Competition between Plasmid-Bearing and Plasmid-Free Organisms in a Chemostat with Pulsed Input and Washout

We consider a model of competition between plasmid-bearing and plasmid-free organisms in the chemostat with pulsed input and washout. We investigate the subsystem with nutrient and plasmid-free organism and study the stability of the boundary periodic solutions, which are the boundary periodic solutions of the system. The stability analysis of the boundary periodic solution yields the invasion threshold of the plasmid-bearing organism. By using the standard techniques of bifurcation theory, we prove that above this threshold there are periodic oscillations in substrate, plasmid-free, and plasmid-bearing organisms. Numerical simulations are carried out to illustrate our results

Periodic Oscillations in a Chemostat Model with Two Discrete Delays

Periodic oscillations of solutions of a chemostat-type model in which a species feeds on a limiting nutrient are considered. The model incorporates two discrete delays representing the lag in nutrient recycling and nutrient conversion. Through the study of characteristic equation associated with the linearized system, a unique positive equilibrium is found and proved to be locally asymptotically stable under some conditions. Meanwhile, a Hopf bifurcation causing periodic solutions is also obtained. Numerical simulations illustrate the theoretical results

Bifurcation Analysis of a Chemostat Model of Plasmid-Bearing and Plasmid-Free Competition with Pulsed Input

A chemostat model of plasmid-bearing and plasmid-free competition with pulsed input is proposed. The invasion threshold of the plasmid-bearing and plasmid-free organisms is obtained according to the stability of the boundary periodic solution. By use of standard techniques of bifurcation theory, the periodic oscillations in substrate, plasmid-bearing, and plasmid-free organisms are shown when some conditions are satisfied. Our results can be applied to control bioreactor aimed at producing commercial producers through genetically altered organisms

Dynamics of a Stochastic Functional System for Wastewater Treatment

The dynamics of a delayed stochastic model simulating wastewater treatment process are studied. We assume that there are stochastic fluctuations in the concentrations of the nutrient and microbes around a steady state, and introduce two distributed delays to the model describing, respectively, the times involved in nutrient recycling and the bacterial reproduction response to nutrient uptake. By constructing Lyapunov functionals, sufficient conditions for the stochastic stability of its positive equilibrium are obtained. The combined effects of the stochastic fluctuations and delays are displayed

Plasmid Instability: Measurement and Use in Antimicrobial Action

The discovery of antibiotics in the early 20th century revolutionised medicine, but quickly bacteria began to demonstrate resistance to these agents. Antibiotic resistance is still on the increase, and soon, if this trend continues, bacterial infections may not be treatable with antibiotics. In an effort to prevent this occurring, the search for novel antibiotics is under way and new antibiotic targets are being considered. One target that has not been studied in depth is the partitioning systems of bacterial plasmids. Disruption of plasmid partition would prevent effective plasmid inheritance, which, in the case of resistance plasmids, would render the host cell antibiotic sensitive. The aim of this study was to determine whether plasmid partitioning is a viable target for a new antibiotic. A series of plasmids that contained genetically altered partitioning systems was used, which provided a range of plasmid stabilities. The plasmids all conferred antibiotic resistance on the host cell, allowing the effect of plasmid instability on a population grown in the presence of antibiotics to be determined. Several different methods of cell culture were used. Simple batch culture experiments allowed the observation that few plasmid-free cells were produced from cells containing plasmids that had a functional system. In contrast, plasmids containing a faulty system were found to be rapidly lost from the host cells. Steady-state chemostat culture was used to provide a simple model of a clinical infection. The formation of equilibria between plasmid-free and plasmid-bearing cells was observed, and the cultures contained a large proportion of plasmid-free cells when the experiments involved unstable plasmids. The slow growth rate of cultures in the chemostat was seen to dramatically affect the inheritance of plasmids relying on random distribution. Finally, cultures were subjected to washout in order to determine their maximum specific growth rate (µmax). While the results from these experiments are not entirely conclusive, there is a strong indication that the growth rate of cultures containing unstable plasmids grown in the presence of antibiotics is reduced

Dynamique de la réponse physiologique d'Escherichia coli à des perturbations maîtrisées de son environnement (vers le développement de nouveaux outils de changement d'échelle)

Les bioréacteurs de grandes dimensions, en raison de phénomènes de transfert limitant, sont le siège d hétérogénéités se traduisant par des gradients locaux de concentration et température. Les microorganismes circulant au sein de ces bioréacteurs subissent donc des fluctuations environnementales qui peuvent affecter leur comportement aux niveaux métaboliques et/ou moléculaires. La réponse microbienne est fonction de la nature, de l intensité, de la fréquence et de la durée de la perturbation. L objectif de ce travail est l étude quantitative de l impact de l intensité, la fréquence et l amplitude d un stress nutritionnel sur le comportement dynamique d Escherichia coli, à savoir des ajouts pulsés de glucose lors de cultures continues en régime permanent. Un effort particulier est consacré au développement et à la validation des outils expérimentaux indispensables pour une caractérisation rigoureuse des dynamiques de réponses transitoires sur des échelles de temps allant de secondes à quelques minutes. Pour permettre le suivi in situ et en temps réel des changements métaboliques et moléculaires, une souche bioluminescente est mise en œuvre. Les réponses transitoires sont caractérisées par les vitesses spécifiques, les rendements, les profils d induction transcriptionnelle, les temps caractéristiques. Selon les différents scenarii réalisés, l ajustement du métabolisme face aux hétérogénéités de substrat est quantifié selon des échelles de temps aux niveaux macroscopiques et/ou moléculaires ; ces résultats originaux contribuent ainsi à l implémentation des connaissances sur les interactions dynamiques entre les phénomènes biologiques et les phénomènes physiques ; l enjeu réside à terme en l amélioration des processus d optimisation et d extrapolation des bioprocédés par l identification et la quantification des dynamiques des phénomènes limitantsIneffective mixing entailing heterogeneity issues within industrial bioreactors have been reported to affect microbial metabolisms at cellular and/or molecular levels. Substrate gradients inside large-scale bioreactors are common environmental fluctuations that microorganisms would have to encouter along with the bioprocess. Depending on intensity, frequency and duration of those fluctuations, microorganisms may respond in a different manner. The objective of this work is to study the impact of intensity, frequency and amplitude of glucose perturbations on the dynamics of Escherichia coli responses. An E. coli bioluminescent strain is used for in situ and real-time monitoring of both metabolic and transcriptional changes. For this purpose, short-term glucose excess was simulated, using pulse-based experiments into glucose-limited chemostat cultures. In addition, an important effort is devoted to the development and validation of technical and mathematical tools in order to acquire quantitative and kinetic data on time scales from seconds to minutes. The transient responses are characterized, using specific rates, yields, transcriptional induction profiles and characteristic response times, and are compared in the different defined perturbation scenarios. The results reflected the fact that short-term heterogeneities of substrate affect both cell metabolism and regulation at macroscopic and/or molecular levels. Quantitative understandings of the dynamics during transient responses to environmental perturbations can thus shed light on the bioprocess optimizationTOULOUSE-INSA-Bib. electronique (315559905) / SudocSudocFranceF

Molecular epidemiology of antibiotic resistance in the commensal Escherichia coli of calves

In animals, the study of antibiotic resistance in bacteria has been focused on organisms that are pathogenic in human or animal hosts. The development of antibiotic resistance in commensal bacteria is also of concern because they may act as a reservoir ofresistance genes. This thesis aimed to determine levels of resistance to veterinary and medical antibiotics in the commensal Escherichia coli of calves, to explore the genotypic diversity of isolates, and to study the molecular mechanism and transfer dynamics ofresistance to apramycin.The antibiotic sensitivity testing of calf faecal E. coli, obtained by weekly sampling, demonstrated that there was resistance to beta-lactams, cephalosporins, streptomycin, trimethoprim, chloramphenicol, tetracycline and sulphamethoxazole. These resistance phenotypes had not been selected for on antibiotic-containing media, indicating a high prevalence of the corresponding resistance determinants.Five hundred and forty three isolates were genotyped by pulsed-field gel electrophoresis. Examination of the patterns generated by restriction with Xbal and analysed with BioNumerics software revealed a total of 55 different genotypes. Ampicillin resistant isolates were more diverse (24 genotypes) than apramycin or nalidixic acid resistant isolates (5 and 2 genotypes respectively). Apramycin resistance (aprR) was conferred by three conjugative plasmids, pUK2001, pUK2002 and pUK2003, of sizes 91, 115 and 181Kb respectively. All aprR plasmids conferred cross-resistance to the medical antibiotics tobramycin and gentamicin. Plasmids pUK2002 and pUK2003 also carried tetracycline and streptomycin resistance. Plasmid pUK2001 demonstrated very high transfer frequencies (4.12x10" during 7 hrs mating), horizontal spread to three different genotypes, and an apparent fitness advantage in vitro.This thesis shows a very high prevalence of antibiotic resistance genes in the commensal faecal flora of food-producing calves. This may have significant implications for the transmission ofresistance genes to human clinical bacteri

Dynamique de la réponse physiologique d’Escherichia coli à des perturbations maîtrisées de son environnement : vers le développement de nouveaux outils de changement d’échelle

Les bioréacteurs de grandes dimensions, en raison de phénomènes de transfert limitant, sont le siège d’hétérogénéités se traduisant par des gradients locaux de concentration et température. Les microorganismes circulant au sein de ces bioréacteurs subissent donc des fluctuations environnementales qui peuvent affecter leur comportement aux niveaux métaboliques et/ou moléculaires. La réponse microbienne est fonction de la nature, de l’intensité, de la fréquence et de la durée de la perturbation. L’objectif de ce travail est l’étude quantitative de l’impact de l’intensité, la fréquence et l’amplitude d’un stress nutritionnel sur le comportement dynamique d’Escherichia coli, à savoir des ajouts pulsés de glucose lors de cultures continues en régime permanent. Un effort particulier est consacré au développement et à la validation des outils expérimentaux indispensables pour une caractérisation rigoureuse des dynamiques de réponses transitoires sur des échelles de temps allant de secondes à quelques minutes. Pour permettre le suivi in situ et en temps réel des changements métaboliques et moléculaires, une souche bioluminescente est mise en œuvre. Les réponses transitoires sont caractérisées par les vitesses spécifiques, les rendements, les profils d’induction transcriptionnelle, les temps caractéristiques. Selon les différents scenarii réalisés, l’ajustement du métabolisme face aux hétérogénéités de substrat est quantifié selon des échelles de temps aux niveaux macroscopiques et/ou moléculaires ; ces résultats originaux contribuent ainsi à l’implémentation des connaissances sur les interactions dynamiques entre les phénomènes biologiques et les phénomènes physiques ; l’enjeu réside à terme en l’amélioration des processus d’optimisation et d’extrapolation des bioprocédés par l’identification et la quantification des dynamiques des phénomènes limitants.---------------------------------------------------------------------------------------------------------------------------------------------------------Ineffective mixing entailing heterogeneity issues within industrial bioreactors have been reported to affect microbial metabolisms at cellular and/or molecular levels. Substrate gradients inside large-scale bioreactors are common environmental fluctuations that microorganisms would have to encouter along with the bioprocess. Depending on intensity, frequency and duration of those fluctuations, microorganisms may respond in a different manner. The objective of this work is to study the impact of intensity, frequency and amplitude of glucose perturbations on the dynamics of Escherichia coli responses. An E. coli bioluminescent strain is used for in situ and real-time monitoring of both metabolic and transcriptional changes. For this purpose, short-term glucose excess was simulated, using pulse-based experiments into glucose-limited chemostat cultures. In addition, an important effort is devoted to the development and validation of technical and mathematical tools in order to acquire quantitative and kinetic data on time scales from seconds to minutes. The transient responses are characterized, using specific rates, yields, transcriptional induction profiles and characteristic response times, and are compared in the different defined perturbation scenarios. The results reflected the fact that short-term heterogeneities of substrate affect both cell metabolism and regulation at macroscopic and/or molecular levels. Quantitative understandings of the dynamics during transient responses to environmental perturbations can thus shed light on the bioprocess optimization

A systems biology approach for the characterization of metabolic bottlenecks in recombinant protein production processes

Tese de doutoramento em Engenharia Química e BiológicaThe main purpose of this thesis is to investigate the influence of recombinant protein production in the reorganization of the metabolic activities and the resulting stress-induced responses in the bacterium Escherichia coli. More specifically, the focus is on the RelA-mediated stringent response, a stress response that is triggered by the sudden lack of intracellular amino acids and that has been associated with the metabolic burden imposed by recombinant processes. To identify the main metabolic bottlenecks in recombinant biosynthetic processes, which include maintenance of recombinant DNA and expression of heterologous genes, a systematic modelling approach is proposed, capable of predicting the amino acid shortages caused by recombinant processes and the consequent activation of the RelA-dependent guanosine pentaphosphate (ppGpp) synthesis. The view of ppGpp as a primarily regulator of gene transcription has been expanded and it is now clear that the response controlled by ppGpp is crucial for cell survival during the adaptation to stressful conditions. Major advances have been achieved to understand this regulatory system governing gene expression in response to environmental growth perturbations, but so far mainly transcriptome and proteome analyses that have been applied to elucidate the stringent control mediated by ppGpp. Metabolomics analysis can provide substantial information on the impact of this stress response at the biochemical level, in particular during recombinant bioprocesses. Therefore, two metabolomics-based approaches were applied: metabolic profiling to evaluate the intracellular metabolic profiles and metabolic footprinting to estimate the profiles of extracellular metabolites. In these metabolomics studies two E. coli strains (E. coli W3110 and the isogenic ΔrelA mutant) were used to investigate the influence of recombinant processes on the host cells’ metabolism, as well as the main metabolic activities affected by the RelA activity under different growth conditions. The mutant strain presented a “relaxed” phenotype that characterized this bacterial system by an acute delay in most metabolic adaptations to transient growth conditions. Most importantly, it was shown that these mutant cells lack metabolic adjustments that are often observed after metabolic burden phenomena. Nevertheless, this cellular system presented major advantages in terms of biomass yield and productivity, which imply a remarkable improvement in recombinant bioprocesses. Thus, alleviating stress responses can be beneficial if they impair the desired quality and quantity of the recombinant product. However, it must be pointed out that this may be an alternative as long as recombinant bioprocesses are designed to achieve a finer balance between strain improvement strategies and culturing conditions.O trabalho realizado no âmbito desta tese teve como principal finalidade a avaliação das alterações metabólicas relacionadas com a produção de proteínas recombinantes em células bacterianas de Escherichia coli e a consequente activação de respostas de stress. Foi evidenciada a resposta restringente promovida pela actividade da enzima RelA, dado ser uma das principais respostas de stress induzidas pelo decréscimo da quantidade de aminoácidos disponíveis no meio intracelular como consequência da expressão de proteínas recombinantes. As diferenças na composição em aminoácidos entre as proteínas da biomassa e recombinantes, têm sido apontadas como principais causas para o desequilíbrio metabólico que conduz à exaustão de alguns metabolitos, nomeadamente de aminoácidos. De modo a explorar estes fenómenos e avaliar o impacto dos processos recombinantes no metabolismo das células hospedeiras, foi proposto um modelo matemático capaz de identificar pontos de estrangulamento na rede metabólica. Estes locais correspondem a vias metabólicas que apresentam limitações na capacidade catalítica e que serão essenciais para compensar o consumo desproporcionado de aminoácidos levado a cabo pela síntese de proteínas recombinantes. Associado a este fenómeno foi considerada a descrição da síntese de nucleótidos guanosina pentafosfato (ppGpp) induzida pela escassez de aminoácidos no meio intracelular. O reconhecimento deste nucleótido como um regulador fundamental na transcrição da informação genética tem sido amplamente descrito e tornou-se evidente que as respostas celulares controladas pelo ppGpp são determinantes para a sobrevivência e adaptação dos organismos a condições adversas. Neste sentido, vários estudos foram elaborados para elucidar o papel do ppGpp no controlo destas respostas de stress e nas alterações fisiológicas que advêm destes processos, nomeadamente ao nível do metabolismo. A análise do metaboloma, em comparação com o transcriptoma ou o proteoma, é capaz de capturar de forma mais directa a relação entre as actividades metabólicas e a fisiologia dos organismos, designadamente em sistema recombinantes. Neste trabalho foram elaborados alguns estudos em que se aplicaram duas abordagens de análise metabolómica distintas: profiling metabólico, que se refere à análise do perfil de metabolitos intracelulares; e footprinting metabólico, que se refere à análise do perfil de metabolitos extracelulares. Nestes estudos foram usadas duas estirpes de E. coli (W3110 e a estirpe isogénica com mutação no gene relA) clonadas com um vector de expressão pTRC-His- AcGP1 que codifica a proteína verde fluorescente AcGFP1, derivada da proteína AcGFP da Aequorea coerulescens. Foram avaliadas as principais alterações metabólicas provocadas pela indução da produção de proteína recombinante e pela actividade catalítica da enzima RelA em diversas condições de crescimento. Comparando os perfis metabólicos das duas estirpes, foram estimadas várias diferenças significativas que se podem revelar críticas durante processos recombinantes. A estirpe mutante revelou um comportamento típico de um fenótipo “relaxado”, que é caracterizado por um retardamento significativo na adaptação do metabolismo a alterações nas condições de crescimento. Não obstante, a estirpe mutante exibiu melhores resultados em termos de rendimento em biomassa e produtividade, o que representa uma vantagem notável para a aplicação destes sistemas bacterianos recombinantes ao nível industrial. Em resumo, a restrição de respostas de stress pode trazer benefícios se a qualidade e quantidade do produto estiverem em causa, mas deve salientar-se que não é uma solução absoluta, sendo que as condições de processamento devem ser também levadas em consideração na implementação destes bioprocessos

Process development for the obtention and use of recombinant glycosidases: expression, modelling and immobilisation

El objetivo general de la presente tesis doctoral es el desarrollo de herramientas para la obtencion, produccion y aplicacion de dos enzimas glicosidicas: �¿-L-arabinofuranosidasa proveniente del hongo Aspergillus niger (Abf) y �À-D-glucosidasa (Bgl), proveniente de la levadura Candida molischiana. Estas hidrolasas se emplean en la liberacion de azucares en procesos de conversion de biomasa y en la industria alimentaria, pero tambien en la sintesis de aminoglicosidos, glicoconjugados y oligosacaridos, compuestos de alto valor anadido para la industria quimico-farmaceutica. Las enzimas se han expresado en la levadura metilotrofica Pichia pastoris, y se han purificado para caracterizar sus propiedades bioquimicas. Asimismo, se ha comprobado su capacidad para catalizar reacciones de transglicosilacion con alto rendimiento. En relacion a su produccion, se ha establecido y validado un modelo basado en restricciones del metabolismo de Pichia pastoris, evaluando su consistencia mediante analisis de flujos metabolicos posibilistico. El modelo permite estimar la tasa de crecimiento y la distribucion de flujos intracelulares a partir de unos pocos flujos extracelulares medidos experimentalmente. Adicionalmente, el modelo se ha extendido para estimar la productividad de proteina recombinante, y se ha empleado para analizar diferentes condiciones de cultivo de las cepas transgenicas que sobreproducen las enzimas Abf y Bgl. Finalmente, las enzimas se han inmobilizado en organosilicas bimodales de la familia UVM-7. Los biocatalizadores resultantes se han caracterizado bioquimica y fisico-quimicamente y se han evaluado en diferentes aplicaciones de interes biotecnologico.Tortajada Serra, M. (2012). Process development for the obtention and use of recombinant glycosidases: expression, modelling and immobilisation [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/16800Palanci