Many spore forming bacteria are the causative agents of severe disease, such as Bacillus anthracis and anthrax. In these cases, the spore often acts as the infectious agent. Spores boast extreme resistance to chemical and UV damage among other bactericidal conditions. This is problematic due to the difficulty and economic costs of decontaminating exposure sites. The present work focuses on identifying and characterizing proteins active within spore germination, with a focus towards understanding the triggering of the major stages of germination. Understanding how each stage is initiated could allow for development of methods that induce these processes to efficiently germinate spores, thus facilitating cheap and effective decontamination. Sequencing of a spore transposon insertion library after exposure to germinants led to the identification of 42 genes with previously uncharacterized roles in spore germination. Fourteen of the genes, encoding proteins associated with the inner spore membrane, were further characterized. Mutants lacking these genes portrayed phenotypes consistent with failure of a GerA receptor-mediated germination response, and these genes affect the earliest stages of germination. Chemical cross-linking was used to characterize protein interactions important for stage II of spore germination. Site-directed in vivo crosslinking indicated that YpeB may exist as a multimer within the dormant spore. Further investigation of individual protein domains using bacterial two-hybrid analysis suggested that both N- and C-terminal domains of YpeB contribute to the formation of a multimer. In addition, the uncharacterized YpeB N-terminal domain was demonstrated to have strong self-association and may mediate self-association within the dormant spore. Additional genes that contribute to efficient initiation of spore germination in a GerA-dependent manner were identified via TnSeq. Chemical cross-linking of dormant spores was implemented to characterize protein interactions leading to stabilization and activation of an important enzyme that contributes to cortex degradation in stage II of germination. The presented studies employed a variety of techniques to provide additional insight into both stages of spore germination with a goal of furthering understanding of specific events that contribute to a loss of spore dormancy. / Doctor of Philosophy / Few bacterial species can undergo a specialized division process leading to the generation of a bacterial endospore. Endospores are dormant cells that boast resistance to a variety of environmental conditions that would otherwise cause bacterial cell death. These resistance traits make endospores immune to traditional bactericidal methods, making decontamination a nontrivial task. Further complicating the matter, spores are often the infectious particle of the associated disease, including hospital acquired diarrhea, infant botulism, anthrax, and many others. Presented work focuses on furthering understanding the process by which a dormant spore returns to a typical growing bacteria cell. Comprehension of major steps in this process may lead to novel methods for spore cleanup in which mechanisms within the spore are subverted to force a return to a typical bacterial cell state.

Muskmelon (<i>Cucumis melo</i> L.) embryos are surrounded by a single layer of endosperm and a two- to four-cell-layered perisperm. The embryonic axis must penetrate the perisperm envelope (perisperm + endosperm) for germination to occur. Radicle emergence could result from increased turgor, weakening of the perisperm tissue, or a combination of both. In a previous report, turgor of the embryonic axis did not increase prior to radicle emergence. This suggests that weakening of the perisperm envelope is a prerequisite for radicle emergence in muskmelon seeds. The changes in cell wall sugars of the perisperm tissue were studied during imbibition using gas chromatography and high performance thin layer chromatography. The major cell wall sugars identified in the micropylar perisperm tissue were glucose, galactose, xylose, and rhamnose. Endo-β-mannanase has been shown to be responsible for endosperm degradation in seeds of Solanaceae, Leguminosae, and Fabaceae. However, the lack of mannose in the cell walls of muskmelon perisperm tissue suggests that this enzyme is not involved in muskmelon seed germination. Structural changes of the micropylar perisperm envelope tissue were visualized during imbibition using electron microscopy. Scanning electron microscope images revealed changes in the perisperm envelope before radicle protrusion. The cell walls of the perisperm envelope tissue were degraded starting 5 h prior to radicle emergence. An Instron was used to measure the mechanical resistance of the perisperm envelope during imbibition. The force and total energy required to penetrate the perisperm tissue of imbibed muskmelon seeds decreased gradually during imbibition. This study confirmed that the perisperm envelope tissue offered mechanical resistance to the expanding embryonic axis. Degradation of the perisperm envelope tissue before radicle protrusion facilitated radicle emergence. / Master of Science

The stage of maturity of seeds at harvest is an important factor that determines seed vigor. Separating seeds from a seed lot composed of many different stages of development can be difficult especially after maximum dry mass is attained. Separating seeds based on their physiological maturity is more challenging than sorting seeds based on their physical properties. Seeds may be non-destructively sorted using chlorophyll fluorescence (CF) as a marker of seed maturity. This study was conducted to test whether CF could be used to remove low vigor immature seeds from muskmelon (Cucumis melo L.'Top Mark') and tomato (Lycopersicum esculentum) seed lots. Light treatments were applied to determine whether the light environment during seed harvesting and processing could affect chlorophyll content and seed vigor. Seeds from nine stages of development were collected from 'TopMark'. Seeds from three stages of fruit development (red ripe, breaker, and mature green) were harvested from tomato cultivar Money Maker and two phytochrome mutants: phytochrome A mutant, fri-1 and phytochrome B mutant, tri-1. The SeedMaster Analyzer (Satake USA Inc., Houston Texas) was used to measure CF and to sort individual seeds according to CF levels. Immature tomato seeds and muskmelon, harvested from green fruits, had the highest CF (p>0.001). Contrary to the results obtained with the other tomato genotypes, the vigor of tri-1 did not change inversely with changing CF levels, rather, seeds with low CF had the same vigor as seeds with high CF. This result may suggest that the presence of phytochrome B exerts an inhibitory influence on vigor in tomato seeds, and that the persistent presence of chlorophyll during seed development does not affect vigor. The light treatments had no consistent effect on seed chlorophyll content or on vigor in either tomato or muskmelon. / Master of Science

Sweet basil (Ocimum basilicum L.) is a warm season herb usually propagated from seeds. Establishment of basil is difficult as seed germination may be limited, particularly during field seeding at cold soil temperatures. The germination of six cultivars (\'Italian Large Leaf\', \'Italian Large Leaf\' 35X, \'Nufar\', \'Genovese\', \'Genovese Compact Improved\' and \'Aroma 2\') of sweet basil seeds were tested on a one dimensional thermo-gradient table over temperatures ranging from 0 to 50"C. At temperatures below 20"C, germination among cultivars was more variable and the mean time to germination (MTG) increased to greater than 25 days for some cultivars. Germination declined sharply and had a sudden termination at high temperatures above 40"C for all six cultivars.  There were statistical differences among the cultivar base temperatures, which ranged between 10.1 and 13.3"C. The optimal and ceiling temperatures for germination were similar and did not differ statistically among the cultivars compared in this study. The average optimal temperature for all cultivars was 35 ± 0"C, while the average ceiling temperature was 43 ± 1.3"C. Stored seeds (> 5 years) had lower seed vigor and lower germination percentage, also lower ceiling temperature compared with the fresh seeds of the same cultivar (\'Italian Large Leaf\'), but the base temperatures were the same for both new and old seeds. Sweet basil (Ocimum basilicum L.) seeds produce a thick layer of mucilage around the pericarp within minutes after hydration. Mucilage is most prevalent among plant species adapted to surviving in arid sandy soils, though its significance in determining ecological fitness is unclear. The mucilage produced by seeds is reported to be composed of cell-wall polysaccharides that are deposited in testa pericarp cells during development. In this study, sweet basil seeds were examined using light and environmental scanning electron microscopy. The mucilage of basil seeds is held together by columnar structures that unfolded from the pericarp and helped hold and stabilize the mucilage to the outer surface. The mucilage was removed using diluted hydrochloric acid to compare performance of seeds with and without mucilage. Mucilage removal did not inhibit seed germination under ideal laboratory conditions but decreased the water content of seeds significantly. The water content of intact seeds was almost 4 times greater than seeds without mucilage. Mucilage enabled seeds cling to an incline board set to a steeper angle than seeds without mucilage. The fully hydrated seeds approached zero water potential, so the mucilage did not prevent seeds from fully hydrating. Soil (media) germination testing showed the seeds with mucilage had higher germination percentage than the seed without mucilage on several different types of media. Seeds with mucilage also had higher survival percentages after 10 days on different types of media. Basil seeds mucilage acts as a reservoir to hold loosely bound water at high water potential so it is available for seed germination and early seedling development. / Master of Science

Bacillus anthracis is the causative agent of the disease anthrax and poses a threat due to its potential to be used as a biological weapon. The spore form of this bacterium is an extremely resistant structure, making spore decontamination exceptionally challenging. During spore germination, nutrient germinants interact with Ger receptors, triggering a cascade of events. A crucial event in this process is degradation of the cortex peptidoglycan by germination-specific lytic enzymes (GSLEs), resulting in cells that are easily killed. This work investigated the regulation of the GSLE SleB by other proteins in the spore. A full understanding of how GSLEs are held inactive in the dormant spore and are activated during germination could lead to development of simplified spore decontamination strategies in which spore germination is the first step. It was found that SleB and YpeB are co-dependent. In the absence of one protein, the other is degraded during sporulation by an unidentified protease(s), although HtrC and SpoIVB are not likely responsible. Specific regions and residues of YpeB were also identified as being important to its relationship with SleB. While some evidence suggests that SleB and YpeB physically interact, a direct interaction was not observed in vivo or in vitro. YpeB was demonstrated to be proteolytically processed by HtrC during germination, resulting in stable products containing the YpeB C-terminus. The presence of inhibitory PepSY domains at the C-terminus of YpeB, coupled with YpeB degradation during germination, may suggest that YpeB processing results in SleB activation. Modification of the predominant YpeB cleavage sites or deletion of htrC reduced proteolysis, but cleavage at other sites still resulted in YpeB instability. Additionally, these changes did not have a significant impact on SleB activity. SleB regulation by other spore proteins was also examined. To test if SleB activation is Ger receptor-dependent, Bacillus subtilis strains lacking Ger receptors and/or GSLEs were germinated via non-nutrient means. Results indicated SleB can be activated independent of these proteins. B. anthracis homologs of the B. subtilis lipoproteins YlaJ and YhcN were also studied, but deletion of these genes did not result in significant changes in SleB stability or activity. / Ph. D.

Master of Science

Man has been concerned with low seed germination for many years. If one could develop economical seed treatments that would break dormancy of new or hard seeds or stimulate old seeds, he would make an important contribution to the agricultural economy. The purpose of this study was to determine the effects of some electrically produced treatment on spinach seed. Seeds were treated between two 6-inch by 6-inch plates, 1-inch apart, at approximately 150 volts rms. Frequencies were 60, 500, and 900 cycles per second. Exposure times were 30, 60, and 90 seconds for each frequency. One sample was treated in an ultrasonic generator at 20 kc for one minute. Another sample was soaked in water for one minute for the wet check. Ultraviolet light at 3654 A and 3129 A was used on other treatments. Four reps consisting of 100 seeds each were germinated in a refrigerator at about 45°F. After 9 days and 13 days the germination of those treated ultrasonically, the wet check, and those exposed to 3129 A was significantly greater than for the other treatments including the dry check. After 31 days all of the treatments germinated above 82% and there were no significant differences for any of the treatments. A field study was conducted in which 102 seeds were planted individually five inches apart in beds with rows ten inches apart. Adverse weather conditions caused poor stands which gave unreliable data. Four reps of 80 seeds each were planted in flats in a greenhouse. After 28 days they were thinned to 10 plants per flat. There were no significant differences in the 28 day emergence or in early growth. Data regarding differences in crop yield were inconclusive. / Master of Science

The effect of fertilizers and the variable weather conditions of temperature and moisture have presented many problems of a scientific nature of the agronomist. Not only has this been true for some time with the scientific agronomist, but of late years these problems have found their place in the working knowledge of the practical farmer from a crop standpoint. Many times the farmer fails to get a stand of certain crops due to bad germinations of the seed. This is attributed to bad seed, disease seed, old seed, to wet land and many other similar reasons. Not until recent years has the farmer attributed the inability to get a good stand of different crops on the land to the effect of fertilizers. Some of the more wide-awake farmers of today have noticed repeatedly that with certain seeds when sown with certain fertilizers a poor germination has always resulted. It has become common practice of many farmers in planting crops in which the seed is sown with the drill to mix the fertilizer with the seed and sow it out the same spout together, thus putting the fertilizer in contact with the seed. Many of the farmers are reporting bad germination from this practice and are asking for information about the effect of certain fertilizers on germination of certain seeds. To be able to give practical information on such problems as these, is the aim for this experiment work. / Master of Science

Ce travail s'intéresse au recyclage des rejets d'eaux usées par les malteries, principalement à l'étape de trempe. L'impact négatif de ces eaux sur la qualité des malts a été mis en évidence: leur réutilisation provoque un retard de prise d'eau par le grain et un retard de la germination. Puis, la caractérisation de ces eaux de trempe a permis d'identifier les inhibiteurs de la germination: il s'agit des intermédiaires d'oxydation et de polymérisation des composés phénoliques. Enfin, un procédé de traitement de ces eaux de trempe par bioréacteur à membrane a été mis au point. Testé aux échelles de laboratoire et pilote - avec ou sans couplage à une étape d'osmose inverse ou de charbon actif - ce procédé s'est révélé efficace pour l'épuration de l'eau et l'élimination des inhibiteurs. La réutilisation de ces eaux traitées dans l'étape de trempe n'a pas eu d'impact sur la qualité du procédé de maltage, et les malts ainsi préparés ont permis d'obtenir des bières de qualités équivalentes / This work is focused on the reuse of malting process wastewaters, especially steep-out waters. The negative impact of these waters on malt quality was demonstrated: their reuse results in a water uptake delay and a germination delay. Then, the characterisation of these steep-out waters allowed to identify germination inhibitors: they are intermediates of oxidation and polymerisation reactions of phenolic compounds. Finally, a membrane bioreactor treatment process of steep-out waters was developed. Studied at laboratory and pilot scales - with and without coupling to reverse osmosis or activated carbon step - this process was efficient for water treatment and for the removal of inhibitors. The reuse of these treated waters at the steeping step didn't have any negative impact on the process performances,and malts obtained allowed to prepare beers of comparable qualities

Bacillus cereus est une bactérie sporulante pathogène largement disséminée dans la nature. Les propriétés de résistance de ses spores aux traitements appliqués dans la chaîne de transformation des aliments font de B. cereus un contaminant à l’origine de toxi-infections alimentaires. La température est considérée comme l’un des facteurs environnementaux majeurs influençant la résistance de la spore. La variabilité des propriétés des spores liée à des modifications profondes dans leur structure contribue à une incertitude sur l’efficacité des processus de décontamination. Ce travail de thèse avait pour objectif de caractériser le mécanisme d’assemblage des enveloppes de la spore en fonction de la température de sporulation, en particulier le rôle de la protéine morphogénétique CotE chez B. cereus. La protéine CotE est retrouvée en abondance dans les spores produites à 20°C, une température suboptimale, par rapport à celles produites à 37°C, température optimale de croissance de la souche ATCC14579. La protéine CotE est détectée dans les tuniques et l’exosporium, structures protectrices de la spore. L’observation en microscopie électronique à transmission de spores d’un mutant DcotE révèle un problème d’assemblage de l’exosporium à 37°C et 20°C, mais également un défaut d’assemblage des tuniques à 20°C, ce qui suggère un rôle fondamental de CotE dans la mise en place de ces deux enveloppes, dépendant de la température de sporulation. Par microscopie à fluorescence, nous avons montré la cinétique de production de la protéine CotE au cours de la sporulation ainsi que sa localisation finale dans la spore mature, qui ne sont pas significativement impactées par la température de formation des spores. Nos résultats suggèrent également que la protéine CotE puisse créer le lien maintenant l’exosporium au contact des tuniques et du cortex. Enfin, nous avons montré que la protéine CotE est impliquée dans la germination et la résistance physique et chimique des spores. / Spores of the pathogenic bacterium Bacillus cereus are widespread in the environment and responsible of foodborne poisonings. Spores are a major concern to public health because of high resistance to treatments applied in food processing operations. Sporulation temperature is a main environmental factor that influences spore resistance properties. The variability of the conditions in which spores are formed during the sporulation process deeply modified their structure and consequently the efficiency of decontamination treatments. The aim of this work was to study the mechanism of spore layers assembly as a function of the sporulation temperature, and more precisely the role of the CotE protein in B. cereus. This morphogenetic protein is more detected in spores formed at 20°C, a suboptimal growth temperature than at 37°C, the optimal growth temperature, of the ATCC14579 strain. Observations in transmission electronic microscopy of DcotE spores revealed an assembly default of the spore exosporium at 37°C and 20°C but also of the spore coat at 20°C, suggesting that CotE has a role in the assembly of both layers, depending of the sporulation temperature. By fluorescence microscopy, we evidenced the kinetics of CotE production during sporulation and its final localization in mature spore, which are not dependent on the temperature of spore formation. Our results suggest that CotE could make a link to maintain the exosporium close to coat and cortex structures. Finally we showed that CotE also plays a role in germination and resistance properties of B. cereus spores to physical and chemical treatments.