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The aim of this study was to identify and measure areas of physiology relevant to the initiation of secondary metabolism and spore formation. It was discovered that the growth kinetics of Saccharopolyspora erythraea in a liquid culture environment were dependent on the type of nutrient limitation the organism was exposed to. Sacc. erythraea was able to produce erythromycin and formed structures which had properties similar to those of spores (designated 'sonic resistant units' in this thesis) in a nitrogen limited medium but, would produce erythromycin but no 'sonic resistant units' in a carbon limited medium. This resulted in two liquid culture systems, one which supported the formation of 'sonic resistant units' and one which did not, and thus enabled the comparison of the conditions which resulted in sporulation or secondary metabolism. The measurement of the rate of DNA, RNA and protein synthesis showed distinct profiles of synthesis in both media. Measurements of these rates in Streptomyces hygroscopicus showed similar profiles in the same media suggesting that the behaviour of the macromolecular synthesis rates in different nutrient limiting conditions might be applicable to streptomycetes in general. A change in macromolecular synthesis rate was implicated in inducing secondary metabolism. Antibiotics were used to modulate these rates in an attempt to induce secondary metabolism. Results from these experiments suggested the possible role of the intracellular concentration of charged and uncharged tRNA in induction of antibiotic synthesis. This relationship implies an analogy between the stringent response and the physiology of the induction of secondary metabolism. The formation of spores ('sonic resistant units') did not appear to be induced by changes in macromolecular synthesis rate or changes in tRNA levels or ratio. However, it was evident that the rates of macromolecular synthesis increased during the production of spores and this implied that it is the action of some intracellular agent that is inducing macromolecular synthesis to fuel the spore forming process.

The present study was initiated with the purpose of determining and comparing the effect of gamma radiation on the capacity of yeast cells to grow and sporulate. Using a new technique by which irradiated and non-irradiated yeast cells could be scored directly, it was found that sporulating yeast cells were more sensitive to radiation than growing cells, and that the inactivation of the capacity of an irradiated yeast cell to sporulate did not affect its ability to grow. Observations on irradiated sporulating cells indicated that spore-formation and reduction division of the nucleus, are closely allied phenomena. A short discussion of a probable mechanism of action of gamma radiation on sporulating yeast is included, together with suggestions for future research. / Thesis / Master of Science (MS)

CwlD has sequence similarities to N-acetyl muramoyl-L-alanine amidases, a class of enzymes known to cleave the bond between the peptide side chain and the N-acetyl muramic acid residue in cortex peptidoglycan formation during sporulation. A major difference between vegetative peptidoglycan and spore peptidoglycan is the presence of muramic-<FONT FACE="Symbol">d</FONT> -lactam (MAL) in spore peptidoglycan. It was previously determined that a <I>cwlD</I> null mutant does not contain muramic-<FONT FACE="Symbol">d</FONT> -lactam in the spore cortex peptidoglycan and the mutant spores were unable to complete germination. Therefore, it is believed that CwlD plays a role in MAL formation during sporulation. However, the specific role of the protein had not been demonstrated. It was also previously found that <I>cwlD</I> is in a two-gene operon with <I>orf1</I>. Orf1 is produced within the forespore with CwlD. The hypothesized role of Orf1 is to inhibit CwlD activity from within the forespore. Muramoyl-L-alanine amidase activity was demonstrated by CwlD <I>in vivo</I>. Therefore, CwlD is carrying out the first step of MAL synthesis, cleaving the peptide side chain while other enzymes are needed to complete MAL formation. Two different forms of CwlD were over-expressed, with and without the protein's signal peptide sequence. Both forms of the protein were purified and in both cases activity was undetectable. Antibodies specific for CwlD were obtained which can be used in future research as a tool to further characterize CwlD activity. A series of <I>B. subtilis</I> <I>cwlD</I> operon mutants were constructed altering the expression patterns of Orf1 and CwlD within the mother cell and forespore compartments. Various resistance properties and the germination ability of the mutant dormant spores were analyzed. It was determined that the absence of just Orf1 or Orf1 and CwlD from within the forespore has no effect on the phenotypes tested. Peptidoglycan from developing mutant forespores was extracted and analyzed throughout sporulation. Evidence was obtained demonstrating that the role of Orf1 is not to inhibit CwlD from within the forespore as hypothesized. / Master of Science

Il est bien connu que des cofacteurs comme le zinc et le cuivre sont essentiels pour la progres-sion de la méiose qui permet la formation de gamètes haploïdes à partir d’une cellule diploïde. Au laboratoire, de graves défauts dans la méiose chez la levure à fission Schizosaccharomyces pombe ont été observés en carence de cuivre, mais peu est connu sur son acquisition durant la différentiation méiotique. Trois transporteurs de cuivre sont connus chez S. pombe, soient Ctr4, Ctr5 et Ctr6. Ils ont été largement caractérisés dans un contexte mitotique, mais nous en connaissons peu sur leur contribution à l’homéostasie du cuivre en méiose. Mes travaux avaient pour objectif de dresser un portrait global des transporteurs de cuivre au cours de la méiose. D'abord, j’ai entrepris d’évaluer le profil d’expression de ces gènes. J’ai mis en évidence des patrons différents d’expression selon les transporteurs. L’expression de ctr4+ et ctr5+ a lieu principalement durant les premières heures de la méiose en carence de cuivre alors que ctr6+ a une expression plus étendue avec un pic durant la phase médiane de la méiose. L’expression de ctr4+ et ctr5+ est dépendante uniquement de Cuf1 alors que l’expression de ctr6+ repose sur les facteurs Cuf1 et Mei4. Les deux protéines Ctr4 et Ctr5 co-localisent à la membrane plasmique rapidement durant les premières heures de la méiose en carence de cuivre. À ce moment, Ctr6 apparaît à la membrane vacuolaire. Après la première division méiotique, Ctr4 et Ctr5 disparaissent alors que Ctr6 transite de la membrane vacuolaire vers la membrane des spores où elle se localise même après la libération des spores. Une délétion des gènes ctr4 et ctr6 affecte grandement l’activité d’enzymes cuivre dépendantes. Notons que dans ce mutant, l’activité superoxide dismutase est abolie et l’activité amine oxydase cuivre est grandement diminuée uniquement dans les premières étapes de la méiose. Mes travaux ont permis de mettre en évidence des profils différents d’expression, de localisation et de contribution à l’activité d’enzymes cuivre-dépendantes. Ces observations suggèrent qu’en cours de méiose la levure à fission voit ses besoins en ion de cuivre modulés et doit adapter ses systèmes d’acquisition et de gestion de cuivre intracellulaire.

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