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Bacteria in the environment face constant stress, due to lack of nutrients or presence of growth inhibiting compounds. As a result, they have developed several strategies to evade unfavourable growth conditions. These range from entering into dormant or quiescent states, through to motility, and biofilm formation. Using the model organism Streptomyces, we investigated how the bacterial cell surface regulates dormancy, biofilm formation, and motility. Dormancy via spore formation allows cells to shut down metabolism in response to poor nutrient conditions. Spores can then be dispersed throughout the environment to encounter favourable conditions. This is an incredibly resilient survival strategy, so long as the spores resuscitate from dormancy and resume growth once favourable conditions are sensed. We established that peptidoglycan remodeling by resuscitation promoting factors is critical for rapid germination of dormant Streptomyces spores, which likely provides a competitive advantage over slower growing microbes in the same environment. Previously it was thought that these proteins produce a signal to stimulate germination in neighbouring cells. We determined that the resuscitation promoting factors are lytic transglycosylases, and were not capable of producing a germination signal on their own. Instead, they function by cleaving the peptidoglycan to make room for new cell growth. This work highlights the importance of peptidoglycan remodeling to the germination process. Biofilms are multicellular communities of microorganisms which are adhered to each other using a protective matrix. Formation of biofilms is thought to be inversely correlated with motility. We established that Streptomyces forms biofilms during the exploratory growth identifying potential extracellular matrix components. These biofilms use sliding motility to expand rapidly across their environment. Components of the biofilm matrix effect colony expansion, suggesting that biofilm formation and motility are intricately linked in Streptomyces. These works demonstrate the importance of surface polymers to the growth and development of Streptomyces. / Thesis / Doctor of Philosophy (PhD) / Bacteria are all around us. In these different environments, whether in the soil, or inside our guts, or in a body of water, they will encounter stress. This can take the shape of nutrient stress, or the presence of growth inhibiting compounds. In response, bacteria can evade these poor conditions by entering into dormancy, analogous to hibernation, by building a biofilm, analogous to building a bunker, or by moving away. The surface of bacterial cells becomes decorated with different polymers as it transitions into one of these three modes of stress evasion. The cell wall holds the cell together and supports its shape, making it the most important surface polymer. I examined how rapid remodeling of the cell wall provides a competitive advantage to cells waking up from dormancy. I also examined the importance of additional polymers to the formation of biofilms that slide across surfaces, away from stressors. These works establish how important the surface of the bacterium is for surviving stressful conditions.

Biologically-mediated subsurface remediation by biofilm communities is a poorly understood process that is spatially and temporally dynamic. Two microbial responses, catabolism and the stress response glutathione-gated potassium efflux (GGKE), to benzene, pentachlorophenol (PCP), or Cd exposure were studied in up-flow sand columns to examine the contribution of each response to the overall functional response of a subsurface biofilm. Benzene was catabolized in the aerobic zone, and did not activate the GGKE response, and exhibited the highest biomass concentrations of all columns. PCP was not catabolized during this study, but was found to elicit two responses, oxidative phosphorylation uncoupling and GGKE, that appeared to be concentration dependent. Oxidative uncoupling was the controlling metabolic response up to 10 mg/L PCP, while the GGKE stress response was activated near 20 mg/L PCP. PCP column biomass did not show long-term biomass detachment, although immediate detachment occurred during initial GGKE activation. Cd column biomass activated the GGKE response as perturbing Cd concentrations increased. Extracellular polymeric substance (EPS)-Cd complexation was a possible detoxification mechanism, as biomass concentrations did not decrease with increasing Cd concentration, and increased as Cd concentrations decreased. Results of this study suggested that the increased exposure of electrophilic contaminants to sand column biomass did not cause biomass detachment. / Master of Science

Tout support immergé dans l’eau est rapidement colonisé par de nombreux organismes micro- et macroscopiques. Ce phénomène séquentiel et complexe appelé biofouling est à l’origine de nombreux préjudices économiques et écologiques, notamment dans le milieu marin. L’interdiction récente de certaines substances toxiques, utilisées comme biocides dans les revêtements antifouling des coques de bateaux notamment, a relancé l’intérêt de rechercher de nouvelles molécules antifouling respectueuses de l’environnement. L’objectif de cette thèse a été de développer et d’amorcer l’étude de la représentativité d’un bio-essai permettant d’évaluer le potentiel antifouling de molécules et de revêtements sur des « biofilms » mono et plurispécifiques in vitro en microplaques grâce à l’utilisation de fluorochromes. Le choix a été fait de se focaliser sur le biofilm primaire car il est envisagé que l’élimination ou la limitation de ce dernier réduisent le biofouling. Cinq souches de bactéries marines pionnières, isolées de la Rade de Toulon et en Bretagne, ont été utilisées afin de comparer l’efficacité anti-adhésion de molécules commerciales et naturelles. Deux dérivés de synthèse de substances naturelles marines (TFA E et Z) ont présenté une activité significative associée à une absence de toxicité sur bactéries, suggérant ainsi un mode d’action anti-adhésion spécifique. En outre, les différences de sensibilité entre souches ont confirmée l’importance de réaliser le bio-essai avec un panel diversifié de bactéries.Afin de voir si les données obtenues en laboratoire reflétaient ce qui se produit dans le milieu naturel, une comparaison entre les résultats du bio-essai en microplaque appliqué à six revêtements et les biofilms qui ont colonisés ces mêmes peintures immergées un mois dans la Rade de Toulon (analysés par cytométrie de flux, microscopie et PCR-DGGE), a été effectuée. Les analyses quantitatives ont suggéré une cohérence entre les deux approches même si l’absence de revêtement d’efficacité intermédiaire et le nombre de systèmes testés limite la portée de nos conclusions / Any surface immersed in water is rapidly colonized by a lot of micro-and macroscopic organisms. Sequential and complex phenomenon known as biofouling, lead to major economic and ecological damages, particularly in the marine environment. The recent ban of toxic substances used as biocides in antifouling coatings for ship hulls notably, has relaunched searching for new molecules antifouling environmentally friendly. The objective of this thesis was to develop and initiate the study of the representativeness of a bioassay allow to assess the potential of molecules and antifouling coatings on "biofilms" mono and multi-species microplate using of fluorochromes. It was decided to focus on the primary biofilm because it is possible that the elimination or limitation of the latter reduce biofouling. Five strains of marine bacteria pioneers, isolated from the Bay of Toulon and Brittany, were used to compare the effectiveness of a settlement of commercial and natural molecules. Two derivatives of natural substances Marine (TFA E and Z) showed significant activity associated with a lack of toxicity in bacteria, suggesting a specific anti-adhesion mechanism. In addition, differences in sensitivity between strains have confirmed the importance of making the bioassay with a various bacteria. To see if the data obtained in the laboratory showed what happens in the natural environment, a comparison between the results of the bioassay in microplate applied to six coatings and biofilms isolated on the same paint immersed for a month in the Bay of Toulon (analyzed by flow cytometry, microscopy and PCR-DGGE) was performed. Quantitative analysis suggested a correlation in between approaches although the absence of effectiency intermediate coating and the number of tested systems limit our conclusions

La connaissance et la maîtrise de la présence d’un biofilm représentent aujourd’hui un challenge important. Dans le contexte d’étude des capacités de développement des biofilms, BioFilm Control fait figure de pionnier grâce à leur test nommé Biofilm Ring Test. Basé sur une sollicitation du biofilm via l’attraction, par un aimant, de microbilles magnétiques au centre du puits, le test évalue la présence de biofilm par l’absence de regroupement des billes au centre du puits à un instant donné. L’enjeu de ce travail est de décliner le BioFilm Ring Test® en un examen dynamique, non destructif et à l’échelle microscopique. Dans le biofilm, la matrice polymérique assure la cohésion entre cellules et confère une protection aux bactéries qui vivent au sein du biofilm. Les propriétés mécaniques de la matrice sont donc un indicateur de l’état local du biofilm. La recherche de ces paramètres permet de pouvoir prédire et contrôler la formation, l’accumulation et la dissémination de bactéries propageant les infections et/ou l’encrassement. Néanmoins, la détermination des propriétés mécaniques des biofilms nécessite des précautions et l’usage d’un vocabulaire homogénéisé et de méthodes unifiées au sein de la communauté. Pour cela, une première partie de ce travail de thèse consiste en la proposition d’un guide de bonnes pratiques quant à la caractérisation mécanique du matériau biofilm. Dans la deuxième partie de ce travail de thèse, une méthodologie pour le suivi de particules micrométriques au sein d’un matériau vivant est développée. Le recours à des techniques d’imagerie telle que la corrélation d’images numériques permet de remonter à la cinématique du mouvement de chacune des microbilles, qui servent de marqueurs au sein des images traitées, par une mesure sans contact. Cette méthode est ensuite appliquée à l’étude de la formation de biofilm. L’originalité de ce travail repose sur la caractérisation de l’évolution de la typologie du mouvement des microbilles métalliques lors de la formation des biofilms. Il s’agit là de discriminer des comportements de billes révélateurs de la genèse d’un biofilm. En tirant parti de l’observation du mouvement de microbilles inertes introduites dans le milieu bactérien, on détecte des changements de typologies de trajectoires qui semblent être reliés à l’activité de bactéries sessiles, adhésion ou formation de matériel extracellulaire. Les résultats montrent que les diverses étapes de la formation de biofilms sont caractérisées, ce qui permet notamment de discriminer la présence ou non d’antibiotiques mélangés avec les bactéries et d’apprécier leur efficacité. Dans une dernière partie, des recherches encore en phase de développement sont exposées. Elles s’intéressent au comportement du biofilm sous sollicitation volumique. Il s’agit dans ce cas d’observer le biofilm en champ lointain et de suivre le déplacement ou la déformation d’un marquage constitué d’un agglomérat de microbilles, plongées dans un champ magnétique. Ces premiers travaux pourront servir d’ébauche à des travaux futurs dans le but de caractériser quantitativement le matériau biofilm. / The control of biofilm formation constitutes an important challenge in many industrial and biomedical applications. In this context, BioFilm Control is a pioneer thanks to its test named BioFilm Ring Test. Based on the immobilisation of magnetic microbeads by adherent cells, the assay allows to detect the presence of biofilm at a given time. The aim of this phD project is to translate the BioFilm Ring Test® into a dynamic, non-destructive and microscopic examination of the biofilm state. Whithin the biofilm, the matrix provides a strong cohesion between cells and therefore increases their resistance against chemical or mechanical stress in comparison to their planktonic counterparts. The mechanical properties of the matrix are therefore an indicator of the local state of the biofilm. The search for these parameters makes it possible to predict and control the formation, accumulation and spread of bacteria that propagate infections and/or biofouling. Nevertheless, the determination of the mechanical properties of biofilms requires precautions and the use of an homogenized vocabulary and methods that are unified within the community. To this end, a first part of this thesis work consists in proposing a guide of good practices for the mechanical characterization of biofilm material. In the second part of this work, a methodology for tracking of micrometric particles within a living material is developed. The use of full field measurement method such as digital image correlation makes it possible to trace the kinematics of the motion of each particle, which is a probe of the local environment. This method is then applied to the study of the biofilm formation, by non-contact measurement. The originality of this work is based on the characterization of the change in the microbeads movement during the biofilm formation steps. The aim is to discriminate bead behaviours that reveal the genesis of a biofilm. By taking advantage of the observation of the movement of inert microbeads embedded into the bacterial environment, we detect changes of type of trajectories which seem to be correlated to the activity of sessiles bacteria, adhesion or formation of extracellular material. The results show that the various stages of the biofilm formation are characterized by a non-destructive test. Especially, It allows to appreciate the efficiency of an antibiotic. In the last part, research still in a development phase is presented. It concerns the behaviour of biofilm under mechanical solicitation. This involves observing the biofilm in the far field and following the displacement or deformation of a pattern consisting of an agglomerate of microbeads immersed in a magnetic field. This initial work can be used as a draft for future work to quantitatively characterize the biofilm material.

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