Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references. Also available on the World Wide Web.

The advantageous association between rhizobia and leguminous plants has motivated numerous studies into the diversity and identity of the associated bacterial symbionts. This, as well as developments in molecular microbiology, has led to major revisions of rhizobial taxonomy. Previous investigations of the rhizobia, associated with various leguminous plants from South Africa, concluded that most of the indigenous strains were related to the genus Bradyrhizobium (Dagutat, 1995; Kruger, 1998). The other rhizobial genera represented, albeit to a lesser extent, were Rhizobium, Sinorhizobium and Mesorhizobium. The major shortcoming of these investigations was the lack of sufficient genotypic characterisation. Since the completion of these initial investigations, a new rhizobial genus and several new species have been described. These developments and the additional isolation of rhizobia, from previously uninvestigated legumes, necessitated a more detailed analysis of the indigenous rhizobia. The aim of this study was therefore to study the diversity of the indigenous strains, focusing particularly on genotypic traits. A selection of indigenous rhizobia was characterised by partial 16S rDNA sequencing, restriction fragment length polymorphism (RFLP) of the 16S-23S intergenic spacer (IGS) region, partial nifH sequencing and nodC RFLP. Based on 16S rDNA sequencing, most of the isolates could be assigned to a specific genus, most being related to the genus Bradyrhizobium. A group of isolates was also related to the genus Methylobacyterium. The IGS- RFLP analyses were sufficiently discriminatory to indicate additional variation among isolates which showed little or no 16S rDNA sequence variation. The nifH phylogenetic groupings correlated well with those obtained by 16S rDNA sequencing. However, nodC RFLP indicated that the indigenous rhizobia carry diverse range nodC genotypes, with only a few showing host-specific associations. In the absence of sequence data of these nodC genotypes, their origin and correspondence to known nodC genes of other rhizibial genera, remain uncertain. Considering the results obtained here and the phenotypic characteristics determined previously, several novel Bradyrhizobium and Mesorhizobium strains were identified, however, their specific status should be validated by DNA homology studies. / Thesis (PhD (Microbiology))--University of Pretoria, 2002. / Microbiology and Plant Pathology / unrestricted

Bacteria in the gastrointestinal tract play an important role in intestinal motility, inflammation, homeostasis, and behavior. Bacteria, through the natural synthesis of neuroactive compounds and secondary metabolites, can modulate the host immune system and communicate with the host along the signaling pathway along the gut-brain axis. Here, we functionally design, develop, test, and characterize a platform for the study of microbial-host interactions using advancements in the field of synthetic biology. First, we describe the engineering of Escherichia coli Nissle to biosynthesize serotonin within the mammalian gut using a native-plasmid optimized approach. Serotonin is crucial for neurotransmission throughout the body and may be playing a role in microbial gut-brain communication. In the gastrointestinal tract, serotonin regulates intestinal motility, cell turnover, intestinal inflammation, and gastrointestinal homeostasis. Upon serial daily oral gavages, our engineered bacterium populates a murine colon to produce serotonin locally in the mucosa layers along the epithelial lining. Changes in host physiology were observed including decreased gastrointestinal motility, increased colonic Muc2 expression, induction of host TPH2, responsible for serotonin biosynthesis in enteric neurons, and upregulation of serotonin receptors HTR3, HTR4, and HTR7 in the colon. Behavioral tests revealed a statistically significant decrease in anxiety and depression in stress-induced environments in mice treated with the engineered bacterium. This work suggests that gut bacteria engineered to modulate host gut-brain axis may have both scientific and clinical uses to study microbial-host interactions and treat gastrointestinal and behavioral mood disorders in humans. Second, we engineered bacteria to produce exogenous butyrate and other SCFAs in the murine gut. Short chain fatty acids (SCFAs) play an important role in intestinal homeostasis, fluid dynamics, inflammation, oxidative stress, and intestinal hypersensitivity and motility. With this development, we characterized the effects of our butyrate-producing bacteria on a high-fat diet and DSS-induced colitis model within the colon. Although energetically burdensome to produce, our strains produced butyrate in the colon at higher density in an actively inflamed colitis model. After 14 days of oral administration, our engineered strain (EcN:B) increased the colon length of normal wild-type mice, in high fat fed mice, and in mice with recovering and actively inflamed DSS-induced colitis. EcN:B increased mucosal barrier thickness, upregulated gene expression of the barrier integrity markers Cldn1, Ocln, Zo1, and altered crypt and villus height during inflammation recovery. Furthermore, as butyrate is known to induce Foxp3+ Regulatory T cells, we saw a 13.01% percent increase in Foxp3+ cells in the colon of mice fed our engineered bacteria. This work suggests that synthetic gut bacteria engineered to produce short chain fatty acids may have future clinical uses to treat patients with inflammatory bowel disease including Crohn’s and Colitis with future potential to serve as a therapeutic for irritable bowel syndrome, idiopathic constipation, obesity, and colorectal cancer. This platform, with the use of synthetic biology to natively engineer Escherichia coli Nissle to produce bioactive compounds in the distal gastrointestinal tract, creates a framework for future characterization of bacterial-host communication and future microbial-based therapeutics.

The Nanoarchaeota are an enigmatic lineage of Archaea found in deep-sea hydrothermal vents and geothermal springs across the globe. These small (~100-400 nm) hyperthermophiles live ectosymbiotically with diverse hosts from the Crenarchaeota. Despite their broad distribution in high-temperature environments, very few Nanoarchaeota have been successfully isolated in co-culture with their hosts and nanoarchaeote genomes are poorly represented in public databases. However, the Nanoarchaeota provide unique insights into the structure and function of symbiosis in the archaeal domain. This study describes novel nanoarchaeotes from multiple geothermal habitats, using a combination of direct cultivation techniques and genomic analysis. A new nanoarchaeote from a New Zealand hot spring, Candidatus Nanoclepta minutus, was isolated in co-culture with its host. Like other terrestrial Nanoarchaeota, Cand. Ncl. minutus harbors genes for gluconeogenesis and archaeal flagella. Zestosphaera tikiterensis, the New Zealand host, was also isolated in pure culture and characterized. Phylogenetic analysis showed that both Cand. Ncl. minutus and Z. tikiterensis are new genera in the Nanoarchaeota and Crenarchaeota, respectively. Metagenome-assembled genomes (MAGs) from the Nanoarchaeota were also recovered from deep-sea hydrothermal vent sites. These MAGs capture a wide range of diversity in the Nanoarchaeota, representing three new species and two novel genera. Key nanoarchaeotal features were identified in the MAGs, including marker genes for archaeal flagella, gluconeogenesis and CRISPR-Cas regions. These studies greatly contribute to our understanding of nanoarchaeotal ecophysiology and provide key insights into the coding potential and diversity of Nanoarchaeota and their hosts.

Thesis (doctoral)--Swedish University of Agricultural Sciences, 2001. / Includes bibliographical references.

Thesis (Ph. D.)--University of Hawaii at Manoa, 2003. / Includes bibliographical references (leaves 162-167). Also available via World Wide Web.

Thesis (Ph. D.)--University of Oregon, 2007. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 60-66). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph. D.)--University of Oregon, 2007. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 70-82). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves 456-600). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves 456-600). Also available on the Internet.