LD2668 .T4 1954 H8 / Master of Science

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Vita. / Digitized by Kansas Correctional Industries

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A method for obtaining purified ungerminated macroconidia is described, and a technique for obtaining 85 to 90% germination of macroconidia under normal nutritional conditions is presented. Macroconidia of Microsporum gypseum release free amino acids into the medium during germination. A single alkaline protease is also found in the germination supernatant fraction. The purified protease is capable of hydrolyzing isolated spore coats in vitro. Phenyl methyl sulfonyl fluoride (PMSF) is an effective inhibitor of the protease. Incorporation of PMSF at 10⁻⁴ M into the germination system inhibits spore germination and the release of free amino nitrogen. Addition of PMSF after germ tube emergence is completed has no effect on subsequent outgrowth. The addition of exogenous purified protease to quiescent spores results in more than a 2.5-fold increase in germinated spores. It is concluded that spore coat proteolysis is an essential event in the germination of dermatophyte macroconidia. A model system to explain macroconidia germination response to inhibition, temperature shift, and addition of protease is presented. Microsporum gypseum macroconidia germinated at 37 C possessed from one to eight nuclei per germinated spore compartment. The distribution of nuclei per spore compartment was the result of a random packaging of nuclei from the available nuclear population. Partial germination inhibition by incubation at 25 C or at 37 C in the presence of 10⁻⁴ M PMSF resulted in an enrichment of germinated spores containing high numbers of nuclei per compartment. The selection of higher nuclear numbers was statistically significant. Compartments possessing high numbers of nuclei appeared to be pre-committed for spore germination and were therefore not sensitive to germination inhibition. The relationship of the germination response to temperature shift is discussed with respect to the organism's natural environment. Biochemical events which occur during macroconidial germination have been studied in M. gypseum. The specific activity levels of various metabolic enzymes have been assayed during germination time periods. The accumulated levels of several of these enzymes, as a function of exogenous carbohydrate source, has been investigated. M. gypseum was found to possess a constitutive glyoxalate shunt, a constitutive glucokinase, a fructose PEP transferase and a mannitol PEP transferase. The integration of endogenous reserve utilization during germination is discussed. The assimilation and conversion of ¹⁴C glucose, ¹⁴C amino acids and ¹⁴C uracil into TCA-precipitable material has been studied during early germination time periods. The time course of pool accumulations is also presented. The de novo synthesis of sporulation and spore germination proteins during spore germination is described. The integration of metabolite assimilation and differential synthesis is discussed. Developmental mutants affected in either sporulation or spore germination have been isolated from M. gypseum with the aid of nitrosoguanidine or as spontaneously-occurring mutants. The levels of several developmental proteins have been assayed during sporulation time periods in these mutants. The spore germination characteristics of two of the mutants are described. The relationship of alkaline protease accumulation to tyrosinase accumulation and spore germination is discussed. A large scale purification procedure for obtaining highly purified fungal chromatin is described. The isolated chromatin has normal ratios of RNA:DNA and non-basic protein:DNA. However, the ratio of basic protein:DNA was extremely low. The possibility of proteolytic degradation of histone during chromatin isolation was unlikely. Chromatin was 40 - 50% as template active as DNA. It is concluded that basic proteins do not represent a major fraction of M. gypseum chromosomal proteins. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Thesis (doctoral)--Universität Zürich, 1906. / Includes bibliographical references.

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Anomalous behaviour of water confined in nanoscale gaps influences many biological and technological processes. However, due to the small size of confining structures, it is historically difficult to manipulate and study water’s dimension-dependent transport character. Experimental studies of nanoconfined water are generally limited to artificial test structures, and/or single-file channels, and so transport behavior of biologically nanoconfined water remains elusive. We utilize poroelastic bacterial spores coated onto a nanomechanical sensor to probe photo-thermal evaporative relaxation in a biological setting and report viscous water, 7 orders of magnitude larger than that of bulk liquid, and via thermodynamic investigations reveal an activation energy close to ice. Overall, these experiments characterize transport behaviour of nanoconfined water in vivo, and highlight the dramatic effects of nanoscale confinement on water that could impact myriad natural and synthetic processes. Following from this work, a hypothesis is pursued in which the bacterial lifecycle is intimately connected with transitions in the physical structure of the internal water. We expand an initial idea proposed in Science, 1960 by J.C. Lewis, N.S. Snell and H.K. Burr that the low water content of the spore core is accomplished through compressive contraction during development3. During sporulation, the genetic material is packaged with chelating chemicals within a special water-responsive, layered coating that electrostatically pulls the water out of the core. Together, these agents produce the extremely dehydrated, hydraulically tensioned, and stable spore-phase organism. During germinative re-awakening, an event lacking a complete mechanistic theory of sensation, the core is rehydrated and the organism subsequently reanimated. This work’s findings regarding the spore’s physically restrained but exchangeable water support the idea that the physical state of the water contributes significantly to tensioning the organisms into a ‘charged’ but dormant configuration. This dormant but spring-loaded phase of the bacterial lifecycle is subject to awakening by agents (nutrient or otherwise) which disrupt surface tension including amino acids, salts, surfactants, and hydrostatic pressures. In the least, it must be acknowledged that the slowed water observed herein enforces slowed biochemistry and thus dormancy. Taken together we present a picture where internal spore water, even that which is exchanged with the external environment, is nanoconfined and slowed under tremendous tension (negative pressure). The mechanism governing this slow water appears to be unlike that any previously described, the majority of which are typically based upon crystalline surfaces, the likes of which are not found in the spore. We consider that the spore water structure itself participates, in certain environments, in the signaling chain of the organism through stabilizing a delicately balanced and highly tensioned architecture. Presently we are working toward testing the hypothesis and expanding our understanding with new methods, including additional structural mutants and expanded biophysical techniques.

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