For all organisms, segregation and proper intracellular localization of DNA are essential processes in ensuring faithful inheritance of genetic material. In prokaryotes, several different mechanisms have developed for efficiently moving chromosomal DNA to proper cellular locations prior to cell division, and the same holds true for bacterial plasmids. Low-copy-number plasmids and bacterial chromosomes encode active partition systems to ensure their inheritance within a bacterial cell population. One of the well-studied models of partition is that of the P1 plasmid in E. coli. The partition system encoded by the P1 plasmid is known as parABS - ParA is the partition ATPase, ParB is the partition site binding protein and parS is the partition site. The goal of this thesis was to investigate the nucleoprotein complexes essential in the P1 plasmid partition reaction. First, I examined how a single ParB dimer can bind its complicated arrangement of recognition motifs in parS to initiate the partition reaction. I then characterized a novel ParA interaction with the host nucleoid that is critical for proper P1 plasmid dynamics in vivo. Finally, I demonstrate how ParA can act as an adaptor between the nucleoid and the partition complex; effectively allowing the plasmid to use the nucleoid as a track for its intracellular movement and localization. My thesis work provides evidence towards a model that explains the P1 plasmid partition mechanism.

The development of biofilms at solid-liquid interfaces has been investigated extensively, whereas the yield of planktonic cells from biofilms has received comparatively little attention. The detachment of single cells from biofilms has been attributed mainly to the erosive action of flowing liquid or the dispersal of cells from within biofilm microcolonies. The result has been an underestimation of the active role that biofilms can play in microbial proliferation through the production and release of planktonic cells to the environment. In this study, the cultivation of Pseudomonas sp. strain CT07 biofilms in conventional flowcells, glass tubes and a novel CO2 evolution measurement system was utilized to show that biofilm-derived planktonic cell yield was initiated within 6 hours of initial surface colonization and increased in conjunction with biofilm development. The magnitude of the yield was influenced by the metabolic activity of the biofilm, which was in turn dependent on environmental conditions, such as carbon availability. The physiologically active region of the biofilm was responsible for the yield of significant numbers of planktonic cells (~107 CFU.cm-2.h-1), whereas a less active biofilm zone was optimized for survival during unfavourable conditions and shown to be responsible for the subsequent re-establishment of biofilm structure, activity and cell yield. Despite the yield of numerically considerable numbers of planktonic cells (~1010 CFU), a carbon balance revealed that the carbon investment required to maintain this yield was insignificant (~1%) compared to the amount of carbon channelled into CO2 production (~54%). Together, these results indicate that biofilm-derived planktonic cell yield represents an efficient proliferation mechanism and support the view that the biofilm lifestyle affords microbes a dual survival-proliferation strategy, where the dominant strategy depends on the prevailing environmental conditions. An alternative model of biofilm development is presented to account for planktonic cell yield during all stages of biofilm development.

Neisseria gonorrhoeae infections are of major concern in areas of low socioeconomic status in both developed and developing nations. N. gonorrhoeae colonizes the genital tract by adhering to mucosal tissues through a number of adhesins, including the colony opacity-associated (Opa) proteins. Despite the random phase-variable expression of Opa proteins, 95% of clinical isolates express Opa variants that bind to the carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1), suggesting an essential role in vivo. Interestingly, even though gonorrhea is characterized by an intense inflammatory response, the organism is capable of evading the adaptive immune response. In previous studies by the Gray-Owen group, it has been established that certain gonococcal Opa variants bind CEACAM1 expressed by CD4+ T helper lymphocytes and, thereby, reduce their activation and proliferation. Since T cells are essential in establishing immune memory, inhibition of T cell function could explain the deficit in immune memory following gonococcal infection. In this thesis, I describe my studies to elucidate how CEACAM1 inhibits T cell activation on a molecular level. In Chapter 2, I demonstrate that outer membrane vesicles (OMVs) naturally shed by OpaCEA-expressing Neisseria sp. effectively inhibit CD4+ T cell activation, implicating a role for OMVs during infection and establishing that the Opa proteins do not have to be expressed in the context of the bacterium in order to elicit an inhibitory effect. In Chapter 3, I describe early steps in the CEACAM1-dependent inhibitory signaling cascade elicited when N. gonorrhoeae binds to CD4+ T cells. Finally, in Chapter 4, I show that a dynamic monomer-dimer equilibrium controls CEACAM1 function in epithelial cells and lymphocytes. Combined, the results presented in this thesis allow the derivation of a model to explain how CEACAM1 controls CD4+ T cell function at a molecular level.

Vibrio vulnificus is an opportunistic human and animal pathogen that is predominantly found in estuarine waters. In aquatic ecosystems, it colonizes filter-feeders, such as oysters, and has been found to form biofilms on the surface of various marine organisms, including plankton, algae, fish, eels, and crustaceans. The bacterium can spontaneously develop a rugose phenotype, which is associated with the production of polysaccharide(s) that impart a raised, wrinkled appearance to cells, copious biofilm formation, and increased stress resistance. Biofilm and rugose colony development, along with pellicle and aggregate formation, are believed to be crucial for the environmental survival and persistence of V. vulnificus. As the biosynthesis of polysaccharide(s) is a key feature linking these physiological processes, the main objectives of this study were to identify polysaccharide loci contributing to survival phenotypes in V. vulnificus and to gain insight into the regulation of these loci. Two polysaccharide loci (brp and rbd) were found to contribute to biofilm formation. The brp locus is regulated by the second messenger c-di-GMP and by at least two transcriptional regulators BrpR and BrpT. Lesions in glycosyltransferases in the locus or in either of the regulators abated the inducing effects of c-di-GMP on biofilm formation. The rbd locus is regulated not by c-di-GMP, but instead by a response regulator (RbdG) belonging to the TCRS family, which is encoded within the locus. The biofilms associated with the expression of the brp and rbd polysaccharides were structurally unique and simultaneous expression of both loci dramatically enhanced pellicle formation. Each locus also provides unique survival characteristics; the development of rugosity and stress resistance could be attributed to brp expression whereas rbd expression augmented aggregate formation. The ability of V. vulnificus to differentially regulate expression of the brp and rbd polysaccharides may allow the bacterium to “fine tune” its biofilm lifestyle to maximally benefit from the characteristics associated with each locus.

HopZ1a is a type III secreted effector (TTSE) protein from Pseudomonas syringae. The goal of this study was to obtain a 3D crystal structure of HopZ1a to provide insight into its biochemical function. The first objective was to obtain HopZ1a protein that was sufficiently abundant and pure for crystallographic studies. Purification conditions were optimized and multiple constructs of HopZ1a were generated using secondary structure prediction programs as well as structural characteristics inherent to TTSEs. Truncations of HopZ1a from the N- and C-terminus led to a soluble, proteolytically resistant construct, HopZ1a66-261. This protein formed granular precipitates in crystallography screens. These conditions will provide the basis for refinement screens aimed at optimizing the HopZ1a crystallization conditions. Overall, the soluble constructs described in this study will provide invaluable tools for future in vitro functional and structural studies of this important family of type III secreted effector proteins.

The genome of B. phytofirmans OLGA172 has been sequenced by Next Generation sequencing methods. Over 42 kbp of its genome surrounding its 3CBA degradative genes, tfdCIDIEIFI, was assembled and annotated. The most important method used was the synteny method, which implies homology between the genes, and descent from a common ancestor (Guttman, 2008). The conserved gene order between B. phytofirmans PsJN, B. xenovorans LB400, and OLGA172 was used as a confirmation of annotation through BLASTn, enabled closing of the gaps in the sequencing data, and allowed prediction of genes further downstream. Though the whole genome is not yet assembled, a very significant region carrying a concentrated area of mobile genetic elements (MGE) has been found to surround the degradative genes in OLGA172. This thesis details the sequence evidence that, upon examination of closely related strains, OLGA172 and its related strain from pristine soils may be the ancestral chlorobenzoate degraders.

In situ bioremediation by Fe(III) reducers is a strategy for clean-up of ground water through reductive immobilization. The dynamics of the community involved is complex and needs to be understood better for improving the bioremediation. Here, we have created a dynamic genome-scale metabolic model of Geobacter sulfurreducens and Rhodoferax ferrireducens, the two primary iron-reducers in subsurface environments, in order to understand the community competition prior to and during uranium- bioremediation. The simulation results suggest that the community competition is modulated by two factors: the ability of G. sulfurreducens to fix nitrogen under ammonium limitation, and a rate vs. yield trade-off between these two organisms. This model will be an important tool for the analyses of more complex microbial communities and the design of effective uranium-bioremediation strategies.

Bone changes composition and structure to adapt to its mechanical environment. Osteocytes are putative mechanosensors responsible for orchestrating the bone remodeling process. Recent in vitro studies showed that osteocytes could sense and respond to substrate strain and fluid shear. However the capacity of osteocytes to sense cyclic hydraulic pressure (CHP) associated with physiological mechanical loading is not well understood. In this study, osteocyte-like MLO-Y4 cells were subjected to CHP of 68 kPa at 0.5 Hz, and the effects of CHP on intracellular calcium concentration, cytoskeleton organization, mRNA expression of genes related to bone remodeling, and osteocyte apoptosis were investigated. The results indicate that osteocytes could sense CHP and respond by increased intracellular calcium concentration, altered microtubule organization, an increase in COX-2 mRNA level and RANKL/OPG mRNA ratio, and decreased apoptosis. Therefore cyclic hydraulic pressure in bone a mechanical stimulus to osteocytes and may play a role in regulating bone remodeling.

Plasmodium falciparum is the causative agent of malaria, one of the world‟s most prevalent infectious diseases. The emergence of strains resistant to current therapeutics creates the urgent need to identify new classes of antimalarials. Here we present and analyse a constraints-based model (iMPMP427) of P. falciparum metabolism. Consisting of 427 genes, 513 reactions, 457 metabolites, and 5 intracellular compartments, iMPMP427 is relatively streamlined and contains an abundance of transport reactions consistent with P. falciparum’s observed reliance on host nutrients. Flux Balance Analysis simulations reveal the model to be predictive in regards to nutrient transport requirements, amino acid efflux characteristics, and glycolytic flux calculation, which are validated by a wealth of experimental data. Furthermore, enzymes deemed to be essential for parasitic growth by iMPMP427 lend support to several previously computationally hypothesized metabolic drug targets, while discrepancies between essential enzymes and experimentally annotated drug targets highlight areas of malarial metabolism that could benefit from further research.

Tuberculosis is a re-emerging global health problem. Bacille Calmette-Guerin (BCG), the available vaccine against the disease, is only effective short term and is associated with adverse reactions clinically. The development of new effective vaccines will require an understanding of virulence, immunogenic factors and the beneficial immune responses induced in the human host. My thesis investigates phoP and whiB3, two genes associated with virulence and immunogenicity in Mycobacterium tuberculosis. Study of PhoP in a natural phoP mutant, BCG-Prague, and in the clinically safe BCG-Japan, shows that over-expression of PhoP increases the immunogenicity of these vaccine strains. In addition, I found that WhiB3 impacts carbon metabolism in BCG-Birkhaug and BCG-Sweden, although the effect of this on virulence in vivo is still unclear. The characterization of genes involved in virulence and immunogenicity allows us to develop novel approaches for improving the efficacy of BCG, which has important implications for future TB vaccine development.