The Rhizobium-legume symbiosis is an opportunistic association between two symbiotic partners that results in the formation of the root nodule. The process depends on the expression of a number of plant and bacterial genes that are considered critical for the establishment and maintainance of the symbiotic state. The merits of a mutational approach to the analysis of symbiosis have been recognized for several years and transposon Tn5 mutagenesis of Rhizobium has led to the identification of several symbiotic genes. This study describes the use of Tn5 mutagenesis for the isolation of symbiotically defective mutants of Bradyrhizobium japonicum. Two classes, auxotrophic and cell surface-altered mutants defective in nodule formation, have been characterized. In B. japonicum USDA 122, histidine auxotrophs that are defective in nodulation have been studied. The mutagenized DNA region has been cloned and the wild-type DNA region isolated by hybridization and complementation. In B. japonicum 61A76, Tn5-induced cell surface-altered mutants have been isolated by selecting for bacteriophage resistance. Several parameters have been used to demonstrate alterations in cell surface components. It has been shown that the Tn5 insertion is not the primary cause of the mutation in two of the characterized mutants. Complementation tests have led to the isolation of a wild-type DNA-containing cosmid, pPS23A, that overcomes the symbiotic defect in one of the mutants. Analysis of the cell surface showed a partial restoration of surface components in the complemented mutant.

No description available.

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Thesis (Ph.D.) -- University of Adelaide, Dept. of Microbiology and Immunology, 1982. / Typescript (photocopy).

Thesis (Ph. D.)--University of Chicago, Department of Biochemistry and Molecular Biology, December 1999. / Includes bibliographical references. Also available on the Internet.

No description available.

No description available.

Malaria persists today as a significant burden for a large part of the world. However, over the past few decades, a concerted effort by governments, non-governmental organizations, researchers, and community health workers worldwide has yielded progress in reducing the deadly impact of this disease. Today, some of these gains are threatened by the rise of antimalarial drug resistance, a recurring problem that has impeded global malaria reduction efforts before. Research on Plasmodium falciprum resistance to the numerous antimalarial compounds used today and in the past has made significant progress on determining which specific mutations modulate drug susceptibility and to what degree they do so. To gain a comprehensive understanding of drug resistance, we need to elucidate how and why it arises. Therefore, it is important to elucidate whether some malaria parasites acquire resistance-conferring mutations faster than others and why the native function of the genetic factors involved lend themselves to modulating drug resistance. For instance, resistance to multiple antimalarial therapies has repeatedly emerged in Southeast Asia. We investigated the long-held hypothesis that this was due to the ability of these parasites to mutate significantly faster than non-Southeast Asian strains. Elucidating whether this hypermutability phenotype accurately represents Southeast Asian parasite evolvability is important, as it can inform when resistance would be expected to next arise, particularly in the Greater Mekong Subregion in Southeast Asia. Here, we have adapted a fluctuation assay to Plasmodium falciparum and determined that some contemporaneous Cambodian parasites exhibit a mild mutator, but not a hypermutator, phenotype. We also show that this is likely driven by mutations in DNA repair genes carried predominantly by multidrug resistant Southeast Asian parasites. One of the most common genes in which drug resistance-conferring mutations occurs is the P. falciparum chloroquine resistance transporter (pfcrt). Mutations in pfcrt are associated with parasite susceptibility to many of the antimalarial compounds that have been used in a clinical setting to date. However, beyond its role in drug resistance, little is known about the native function of PfCRT. To facilitate the study of pfcrt, we have designed a zinc-finger nuclease (ZFN)-based gene engineering system that introduces a single double-strand break in intron 1 of pfcrt. Our ZFN strategy enables replacing nearly any endogenous pfcrt locus with a user-defined recombinant pfcrt allele. We show that our method of pfcrt allelic replacement is fast, efficient, and reliable. We used this system to generate a unique mutant parasite encoding a pfcrt-L272F mutation, which enlarges the parasite digestive vacuole, the lysosome-like organelle used to catabolize host-derived hemoglobin for amino acid salvage. Our results provide clear evidence that PfCRT is associated with the terminal steps of hemoglobin degradation, overall parasite fitness, and the balance of osmolytes across the digestive vacuole membrane. Bringing clarity to the native function of PfCRT can reveal how and why this single genetic factor has been and continues to be involved in the resistance to many different antimalarial compounds.

Typescript (photocopy) / xi, 115 leaves, [24] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1982