Universiẗat, Diss., 2005--Kiel.

Paenibacillus velaei, sp. nov. is a soil bacterium capable of producing an unusually large amount of exopolysaccharide (EPS). The EPS contains glucose, mannose, galactose and fucose in a molar ratio of 4:2:1:1. The molecular weight of the EPS is higher than 2x106. The viscosity of 1% EPS is 1300 cP when measured at a shear rate of 1 sec-1. Physiological parameters for optimal production of the EPS were studied and it was found that 1.4 g dry weight per 1 l of medium was produced when the bacteria were grown at 30EC and the pH adjusted at 7± 0.2 in a medium containing glucose as the carbon source. Growing the bacteria on different carbon sources did not alter the quantity or the composition of the EPS produced. No toxicity effects were observed in mice or rats when EPS was administered in amounts ranging from 20 to 200 mg per kg body weight. The data obtained from physical, chemical and biological properties suggest that the EPS may be employed in several industrial and environmental applications. It is an excellent emulsifier, it holds 100 times its own weight in water, it is not toxic, and it can be used to remove mercury, cadmium and lead from aqueous solutions.

This thesis is an investigation of the exopolysaccharides produced by Lactococcus lactis subsp. Cremoris JFR1 and the hyphal cell wall glucans of Candida albicans. L. lactis is an important organism in the dairy industry for the production of fermented dairy products and the exopolysaccharides have been shown to add textural qualities to the foods. C. albicans is a fungal pathogen responsible for the common yeast infection and many post-surgery complications in hospitals and can grow in both the yeast and hyphae form. Through a series of GC-MS, NMR and chemical degradation experiments three unique polysaccharides are discovered in the L. lactis samples giving a molecular basis to the textural qualities provided by these molecules. Additionally, several unique structural features are discovered on the C. albicans hyphal glucan providing possible explanations for the differing immune responses elicited by the hyphae form of the fungus.

Zugl.: Hohenheim, Univ., Diss., 2007

Zugl.: Braunschweig, Techn. Univ., Diss., 2008

Zugl.: Hohenheim, Univ., Diss., 2007

Techn. University, Diss., 2005--München.

Myxococcus xanthus, a model organism for studying development and Type IV pili (T4P), harbors three Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) on its chromosome. CRISPR systems, which function as an adaptive immune system in prokaryotes, are classified into three types based on CRISPR-associated genes. Evidence suggests that these three types mediate immunity slightly differently. M. xanthus CRISPR1 and CRISPR2 are Type I systems while CRISPR3 is a Type III-B system. In a genetic screen, a mariner transposon insertion in the 13th spacer of CRISPR3 (3SP13) was found to restore exopolysaccharide (EPS) production to a pilA mutant. Since the deletion of CRISPR3 failed to suppress a pilA mutation and expression of CRISPR3 from a heterologous promoter led to pilA suppression, it was concluded that the 3SP13 transposon insertion is a gain-of-function mutation. Deletion of the adjacent Repeat Associated Mysterious Proteins (RAMP) genes indicated that they are essential for the 3SP13 transposon insertion to suppress pilA, providing evidence that Type III-B systems may be involved in the regulation of chromosomal genes. We suggest that one of the spacers, once expressed and processed, may inhibit the expression of a negative regulator of EPS production in M. xanthus. / Master of Science

Les microalgues rouges de l’espèce Rhodella violacea produisent un exopolysaccharide soluble dans le milieu de culture. Au cours de ce travail, les conditions optimales de production ont été déterminées en étudiant l’activité photosynthétique et le milieu de culture des microalgues. La croissance cellulaire et la production d’EPS de R. violacea sont optimales avec un milieu de culture f/2 modifié, supplémenté en azote et en phosphore. L’irradiance optimale est de 420 μmol de photons.m-2.s-1, sous une température de 24°C avec un pH de 8,3. La culture de la microalgue en photobioréacteur de 5 L a permis la production optimale de 0,5 g.L-1 de polymère. Le polysaccharide produit est un protéoglycane de type xylane sulfaté et de haute masse molaire (1,2 106 g.mol-1), contenant également du rhamnose, du glucose, de l’arabinose, du galactose et de l’acide glucuronique. L’effet antiparasitaire des polymères de microalgues et de macroalgues a été testé sur des microsporidies, in vitro avec des fibroblastes de prépuce humain infestés par Encephalitozoon cuniculi et in vivo contre la nosémose des abeilles causée par Nosema ceranae. L’exopolymère de R. violacea empêche la croissance des microsporidies in vitro et in vivo, et conduit à une diminution de la mortalité des abeilles. D’autres molécules testées ont également une action antiparasitaire, les EPS de Porphyridium purpureum et marinum, et des carraghénanes sulfatés permettent également de diminuer la croissance des microsporidies et la mortalité des abeilles. / Red microalgae from Rhodella violacea species product a soluble exopolysaccharide release in the media. In this study, optimal culture conditions for exopolysaccharide production were investigated, following photosynthesis activity and culture conditions. This study allowed us to determinate R. violacea optimal media for growth and exopolysaccharide production, which is f/2 media supplemented in nitrogen and phosphorus. Optimal physicochemical parameters are an irradiance of 420 μmol photons.m-2.s-1, a temperature of 24°C, and a pH of 8.3. Photobioreactor of 5 L used to cultivate R. violacea in optimal conditions, gave 0.5 g.L-1 of EPS. Structural analysis of the EPS revealed the production of a proteoglycan, principally composed by xylose, sulfated and with a high molecular mass (1.2 106 g.mol-1). The polymer is complex, as it contains different monosaccharide: rhamnose, galactose, arabinose and glucuronic acid. The antiparasitic effect of polymers from microalgae, and macroalgae were investigated on microsporidia, in vitro against Encephalitozoon cuniculi using Human foreskin fibroblasts, and in vivo against Nosema ceranae using bees. Exopolysaccharide from R. violacea decreases microsporidia growth in vivo and in vitro. In addition, in vivo the polymer allows decrease in bees’ mortality. Polysaccharide from others origins also have antiparasitic effet, such as exopolymer from Porphyridium purpureum and marinum, and sulphated carragheenans which reduce microsporidia growth, and decrease bees’ mortality.

Les exopolysaccharides - des polymères de sucres exportés - sont impliqués dans des fonctions essentielles de la physiologie bactérienne. Ce sont en effet des composants majeurs, respectivement, de la paroi bactérienne, des polymères secondaires attachés à cette paroi, des capsules, et de la matrice du biofilm. Bacillus thuringiensis est une bactérie entomopathogène, appartenant au groupe Bacillus cereus, capable de former un biofilm à l’interface air-liquide. Ce biofilm comporte deux structures distinctes: une pellicule flottant sur le milieu de culture et, en périphérie, en continuité avec celle-ci, un anneau adhérent sur les surfaces solides. Pour identifier les exopolysaccharides constitutifs de la matrice du biofilm chez cette bactérie, j'ai recherché, dans le génome séquencé de la souche 407 de B. thuringiensis, les différents loci chromosomiques susceptibles d'être impliqués dans la production de ces exopolymères. Deux loci ont étés identifiés, que nous avons appelé eps1 et eps2. Le locus eps1 avait déjà été décrit comme n'ayant aucun rôle dans la formation des biofilms chez B. cereus et sa fonction était restée inconnue. Nous avons montré que l'exopolysaccharide Eps1, dépendant du locus eps1, forme une capsule en phase stationnaire et en condition d'hypoxie. Cette capsule, qui présente des propriétés adhésives importantes sur des surfaces biotiques et abiotiques, permet l'adhésion du biofilm sur les surfaces solides et est requise pour la formation de l'anneau du biofilm. En accord avec ces résultats, nous avons observé que Eps1 n'est présent que dans l'anneau du biofilm. En revanche, l'exopolysaccharide Eps2 dépendant du locus eps2 est un élément essentiel de la matrice du biofilm et est nécessaire pour la formation de la pellicule. Enfin, à l'aide de marqueurs fluorescents, nous avons montré que deux souches mutantes capables de ne produire, respectivement, que Eps1 ou Eps2, se distribuent de façon hétérogène dans le biofilm lorsqu'elles sont mises en co-culture. En effet, la souche ne produisant que Eps1 est localisée dans l'anneau tandis que la souche ne produisant que Eps2 est localisée dans la pellicule. L'étude de la régulation de la transcription des loci eps1 et eps2 montre que ces deux loci sont régulés de façon identique. Le répresseur SinR, qui contrôle la formation de la composante protéique de la matrice du biofilm chez B. thuringiensis, n'a aucun effet sur la transcription de eps1 et eps2 chez cette bactérie. En revanche, cette transcription est activée par Spo0A et réprimée par AbrB. Enfin, le régulateur CodY réprime l’expression de ces loci en phase exponentielle, mais stimule cette expression en phase stationnaire tardive. Nos résultats montrent également un rétrocontrôle négatif d’Eps2 sur la production d’Eps1, suggérant l'existence d'une bascule ne permettant la production, au niveau d'une cellule isolée, que d'un seul de ces exopolysaccharides. / Exopolysaccharides - polymers of exported sugars - are involved in essential functions of bacterial physiology. Exopolysaccharides are in fact major components, of the bacterial wall, secondary polymers attached to this wall, the capsules, and finally the biofilm’s matrix. Bacillus thuringiensis is an entomopathogenic bacterium of the cereus group, capable of forming a biofilm at the air-liquid interface. This biofilm has two distinct structures: a floating pellicle on the culture medium and, at the periphery, in continuity with the pellicle, a ring adhering to the solid surfaces. To identify the exopolysaccharides, which constitute the biofilm matrix in B. thuringiensis, I investigated the various chromosomal loci in the sequenced genome of B. thuringiensis strain 407. Two loci have been identified and were called eps1 and eps2. To date, no role in the formation of biofilms in B. thuringiensis had been attributed to eps1 locus. Our data showed that the exopolysaccharide Eps1, depending on the eps1 locus, forms a capsule in the stationary phase and in hypoxic conditions. This capsule, which has significant adhesive properties on biotic and abiotic surfaces, allows adhesion of the biofilm to the solid surfaces, thus forming of the biofilm ring. Consistently with these results, we observed that Eps1 is present only in the biofilm ring. We found that Eps2 exopolysaccharide depending on the eps2 locus is an essential element of the biofilm matrix and is necessary for the formation of the pellicle. We have shown using fluorescent markers that two mutant strains capable of producing only type of exopolysaccharides Eps1 or Eps2 are distributed heterogeneously in the biofilm when they are cocultured. The mutant strain producing only Eps1 is localized in the ring while the mutant strain producing only Eps2 is located in the pellicle. Our data show that the transcription of eps1 and eps2 loci is regulated identically by the same set of regulators. The SinR repressor, which controls the formation of the protein component of the biofilm’s matrix in B. thuringiensis, has no effect on the transcription of eps1 and eps2 in this bacterium. The transcription is activated by Spo0A and repressed by AbrB. The CodY regulator represses the expression of these loci in exponential phase, but activates it in the late stationary phase. Our results also show a negative feedback from Eps2 on the production of Eps1, suggesting the existence of a switch, allowing only one of these exopolysaccharides to be produced in an isolated cell.