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The Lycopodiaceae is an ancient and cosmopolitan family of fern allies that include an estimated 35 Huperzia species occurring throughout the South-East Asian and Australian region. Thirteen species naturally occur in Australia and are found mainly in the tropical rainforests of far north Queensland. Over the past decade, there has been renewed interest in Huperzia and their respective Huperzine alkaloid concentrations following the discovery of Huperzine A (HupA) and Huperzine B (HupB) alkaloids in H. serrata (Thunb. ex Murray) Trevis. Both alkaloids are of pharmaceutical interest since they are highly selective and potent reversible inhibitors of acetylcholine esterase. Huperzine alkaloid concentrations of Australasian Huperzia have not been well documented, and no prior studies have been undertaken to investigate the amenability of Australasian Huperzia to alternative propagation techniques such as axenic culture. / This research presents an extensive screen of 16 Australasian Huperzia species to investigate their Huperzine alkaloid concentrations. HupA (0.032 to 1.012 mg g-1 DW) was detected in ten out of the sixteen Huperzia species examined, while HupB (0.008 to 0.339 mg g-1 DW) was detected in eight. From this extensive study, H. elmeri (Herter) Holub was observed as the species with the greatest potential to yield high Huperzine-containing individuals. In addition, the screen established that Australasian Huperzia generally contain higher HupA levels than H. serrata, the main source of commercial HupA, which on average only contains 0.082 mg g-1 DW HupA. The fractionation and spectrometric analysis of alkaloids as part of the screen led to the discovery of three Huperzine alkaloids co-occurring within the same plant: HupA, HupB and Huperzine C, isolated from an individual of an Australian H. carinata (Desv. Ex Poir.) Trevis. / The potential of establishing axenic cultures of Australasian Huperzia was also investigated in this research. Actively growing axenic cultures of H. carinata, H. squarrosa (C.Forster) Trevisan, H. phlegmaria (L.) Rothm. and H. phlegmarioidies (Gaudich.) Rothm., together with callus and cell suspension cultures of H. carinata and H. phlegmaria, were successfully established. The results suggest that culturing in total darkness is essential to allow for optimal callus and cell suspension growth. In addition, this study also investigated the possibilities of germinating various Huperzia spores, by both symbiotic and asymbiotic means. Germination of H. squarrosa spores was achieved by both symbiotic and asymbiotic means, and was only observed in cultures which were kept in the dark, implying that there is a form of photo-inhibition mechanism preventing spores from germinating when they are exposed to light. Beneficial effects of various types of spore treatments prior to sowing, in terms of increased spore germination was also observed. / In conclusion, the results presented suggest that Australasian Huperzia are indeed a potentially valuable resource for Huperzine alkaloids. The investigations into the conditions required for the successful introduction and maintenance of Australasian Huperzia in axenic culture has also further extended our understanding of these plants, and their amenability towards axenic culture conditions as a means of alternative propagation.

Spores of various strains of Bacillus cereus are the causative agents of emetic and diarrheal foodborne illnesses. Typically, spores will survive thermal treatments that destroy vegetative cells, and then go on to germinate to form the vegetative cells that are associated with toxin production. The spore has to germinate in order to develop into the vegetative cells that produce toxins, hence a thorough understanding of the proteins and molecular mechanisms that underpin spore germination are of great significance from spore control perspectives. A major objective of this thesis was to use molecular genetic and fluorescence microscopy techniques to characterise the location and function of the GerP proteins in Bacillus cereus 14579. The GerP proteins have been identified from mutagenesis studies across the Bacilli as being implicated in spore germination, most likely by impacting upon the permeability of the spore coat. Data presented in this thesis reveal that the various GerP proteins all localise to the same inner-coat vicinity within the spore, as determined via the super-resolution ellipsoid localisation microscopy technique. The study also reveals that only the GerPA protein is required for the localisation of the other GerP proteins in the developing spore. A number of other coat and or germination associated proteins in B. cereus 14579 were examined in the course of this work. These include the GerN and GerT antiporters, which are both shown to have an involvement in inosine mediated spore germination in this strain. However, hypothetical interactions between antiporter proteins and the ‘linker-like’ N-terminal domain of the GerIA inosine-responsive germinant receptor protein appear unlikely since spores engineered with a truncated GerIA receptor subunit germinate normally. The protein encoded at locus BC1245 was also examined in this work, since it too had been implicated in spore germination. Data presented in this thesis indicate that this is not the case, and that the protein is a component of the spore coat. Overall, the work conducted in this project contributes to knowledge of spore assembly, spore structure and mechanisms that underpin germination, which ultimately, should permit the development of improved methodologies for spore control.

<p>The content and utilization of carbohydrate during the yeast life cycle were studied with special reference to spore germination. The experiments were designed to investigate the nutritional requirements, the changes in the carbohydrate content and dry weight, the respiratory activities, the possible substitution of exogenous glucose by other carbon sources, the effect of temperature treatments, the uptake and distribution of exogenous glucose and the effect of inhibitors of carbohydrate metabolism on germination.</p> <p>The experiments showed the relative importance of endogenous and exogenous carbohydrates, the kind of respiratory activity and the fate of exogenous glucose carbon during germination. The pathway of carbon metabolism essential for germination was also indicated.</p> <p>The present work is the first attempt to compare qualitatively and quantitatively the carbohydrate content of yeast during the three major· phases of life cycle, viz., growth, sporulation and germination. Quantitative data on uptake and distribution of exogenous glucose during germination of yeast ascospores are provided. From an analysis of the results an attempt is made to assess the role, of carbohydrate in the germination of yeast ascospores.</p> / Doctor of Philosophy (PhD)

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.

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.

Bacillus anthracis is a spore-forming bacterium that is included on the list of select agents compiled by the Centers for Disease Control. When a B. anthracis spore germinates, a protective layer of peptidoglycan known as the cortex must be depolymerized by germination-specific lytic enzymes (GSLEs) before the bacterium can become a metabolically active vegetative cell. By exploiting cortex lytic enzymes it may be possible to control germination. This could be beneficial in elucidating ways to enhance current decontamination methods. In this work we created in-frame deletion mutants to study not only the role of one GSLE, SleL, but by creating multi-deletion mutants, we were able to analyze how the protein cooperates with other lytic enzymes to efficiently hydrolyze the cortical PG. We determined that SleL plays an auxiliary role in complete peptidoglycan hydrolysis, secondary to cortex lytic enzymes CwlJ1, CwlJ2, and SleB. The loss of sleL results in a delay in the loss of optical density during germination. However, spores are capable of completing germination as long as CwlJ1 or SleB remains active. HPLC analysis of muropeptides collected from B. anthracis sleL strains indicates that SleL is an N-acetylglucosamidase that acts on cortical PG to produce small muropeptides which are quickly released from the germinating spore. By analyzing the in vitro and in vivo activities of SleL we confirmed the enzymatic activity of the protein, characterized its substrates, and studied the roles of its putative LysM domains in substrate binding and spore-protein association. We were able to show that purified SleL is capable of depolymerizing partially digested spore PG resulting in the production of N-acetylglucosaminidase products that are readily released as small muropeptides. In vitro, loss of the LysM domain(s) decreases hydrolysis effectiveness. The reduction in hydrolysis is likely due to LysM domains being involved in substrate recognition and PG binding. When the SleL derivatives are expressed in vivo those proteins lacking one or both LysM domains do not associate with the spore, suggesting that LysM is involved in directing protein localization. / Ph. D.

Clostridium difficile is a Gram-positive bacterium that causes a toxin-mediated disease, typically in individuals whose normal intestinal flora has been compromised by antibiotic therapy. C. difficile is naturally resistant to many antibiotics and produces spores that can withstand harsh environmental conditions and many disinfectants, making the infection difficult to clear and easy to spread. The infection begins when spores from the environment are ingested and germinate upon exposure to taurocholate and glycine in the digestive tract. This germination process is required to initiate infection and thus represents a good target for the development of novel therapeutics. Although spore germination is necessary for disease transmission, the molecular mechanisms regulating this process are poorly understood. Germination relies on sensing a germinant and triggering degradation of the cortex layer of the spore, which is important for spore resistance. Once the cortex is degraded, the spore can undergo outgrowth to a vegetative cell and secrete toxins to cause disease symptoms. There are several discrete steps to the proteolytic cascade that ultimately lead to cortex hydrolysis. First, the pseudoprotease CspC acts as a germinant receptor for the bile salt taurocholate; CspC then relays this signal to the subtilisin-like serine protease, CspB. CspB is required for efficient cleavage and activation of the cortex hydrolase. SleC. Upon proteolytic activation of SleC, cortex hydrolysis can proceed, which allows subsequent outgrowth. To better understand the mechanistic basis of the germination process, we solved the 1.6 Å structure of the required germination protease, CspB, from C. perfringens (a related pathogen). This structure revealed that CspB is comprised of three domains: an associated prodomain, a subtilase domain, and a jellyroll domain. Our work significantly advanced our understanding of the proteolytic cascade that leads to germination; in particular the structure and function of the CspB protease, and the role of its three domains. We have described the four domains of the cortex hydrolase, SleC, and how they contribute to the activity of SleC. We have recently obtained diffraction-quality crystals of the pseudoprotease, CspC, from an organism more closely related to C. difficile, C. bifermentans. Our latest work, focusing on the germination receptor, CspC, has brought us closer to a three-dimensional structure of this protein, which will likely reveal how it binds ligands and functions in germination.

Fungal infections of animals have become an increasingly important global issue. White-Nose Syndrome is an ongoing fungal epizootic of North American hibernating bats, caused by epidermal infections of the fungus, Pseudogymnoascus destructans. Infections emerged early in 2006 in New York State and have since spread to 35 US States and seven Canadian Provinces, with rates of mortality exceeding 90% in some bat colonies. As an emerging outbreak in North America, the transmission of P. destructans is assumed to occur in a radial fashion outwards from the point of origin. In addition, the factors that may influence P. destructans transmission have been postulated, but not tested before. Lastly, as reproduction is assumed to be strictly clonal in North America, invasive populations should have low genetic diversity, and may even accumulate deleterious mutations over time. The aim of my PhD research is to test these assumptions regarding the spread, evolution, and adaptation of P. destructans using combination of genotyping methods. My results showed how P. destructans isolates have shifted in terms of phenotypes and physiological capabilities since being introduced. In addition, I describe patterns of connectivity across the landscape, which are more consist with the level of anthropogenic activity than variation in climate. The mutations common to all invasive strains of P. destructans are associated with adaptations that have occurred since being introduced from Europe, some with relevant metabolic functions that fit their pathogenic lifestyle. Together, my results revealed significant phenotypic and genotypic changes during the spread of P. destructans in North America. The factors identified here that influence the phenotypic and genotypic changes should help developing better management strategies against the White-Nose Syndrome pathogen. / Thesis / Doctor of Philosophy (PhD)