Sporulation in Bacillus subtilis is triggered by starvation for carbon and nitrogen sources. The process of endospore formation involves a highly orchestrated program of gene expression resulting in morphological change. A key early event is the asymmetric sporulation division, which yields the smaller prespore and larger mother cell. The transcription factor σF becomes active in the prespore, and directs the transcription of approximately 50 genes. One of those genes, paramount to this study, is spoIIR. The SpoIIR protein is exported, and localizes to the inter-membrane space of the sporulation septum, which may be mediated directly by SpoIIGA. SpoIIR and SpoIIGA are essential for the activation of σE from its inactive precursor, pro-σE. Pro-σE, encoded by spoIIGB, is part of a two-gene operon that also includes spoIIGA located immediately upstream. SpoIIGA is an integral membrane protein, and is an aspartyl protease that cleaves 27 residues from the N-terminus of pro-σE to yield active σE. The N-terminal portion of SpoIIGA contains five membrane-spanning hydrophobic domains. The C-terminal portion lies within the mother cell cytoplasm, and contains the proteolytic domain with residue D183 acting as the catalytic aspartate. Activation of the proteolytic domain of SpoIIGA is dependent on signaling through SpoIIR at the septum. The interaction with SpoIIR is thought to cause a conformational change in the proteolytic domain of SpoIIGA, which activates it. Normally, σE only becomes active in the mother cell. The current model for σE compartmentalization suggests that before septation SpoIIGA is evenly distributed throughout the entire cell membrane. Once septation occurs, there is a higher amount of SpoIIGA in the mother cell than in the prespore. SpoIIGA is then concentrated in the septum and the mother cell outcompetes the prespore for the limited amount of SpoIIR available. We are investigating the role of SpoIIGA in compartmentalization of σE. To accomplish this, a spoIIGA-gfp translational fusion was used. The fusion was introduced into a B. subtilis SpoIIGA mutant (SpoIIGA49) that lacks functional SpoIIGA because of a point mutation, G100R. The mutation is located within the fourth membrane-spanning domain. The spoIIGA-gfp fusion was also introduced into a spoIIGA knockout strain (spoIIGA-null) in order to assess the effect of SpoIIGA-GFP on sporulation without influence from SpoIIGA-G100R. The SpoIIGA49 strain that expressed spoIIGA-gfp in the prespore from the σF-directed promoter, PspoIIQ showed a weak GFP signal in the prespore, and restored sporulation to parental levels. The same fusion also showed a weak prespore GFP signal in the spoIIGA-null background, however sporulation was not restored. This result suggests that the fusion protein could interact with SpoIIGA-G100R across the septum through SpoIIR, restoring proteolytic activity to SpoIIGA-G100R. In both cases, fluorescence was only detected after σE had become active. Expression of spoIIGA-gfp from its natural promoter also largely complemented SpoIIGA49, but only partially complemented spoIIGA-null. Again, GFP fluorescence was weak, and was only detected after σE had become active. Possible explanations for the poor fluorescence are: 1, GFP function is impaired. 2, SpoIIGA-GFP is present at low levels. To assess the amount of protein present, western blot analysis was performed using anti-GFP antibodies. The results indicated weak expression. When spoIIGA-gfp was expressed from PspoIIG, protein was detected two hours after entry into stationary phase (T2), which was before GFP fluorescence was detected, with or without SpoIIGA-G100R. Detection of SpoIIGA-GFP from PspoIIQ occurred by T4 in with and without influence from SpoIIGA-G100R. Because PspoIIQ requires σF to be active and PspoIIG is active prior to septation, it was expected that SpoIIGA-GFP expressed from PspoIIG would be detected earlier. Weak bands representing SpoIIGA-GFP were observed which suggests low levels of SpoIIGA-GFP. Overall, the PspoIIG promoter appeared to drive more expression of spoIIGA-gfp before septation than PspoIIQ in the prespore alone. / Microbiology and Immunology

The survival of all organisms depends on their ability to perform certain enzymatic activities and the ability to construct certain structures. In prokaryotes, enzymes are required for the final reactions of peptidoglycan (PG) synthesis, the structural element of the bacterial cell wall. These proteins, known as penicillin-binding proteins (PBPs), are identified through the presence of conserved motifs within their functional domains. The Class A high-molecular weight PBPs are bifunctional, performing the penicillin-sensitive transpeptidase activity and the glycosyl transferase (GT) activity required for the polymerization of the glycan strands. The Class A PBPs in Bacillus subtilis are PBP1, PBP4, PBP2c, and PBP2d (YwheE) and they are encoded by ponA, pbpD, pbpF, and pbpG (ywhE), respectively. These proteins appear to be somewhat functionally redundant because removal of one or more does not cause any noticeable change in phenotype. However, the loss of PBP1 has previously been demonstrated in B. subtilis to cause a decreased growth rate and changes in morphology of vegetative cells, both of which are increased upon the additional loss of PBP4. Furthermore, the loss of sporulation-expressed Class A PBPs, PBP2c and 2d, causes a 10,000-fold decrease in the production of heat resistant spores. This double mutant is shown to have changes in the structural parameters of cortex PG that appear minor when compared to other strains, but are coupled with a large defect on the deposition of cortex PG, apparently from the synthesis of an abnormal germ cell wall. The Class A PBPs are believed to be the only proteins capable of performing the GT activity and it is therefore believed that cell viability requires the presence of at least one functional Class A PBP. This requirement has been demonstrated in other organisms, but a B. subtilis strain lacking all Class A PBPs is viable. The phenotypical changes seen in the PBP1 mutant are exacerbated in this strain. The GT activity remaining in this strain is sensitive to the antibiotic moenomycin in vitro whereas it appears resistant in vivo. Identification of the protein(s) performing this novel GT activity will rely on the demonstration of the GT activity in vitro. / Ph. D.

Clostridium perfringens is a significant human pathogen, causing 250,000 cases of food poisoning in addition to several thousand potentially lethal cases of gas gangrene each year in the United States. Historically, work in this field has centered around toxin production, as C. perfringens can produce over 13 toxins. This work expands the knowledge of the starvation-response of C. perfringens, which includes several potential virulence factors, sporulation, motility and biofilm formation. Sporulation protects cells from a variety of stresses, including starvation. Efficient sporulation requires the transcriptional regulator CcpA, mediator of catabolite repression. Sporulation is repressed by glucose, but, surprisingly, in a CcpA-independent fashion. C. perfringens cells in a biofilm are resistant to a number of environmental stresses, including oxygen and antibiotics. Biofilm formation is repressed by glucose, and other carbohydrates, independently of CcpA. Gliding motility, a type four pili (TFP)-dependent phenomenon, affords C. perfringens with a mechanism for moving across a solid surface in response to carbohydrate starvation, while carbohydrates supplementation at high levels delay the initiation of the motility response. CcpA is required for the proper initiation of motility, a ccpA^-C. perfringens strain showed a considerable increase in the time to initiation of motility on lactose and galactose, and was unable to move at all in the presence of glucose. Gliding motility represents the most significant finding of this work. TFP were previously undescribed in any Gram-positive bacterial species, and this work produced genetic evidence suggesting their presence in all members of the clostridia, and physical evidence for TFP-dependent gliding motility in a second species, C. beijerinckii. / Ph. D.

Bacterial spore peptidoglycan (PG) is very loosely cross-linked relative to vegetative PG. Theories suggest that loosely cross-linked spore PG may have a flexibility which contributes to the attainment of spore core dehydration. The structure of the PG found in fully dormant spores has previously been examined in wild type and many mutant strains. These analyses showed little correlation between the degree of spore PG cross-linking and core dehydration. However, these studies only examined the structure of PG from dormant spores and did not allow for the structural analysis of spore PG during sporulation when actual spore PG synthesis and core dehydration occur. Structural analyses of developing spore PG from wild type Bacillus subtilis and eight mutant strains are included in this study. Structural analyses of developing spore PG suggest the following: a) the germ cell wall PG is synthesized first next to the inner forespore membrane; b) cross-linking is relatively high in the first 10% of spore PG synthesized; b) a rapid decrease in cross-linking is observed during synthesis of the next 20% of the spore PG; and c) this decrease is followed by an eightfold rise in the degree of cross-linking during synthesis of the final 70% of the spore PG. This increasing gradient of cross-linking was previously predicted to contribute to the attainment of spore core dehydration. However, analyses of mutant strains indicate this cross-linking gradient is not required for the attainment of spore dehydration. / Master of Science

Clostridium perfringens is a versatile pathogen capable of causing a wide array of diseases, ranging from clostridial food poisoning to tissue infections such as gas gangrene. An important factor in virulence as well as in the distribution of C. perfringens is its ability to form an endospore. The symptoms of C. perfringens food poisoning are directly correlated to the release of an enterotoxin at the end of the sporulation process. The sporulation process in C. perfringens is subject to carbon catabolite repression (CCR) by sugars, especially glucose. CCR is a regulatory pathway that alters transcription based on carbon source availability. In Gram-positive bacteria, the HPr kinase/phosphatase is responsible for this nutritional sensing by phosphorylating or dephosphorylating the serine-46 residue of HPr. HPr-Ser-P then forms a complex with the transcriptional regulator CcpA to regulate transcription. We were able to show here that purified recombinant C. perfringens HPr kinase/phosphatase was able to phosphorylate the serine-46 residue of HPr. When the codon for this serine residue is mutated through PCR mutagenesis to encode alanine, phosphorylation could not take place. We have also shown that in gel retardation assays, CcpA and HPr-Ser-P were able to bind to two DNA fragments containing putative C. perfringens CRE-sites, sequences where CcpA binds to regulate transcription. The genome sequence of a food poisoning strain of C. perfringens was searched for potential CRE-sites using degenerate sequences designed to match those CRE-sites CcpA was shown to bind. DNA fragments containing these newly identified CRE-sites were then used in gel retardation assays to determine whether CcpA binds to these CRE-sites, making them candidates for CCR regulation. These results, combined with comparisons of metabolic characteristics of a ccpA- strain versus wild-type C. perfringens, provide evidence that CcpA participates in the regulation of carbon catabolite repression in the pathogenic bacterium C. perfringens / Master of Science

Penicillin-binding proteins (PBP's) are membrane-associated enzymes involved in the polymerization of peptidoglycan. PBP's are divided into three classes based upon their molecular weights and functional domains. Gene expression is regulated in the two differentiated cells in Bacillus subtilis, the mother cell and the forespore, by coordinated expression of different sigma factors that recognize specific promoters in each compartment. The functional and compartmental specificity of individual penicillin-binding proteins from the different classes of PBP's were examined during sporulation in B. subtilis. Analyses of three class A high molecular weight PBP's indicated that pbpF and pbpG must be expressed in the forespore to carry out their specific role during spore peptidoglycan synthesis. Expressing pbpD in either the forespore or the mother cell could not complement for the loss of pbpF and pbpG, suggesting that there must be additional sequence information in PBP2c and PBP2d that allows them to carry out their specific role during germ cell wall synthesis. Analyses of a low molecular weight PBP, PBP5*, suggested that expressing dacB in either the mother cell or in the forespore could regulate the level of spore peptidoglycan cross-linking to what is typical of wild type spore peptidoglycan. / Master of Science

Clostridium difficile is a Gram positive pathogen of significant importance in the UK, Europe and the USA. No vaccine has been developed and current treatments are focused on hospital management and the use of antibiotics. The disease is spread in hospitals in the spore form and the role of spores in C. difficile infecton is poorly understood. In this project spores of C. difficile have been characterised. The proteins from the outermost layers of the spore were identified and the genes cloned. Three of these surface proteins have unique enzymatic properties that maybe important for symptoms of disease. The ability of C. difficile spores to adhere to intestinal cells was found to be far greater than with live cells and through this we have identified that the spore may play an important role in colonisation. The regulation of spore coat gene expression during sporulation was also examined and temporal phases of genes expression identified. A major part of this project was to develop a mucosal vaccine to C. difficile. The approach used was to clone the C-terminus of toxin A onto the surface of Bacillus subtilis spores and use these recombinant spores to immunise mice and hamsters. We found that oral delivery of these spores conferred 75% protection to C. difficile infection in a hamster model of infection. Further, parenteral immunisation of the same antigens (toxin A and B) failed to generate mucosal responses and this showed that mucosal immunisation is critical for good protection. Finally, we found that antibodies to the C-terminus of toxin A were cross reactive to the C-terminus of toxin B. This showed that mucosal delivery of just the C-terminus of toxin A is sufficient to confer protection in an animal model of infection. The outcome of this work is that we have shown the parameters for successful immunisation and vaccination against C. difficile.

Genome transcription is much more widespread than has been traditionally thought because our view of a "gene" or "transcription unit" has changed dramatically over the past 4 to 5 years with the identification of many different non-coding ribonucleic acids. In the yeast, Saccharomyces cerevisiae, meiosis and sporulation are an important part of the life cycle and IME4 gene expression is required for these processes. IME4 sense transcript levels of expression are influenced by the level of its complementary non-coding antisense strand by mechanisms that are currently unknown. The a1-alpha2 heterodimer binding in the downstream 3' region of IME4 is one component required for repression of IME4 antisense transcription. However, this thesis shows that the general regulatory protein Reb1 is also required in this system. Reb1 involvement is most likely to create a nucleosome-free zone in the promoter region of the IME4 antisense strand therefore contributing to transcription.

A mancha-marrom de Alternaria causada pelo fungo Alternaria alternata f.sp. citri patótipo \"tangerina\" é uma das principais doenças que acometem a cultivo de tangerinas (Citrus reticulata) e alguns de seus híbridos. A infecção ocorre principalmente em folhas e frutos jovens que senescem prematuramente. Para realização de estudos com a finalidade de obtenção de cultivares resistentes, ou para a compreensão dos processos de interação planta-patógeno, é necessária a produção massal de esporos in vitro. Este trabalho, no primeiro momento, buscou desenvolver um método eficaz de obtenção de esporos do fungo em meio de cultura. Para isso, comparou os efeitos de diferentes meios de cultura (BDA e CaCO3-A) e tipos de iluminação (LED e NUV). Além do número de esporos, características como germinação, pigmentação, formação de apressórios e patogenicidade foram avaliadas. Descobriu-se que o melhor método de produção de esporos consistiu da manutenção de colônias em meio CaCO3-A por 3 dias sob LED e em seguida por 4 dias sob NUV, com fotoperíodo de 12 h. A partir de então, no segundo momento, realizaram-se testes com técnicas de microscopia e bioquímica fitopatológica para analisar a interação entre o patógeno e folhas das cultivares suscetíveis Ponkan e Murcott e da resistente Fremont. Notou-se por meio de microscopia eletrônica de varredura (MEV) que, independentemente da cultivar, o fungo germinou sobre as folhas, formou apressórios e entrou em cavidades estomáticas. Não foi observada, porém, a colonização de tecidos por meio de microscopia de luz. As cultivares suscetíveis responderam à inoculação com acúmulo de peróxido de hidrogênio e reação de hipersensibilidade. A cultivar resistente, por sua vez, não apresentou reação de hipersensibilidade e aumentou a síntese de enzimas relacionadas ao estresse oxidativo (SOD e CAT) e eliminação de espécies reativas de oxigênio. Como se trata de um fungo necrotrófico capaz de detoxificar espécies reativas de oxigênio, presumiu-se que a reação de hipersensibilidade e a morte celular nas cultivares suscetíveis favorecem a infecção e colonização do patógeno, enquanto que na cultivar resistente a ausência de morte celular e detoxificação de espécies reativas de oxigênio podem estar envolvidas na sua capacidade de defesa. Nas cultivares suscetíveis, verificou-se, por meio de microscopia eletrônica de transmissão (MET), plasmólise e fragmentação de membranas plasmáticas, local indicado como sítio de ação da toxina ACT produzida pelo patógeno. As células da cultivar resistente não apresentaram plasmólise ou alterações nas membranas plasmáticas em decorrência da inoculação do patógeno. / Alternaria brown spot caused by Alternaria alternate f.sp. citri pathotype \"tangerine\" is a important disease that affects tangerine (Citrus reticulate), and some of its hybrids. The infection happens mainly in young leaves and fruits and may cause premature senescence of tissues. To carry out studies in order to obtain resistant cultivars, or to understand the processes of plant-pathogen interaction, it is necessary abundant in vitro spores production. This work, in the first moment, aimed to develop an efficient method for in vitro spore production of the fungus. It was compared the effects of different culture media (BDA and CaCO3-A) and light (LED and NUV). Besides the number of spores, characteristics such as germination, pigmentation, apressoria formation and pathogenicity were investigated. It was found that the best method of spore production consisted of maintaining colonies on CaCO3-A medium for 3 days under LED and then for 4 days under NUV, with photoperiod of 12 h. Thus, in the second moment, assays were carried out using microscopy and phytopathological biochemistry techniques to analyze the interaction between the pathogen and leaves of the susceptible cultivars Ponkan and Murcott, and the resistant Fremont. It was noted by scanning electron microscopy (SEM) that independently of the cultivar, the fungus germinated on the leaves, formed apressoria and grew into stomatal cavities. However, colonization of tissues was not observed by light microscopy. Susceptible cultivars responded to inoculation with accumulation of hydrogen peroxide and hypersensitivity reaction. The resistant cultivar did not show hypersensitivity reaction, but increased the synthesis of enzymes (CAT and SOD) related to oxidative stress and elimination of reactive oxygen species. As a necrotrophic pathogen able to detoxify reactive oxygen species, it was assumed that the hypersensitivity reaction and cell death in the susceptible cultivars contribute to infection and colonization of the pathogen. On the other hand, the absence of cell death and detoxification of reactive oxygen species verified in the resistant cultivar may be involved on its capacity of defense. Cell plamolysis and plasma membranes fragmentation, indicated as the site of action of the ACT toxin produced by the pathogen, were verified in the susceptible cultivars by transmission electron microscopy (TEM). The cells of the resistant cultivar did not show plasmolysis or alterations in the plasma membranes due to pathogen inoculation.

A divisão celular em B. subtilis inicia-se pela formação de um complexo multiprotéico, o divisomo, no sítio onde a bactéria irá se dividir. FtsZ é a primeira proteína a se localizar no futuro sitio de divisão, formando uma estrutura em anel (anel Z) que se estende por toda a circunferência da célula. O anel Z funciona como um arcabouço responsável por recrutar outras quinze proteínas de divisão que irão participar da montagem do divisomo. Nesta tese, utilizamos abordagens quantitativas e qualitativas de microscopia de fluorescência vital para estudarmos duas questões ainda não esclarecidas sobre o funcionamento do divisomo. A primeira delas é como o divisomo é montado. Para estudarmos a montagem do divisomo nós realizamos ensaios de co-localização entre o anel Z (FtsZ-mCherry) e as proteínas ZapA, EzrA, FtsW, FtsL, YpsB , DivIVA, e MinC fusionadas a GFP. Quanto maior a freqüência de co-localização entre FtsZ e outra proteína de divisão, mais inicial é a participação da proteína na formação do divisomo. Portanto, a medida da freqüência de co-localização entre o anel Z e as proteínas componentes do divisomo permite que se deduza uma cinética da montagem deste complexo. Estes ensaios demonstraram uma freqüência de co-localização de 97,33% para ZapA; 98,31% para EzrA; 83,90% para FtsW; 78,43% para FtsL; 50% para YpsB; 41,7% para DivIVA e 31,64% para MinC. Estes resultados sugerem que o divisomo seja formado em três etapas. ZapA e EzrA se associam ao divisomo imediatamente após a formação do anel Z, em seguida FtsW e FtsL são recrutados para o divisomo, e por último YpsB, DivIVA, MinC associam-se ao divisomo. A segunda questão que investigamos nesta tese foi o mecanismo da mudança de posição do divisomo que ocorre durante a esporulação em B. subtilis. Na fase de esporulação a célula divide-se assimetricamente, com a formação do septo próxima a um dos pólos. Durante o crescimento vegetativo a divisão não ocorre próxima aos pólos por causa da ação das proteínas MinC, MinD e DivIVA, importantes reguladores espaciais da divisão. MinCD e DivIVA são inibidores da formação do anel Z que durante o crescimento vegetativo se localizam nos pólos das células.. Uma hipótese para explicar o uso dos sítios polares para a divisão durante a esporulação seria que as proteínas MinCD e DivIVA seriam removidas dos pólos celulares. Para testarmos esta hipótese, estudamos a localização das proteínas MinCD e DivIVA durante a esporulação. Nossos resultados demonstraram que MinCD e DivIVA se re-localizam e saem dos pólos celulares durante a esporulação. Porém esta dinâmica ocorre após a formação do anel Z assimétrico, sugerindo que o anel Z seja insensível a estes inibidores durante a esporulação. Por ensaios genéticos em B. subtilis demonstramos que a proteína SpoIIE, conhecida como provável proteína responsável por promover a formação do septo assimétrico, seja capaz de contrapor a ação de MinC no início da esporulação. Dessa maneira nós propomos um novo modelo de mudança da divisão simétrica para assimétrica durante a esporulação, diferentemente da simples saída do complexo MinCD dos pólos como é proposto na literatura. / Bacillus subtilis division begins through the formation of a multiprotein complex, the divisome, at the site of division. FtsZ is the earliest known protein to localize to the future division site where the protein forms a ring-like structure (Z-ring) that extends around the circumference of the cell. The Z-ring functions as a scaffold and recruits about fifteen other division proteins that compose the divisome. In this work, we used quantitative and qualitative methods of vital fluorescence microscopy to study two questions that have not been elucidated about the divisome dynamics. The first is how divisome is assembled. To address that problem, we made co-localization between Z-ring (FtsZ-mCherry) and proteins ZapA, EzrA, FtsW, FtsL, YpsB, DivIVA, and MinC fused to GFP. Higher is the match between GFP fusions to Z-ring, earlier is the assembly of division proteins to divisome. Therefore, the co-localization frequency between Z ring and divisome proteins will allow us to deduce the assemble kinetics of the divisome. This assays showed a co-localization frequency of 97,33% for ZapA; 98,31% for EzrA; 83,90 for FtsW; 78,43% for FtsL; 50% for YpsB; 41,7% for DivIVA and 31,64% for MinC. This data suggests that the divisome does not assemble in two but in three steps. ZapA and EzrA assemble into the divisome immediately after Z ring formation, secondly FtsW and FtsL were recruited to the divisome, and finally YpsB, DivIVA, MinC associated with the divisome. The second question that we investigated in this work is the mechanism responsible for change the divisome position that occurs during sporulation in B. subtilis. In sporulation the cell divides asymmetrically, with a septum formation near poles. During vegetative grown the divisiome does not occur near poles because of MinC, MinD and DivIVA action, relevant for spatial regulation of division. MinCD and DivIVA are inhibitors of Z ring formation that during vegetative growth are located at poles. A hypothesis to explain the use of polar sites for division during sporulation would be that MinCD and DivIVA would be removed from cellular poles. To test this hypothesis, we studied the location of MinCD and DivIVA proteins during sporulation. Our results demonstrated that MinCD and DivIVA re-localize and leave to cell poles during sporulation. However this process occurs after asymmetric Z ring formation, suggesting that Z ring would be unresponsive to this inhibitors during sporulation. Through genetics assays in B. subtilis we demonstrated that SpoIIE protein, known to probably play a role in asymmetric septum formation, would be able to contrapose MinC action during early sporulation. Therefore, we propose a novel model for change the symmetric to asymmetric division during sporulation, unlike the release of MinCD from pole proposed in the literature.