Kött är skelettmuskler från olika djur som till exempel gris, nötdjur eller får. På kött kan många bakterier tillväxa. Pseudomonas är ett släkte bakterier som vanligen orsakar att mat blir dålig. De finns i vår omgivning och kan ge problem bland annat på grund av biofilmbildning inom sjukvård och på livsmedelsindustrier. Slakteriet KLS Ugglarps har tidigare sett att vissa styckningsdetaljer av gris blivit missfärgade med en blå färg och ville ta reda på orsaken till detta. Berörda delar var främst karré och kotlett. Pseudomonas aeruginosa hade tidigare hittats i lokalerna och misstänktes även i detta fall.Kött, både med och utan missfärgningar, undersöktes med hjälp av olika mikrobiologiska metoder för att se vilken bakterie som var orsaken till den blå färgen. Renodling och isolering utfördes och analyser gjordes med hjälp av API 20NE, gramfärgning och oxidastest för att kunna identifiera bakterien och ett referensisolat användes som kontroll. Det visade sig att det inte var P. aeruginosa utan istället en Pseudomonas fluorescens och denna kunde säkerställas som orsaken till blåfärgen genom att den isolerade bakterien från köttet ympades till sterilt kött och då gav en blå färg igen efter inkubering. En smittspårning utfördes i produktionslokalen för att se om bakterien kunde hittas innan uppstart samt under produktionens gång. Proverna visade vid odling att det fanns ytor i lokalen som var odlingspositiva för Pseudomonas. / Meat is skeletal muscle from different animals, such as pigs, cattle or sheep. Pseudomonas are bacteria that may cause food spoilage. The bacteria live in our environment and can cause problems due to biofilm formation in hospitals and industries. The slaughterhouse KLS Ugglarps has found that some pig cuttings have become discolored with at blue color and they wanted to find out what caused it. Pseudomonas aeruginosa had previously been found in the production area and was suspected as the cause.Meat, with and without discoloration, was investigated using various microbiological methods to see which bacterium cause the blue color. Different colonies were isolated and identified. The methods used were API 20NE, gram staining and oxidation test. A reference isolate was used as control. It was found that it was not P. aeruginosa but instead Pseudomonas fluorescens that caused the blue color. This was confirmed by applying the isolated bacteria to sterile meat and the blue color did appear after incubation. A screening for the source of contamination was performed in the production area to see if the bacterium could be found before start-up and during production. The samples taken showed that there were bacteria at both time points and that the production surfaces at the beginning of the production line were more prone to contamination than the other surfaces.

When provided with spatial niches by growth in static nutrient medium, Pseudomonas fluorescens diversifies through adaptive radiation into several well-defined phenotype classes. One of these classes, named wrinkly spreader (WS) for its morphology on agar medium, forms a biofilm at the air-liquid interface through mutations in one of several loci including the genes wspF and awsX. These genes code for negative regulators of di-guanylate cyclases (DGCs). These DGCs catalyse synthesis of cyclic-di-GMP, a second messenger, overproduction of which effects physiological changes leading to overproduction of a cellulose polymer and the WS phenotype. Intriguingly, a diverse range of wspF mutations leads to diversity both in colony morphology and strain fitness.In this study, I investigate genetic and fitness diversity in wrinkly spreaders with the aim of identifying the causal factors that link genetic diversity and physiological factors with diversity in fitness. I approach the subject from several directions, examining the historical context of genetic diversity in wspF and awsX, distribution of control over output in the Wsp pathway and overall fitness effects of different causal factors. I investigate the genetic basis of wrinkly spreader evolution through generation of a large number of novel WS strains and exploration of the distribution of mutations in the wspF and awsX genes. In combination with this I calculate estimates of the past rates of mutation in these genes, derived from a phylogenetic investigation of a group of orthologues. I examine the response of the Wsp pathway to change in WspF function through a novel computational analysis that is capable of revealing valuable information on control in a biological system based purely on model structure. In addition I show how this analysis can be refined through specification of broad estimates of system parameters, thereby avoiding issues related to over-reliance on specific parameter values. Finally, I investigate the fitness implications of these factors, as well as a variety of others, through assays of fitness in a group of WS strains combined with machine learning analyses of predictive relationships between protein and mutation characteristics and experimentally measured strain fitness, and consider the implications of this analysis in the context of intermediate physiological effects.I find that mutations in the WspF protein that lead to the WS phenotype tend to be located in regions of historically strong conservation, the first time that any such pattern to WS mutations has been identified. Mutations in AwsX, on the other hand, do not fit such a pattern. Computational analysis of the Wsp pathway shows that, regardless of model parameters, pathway output is always more sensitive to changes in methylesterase activity by WspF than to changes in phosphorylation of WspF, which may explain the greater frequency of mutations fixed in vivo seen in the methylesterase domain. Despite these patterns, none of a wide range of mutation and sequence-based biochemical characteristics, including local rates of past evolution and size and position of mutations, exhibited any predictive power over WS fitness. Overall, the findings in this study point towards an essential role for complex pleiotropic effects in strongly modulating the fitness effect of different mutations in wspF.

Ecological processes have the potential to influence evolution through their effects on selection. This thesis explores the effects of two ecological factors - competition and ecological opportunity. Intraspecific (within-species) competition is often expected to drive adaptation and diversification by increasing selection for the use of novel resources, thereby alleviating the detrimental effects of competition. However, this is not always the expected outcome; theory suggests that intraspecific competition can also drive convergent evolution. On the other hand, interspecific (between-species) competition is usually expected to impede adaptation and diversification because competitor species occupy potential available niches, preventing the focal species from diversifying to do so. In this thesis, I review previous experimental studies exploring the effects of competition on adaptive diversification, and then directly test these effects using experimental evolution of the bacterium Pseudomonas fluorescens. I confirm that intraspecific competition drives adaptive diversification, while the effects of interspecific competition are varied. Strong interspecific competition impedes adaptation and diversification, while the presence of weak, non-diversifying interspecific competitors drives diversification through increased resource competition. The presence of ecological opportunity is essential for adaptation and diversification, and so variation in attributes of those opportunities is expected to have important effects on the dynamics of adaptive evolution. In another evolution experiment with P. fluorescens, I tested the effects of variation in ecological opportunity on adaptive evolution and found that the type and arrangement of ecological opportunities drives adaptation but, in this system, not diversification. I also show that ecological opportunity drives differences in the degree of parallel evolution at the phenotypic and genotypic level. Finally, I explore some unexpected genetic changes identified in one of these evolved populations - two synonymous mutations that conferred fitness benefits, and show that the observed fitness improvements are the result of increased gene expression. I have shown that ecological processes can play an important role in shaping the evolutionary trajectories taken by populations. Understanding the interactions between ecological and evolutionary processes is vital for our understanding of evolutionary dynamics as a whole, and the studies laid out in this thesis represent valuable contributions to this field of study.

Little is known concerning the production and significance of bacterial pigments. There is seemingly an open field for studying the effects produced by varying the nutritive content of culture media upon which organisms are grown. This has led to an especial interest in, and the purpose of this investigation.

Utilization of cyanide as a nutritional nitrogen source in P. fluorescens NCIMB 11764 was shown to involve a novel metabolic mechanism involving nonenzymatic neutralization outside of cells prior to further enzymatic oxidation within. Several cyanide degrading enzymes were produced by NCIMB 11764 in response to growth or exposure to cyanide, but only one of these cyanide, oxygenase (CNO), was shown to be physiologically required for assimilation of cyanide as a growth substrate.

Proteolytic enzymes and a glycolytic enzyme used in dishwashing detergents and by the starch conversion industry were examined for their ability to remove bacteria attached to black buna-N rubber. Pure culture and mixed culture biofilms of Pseudomonas fluorescens and Listeria monocytogenes were treated with the proteolytic enzymes Purafect® (Genencor International), Durazym™, and Savinase® and the glycolytic enzyme Termamyl® (Novo Nordisk BioChem North America). Compared to controls, none of the enzyme treatments were able to significantly remove P. fluorescens cells adherent in pure culture (p>0.05). Durazym™, Purafect®, and Termamyl® did significantly reduce the number of adherent cells of L. monocytogenes grown in pure culture. Treatment with Purafect® reduced the number of attached cells of both P. fluorescens and L. monocytogenes when grown in mixed culture. Material which absorbs at 280 nm was released from both pure and mixed culture biofilms when all three proteolytic enzymes were used. No survivors remained after planktonic (free floating) cells of both P. fluorescens and L. monocytogenes were incubated with Purafect®. Reduction in overall numbers of P. fluorescens and L. monocytogenes attached in mixed culture by Purafect® appeared to involve a combination of release of proteinaceous material followed by bactericidal effects on exposed cells. / Master of Science

Ce travail s’inscrit dans l’étude de l’incidence de sidérophores sur la physiologie de la plante. Il décrit plus précisément, à l’échelle phénotypique et moléculaire, l’impact de la pyoverdine produite par la souche bactérienne bénéfique Pseudomonas fluorescens C7R12 sur la croissance, la réponse immunitaire et l’homéostasie du fer chez Arabidopsis thaliana. Le lien fonctionnel entre immunité et homéostasie du fer a été abordé de façon plus spécifique via l’analyse du mode d’action de l’acide β-aminobutyrique (BABA), un potentialisateur des réponses de défense de la plante. En conditions de fer limitantes, afin de pourvoir à la carence, Pseudomonas fluorescens libère la pyoverdine dans le sol sous sa forme non chélatée (apo-pyo). Le complexe fer-pyoverdine (ferri-pyo) est ensuite internalisé par la bactérie. Nous avons vérifié que l’apo-pyo est assimilée par des plantes d’A. thaliana cultivées dans un milieu contenant ou non du fer. De façon remarquable, l’apo-pyo restaure le phénotype de croissance des plantes carencées en fer. Une analyse transcriptomique a révélé que chez ces dernières, l’apo-pyo induit fortement l’expression de gènes associés à la croissance, l’import et la redistribution du fer in planta. En revanche, une répression de l’expression de gènes de défense s’opère. De façon concordante, l’effet promoteur de croissance de l’apo-pyo chez les plantes carencées est strictement dépendant de l’expression des gènes IRT1 et FRO2 codant deux protéines majeures de l’import de fer. De plus, une moindre résistance de ces plantes à Botrytis cinerea a été relevée. L’incidence négative de l’apo-pyo sur les défenses s’accompagne d’une surexpression du facteur de transcription HBI1 jouant un rôle clé dans la régulation de la balance croissance/défense. L’ensemble de ces événements n’a pas été observé chez les plantes cultivées dans un milieu enrichi en fer, démontrant que les effets de l’apo-pyo chez A. thaliana sont conditionnés par le statut en fer de la plante. En parallèle, l’étude du mode d’action du BABA a indiqué que ce potentialisateur de l’immunité est un chélateur très efficace du fer. En conséquence, appliqué à des plantes d’A. thaliana, le BABA déclenche une carence en fer transitoire. Nous avons émis l’hypothèse que cette carence pourrait constituer un signal plaçant la plante en veille défensive. En accord avec cette assomption, les plantes carencées en fer présentent une résistance accrue à B. cinerea et produisent des métabolites secondaires associés aux défenses dont l’accumulation est également induite par le BABA. Ainsi, la carence en fer transitoire occasionnée par le BABA pourrait constituer l’une des composantes de son effet potentialisateur sur l’immunité. En conclusion, ce travail apporte des premiers éléments explicatifs quant à l’incidence de la pyoverdine sur des traits physiologiques de la plante et rapporte un mode d’action orignal du BABA. Plus généralement, il renforce le concept encore naissant de l’existence de régulations croisées entre les voies de signalisation associées à la croissance, l’immunité et l’homéostasie du fer chez les plantes. / Siderophores are strong iron chelators produced by bacteria under iron deficiency conditions. In the present work, we studied the impact of the siderophore pyoverdine, produced by the plant growth promoting rhizobacteria Pseudomonas fluorescens C7R12, on plant physiology from phenotypic to molecular effects with a specific focus on plant growth, immune response and iron homeostasis. Based on our analysis of the mode of action of the non-protein amino acid β-aminobutyric acid (BABA), a priming inducer in plants, we studied more specifically the functional link between iron homeostasis and plant immunity. Under iron deficiency, P. fluorescens excretes the iron free form of pyoverdine (apo-pyo) in the soil. Once chelated with iron (ferri-pyo), the complex is internalized by the bacteria. We demonstrated that Arabidopsis thaliana plants treated by apo-pyo in a medium containing or not iron (Fe 25 or Fe 0) also internalize pyoverdine. Moreover, we observed that under iron deficiency, pyoverdine treated plants did not display the growth reduction induced by iron deficiency. In accordance with this phenotype, a microarray analysis revealed that the expression of genes related to growth and development was induced, as well as genes related to iron uptake and transport in planta. In contrast, the down regulation of the expression of genes related to defense was observed. Correspondingly, we demonstrated that the growth improvement induced by apo-pyo under iron deficiency depends on the expression of IRT1 and FRO2, two major genes involved in iron uptake mechanisms. Of interest, the resistance to Botrytis cinerea conferred by iron deficiency was lost following apo-pyo treatment. The overexpression of the HBI1 transcription factor, known to be involved in the growth-defense tradeoff, can be linked to the above observations. These apo-pyo effects were not observed after treatment of plants under sufficient iron conditions, indicating that in A. thaliana apo-pyo effects are dependent on the plant iron status. In the same time, the analysis of the mode of action of BABA that potentiates plant defense responses demonstrated that BABA is a powerful iron chelator. BABA treatment in A. thaliana triggered a transient iron deficiency response. Based on this assessment, we assume that iron deficiency response and priming of defense may be connected. In accordance with this hypothesis, we showed that plants cultivated under iron deficiency and BABA treated plants both displayed resistance to B. cinerea and produced secondary metabolites associated to defense. Hence, the BABA priming effects on plant defense may be due to the induction of transient iron deficiency. To conclude, this work draws first explications on pyoverdine effects on plant physiology and presents an original mode of action contributing to the priming effects of BABA. In a larger view, this work supports the recent concept of the existence of a cross-regulation between growth, immunity and iron homeostasis in plants.

No description available.

Pseudomonas fluorescens est un bacille à Gram négatif ubiquitaire qui possède un fort potentiel adaptatif. En milieu hospitalier, cette bactérie est fréquemment retrouvée dans les solutions injectables, qu'elle contamine, mais émerge également comme agent pathogène responsable d'infections nosocomiales. Cependant, actuellement peu d'études permettent de déterminer le réel potentiel de virulence de cette bactérie, qui présente pourtant de nombreux traits relatifs à un pathogène opportuniste. <br />Afin d'appréhender le pouvoir pathogène de cette espèce, nous avons caractérisé et étudié une souche clinique, P. fluorescens MFN1032, isolée d'un patient atteint d'une infection pulmonaire. Cette souche est capable de pousser à 37°C et produits des facteurs de virulence. <br />Dans un premier temps, notre intérêt s'est particulièrement porté sur l'activité hémolytique de la souche, l'objectif étant de déterminer le ou les facteurs impliqués dans cette activité. Nous avons caractérisé une phospholipase C, PlcC, qui appartient à une classe de phospholipase C distincte de celles actuellement répertoriées. PlcC est impliquée dans l'activité hémolytique de MFN1032. Les résultats montrent que PlcC est étroitement liée à la production de biosurfactants, qui participent à l'activité hémolytique, via le régulateur transcriptionnel GntR.<br />D'autre part, le potentiel d'adaptation et de virulence de la souche est favorisé par des mécanismes de variations de phénotypes. Ainsi, différents variants phénotypiques ayant émergé de la souche MFN1032 ont été étudiés et comparés à la souche « sauvage », et les résultats montrent que l'activité hémolytique est directement concernée par cette variation. <br />D'un point de vue moléculaire, le phénotype d'une partie des variants est restauré par une complémentation en trans des gènes gacA ou gacS. La surexpression d'un des gènes gac diminue l'émergence des autres variants non complémentables par les gènes gac et présentant un phénotype « hyperadhérent ».<br />Le regroupement des différents résultats suggère que PlcC et GntR pourraient participer au processus d'émergence des variants hyperadhérents.

El fuego bacteriano, causado por Erwinia amylovora, es una enfermedad muy importante a nivel comercial y económico porque afecta a plantas de la familia de las rosáceas y es especialmente agresiva en manzano (Pyrus malus) y peral (Pyrus communis), así como en plantas ornamentales (Crataegus, Cotoneaster o Pyracantha). Esta enfermedad está distribuida por todo el mundo en zonas climáticas templadas de Amércia del Norte, Nueva Zelanda, Japón, Israel, Turquí y Europa. En España, el fuego bacteriano fue detectado por primera vez en 1995 en el norte del País (Euskadi) y más tarde en nuevos focos aparecidos en otras áreas. La enfermedad puede ser controlada comercialmente mediante la aplicación de pesticidas quimicos (derivados de cobre, antibioticos). Sin embargo, muchos de los productos químicos presentan baja actividad o causan fitotoxicidad, y la estreptomicina, el producto más eficaz, esta prohibido en muchos países, incluyendo España. Por tanto, en ausencia de apropiados agentes químicos, el control biológico se contempla como una buena alternativa. En el presente trabajo, un agente de control biológico, Pseudomonas fluorescens EPS62e, ha sido seleccionada de entre 600 aislados de las especies P. fluorescens y Pantoea agglomerans obtenidos de flores, frutos y hojas de plantas de la familia de las rosáceas durante una prospección llevada a cabo en varias áreas geográficas de España. La cepa ha sido seleccionada por su capacidad de suprimir la infecciones producidas por E. amylovora frutos inmaduros, flores y brotes de peral en condiciones de ambiente controlado, presentando unos niveles de control similares a los obtenidos mediante el control químico usando derivados de cobre o antibióticos. La cepa además ha mostrado la capacidad de colonizar y sobrevivir en flores y heridas producidas en frutos inmaduros en condiciones de ambiento controlado pero también en flores en condiciones de campo. La exclusión de E. amylovora medinate la colonización de la superficie, el consumo de nutrientes, y la interacción entre las células del patógeno y del agente de biocontrol es la principal causa de la inhibición del fuego bacteriano por la cepa EPS62e. Estas características constituyen aspectos interesantes para un desarrollo efectivo de la cepa EPS62e como un agente de biocontrol del fuego bacteriano en condiciones comerciales. / Fire blight, caused by Erwinia amylovora is a serious disease of rosaceous plants with great commercial and economic interest that is distributed over the world. Disease may be controlled commercially by the application of chemicals (copper compounds, antibiotics). Many chemical agents have low activity or cause phytotoxicity, and streptomycin, the most effective antibiotic, is not approved for use in many countries, including Spain. Therefore, in the absence of suitable chemical control agents, biological control could provide a useful alternative. In the present work, a biological control agent, Pseudomonas fluorescens EPS62e, has been selected among 600 isolates of P. fluorescens and Pantoea agglomerans obtained from flowers, fruits and leaves of rosaceous plants in a survey performed through several geographic areas of Spain. This strain has been selected for its capacity to suppress immature fruit, blossom and shoot infections caused by E. amylovora, under controlled environment conditions, providing control levels similar to chemical control with copper or antibiotic compounds. The strain has also shown the capacity to colonize and survive well in flowers and wounds on immature fruit under controlled environment conditions but also in flowers under natural conditions. Pre-emptive exclusion of the pathogen E. amylovora by surface colonization and nutrients depletion, and cell-to-cell interaction appear to be the main mechanisms of biocontrol. These characteristics constitute interesting traits for an effective development as a fire blight biological control agent under commercial conditions.