Thesis (Ph. D.)--University of Hawaii at Manoa, 2001. / Includes bibliographical references (leaves 117-130). Also available on microfiche.

Thesis (Ph. D.)--University of Hawaii at Manoa, 2003. / Includes bibliographical references (leaves 118-142).

Quorum sensing is a cell density-dependent bacterial gene regulatory mechanism used for the expression of colonization-related genes. The symbiotic relationship between the luminescent bacterium Vibrio fischeri and the Hawaiian bobtail squid Euprymna scolopes serves as a model system to study the molecular processes underlying bacterial colonization. This system is especially well-suited for the investigation of the impact of quorum sensing on colonization because (i) it is an easily accessible, natural, two-species colonization model, and (ii) quorum sensing regulates luminescence expression in V. fischeri, which allows the non-invasive detection of quorum-sensing activity both in culture and in symbiosis. While the impact of one of V. fischeri's quorum-sensing systems, lux, on luminescence expression and symbiotic competence has been extensively studied, little was known about other putative systems. The results of this study demonstrate that the V. fischeri ain system is essential for both maximal luminescence expression and symbiotic competence. The ain system predominantly induces luminescence expression at intermediate cell densities, which occur in culture, while the lux system is responsible for luminescence expression at the high cell densities found in symbiosis, suggesting the sequential induction of luminescence gene expression by these two systems. Furthermore, the ain quorum sensing system is important for the processes underlying colonization initiation, while the impact of the lux system is apparent only in later stages of the symbiosis, indicating distinct functions of these two systems during the colonization process. A global transcriptome. analysis of quorum-sensing mutants revealed that ain quorum sensing represses motility gene expression, providing a likely explanation for the initiation defect. Although it has been known that many bacterial species possess multiple quorum-sensing systems, this is the first study demonstrating that two quorum-sensing systems are employed to specifically regulate functions important at distinct cell densities occurring during the colonization process.

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

All animals exist in lifelong relations with a complement of bacteria. Because of the ubiquity of these symbioses as well as the derived biomedical applications, the study of both beneficial and pathogenic host-microbe associations has long been established. The monospecific light organ association between the Hawaiian sepiolid squid Euprymnascolopes and the marine luminous bacterium Vibrio fischeri has been used as a experimental model for the study of the most common type of animal-bacterial interaction, i.e., the association of coevolved Gram-negative bacteria with the extracellular apical surfaces of polarized epithelia. A fundamental step for understanding the mechanisms of host-symbiont associations lies in defining the genetic components involved; specifically defining changes in host gene expression. The studies presented in this dissertation identify and characterize V. fischeri-induced changes in host gene expression at both the transcript and protein level.

The primary contributor to Vibrio fischeri aminopeptidase activity is aminopeptidase N, PepN. Colonization assays revealed the pepN mutant strain to be deficient at forming dense aggregates and populating the host’s light organ compared to wildtype within the first 12 hours of colonization; however the mutant competed normally at 24 hours. To address the role of PepN in colonization initiation and establish additional phenotypes for the pepN mutant strain, stress response and other physiological assays were employed. Marked differences were found between pepN mutant and wildtype strain in response to salinity, acidity, and antibiotic tolerance. This study has provided a foundation for future work on identifying a putative role for V. fischeri PepN in regulating stress response.