BIOSENSING SYSTEMS FOR THE DETECTION OF BACTERIAL QUORUM SENSING MOLECULES: A TOOL FOR INVESTIGATING BACTERIA-RELATED DISORDERS AND FOOD SPOILAGE PREVENTION

Quorum sensing enables bacteria to communicate with bacteria of the same or different species, and to modulate their behavior in a cell-density dependent manner. Communication occurs by means of small quorum sensing signaling molecules (QSMs) whose concentration is proportional to the population size. When a QSM threshold concentration is reached, certain genes are expressed, thus allowing control of several processes, such as, virulence factor production, antibiotic production, and biofilm formation. Not only many pathogenic bacteria are known to produce QSMs, but also QSMs have been identified in some bacteria-related disorders. Therefore, quantitative detection of QSMs present in clinical samples may be a useful tool in the investigation and monitoring of bacteria-related diseases, thus prompting the use of QSMs as biomarkers of disease. Herein, we have developed and utilized whole-cell biosensing systems and protein based biosensing systems to detect QSMs in clinical samples, such as, saliva, stool, and bowel secretions. Additionally, since bacteria are responsible for food spoilage, we employed the developed biosensing systems to detect QSMs in food samples and demonstrated their applicability for early identification of food contamination. Furthermore, we have utilized these biosensing systems to screen antibacterial compounds employed for food preservation, namely, generally regarded as safe (GRAS) compounds, for their effect on quorum sensing

Consequences of biofilm architecture on Vibrio cholerae ecology and life history

The diversity of microbes and the environments they inhabit are staggering. In many of these environments, bacteria have evolved to form sessile surface attached communities called biofilms. These biofilms have wide reaching impacts from importance in global carbon cycling, to persistent catheter infections, to biofouling and wastewater treatment. While many species of microbes form biofilms to survive in their environment, the architectures of these structures vary widely between organisms. Even though a great deal of work has been done to understand bacterial communities and their functions, little work has examined how the spatial aspects of biofilm architecture can affect the ecology of a species. Vibrio cholerae is a marine bacterium that has been at the forefront of understanding biofilm architecture at the single cell level. Here, we use confocal microscopy and microfluidics to understand the impacts that biofilm architecture have on V. cholerae’s ability to exist in a wealth of environments. We examine its capacity for intra-strain competition, predation protection, and multispecies community assembly through the lens of biofilm architecture. This thesis establishes how the architecture of a biofilm is a critical component when understanding the ecology of a microbe and should be considered along with more conventional traits

Biofouling and its control for in situ lab-on-a-chip marine environmental sensors

Biofouling is the process by which biological organisms attach to surfaces in an aqueous environment. This occurs on nearly all surfaces in all natural aquatic environments, and can cause problems with the functioning of scientific equipment exposed to the marine environment for extended periods. At the National Oceanographic Centre in Southampton (NOCS), the Centre for Marine Microsystems (CMM) is developing lab-on-chip micro-sensors to monitor the chemical and biological environment in situ in the oceans. Due to the long periods (up to several months) that these sensors will be deployed, biofouling by microbial biofilms is an important concern for the efficient running of these sensors. The aim of this project was therefore to determine the potential level of fouling within the sensors and to investigate the potential use of low-concentration diffusible molecules (LCDMs) to remediate biofouling.Many of the sensors in development by CMM are designed to sense specific chemical species and they use various chemical reagents to achieve this. The effects of some of these reagents on the formation of biofilms by mixed marine communities were investigated. It was shown that Griess reagent and ortho-phthadialdehyde (OPA), used to sense nitrites and ammonium respectively, effectively stop biofilm formation by killing microorganisms before they can attach to surfaces.Biofouling on two different polymers, cyclic olefin copolymer (COC) and poly (methyl methacrylate) (PMMA), used in the construction of micro-sensors, was compared with biofouling on glass. No differences were observed between COC and PMMA, however a small but significant difference in surface coverage was observed between glass and COC at the early stages of exposure to the marine environment. The lack of differences between the two polymers suggests that biofouling is not an important consideration when deciding whether to construct sensors from COC or PMMA. However, the larger degree of fouling on hydrophobic COC compared with hydrophilic glass indicates a potential use of surface modifications as an antifouling strategy.The effects on biofouling of the LCDMs nitric oxide (NO), cis-2-decenoic acid (CDA) and patulin, were investigated to evaluate their potential for anti-fouling in marine micro sensors. All three molecules were shown to reduce the formation of biofilms by mixed marine communities, but colony counts suggested that the effect of patulin was due to toxicity as opposed to a physiological effect. Investigation of biofilm growth in the light and the dark revealed that there was less biofilm formation in the light that the dark and this effect was determined to be due to an interaction with the polystyrene growth substratum.Analysis of the biofilm communities grown in the presence of LCDMs by denaturing gradient gel electrophoresis (DGGE), showed no clear differences in community profiles depending on the LCDMs. However those biofilms grown in the light appeared to have a greater proportion of Alphaproteobacteria than those grown in the dark.Further study is needed to determine the level of fouling and the applicability of LCDMs in real micro-sensor systems. However, this study has shown that LCDMs have the potential to remediate, at least in part, the biofouling of marine micro-sensors

Biofouling and its control for in situ lab-on-a-chip marine environmental sensors

Biofouling is the process by which biological organisms attach to surfaces in an aqueous environment. This occurs on nearly all surfaces in all natural aquatic environments, and can cause problems with the functioning of scientific equipment exposed to the marine environment for extended periods. At the National Oceanographic Centre in Southampton (NOCS), the Centre for Marine Microsystems (CMM) is developing lab-on-chip micro-sensors to monitor the chemical and biological environment in situ in the oceans. Due to the long periods (up to several months) that these sensors will be deployed, biofouling by microbial biofilms is an important concern for the efficient running of these sensors. The aim of this project was therefore to determine the potential level of fouling within the sensors and to investigate the potential use of low-concentration diffusible molecules (LCDMs) to remediate biofouling.Many of the sensors in development by CMM are designed to sense specific chemical species and they use various chemical reagents to achieve this. The effects of some of these reagents on the formation of biofilms by mixed marine communities were investigated. It was shown that Griess reagent and ortho-phthadialdehyde (OPA), used to sense nitrites and ammonium respectively, effectively stop biofilm formation by killing microorganisms before they can attach to surfaces.Biofouling on two different polymers, cyclic olefin copolymer (COC) and poly (methyl methacrylate) (PMMA), used in the construction of micro-sensors, was compared with biofouling on glass. No differences were observed between COC and PMMA, however a small but significant difference in surface coverage was observed between glass and COC at the early stages of exposure to the marine environment. The lack of differences between the two polymers suggests that biofouling is not an important consideration when deciding whether to construct sensors from COC or PMMA. However, the larger degree of fouling on hydrophobic COC compared with hydrophilic glass indicates a potential use of surface modifications as an antifouling strategy.The effects on biofouling of the LCDMs nitric oxide (NO), cis-2-decenoic acid (CDA) and patulin, were investigated to evaluate their potential for anti-fouling in marine micro sensors. All three molecules were shown to reduce the formation of biofilms by mixed marine communities, but colony counts suggested that the effect of patulin was due to toxicity as opposed to a physiological effect. Investigation of biofilm growth in the light and the dark revealed that there was less biofilm formation in the light that the dark and this effect was determined to be due to an interaction with the polystyrene growth substratum.Analysis of the biofilm communities grown in the presence of LCDMs by denaturing gradient gel electrophoresis (DGGE), showed no clear differences in community profiles depending on the LCDMs. However those biofilms grown in the light appeared to have a greater proportion of Alphaproteobacteria than those grown in the dark.Further study is needed to determine the level of fouling and the applicability of LCDMs in real micro-sensor systems. However, this study has shown that LCDMs have the potential to remediate, at least in part, the biofouling of marine micro-sensors

PATTERNED BIOFILM FORMATION TO INVESTIGATE BACTERIA-SURFACE INTERACTIONS

Bacterial adhesion to surfaces and subsequent formation of microcolonies play important roles in biofilm formation, which is a major cause of chronic infections and persistent biofouling. Despite the significance, mechanistic understanding of biofilm formation is still hindered by the structural heterogeneity in biofilms; and effective control of biofilm formation remains challenging. Biofilm formation is a dynamic process that involves numerous changes in bacterial gene and protein expression. These changes are highly sensitive to environmental factors such as surface chemistry, topography, charge, and hydrophobicity. To better control biofilm morphology and specifically investigate the effects of these factors, a platform was developed in this study to obtain patterned biofilm formation using surfaces with well-defined patterns of chemistry and topography. By modifying surfaces with systematically varied square patterns of self-assembled monolayers (SAMs) of functional alkanthiols, the size of cell clusters and inter-cluster distance were well controlled. By following biofilm formation of Escherichia coli on these surfaces, it was found that multicellular connections were formed between adjacent cell clusters when the clusters were within a threshold distance (10 µm); and such connections were influenced by the size of interacting cell clusters. It was also found that the connections were formed by active interactions of cell clusters, rather than nonspecific binding of planktonic cells on the bioinert background. Interestingly, the mutants of luxS and motB exhibited major defects in interaction between cell clusters. The phenotype of the luxS mutant was successfully restored by both complementing the luxS gene on a plasmid and by adding the precursor of autoinducer-2 (AI-2) signal in the culture. These results suggest that AI-2 mediated quorum sensing and motility are involved in the interaction among cell clusters. Based on these findings, a model was proposed to explain the intrinsic heterogeneity in biofilm structures. Consistently, cells attached between interacting clusters were found to be more sensitive to the antibiotic ampicillin. Besides surfaces with patterns of surface chemistry, poly(dimethylsiloxane) (PDMS) surfaces with microtopographic patterns of different shapes, dimensions and inter-pattern distances were used to understand the effects of surface topography on bacteria-surface interactions and biofilm formation. E. coli was found to preferentially attach and form biofilms in the valleys between square shaped plateaus. In addition, there appeared to be a threshold dimension of a plateau to allow bacterial attachment and biofilm formation on top of the plateaus. The threshold was found to be 40 µm × 40 µm for inverted patterns used in this study. Inspired by this finding, we created PDMS surfaces with hexagon shaped patterns and found that the ones with 15 µm side width and 2 µm inter-pattern distance can reduce biofilm formation by 7-fold compared to flat PDMS surfaces. These results were integrated with additional tests to better understand the resistance of biofilm cells to antibiotics. Specifically, the biofilm formation of fluorescently labeled donors and recipients on PDMS surfaces with square shaped microtopographic patterns was followed to investigate the effects of cell density on bacterial conjugation. PDMS surfaces with microtopogrpahic patterns were found to promote both biofilm formation and bacterial conjugation. This result was found to be due to the aggregation of biofilm cells on the side of plateaus, providing hot spots for bacterial conjugation. Bacterial motility was also found to play an important role in biofilm formation and bacterial conjugation. Collectively, these results are helpful for understanding the mechanism of biofilm formation and associated drug resistance, as well as the design of nonfouling surfaces

Biosensors for Environmental Monitoring

Real-time and reliable detection of molecular compounds and bacteria is essential in modern environmental monitoring. For rapid analyses, biosensing devices combining high selectivity of biomolecular recognition and sensitivity of modern signal-detection technologies offer a promising platform. Biosensors allow rapid on-site detection of pollutants and provide potential for better understanding of the environmental processes, including the fate and transport of contaminants.This book, including 12 chapters from 37 authors, introduces different biosensor-based technologies applied for environmental analyses

Integration of advanced off-line and on-line systems for the monitoring of surface and drinking water quality

An optimal strategy for monitoring water quality should be based on a combination of different technologies depending on the particularities of the system being observed. The final solution will be a combination of laboratory techniques, on-line instruments and statistical methods. In this case, the investigation was based on the Llobregat River basin in order to monitor natural and drinking waters after treatment in the Sant Joan Despi Water Treatment Plant (Barcelona). The thesis includes an optimization method for laboratory analysis of pharmacueticals in surface waters. 23 of the 28 compounds tested were detected. The highest concentrations were obtained for ß-blockers metoprolol and sotalol; the antibiotic ofloxacin; and lipid regulator gemfibrozil. Within the group of estrogens, estrone and estrone-3-sulfate were identified. The latter showed concentrations in some points high enough to induce estrogenic effects on aquatic organisms. A series of indexes have been developed for the assessment of the risks posed by certain substances found in the Llobregat River to the ecosystems. The methodology is based on comparison of average concentrations with the higher concentrations that have been proved to show no effect on the environment. According to the results, the studied metals (barium, copper, nickel and zinc) had higher rates than 1 for aquatic organisms. For the organic compounds, the most significant indexes are referred to the pesticides terbuthylazine, diazinon and MCPA; and the antibiotics ciprofloxacin and clarithromycin. When the relationship is established to the legal threshold, chlorpyrifos and lindane showed indexes above 1 over some months. The work also includes the development of indexes for measuring the potential danger of these substances to human health. The methodology considers the systemic and carcinogenic effects caused by the ingestion of water based on data from the World Health Organization (WHO) and the Information System Risk Assessment (RAIS). Over 5 years, systemic RAIS index drops from 0.64 to 0.42 for surface water and from 0.61 to 0.31 for potable water; the carcinogenic index is insignificant for the water inlet and varies from 4.2x10-05 to 7.4x10-06 for drinking water; WHO systemic index ranges from 0.41 to 0.16 for surface water and from 0.61 to 0.31 for potable water. All indices are below the thresholds, except for the carcinogenic risk in treated water in 2008 and 2009, where the rate is slightly above the limit. One of the technologies being explored to provide useful information for operators of water is UV-Visible spectrophotometry. A probe based on this technology along with statistical methods have been used to obtain a multivariate model that will predict the origins of water in the drinking water network of Barcelona. The analysis of the combination of the spectral fingerprints with conductivity, boron and fluoride was performed to improve predictability. The information that reports on the physical and chemical parameters in the water can be combined with toxicological information. An automatic biosensor was tested to measure its response to a series of priority pollutants. EC50 values (effective concentration that causes a 50% decrease in the activity; in mg L-1) were calculated for nonylphenol (0.03 and 0.06 for 15 and 30 min), triclosan (0.13 and 0.13), terbuthylazine (2.88 and 2.74), dimethoate (6.80 and 6.20), diclofenac (10.26 and 9.82), DBSS (50 and 39), diazinon (193 for 15 min), propanil (1594 for 15 min) and MCPA (2.0 for 15 min). For heavy metals, results were obtained with copper (II) (10.61 and 4.68), nickel (II) (317 and 222), chromium (III) (190 and 123) and iron (III) (52 for 15 min).Una estrategia óptima para monitorizar la calidad del agua se debe basar en una combinación de diferentes tecnologías en función de las particularidades del sistema a supervisar. La solución final será una combinación de técnicas de laboratorio, equipos en continuo, y métodos estadísticos. En este caso, la investigación se ha basado en el río Llobregat, para monitorizar las aguas naturales o potables después de su paso por la potabilizadora de Sant Joan Despí (Barcelona). La tesis incluye una optimización del método para el análisis en laboratorio de fármacos en aguas superficiales. Se detectaron 23 de los 28 compuestos analizados. Las concentraciones más altas se obtuvieron para los ß-bloqueantes metoprolol y sotalol; el antibiótico ofloxacina; y el regulador lipídico gemfibrozilo. Dentro del grupo de los estrógenos, se identificaron estrona y estrona-3-sulfato . Esta última mostró concentraciones en algunos puntos suficientes para inducir efectos estrogénicos en los organismos acuáticos. Para la evaluación del riesgo para los ecosistemas de las sustancias que se encuentran en el río Llobregat, se han desarrollado una serie de índices. La metodología se basa en la comparación de las concentraciones medias con las concentraciones más altas que no tienen efecto en el medio ambiente. Según los resultados, los metales estudiados (bario, cobre, níquel y zinc) dieron índices superiores a 1 para los organismos acuáticos. En cuanto a los compuestos orgánicos, los índices más significativos son los referidos a los pesticidas terbutilazina, diazinón y MCPA; y a los antibióticos claritromicina y ciprofloxacina. Cuando la relación se establece con el umbral legal, clorpirifos y lindano mostraron índices superiores a 1 en algunos meses. Asimismo, se han desarrollado índices para medir el peligro potencial de estas sustancias en la salud humana. La metodología considera los efectos sistémicos y cancerígenos causados por la ingestión oral de agua basados en los datos de la Organización Mundial de la Salud (OMS) y el Sistema de Información de Evaluación de Riesgos (RAIS). En 5 años, el índice sistémico RAIS desciende de 0,64 a 0,42 para aguas superficiales y de 0,61 a 0,31 para agua potable; el índice cancerígeno es insignificante para el agua de entrada y varía de 4.2x10-05 a 7.4x10-06 para agua potable; el índice sistémico OMS va de 0,41 a 0,16 para aguas superficiales y de 0,61 a 0,31 para agua potable. Todos los índices se encuentran por debajo de los umbrales, a excepción del riesgo cancerígeno en el agua tratada durante 2008 y 2009, donde el índice está ligeramente por encima del umbral. Una de las tecnologías que se explora para obtener información útil para los operadores de agua es la espectrofotometría Ultravioleta-Visible. En la red de agua potable de Barcelona, se utilizó una sonda basada en esta tecnología junto a métodos estadísticos, para obtener un modelo multivariante que sirva para predecir los orígenes del agua. Con el fin de mejorar la capacidad de predicción, se realizó el análisis de la combinación de las huellas espectrales con los parámetros: conductividad, flúor y boro. La información que refleja los parámetros físicos y químicos en el agua puede ser combinada con información toxicológica. Un biosensor automático ha sido probado para medir su respuesta a una serie de contaminantes prioritarios. Se calcularon los valores de EC50 (concentración efectiva que causa una disminución del 50% de la actividad en mg L-1) para nonilfenol (0,03 y 0,06 para 15 y 30 min), triclosán (0,13 y 0,13), terbutilazina (2,88 y 2,74), dimetoato (6,80 y 6,20), diclofenaco (10,26 y 9,82), DBSS (50 y 39), diazinon (193 para 15 min), propanil (1594 para 15 min) y MCPA (2,0 para 15 min). Para los metales pesados, los resultados se obtuvieron con cobre (II) (10,61 y 4,68), níquel (II) (317 y 222), cromo (III) (190 y 123) y hierro (III) (52 para 15 min)

DEVELOPMENT OF SYNTHETIC CHEMOTAXIS BASED BACTERIAL BIOREPORTERS

With increasing industrialization and the increasing amount of chemical compounds produced every day, the need of a proper monitoring of the environment is crucial. The use of biosensors based on living microorganisms is an interesting alternative to common chemical analysis. Since microorganisms are easy and cheap to produce, and thanks to their small size, they can be implemented in miniaturized portable devices, which may be used directly in the field. Common whole-cell bacterial bioreporters produce an easy detectable signal by induction of expression of a reporter protein in presence of either a specific molecule or a general stress reaction. Whole-cell bioreporter analysis is robust, but requires a couple of hours to obtain a clear reporter signal. In this thesis, we envisioned to develop bacterial biosensors based on a different physiological response than de novo gene expression that could lead to a faster response time while keeping target sensitivity and specificity. In particular, we tried to exploit chemotaxis, the behaviour of motile bacteria to sense their environment and swim in the direction of or away from chemical compounds. Chemotaxis is rapid (sec–min scale) and some species show naturally chemotaxis towards compounds of environmental interest. In Chapter 2, we quantified bacterial chemotaxis from direct measurements of cellular motility. We developed a microfluidic chip, which generated a stable attractant gradient and into which motile bacteria could be added. The bacteria sensed the chemical gradient and accumulated where the concentration of attractant is highest. Accumulation of cells was quantified over time by epifluorescence microscopy. As a proof of concept, we used chemotaxis of Escherichia coli towards serine, aspartate and methylaspartate. Notably, E. coli accumulated to 10 µM serine within 10 minutes, but showed maximum accumulation to 100 µM serine after 20 minutes or longer. Secondly, we quantified chemotaxis of Cupriavidus pinatubonensis JMP134 to 2,4-dichlorophenoxyacetate, a commonly used herbicide. Unfortunately, accumulation of JMP134 was not very sensitive and could only be observed with 1 mM 2,4- dichlorophenoxyacetic acid or higher. Steady-state chemodynamic and chemotaxis modelling was used to support the observed cellular response in the microfluidic chip as a function of attractant concentration. In Chapter 3, we wanted to facilitate the manipulation of the microfluidic chip by the development of a different chip that integrates valves inside the structure. This could facilitate the control of the liquid flow inside the chip, as well as enable sample exchange. By focusing on individual cell movement, we expected we might achieve very short response times upon addition of attractants. The gradient was generated by alternating valve opening and motile E. coli were inserted in the middle of this pre-established gradient. Individual cell trajectories were monitored in the few first minutes of response. Contrary to our expectations, no significant difference in trajectory characteristics was measured in presence compared to absence of a gradient. Mathematical simulations of single cell chemotaxis response suggested that more time is required to observe cell accumulation, or that cells would have to be introduced closer to the source. In Chapter 4, I focused on chemotaxis responses at the molecular and single cell level by measuring CheY–CheZ interactions. I fused two non-fluorescent parts of the green fluorescent protein (Gfp) to CheY and CheZ, components of chemotaxis pathway. I could demonstrate that Gfp fluorescent foci appear in single cells as a genuine interaction between CheY and CheZ. By mutant analysis, I showed that foci form mostly at the motor complex and less frequently at the sites of chemoreceptors. Not completely unexpected, the reformed split-Gfp was relatively stable and little dynamics in position or fluorescent intensity of foci was detected. However, single cell analysis indicated that the turnover of split-Gfp is more important immediately after addition of 100 µM nickel as repellent. In Chapter 5, I attempted to measure chemotaxis response through pH changes at single cell levels. Notably, the flagellar motor is powered by an influx of protons through the cytoplasmic membrane. I deployed the pH-sensitive fluorescent protein pHluorin, either expressed inside the cytoplasm or in the periplasm of E. coli, to measure pH differences in chemotactically active cells. For this, I used a modified agarose-block test as attractant and recorded fluorescent changes in accumulating cells. Interestingly, a 100 µM serine source induced an increase of pH in the cytoplasm and a decrease in the periplasm in cells close to the source, but not in cells further away. This suggests an active export of protons from the cytoplasm to the periplasm during chemotaxis in order to compensate for the increased flux needed for the flagellar motors. Finally in chapter 6, I attempted to change E. coli chemotaxis specificity by introducing receptors coming from Pseudomonas putida. I focused on two P. putida receptors, one for benzoate and the other for toluene. I demonstrated expression of both receptors in E. coli – although we cannot be completely certain that the proteins inserted into the membrane. Agarose-block tests with serine, toluene and benzoate, in comparison to no attractants, showed that E. coli cell accumulation close to a source of toluene was significantly higher in strains expressing the toluene receptor. E. coli without benzoate receptor accumulated as well as those with benzoate receptor near sources with benzoate. In this work, we investigated different approaches to exploit chemotaxis in order to produce biosensing signals. Our results are promising and show that functional biosensors based on chemotaxis may be achieved in a variety of ways. -- En raison de l’avancée de l’industrialisation et de l’augmentation du nombre et de la quantité de composés chimiques produits chaque jour, la surveillance de la pollution touchant notre environnement est cruciale. L’utilisation de biosenseurs basés sur des micro-organismes vivants est une alternative intéressante aux analyses chimiques couramment utilisées. Ces micro-organismes sont faciles et peu coûteux à produire et, grâce à leur petite taille, ils peuvent être facilement implémentés dans des appareils miniaturisés et portables pouvant être utilisés directement sur le terrain. Les biorapporteurs bactériens usuels produisent un signal facilement détectable conséquence de l’induction de l’expression d’une protéine rapportrice en présence d’une molécule spécifique ou en réponse à un stress. Les biorapporteurs sont des outils d’analyse robustes mais requièrent quelques heures pour obtenir un signal clair. Dans cette thèse, nous avons eu pour but de développer des biosenseurs bactériens basés sur une autre réponse physiologique que l’expression de novo de gènes, ceci dans le but de produire une réponse plus rapide tout en conservant la sensibilité et la spécificité pour la molécule cible. En particulier, nous avons exploité le chimiotactisme, soit le comportement des bactéries mobiles qui peuvent sentir leur environnement et se déplacer pour s’approcher ou s’éloigner de composés chimiques. Le chimiotactisme présente l’avantage de fournir une réponse rapide, prenant de quelques secondes à quelques minutes. De plus, certaines espèces bactériennes sont connues pour montrer naturellement une attraction pour des composées d’intérêt environnemental comme des polluants. Dans le chapitre 2, nous avons quantifié le chimiotactisme bactérien par mesure directe de la mobilité cellulaire. Nous avons développé une puce micro-fluidique qui génère un gradient stable d’attractant dans lequel des bactéries mobiles peuvent être ajoutées. Les bactéries sentent le gradient chimique et s’accumulent là où la concentration d’attractant est la plus élevée. Cette accumulation de cellules est quantifiée au cours du temps par microscopie à épifluorescence. Comme preuve de concept, nous avons utilisé le chimiotactisme d’Escherichia coli vers la sérine, l’aspartate et le méthylaspartate. E. coli est attiré par 10 µM de sérine en 10 minutes, mais montre une accumulation maximale avec 100 µM de sérine après au moins 20 minutes. Nous avons également quantifié le chimiotactisme de Cupriavidus pinatubonensis JMP134 pour le 2,4- dichlorophenoxyacetate, un herbicide communément utilisé. Malheureusement JMP134 n’était pas très sensible et la réponse n’a pu être observée qu’avec un minimum de 1 mM de 2,4- dichlorophenoxyacetate. La modélisation mathématique du chimiotactisme en fonction de la concentration d’attractant a été utilisée pour appuyer les résultats obtenus expérimentalement en utilisant la puce micro-fluidique. Dans le chapitre 3, nous avons voulu faciliter la manipulation de la puce micro-fluidique en développant un autre type de puce qui intègre des valves à l’intérieure de leur structure. Cela facilite le contrôle du flux de liquide dans la puce, et permet ainsi un potentiel échange d’échantillons. En se focalisant sur le mouvement de cellules individuelles, nous nous attendions à obtenir un temps de réponse plus court après l’ajout d’attractant. Le gradient d’attractant est généré par des ouvertures alternées des valves et des cellules d’E. coli sont ensuite insérées au milieu du gradient préétabli. Les trajectoires de cellules individuelles sont enregistrées pendant les premières minutes de réponse. Contrairement à nos attentes, aucune différence significative dans les caractéristiques des trajectoires n’a été mesurée en présence ou en absence de gradient. Des simulations mathématiques de la réponse chimiotactique de cellules individuelles suggère la nécessitée d’un plus long temps d’observation ou encore d’introduire les cellules plus proche de la source d’attractant. Dans le chapitre 4, nous nous sommes focalisés sur le chimiotactisme aux niveaux cellulaire et moléculaire en mesurant l’interaction entre deux acteurs de la signalisation. Pour cela, deux parties non-fluorescentes de la protéine fluorescente verte (GFP) ont été fusionnées aux protéines CheY et CheZ, composants de la signalisation du chimiotactisme. J’ai pu démontré que les foci de fluorescence apparaissant dans les cellules individuelles montrent l’interaction entre CheY et CheZ. Par l’analyse de mutants, j’ai montré que les foci se forment principalement au niveau du moteur des flagelles et moins fréquemment au niveau des récepteurs. La GFP reformée lors de l’interaction de CheY et CheZ est relativement stable et se révèle peu dynamique dans la position ou dans l’intensité de fluorescence des foci. Néanmoins, l’analyse des cellules individuelles indique que le turnover de la GFP est plus important immédiatement après ajout de 100 µM de nickel, utilisé comme substance répulsive. Dans le chapitre 5, j’ai mesuré la réponse chimiotactique à travers les changements de pH au niveau des cellules individuelles. Le moteur des flagelles est énergisé par un influx de protons à travers la membrane cytoplasmique. J’ai donc exprimé une protéine fluorescente sensible au pH, la pHluorin, soit dans le cytoplasme ou le périplasme d’E. coli et mesuré les différences de pH dans des cellules actives pour le chimiotactisme. Pour cela, j’ai utilisé un bloc d’agarose contenant la source d’attractant et mesuré les changements de fluorescence des cellules attirées. Une source de 100 µM de serine induit une augmentation du pH dans le cytoplasme et inversement, une diminution dans le périplasme dans les cellules proches de la source mais pas dans les cellules plus éloignées. Cela suggère un export actif de protons depuis le cytoplasme dans le périplasme pendant le chimiotactisme afin de compenser l’augmentation de l’influx de protons nécessaire à la rotation des flagelles. Finalement dans le chapitre 6, j’ai changé la spécificité du chimiotactisme d’E. coli en y introduisant des récepteurs venant de Pseudomonas putida. Je me suis intéressée à deux récepteurs de P. putida, l’un liant le benzoate et l’autre le toluène. J’ai démontré que l’expression des deux récepteurs se fait de façon correcte chez E. coli, même si l’on ne peut pas être complétement certain que les protéines soient correctement repliées ou encore bien insérées dans la membrane. Les tests par bloc d’agarose contenant la sérine, le toluène ou le benzoate en comparant à l’absence d’attractant, montrent que l’accumulation d’E. coli proche de la source est significativement plus important pour la souche exprimant le récepteur pour le toluène. D’autre part, la souche n’exprimant pas le récepteur au benzoate s’accumule aussi bien que la souche avec récepteur autour d’une source de benzoate. Dans ce travail, nous avons investigué différentes approches pour exploiter le chimiotactisme dans le but de produire des signaux par des biosenseurs bactériens. Nos résultats sont prometteurs et montrent que des biosenseurs fonctionnels basés sur le chimiotactisme peuvent être obtenu par différentes approches