystems

Natural microbiota are essential to the health of living systems - from the human gut to coral reefs. Although advances in DNA sequencing have allowed us to catalogue many of the different organisms that make up these microbial communities, significant challenges remain in understanding the complex networks of interspecies metabolic interactions they exhibit. These interactions are crucial to community stability and function, and are highly context-dependent: the availability of different nutrients can determine whether a set of microbes will interact cooperatively or competitively, which can drastically change a community’s structure. Disentangling the environmental factors that determine these behaviors will not only fundamentally enhance our knowledge of their ecological properties, but will also bring us closer to the rational engineering of synthetic microbiomes with novel functions. Here, I integrate modeling and experimental approaches to quantify the dependence of microbial communities on environmental composition. I then show how this relationship can be leveraged to facilitate the design of synthetic consortia. The first chapter of this dissertation is a review article that introduces a framework for cataloguing interaction mechanisms, which enables quantitative comparisons and predictive models of these complex phenomena. The second chapter is a computational study that explores one such attribute – metabolic cost – in high detail. It demonstrates how a large variety of molecules can be secreted without imposing a fitness cost on microbial organisms, allowing for the emergence of beneficial interspecies interactions. The third chapter is an experimental study that determines how the number of unique environmental nutrients affects microbial community growth and taxonomic diversity. The integration of stoichiometric and consumer resource models enabled the discovery of basic ecological principles that govern this environment-phenotype relationship. The fourth chapter applies these principles to the design of engineered communities via a search algorithm that identifies environmental compositions that yield specific ecosystem properties. This dissertation then concludes with extensions of the modeling methods used throughout this work to additional model systems. Future work could further quantify how microbial community phenotypes depend on each of the individual factors explored in this thesis, while also leveraging emerging knowledge on interaction mechanisms to design synthetic consortia

ELECTROPHORETIC REGISTERS: CORRECCIÓN DE LÍNEA BASE, ALINEAMIENTO, ELIMINACIÓN DE RUIDO Y CONCENTRACION DE ENERGÍA

Este artículo presenta una nueva metodología para lacorrección de línea base, el alineamiento de picos, la eliminación de ruido y la clasificación de datos electroforéticos (DER). Las técnicas matemáticas usadas fueron: corrección linea base L2, análisis wavelet,alineamiento Nedleman and Wusch y análisis multi energía que fue utilizada con el propósito de ubicar información importante en los aminoácidos presentes en los datos electroforéticos. Adicionalmente,este artículo evidencia que es posible corregir la línea base, eliminar ruido y alinear datos electroforéticos sin cambiar sus características originales de energía y formas de onda. Se utilizaron 35 señales, en las cuales el porcentaje de error en cuanto a corrección de línea base fue de 0%, en alineamiento fue de 1.23%, y de clasificación fue de 3.5±1.2.Finalmente, se muestra que es posible utilizar esta metodología sin importar las características de las señales originales, lo que permite que sea un sistema de múltiples soluciones en los datos electroforéticos,cromatográficos y otros

Induction of antibiotic tolerance in bacteria by self-produced and inter-species signaling

Thesis (Ph.D.)--Boston UniversityThough most bacteria within a population are killed by high concentrations of antibiotics, tolerant bacteria survive and can re-grow once antibiotics are removed. Bacterial persisters are dormant cells within an isogenic bacterial population that are tolerant to antibiotic treatment and have been implicated in chronic and recurrent infections. Tolerant and persistent bacteria are generated heterogeneously within populations, and a complete understanding of the processes by which these cells are formed remains elusive. However, there is increasing evidence that bacterial communication by chemical signaling plays a role in establishing population heterogeneity. Here I show that bacterial communication induces persistence in Escherichia coli using the self-produced signaling molecule indole. Indole-induced persister formation was monitored using microfluidics, and oxidative stress and phage-shock pathways were determined to play a role in this phenomenon. I propose a model in which indole signaling "inoculates" a bacterial sub-population against antibiotics by activating stress responses, leading to persister formation. Having demonstrated that communication using the signaling molecule indole controls persistence in the intestinal bacterium E. coli, I sought to determine whether indole could be used as an interspecies signal to control antibiotic tolerance in mixed microbial communities. The common bacterial pathogen Salmonella typhimurium was chosen for these experiments because this species, though closely related to E. coli, does not produce indole. The results demonstrated that indole signaling by E. coli induces tolerance to antibiotics in S. typhimurium. Further, the data suggest that indole-induced tolerance in S. typhimurium is mediated at least in part by the phage shock and oxidative stress response pathways, which were previously implicated in control of indole-induced persistence in E. coli. I used C. elegans as a simple in vivo model for gastrointestinal infection with S. typhimurium, demonstrating that indole signaling increased Salmonella tolerance and altered heterogeneity of infection in this system. These results suggest that antibiotic tolerance in pathogens may be induced by interception of bacterial signals in the host environment

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

Understanding Fermentative Glycerol Metabolism and its Application for the Production of Fuels and Chemicals

Due to its availability, low-price, and higher degree of reduction than lignocellulosic sugars, glycerol has become an attractive carbon source for the production of fuels and reduced chemicals. However, this high degree of reduction of carbon atoms in glycerol also results in significant challenges in regard to its utilization under fermentative conditions. Therefore, in order to unlock the full potential of microorganisms for the fermentative conversion of glycerol into fuels and chemicals, a detailed understanding of the anaerobic fermentation of glycerol is required. The work presented here highlights a comprehensive experimental investigation into fermentative glycerol metabolism in Escherichia coli, which has elucidated several key pathways and mechanisms. The activity of both the fermentative and respiratory glycerol dissimilation pathways was found to be important for maximum glycerol utilization, a consequence of the metabolic cycle and downstream effects created by the essential involvement of PEP-dependent dihydroxyacetone kinase (DHAK) in the fermentative glycerol dissimilation pathway. The decoupling of this cycle is of central importance during fermentative glycerol metabolism, and while multiple decoupling mechanisms were identified, their relative inefficiencies dictated not only their level of involvement, but also implicated the activity of other pathways/enzymes, including fumarate reductase and pyruvate kinase. The central role of the PEP-dependent DHAK, an enzyme whose transcription was found to be regulated by the cyclic adenosine monophosphate (cAMP) receptor protein (CRP)-cAMP complex, was also tied to the importance of multiple fructose 1,6-bisphosphotases (FBPases) encoded by fbp, glpX, and yggF. The activity of these FBPases, and as a result the levels of fructose 1,6-bisphosphate, a key regulatory compound, appear to also play a role in the involvement of several other enzymes during fermentative glycerol metabolism including PEP carboxykinase. Using this improved understanding of fermentative glycerol metabolism as a platform, E. coli has been engineered to produce high yields and titers of ethanol (19.8 g/L, 0.46 g/g), co-produced along with hydrogen, and 1,2-propanediol (5.6 g/L, 0.21 g/g) from glycerol, demonstrating its potential as a carbon source for the production of fuels and reduced chemicals

Phytochemical Investigation and Bioactivity Assessment of Medicinal Plant from Northern Nigeria

Boswellia dalzielii (Burseraceae) has ethnopharmacological importance and is claimed to have anti-infection and immunomodulatory effects. In the Northern part of Nigeria, a region with a tropical dry climate, an aqueous infusion of this plant is used in the treatment of infections and tumours. The traditional formulation method was mimicked under laboratory conditions, and the effect of temperature and the impact of endophytic microbes present in aqueous infusion of B. dalzielii was also investigated. Activity-guided fractionation against Staphylococcus aureus and its methicillin-resistant strain resulted in the identification of two antibacterial compounds namely gallic acid and pyrogallol. The Minimum Inhibitory Concentration for pyrogallol and gallic acid against S. aureus growth are 508 and 753 μM, while against MRSA growth are 254 and 2032 μM, respectively. A growth Inhibition assay showed the activity of gallic acid as bacteriostatic, and pyrogallol as bacteriocidal against tested microorganisms. Interestingly, the bacteriocidal compound was found to arise by conversion of gallic acid by the endophyte Enterobacter cloacae. In addition, Pantoea spp was also isolated from the bark of B. Dalzielii. The sequences of both E. cloacae and Pantoea spp are deposited in the GenBank nucleotide database under the accession number MH764584 and MH764583, respectively. Similarly, activity-guided fractionation of B. Dalzielii bark against breast cancer cell line (MCF7) using MTT cytotoxicity assay resulted in the identification of a cytotoxic compound, catechol, and the half maximal effective concentration (EC50) observed was 86μM. The growth inhibition effect of catechol was observed to be time- and concentration- dependent. Endophytic Klebsiella pneumonia species (strain A and B) were shown to be responsible for bioconversion of protocatechuic acid to catechol. In addition, Pantoea agglomerans was also isolated from the bark of B. dalzielii. The sequences of Klebsiella pneumonia A, Klebsiella pneumonia B and Pantoea agglomerans are deposited in the GenBank nucleotide database under the accession number MH762022, MH762023 and MH762024, respectively. All isolated compounds were identified using HPLC, TLC, NMR, FTIR and HRMS.TETFUN

Book of Abstracts of MICROBIOTEC09

Sítio da conferência: http://www.deb.uminho.pt/microbiotec09/This book contains the abstracts presented at the 3rd joint meeting of the Portuguese Society of Microbiology and The Portuguese Society of Biotechnology - MicroBiotec09, held in Vilamoura, Portugal, over 3 days, from the 28th to the 30th of November, 2009. MicroBiotec09 comes in the sequence of previous conferences organized by each society, since 1982, date of the I Encontro Nacional de Biotecnologia (Lisbon), till 2005, date of the first joint meeting - MICRO'05 + BIOTEC'05 (Póvoa de Varzim). Following this joint meeting, another - MICRO 07 + BIOTEC 07 + XXIII JPG took place in Lisbon (2007). MicroBiotec09 is a joint organization of “Sociedade Portuguesa de Biotecnologia”, “Sociedade Portuguesa de Microbiologia”, Institute for Biotechnology and Bioengineering (Universidade do Minho – Departamento de Engenharia Biológica) and Centro de Recursos Microbiológicos (Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia – Departamento de Ciências da Vida). MicroBiotec09 brings together both young and established researchers and end users to discuss recent developments in different areas of Biotechnology and Microbiology. The conference program has thus been divided in 8 major sessions: Microbial Physiology, Molecular Biology and Functional Genomics; Cell and Tissue Engineering, Biomaterials and Nanobiotechnologies; Clinical Microbiology and Epidemiology; Environmental Microbiology and Biotechnology; Health and Pharmaceutical Biotechnology; Cellular Microbiology and Pathogenesis; Industrial and Food Microbiology and Biotechnology; Bioinformatics, Comparative Genomics and Evolution. A special session to celebrate the 200th anniversary of Charles Darwin's birth and the 150th anniversary of the publication of his landmark work “On the Origin of Species by Means of Natural Selection” will also take place. A total of 295 abstracts are included in the book, consisting of 6 invited lecturers, 10 oral presentations and 44 short oral presentations given in 3 parallel sessions, along with 4 slots for viewing poster presentations. All abstracts have been reviewed and we are grateful to the members of scientific and organizing committees for their evaluations. It was an intensive task since 328 submitted abstracts were received. It has been an honor for us to contribute to setting up MicroBiotec09 during an intensive period of 6 months. We wish to thank the authors who have contributed to yield a high scientific standard to the program. We are thankful to the sponsors who have contributed decisively to this event. We also extend our gratefulness to all those who, through their dedicated efforts, have assisted us in this task. On behalf of the Scientific and Organizing Committees we wish you that together with an interesting reading, the scientific program and the social moments organized will be memorable for all.Fundação para a Ciência e a Tecnologia (FCT