Sinorhizobium meliloti (Ensifer meliloti) is a bacterium that will exist saprotrophically in the soil and rhizosphere or as a differentiated bacteroid inside root nodules of the legume genera Medicago, Melilotus, and Trigonella. It exists in symbiosis when inside a host plant and will fix gaseous N2 into ammonium for the plant. In return, a population of the bacteria is harbored inside the plant where it can proliferate beyond what would be possible in the rhizosphere or bulk soil. This symbiosis is a defining feature of the Fabaceae (legume) family, a clade that diverged approximately 60 million years ago and is now the 5th largest plant family by species count. Each legume species pairs with one or several strains of bacteria, referred to broadly as rhizobia. The rhizobia identify their proper host plant by a cocktail of secondary metabolites called flavonoids released from specific parts of the roots. Initiation of the symbiosis may only occur at the tips of young root hairs. Therefore, the means rhizobia take to localize themselves to these sites must be the inceptive step in the symbiotic interaction. The studies here examine the mechanisms and priorities rhizobia use to achieve this goal. Movement of bacteria is referred to as motility and is achieved via (in rhizobia, multiple) rotating flagella, proteinaceous extracellular appendages that propel the cell through liquid environments. On their own, flagella may only move but not guide the cell. Navigation is achieved through sensors that detect chemical attractant or repellent cues in the environment and an intracellular signaling system that relays information to appropriately control locomotion. This sensing is called chemotaxis. A research focus is directed on the sensing aspect of chemotaxis to understand which chemical compounds are the preferred attractants for S. meliloti. An emphasis is placed on those compounds released from germinating host seeds. Chapter 2 spearheads our research goals by examining the chemotactic potential of host-derived flavonoids, the compounds that induce the symbiotic signaling in the rhizobial symbiont. While a logical place to start, this study reveals that our strain of rhizobia is not attracted to flavonoids. We determined that the best chemoattractants are hydrophilic in nature and that hydrophobic compounds, such as flavonoids, are not effective chemoattractants. In addition, we discuss the nature of chemotactic agents and symbiosis inducers to fortify our understanding of how classes of compounds contribute to the rhizobia-plant interaction. In chapter 3, we characterize the sensor protein, McpV, and its ligand profile for carboxylates. The protein is first screened using a high-throughput assay to test numerous possible ligands simultaneously. We confirm positive reactions using direct binding studies and quantify dissociation constants. Then, the phenotypic response to these ligands is measured using capillary chemotaxis assays, and the role mcpV plays in this response is confirmed using deletion mutants. Last, the symbiotic context is addressed by quantifying these ligands in exudates of the host alfalfa. These experiments show that McpV is a chemotactic sensor dedicated to detecting 2 – 4 C monocarboxylates. Only one of the compounds found in the ligand profile, glycolate, was detected in seed exudates, so the contribution of McpV to host sensing is yet to be expounded. Chapter 4 follows the model of chapter 2 but is complicated when the ligand screen used previously gives ambiguous results. Using direct binding studies, we were able to confirm the true ligand amidst numerous false positives. Analytical gel filtration suggests that McpT exists as a dimer regardless of ligand binding. Capillary chemotaxis assays quantified the responses mediated by McpT to di- and tri-carboxylates, which were slightly weaker, but still on-par with the responses to McpV ligands. Strains with mcpT deletions showed strongly reduced, but in some cases, not abolished, chemotaxis to carboxylates. Chapter 5 examines McpX – the chemoreceptor already known to be a sensor of quaternary ammonium compounds. This is a structural investigation into the binding of McpX to its ligands. A crystal structure of the ligand binding region of the protein is resolved to understand how ligands fit into the binding pocket of McpX and what determines its structurally diverse ligand profile. The contribution of certain residues to ligand binding are further probed using direct binding studies on single point variants of McpX. The analysis of chemoreceptor functions hint at what kinds of molecules are most important to bacterial survival and reproduction. Knowing what the bacterium is tuned to seek out grants understanding of what niches they prefer, and how they thrive in those niches. For S. meliloti and other rhizobia, the preeminent niche is one in symbiosis with a host plant. The sum of this knowledge we have accrued with S. meliloti lends itself to agricultural goals of soil enrichment, legume inoculation, nutrient cycling, and environmentally safe and efficient crop fertilization. / Doctor of Philosophy / Sinorhizobium meliloti and other soil-dwelling bacteria termed rhizobia are crucial to the cultivation of leguminous crops such as alfalfa, soy, pea, lentil, peanut, and many more. The bacterium can be internalized by the plant host's roots where it will supply the plant with nitrogen. This is a great boon to crops when they need to accumulate more protein in seed stores, or for plants that survive in nutrient depleted soils. The bacterium must begin seeking out the host plant by sensing chemical cues. It can navigate to the proper location by using a process called chemotaxis. This process is centered around chemoreceptors that can be likened to the nose of the bacterium. Using these chemoreceptors, the bacterium will seek out compounds that benefits it – these are usually food sources. Identifying what each individual chemoreceptor senses allows us to understand what the bacterium needs to seek out to survive. We correlate this information with compounds that the plant secretes and find that many chemoreceptors have evolved to sense signals that will lead the bacterium to a plant root. This interaction is a key part of how the symbiosis is propagated and ultimately benefits the agriculture of leguminous plants.

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Peritrichously flagellated bacteria carry out chemotaxis by modulating the frequency of switching between smooth swimming and tumbling. The tumbling frequency is controlled by a signal transduction cascade in which transmembrane receptors modulate the activity of a histidine kinase CheA that transfers phosphate to its cognate response regulator CheY. The proteins of the chemotaxis signaling cascade are localized to clusters found primarily at the poles of cells. In this work, the localization of the CheZ protein, a phosphatase that dephosphorylates CheY~P, is examined. Using a CheZ-GFP fusion protein, we show that CheZ was localized to the polar receptor patch via interaction with the short form of CheA (CheAS). Aromatic residues of CheZ near one end of the elongated CheZ four-helix bundle were determined to be critical for localization. Aliphatic residues in CheAS were also determined to be critical for CheZ localization to the receptor patch and substitution of these residues conferred a tumble bias to swimming cells. A mechanism of CheZ localization is proposed in which the CheZ apical loop interacts with a binding site formed by dimerization of the P1 domain of CheAS. The potential role of CheZ localization as a means of coordinating the rotation state of peritrichously distributed flagella is discussed.

The overall goal of this work was to develop and utilize microfluidic models for investigating bacterial chemotaxis and biofilm formation - phenotypes that play key roles in bacterial infections. Classical methods for investigating chemotaxis and biofilm formation have many limitations and drawbacks. These include being unsuitable for investigating the effect of chemorepellents, non-quantitative readouts, and not accounting for interaction between hydrodynamics and biofilm formation. The novel microfluidic model systems for chemotaxis and biofilm formation developed in this study addresses these drawbacks. Chemotaxis model system development was done in three stages. We first developed two static chemotaxis model systems - the two fluorophore chemotaxis agarose plug assay and the mu Plug assay - for rapidly determining the extent of chemotaxis in a qualitative manner. A key feature of these model systems was the incorporation of dead cells and differential labeling with green and red fluorescent proteins for partitioning the effects of movement due to fluid flow from chemotaxis. The static systems were used to rapidly screen a wide range of conditions for use in the flow-based mu Flow chemotaxis model system. The effect of four major variables - cell preparation method, gradient strength, flow rate in the device, and imaging position - that influence the chemotactic response in the mu Flow was characterized using the repellent taxis from Ni^2 gradients as the model chemoeffector. Using the mu Flow chemotaxis device, we investigated the chemotaxis of Escherichia coli RP437 to different signals that are present in the human gastrointestinal tract and are likely to be mediators of infection through their effect on chemotaxis. Our data show that the bacterial signal indole is a repellent, while the signals autoinducer-2 (AI-2) and isatin are attractants for E. coli RP437. However, cells exposed to a competing gradient of indole and either AI-2 or isatin, attracts E. coli. The ?Flow device was also used to refute a long-standing view on how the repellent Ni2 is sensed in E. coli. Our data show that only the Tar chemoreceptor is needed for sensing Ni^2 and the nickel binding protein, NikA, and the Ni^2 transport system proteins, NikB and NikC, are not required for repellent taxis from nickel. A microfluidic biofilm model was also developed in this study and used in conjunction with a mathematical model to investigate biofilm formation and quorum sensing in closed systems (where biofilm growth and hydrodynamics are interdependent). The mathematical model predictions were experimentally validated using Pseudomonas aeruginosa PA14 in a microfluidic biofilm system at various flow rates.

One major barrier that prevents cartilage integration following mosaic arthroplasty is the presence of a zone of chondrocyte death (ZCD) that is generated upon osteochondral graft harvest, which can extend up to 400 μm into the cartilaginous portion of the graft. In order for cartilage integration to occur, chondrocytes must be present at the graft periphery; however chondrocyte migration through the ZCD to the graft periphery is inhibited by the dense extracellular matrix (ECM) of cartilage. The purpose of this study was to develop a method for increasing the number of chondrocytes within the ZCD and at the periphery of a cartilage graft. This method used a combination of collagenase treatment (as a means of degrading the ECM within the ZCD) and chondrocyte chemotaxis (as a means of improving chondrocyte migration into the ZCD and to the cartilage periphery). Results indicate that treating bovine articular cartilage with 0.6 % collagenase for 10 min decreased with extent of the ZCD by approximately 35% (collagenase: 109 ± 13 μm; control: 175 ± 13 μm). Each of the chemotactic agents tested (PDGF-bb, bFGF, and IGF-I) were found to induce bovine chondrocyte chemotaxis at concentrations of 25 ng/mL in modified Boyden chamber experiments. However, in bovine articular cartilage samples that were pre-treated with collagenase (0.6% for 10 min), supplementation with 25 ng/mL of either PDGF-bb or bFGF had no apparent effect on the ZCD relative to samples treated only with collagenase (PDGF-bb: 85 ± 10 μm; bFGF: 88 ± 10 μm). Alternatively, bovine articular cartilage samples pre-treated with collagenase (0.6% for 10 min) and supplementation with 25 ng/mL IGF-I resulted in an approximately 65% reduction in the ZCD relative to samples treated only with collagenase (IGF-1: 38 ± 5 μm). Thus, treating osteochondral grafts with collagenase and IGF-1 induces chondrocyte repopulation of the zone of chondrocyte death generated by osteochondral graft harvesting, and could enhance cartilage integration after implantation. / Thesis (Master, Chemical Engineering) -- Queen's University, 2009-12-21 20:16:05.815

Directed immune cell migration plays important roles in immunosurveillance and immune responses. Understanding the mechanisms of immune cell migration is important for the biology of immune cells with high relevance to immune cell trafficking mediated physiological processes and diseases. Immune cell migration can be directed by various guiding cues such as chemical concentration gradients (a process termed chemotaxis) and direct current electric fields (dcEF)(a process termed electrotaxis). Microfluidic devices that consist of small channels with micrometer dimensions have been increasingly developed for cell migration studies. These devices can precisely configure and flexibly manipulate chemical concentration gradients and electric fields, and thus provide powerful quantitative test beds for studying the complex guiding mechanisms for cell migration. In the research of this thesis, a PDMS-based and a glass-based microfluidic devices were developed for producing controlled dcEF and these devices were used to analyze electrotaxis of activated human blood T cells. Using both devices, we have successfully demonstrated that activated human blood T cells migrate toward the cathode of the applied dcEF. Furthermore, a novel microfluidic device was developed to configure better controlled single or co-existing chemical gradients and dcEF to mimic the complex guiding environments in tissues and this device was used to investigate the competition of chemical gradients and dcEF in directing activated human blood T cell migration.

Directed immune cell migration plays important roles in immunosurveillance and immune responses. Understanding the mechanisms of immune cell migration is important for the biology of immune cells with high relevance to immune cell trafficking mediated physiological processes and diseases. Immune cell migration can be directed by various guiding cues such as chemical concentration gradients (a process termed chemotaxis) and direct current electric fields (dcEF)(a process termed electrotaxis). Microfluidic devices that consist of small channels with micrometer dimensions have been increasingly developed for cell migration studies. These devices can precisely configure and flexibly manipulate chemical concentration gradients and electric fields, and thus provide powerful quantitative test beds for studying the complex guiding mechanisms for cell migration. In the research of this thesis, a PDMS-based and a glass-based microfluidic devices were developed for producing controlled dcEF and these devices were used to analyze electrotaxis of activated human blood T cells. Using both devices, we have successfully demonstrated that activated human blood T cells migrate toward the cathode of the applied dcEF. Furthermore, a novel microfluidic device was developed to configure better controlled single or co-existing chemical gradients and dcEF to mimic the complex guiding environments in tissues and this device was used to investigate the competition of chemical gradients and dcEF in directing activated human blood T cell migration.

Ulm, Univ., Diss., 2008.