The fluffy gene of Neurospora crassa is required for asexual sporulation. It encodes an 88 kDa polypeptide containing a typical fungal Zn2Cys6 DNA binding motif. To identify the target genes on which FL may act, I sought to identify target sequences to which the FL protein binds. Several strategies were attempted to obtain purified FL protein. Purification was achieved by expressing the DNA binding domain of FL in Escherichia coli as a fusion with glutathione S-transferase followed by affinity purification using glutathione sepharose chromatography. DNA binding sites were selected by in vitro binding assays. Comparison of the sequences of selected clones suggested that FL binds to the motif 5??-CGG(N)9CCG-3??. A potential binding site was found in the promoter region of the eas (ccg-2) gene, which encodes a fungal hydrophobin. In vitro competitive binding assays revealed a preferred binding site for FL in the eas promoter, 5??-CGGAAGTTTCCTCCG-3??, which is located 1498 bp upstream of the eas translation initiation codon. In vivo experiments using a foreign DNA sequence tag confirmed that this sequence is a target site for FL regulation. Using Saccharomyces cerevisiae as an experimental system, I demonstrated that the C-terminal portion of FL functions in transcriptional activation. Microarray analysis was performed to study the role of fl in gene regulation on a large scale. mRNA levels in a fl mutant were compared to those in a strain overexpressing the fl gene. Experiments with cDNA microarray containing 13% of the total number of predicted N. crassa genes revealed 122 genes differentially expressed in response to overexpression of fl. Among these, eas displayed the greatest level of response. The cDNA microarray approach also revealed a number of genes that may be indirectly regulated by fl but may be involved in development. This information provides a foundation for further analysis of the role of fl in conidial development.

The fluffy gene of Neurospora crassa is required for asexual sporulation. It encodes an 88 kDa polypeptide containing a typical fungal Zn2Cys6 DNA binding motif. To identify the target genes on which FL may act, I sought to identify target sequences to which the FL protein binds. Several strategies were attempted to obtain purified FL protein. Purification was achieved by expressing the DNA binding domain of FL in Escherichia coli as a fusion with glutathione S-transferase followed by affinity purification using glutathione sepharose chromatography. DNA binding sites were selected by in vitro binding assays. Comparison of the sequences of selected clones suggested that FL binds to the motif 5??-CGG(N)9CCG-3??. A potential binding site was found in the promoter region of the eas (ccg-2) gene, which encodes a fungal hydrophobin. In vitro competitive binding assays revealed a preferred binding site for FL in the eas promoter, 5??-CGGAAGTTTCCTCCG-3??, which is located 1498 bp upstream of the eas translation initiation codon. In vivo experiments using a foreign DNA sequence tag confirmed that this sequence is a target site for FL regulation. Using Saccharomyces cerevisiae as an experimental system, I demonstrated that the C-terminal portion of FL functions in transcriptional activation. Microarray analysis was performed to study the role of fl in gene regulation on a large scale. mRNA levels in a fl mutant were compared to those in a strain overexpressing the fl gene. Experiments with cDNA microarray containing 13% of the total number of predicted N. crassa genes revealed 122 genes differentially expressed in response to overexpression of fl. Among these, eas displayed the greatest level of response. The cDNA microarray approach also revealed a number of genes that may be indirectly regulated by fl but may be involved in development. This information provides a foundation for further analysis of the role of fl in conidial development.

Doctor of Philosophy / Genetics Interdepartmental Program / Revathi Govind / Clostridium difficile is a gram-positive anaerobic, motile, spore-forming opportunistic bacterium. It is a nosocomial pathogen, and the symptoms of C. difficile infection (CDI) range from mild diarrhea to life-threatening pseudomembranous colitis and toxic megacolon. Antibiotic use is the primary risk factor for the development of CDI as it disrupts the healthy protective gut flora which enables C. difficile to colonize and establish in the colon. C. difficile damages the host tissue by secreting toxins and disseminates in the environment by forming spores. The two-major toxin-encoding genes, tcdA, and tcdB are located within a 19.6 kb pathogenicity locus (PaLoc), which also includes the gene encoding an RNA polymerase sigma factor TcdR, that is essential for toxin gene expression. We created a site-directed mutation in tcdR in the epidemic-type C. difficile R20291 strain and found that disruption of tcdR affected sporulation in addition to toxin production. Spores of the tcdR mutant were more heat- sensitive and required nearly three-fold higher taurocholate to germinate when compared to the wild-type (WT). Transmission Electron Microscopic analysis of the tcdR mutant spores also revealed a weakly assembled exosporium. Consistent with our phenotypic assays, our comparative transcriptome analysis also showed significant downregulation of sporulation genes in the tcdR mutant when compared to the WT strain. Our findings on tcdR suggest that the regulatory networks of toxin production and sporulation in C. difficile R20291 strain are interlinked with each other. Transcriptome analysis revealed the sin operon to be significantly downregulated in the tcdR mutant which made us hypothesize the link between sin operon regulation and sporulation. The sin locus coding SinR (113 aa) and SinI (57 aa) is responsible for sporulation inhibition in B. subtilis. SinR in B. subtilis mainly acts as a repressor of its target genes to control sporulation, biofilm formation, and autolysis. SinI is an inhibitor of SinR, and SinI/SinR interaction determines whether or not the SinR can inhibit target gene expression. The C. difficile genome carries two sinR homologs in the operon, and we named it as sinR and sinR’, coding for SinR (112 aa) and SinR’ (105 aa), respectively. To identify the regulation of sin on sporulation, we created a site-directed mutation in the sin locus in two different C. difficile strains R20291 and JIR8094. Comparative transcriptome analysis of the sinRR’ mutants revealed their pleiotropic roles in controlling several essential pathways including sporulation, toxin production, and motility (STM) in C. difficile. We performed several genetic and biochemical experiments, to prove that SinR regulates transcription of crucial regulators in STM pathways, which includes sigD, spo0A, and codY. Unlike B. subtilis, SinR’ acts as an antagonist of SinR and SinR’/SinR determines SinR activity. Our in vivo experiment using hamster model also demonstrated the importance of sin locus for successful C. difficile colonization. Our findings above reveal that sin locus acts as a central link that regulates essential pathways including sporulation, toxin production, and motility, which are critical for C. difficile pathogenesis. The final section of this dissertation analyzes a variant codY gene in the epidemic C. difficile R20291 strain. In this strain the CodY, a global nutrient sensor-regulator carry a missense mutation where the 146th tyrosine residue is replaced with asparagine (CodY[superscript Y146N]). Our preliminary study with the mutated CodY[superscript Y146N] suggested its differential role in its regulatory activity. Further analysis of CodY[superscript Y146N] might give some possible clues behind the hypervirulent nature of epidemic R20291 strain. Taken together, studies performed on both tcdR and sinR mutants reveal a significant amount of crosstalk occurring between the powerful regulators of STM pathways under the directionality of TcdR and SinR in determining their ultimate cell fate. Our findings on CodY[superscript Y146N] suggest how the bacteria could switch to a hypervirulence mode by manipulating one of its vital regulators like CodY.

B. anthracis alters its virulence gene expression profile in response to a number of environmental signals, including levels of bicarbonate and CO2. Virulence plasmid pXO1 is important to Bacillus anthracis pathogenicity as it carries the genes encoding the anthrax toxin and virulence regulatory factors. Induction of toxin and other virulence genes requires the pXO1-encoded AtxA regulatory protein. The cytochrome c maturation system influences the expression of virulence factors in Bacillus anthracis. B. anthracis carries two copies of the ccdA gene, encoding predicted thiol-disulfide oxidoreductases that contribute to cytochrome c maturation. Loss of both ccdA genes results in a reduction of cytochrome c production, an increase in virulence factor expression, and a reduction in sporulation efficiency. pXO1 also carries a gene encoding an Hfq-like protein, pXO1-137. Loss of pXO1-137 results in significant growth defects and reductions in toxin gene expression only when grown under toxin inducing conditions. Similarly, loss of a small RNA on pXO1, sRNA-1, results in similar growth defects and reductions in toxin gene production. Both increased and decreased expression of pXO1-137 and sRNA-1 result in growth defects suggesting narrow functional set points for Hfq and sRNA levels.

The process of sporulation in Bacillus subtilis serves as a paradigm for study of sporulation in all Bacillus and Clostridium species. During the process of sporulation there is a sequential and cell type specific activation of RNA polymerase sigma factors. The asymmetric sporulation septum forms, which divides the cell into the smaller prespore and the larger mother cell. Then, óF becomes active in the prespore and óE becomes active in the mother cell. Following completion of engulfment of the prespore by the mother cell, óG becomes active in the prespore and finally óK becomes active in the mother cell. At the time the sporulation septum forms only the 30% origin proximal portion of the chromosome is within the prespore. It is the responsibility of the SpoIIIE translocase to pump the remaining chromosome destined for the prespore into the prespore. We hypothesize that activation of óG does not occur until the complete chromosome has been translocated into the prespore, and the prespore has been engulfed by the mother cell. Our first method of investigation was to increase the time required to complete translocation of the chromosome into the prespore. Strains in which large inserts of foreign DNA have considerably increased the genome size were obtained and characterized. The strains have shown a delay in the completion of engulfment, which still occurred before the activation of óG. Activation was identified by visualization of GFP from a óG-directed promoter indicating translation of a product transcribed by óG. We have also shown that the terminus region of the chromosome entered the prespore shortly before the completion of engulfment. It was determined that the increased genome size did not result in a delay of the transcription of the structural gene for óG,spoIIIG. Using a strain in which the activity of the SpoIIIE translocase was reduced 2.5 fold we used a óG-directed GFP reporter to study óG activation under both time course and time lapse conditions. We again found that óG only became active following the completion of engulfment. Our second avenue of investigation was to change the site of replication termination. A strain was used in which several terminus associated genes have been relocated from their usual location near the terminus (172°) resulted in termination of replication occurring at 145° on the chromosome. In this strain, it has been found that, under both time course and time lapse microscopy conditions, óG activation still occurred only following completion of engulfment. The translocation of the chromosome in this strain was studied by time lapse microscopy using a óF- directed reporter and it was determined that the origin proximal region, the site of replication termination, and the traditional terminus region, were all translocated into the prespore prior to the completion of engulfment. The results support the hypothesis that there is a strong link requiring complete translocation of the chromosome, followed by engulfment of the prespore, before óG becomes active. / Microbiology and Immunology

Formation of spores by Bacillus subtilis is a primitive system of differentiation that has become a paradigm for studying cell differentiation in prokaryotes. Differential gene expression commences soon after the single, asymmetric sporulation division through the activation of different RNA polymerase sigma factors, sigma F in the smaller prespore and sigma E in the larger mother cell. sigma E activation relies on an inter-cellular signaling emanating from sigma F-directed gene expression. Formation of the asymmetric division septum and compartmentalized activity of both sigma factors occur prior to chromosome partitioning. At the time of septation, only 30% of the chromosome destined to be in the prespore is actually present in that compartment and the remaining 70% is in the mother cell. Thus, both cell types contain unequal DNA content. This study focused on the effect of this genetic asymmetry on sigma F-directed gene expression, and exploited this effect in order to study aspects of sigma F to sigma E inter-compartmental signaling. Perturbed signaling resulted in the discovery of a novel twin-spore forming morphology, which was further characterized. A DNA translocase is required to translocate the remaining portion of the chromosome from the mother cell to the prespore. The replication terminus region of the chromosome was observed to be the last to enter the prespore and thus, sigma F-directed genes showed delayed and reduced expression when moved to a terminus-proximal location. The studies indicate that this positional regulation of sigma F-directed gene expression is attributed to both delayed entry and inhibition in sigma F activity at late stages of sporulation. Moreover, the next prespore-specific sigma factor, sigma G, could have a role in inhibiting sigma F. The link between sigma F and sigma E activation is the spoIIR locus, which is transcribed in the prespore from a sigma F-directed promoter soon after the formation of the asymmetric septum. Inactivation of the structural genes for sigma F or sigma E or SpoIIR results in the formation of a second septum at the opposite pole; development proceeds no further, resulting in an "abortively disporic" phenotype. The second septum is formed about 20 min after the first, and sigma E activity is required to prevent its formation. As a sigma F-directed gene, spoIIR is subject to `positional regulation': a delay in spoIIR expression caused by moving it from its origin proximal position to the chromosome terminus, is sufficient to delay sigma E activation and block spore formation, giving the abortively disporic phenotype. The effects of delaying and enhancing spoIIR expression were tested. The changes delayed sigma E activation, and many organisms formed a septum at both ends. However, both prespores in these organisms were able to develop into mature spores (twins). Extra rounds of chromosome replication occured during twin formation, so that each twin had a chromosome and the mother cell had either one or two chromosomes. This over-initiation of chromosome replication is a prerequisite for twin spore formation. Moreover, the studies showed that mother cells of twin forming organisms were longer than those containing single spores; image analysis showed that mother cell length correlates with chromosome content. In contrast to twin spore formation, during normal spore development, there is usually one copy of the chromosome in the prespore and one in the mother cell, with no growth of either compartment. Therefore, the system allowed investigating regulation of chromosome replication and growth of the mother cell. The studies showed that replication and growth are permitted because of the absence of active sigma E and of reduced levels of transcription directed by the master regulator for entrance to spore formation, Spo0A. The results indicate that the burst of Spo0A-directed expression along with activation of sigma E provide mechanisms to block replication and growth of the mother cell. / Microbiology and Immunology

The bacterium Clostridium perfringens is a gram-positive anaerobe responsible for many diseases in man and other animals, the most common of which is acute food poisoning (AFP). It is estimated that nearly 240,000 cases of AFP occur each year in the U.S. The C. perfringens spore plays an important role in this infection. The heat resistance of the spore allows the organism to survive the cooking process, grow in the cooling food, and infect the victim. Despite the occurrence of this disease and the importance of the spore to this process, little work has been performed to determine how heat resistance is obtained and maintained by C. perfringens spores. In this work we study the spores and sporulation process of C. perfringens to determine what factors are most important in the formation of a heat resistant spore. We analyzed the spores produced by nine wild-type strains, including five heat-resistant food poisoning isolates and four less heat-resistant environmental isolates. We determined that threshold core density and a high ratio of cortex peptidoglycan relative to germ cell wall were necessary components of a highly heat-resistant spore. In order to test these observations, we constructed two mutant strains. The first could not achieve the necessary level of core dehydration and rapidly lysed in solution. The second mutant had a reduced amount of cortex relative to germ cell wall, and suffered a corresponding decrease in heat resistance as compared to our wild-type strains. The mutant strains supported the observations drawn from our wild-type strains. Dipicolinic acid is a major component of bacterial spores and is necessary for spore heat resistance. The Cluster I clostridia, including C. perfringens, lack the known DPA synthase operon, spoVF. We developed an in vitro assay for detecting DPA synthetase activity and purified the active enzyme from sporulating C. perfringens crude extract and identified the proteins with mass spectrometry. These results identified the electron transfer flavoprotein alpha chain (EtfA) as the DPA synthase of C. perfringens. Inactivating the etfA gene in C. perfringens resulted in a strain that could begin, but not complete, the sporulation process and produced dramatically lower amounts of DPA than the wild-type. The purified enzyme was shown to produce DPA in vitro and utilized FAD as a preferred cofactor. The results of this research may lead to future techniques to decrease the occurrence of the diseases caused by C. perfringens spores and treatments which may carry over to the diseases caused by similar organisms. / Ph. D.

Moorella thermoacetica est une bactérie d’altération anaérobie thermophile sporulée produisant les spores les plus thermorésistantes isolées à ce jour en industrie agroalimentaire. Les spores de M. thermoacetica peuvent survivre aux traitements d’appertisation appliqués dans l’industrie de la conserve et représente la principale cause d’altération de conserves peu acides après incubation à 55 °C. Les mécanismes mis en place pendant la sporulation, ainsi que l’impact des facteurs environnementaux sur la sporulation et les propriétés des spores de Moorella sont largement méconnus.L’objectif de ces travaux était donc de caractériser l’impact de la température sur les caractéristiques de sporulation et de résistance des spores ainsi que de déterminer les mécanismes moléculaires impliqués dans la sporulation. Nous avons montré que la capacité de sporulation est meilleure lorsque la sporulation est effectuée à la température optimale (55 °C) en comparaison avec les températures limites basse et haute (45 °C et 65 °C, respectivement). De plus, les spores de M. thermoacetica produites à 45 °C sont plus sensibles à la chaleur humide et aux biocides que les spores produites à 55 °C. La structure des spores ainsi que leur composition protéique varient en fonction de la température de sporulation. Une étude RNAseq menée au cours de la sporulation de M. thermoacetica en conditions optimales régulées a montré que la plupart des 167 gènes de sporulation, identifiés in silico dans le génome de M. thermoacetica ATCC39073, est sur-exprimée pendant la sporulation. Ces résultats suggèrent que les mécanismes décrits chez d’autres espèces endosporulées sont conservés chez Moorella.L’ensemble des données acquises montrent que la température joue un rôle essentiel dans les caractéristiques de sporulation et de résistance des spores de M. thermoacetica mais également que les mécanismes moléculaires impliqués dans la sporulation semblent conservés au regard des données disponibles pour d’autres espèces. / Moorella thermoactica is a spoilage anaerobic and thermophilic spore-former producing the most highly heat-resistant spores isolated so far in food industry, which enables the bacteria to survive the sterilization process applied in cannery. M. thermoacetica is the main cause of low acid canned food spoilage after incubation at 55 °C. Little is known about sporulation mechanisms and spore properties according to environmental conditions.In this work, we aimed at characterizing the impact of environmental conditions on sporulation and spore resistance properties, as well as describing the molecular mechanisms underlying sporulation. We showed that sporulation capacities are higher when sporulation is performed at the optimal temperature (55 °C) that at limit temperatures (45 °C and 65 °C). Besides, spores are less resistant to wet heat and biocides when formed at 45 °C than at 55 °C. We showed that the ultrastructure and spore protein composition varied according to sporulation temperature. Moreover, the study of gene expression by RNAseq during sporulation in optimal regulated conditions showed that most of the 167 genes involved in the sporulation process and identified in silico in M. thermoacetica ATCC 39073 genome, were up-regulated during sporulation, suggeting that the mechanisms described in other endospore-formers are conserved in Moorella.Altogether, our results showed that sporulation temperature strongly impacts sporulation and spore properties of M. thermoacetica and that sporulation mechanisms tend to be conserved in Moorella considering data available on other endospore-formers.