The bacterium Sinorhizobium (Ensifer) meliloti is a member of the Rhizobiaceae family and can enter a mutualistic, diazotrophic relationship with most plants of the genera Medicago, Melilotus, and Trigonella. Medicago sativa (alfalfa) is an agriculturally important legume that hosts S. meliloti and allows the bacterium to invade the plant root and begin fixing nitrogen. Prior to invasion, S. meliloti exists as a free living bacterium and must navigate through the soil to find alfalfa, using chemical signals secreted by the root. Alfalfa is the 4th most cultivated crop in the United States, therefore, identification of plant host signals that lure S. meliloti, and identification of the bacterium's chemoreceptors that perceive the signals can aid in propagating the symbiosis more efficiently, thus leading to greater crop yields. Investigations here focus on discovering alfalfa derived attractant signals and matching them to their respective chemoreceptors in S. meliloti. We have determined the chemotactic potency of alfalfa seed exudate and characterized and quantified two classes of attractant compounds exuded by germinating alfalfa seeds, namely, amino acids and quaternary ammonium compounds (QACs). At all points possible, we have compared alfalfa with the closely related non-host, spotted medic (Medicago arabica). The chemotactic potency of alfalfa seed exudate is the same as spotted medic seed exudate, however, the attractant compositions are chemically different. The amount of each proteinogenic amino acid (AA) exuded by spotted medic is slightly greater than the amounts exuded by alfalfa. In addition, the five QACs studied are exuded in various amounts between the two Medicago species. In comparison, the total amount of proteinogenic AAs exuded be alfalfa and spotted medic are 2.01 μg/seed and 1.94 μg/seed respectively, and the total amount of QACs exuded are 249 ng/seed and 221 ng/seed respectively. By performing a chemotaxis assay with synthetic AA mixtures mimicking the amounts exuded from the medics, it was found that the AA mixtures contribute to 23% and 37% of the responses to alfalfa and spotted medic exudates, respectively. The chemoreceptor McpU was found to be the most important chemoreceptor of the eight for chemotaxis to the whole exudates and the AA mixtures. Furthermore, McpU is shown to mediate chemotaxis to 19 of 20 AAs excluding aspartate. McpU directly interacts with 18 AAs and indirectly mediates chemotaxis to glutamate. Through single amino acid residue substitutions, it is determined that McpU directly binds to amino acids in the annotated region called the Cache_1 domain, likely utilizing residues D155 and D182 to interact with the amino group of AA ligands. In all, McpU is a direct sensor for AAs except for the acidic AAs aspartate and glutamate. Work is presented to show that the QACs betonicine, choline, glycine betaine, stachydrine, and trigonelline are potent attractants for S. meliloti, McpX is the most important chemoreceptor for chemotaxis to these QACs, and we demonstrate the binding strength of McpX to the QACs with dissociation constants ranging from low millimolar to low nanomolar, thus making McpX the first observed bacterial MCP that mediates chemotaxis to QACs. Overall, we match medic derived AAs with McpU and QACs with McpX. These results can aid in optimizing chemotaxis to the host derived attractants in order to propagate the symbiosis more efficiently resulting in greater crop yields. Chapter 2 characterizes the function of the S. meliloti Methyl accepting Chemotaxis Protein U (McpU) as receptor for the attractant, proline. A reduction in chemotaxis to proline is observed in an McpU deletion strain, but the defect is restored in an mcpU complemented strain. Single amino acid substitution mutant strains were created, each harboring a mutant mcpU gene. The behavioral experiments with the mutants display a reduction in chemotaxis to proline when aspartate 155 and aspartate 182 are changed to glutamates. The periplasmic region of wild type McpU was purified and demonstrated to directly bind proline with a dissociation constant (Kd) of 104 μM. The variant McpU proteins show a reduction in binding affinity confirming McpU as a direct proline sensor. Chapter 3, describes the development of a high-throughput technique that is able to observe chemotaxis responses in ten separate chemotaxis chambers all at once. This procedure also allows for real time observations at intervals of two minutes for however long the experiment is scheduled. Using this new method it was found that McpU and the Internal Chemotaxis Protein A (IcpA) are the most involved with chemotaxis to seed exudates followed by McpV, W, X, and Y. The amounts of each proteinogenic amino acid (AA) in host and non-host seed exudates are quantified, which reveals that similar amounts are exuded from each species. It is shown that McpU is the most important receptor for chemotaxis toward synthetic mixtures that mimic the amounts seen in the exudates. Chapter 4 further investigates the role of McpU in sensing amino acids using the high-throughput technique developed in Chapter 3. It is shown that McpU is important for chemotaxis to all individual proteinogenic amino acids except the acidic AA, aspartate. In vitro binding experiments confirm that McpU directly interacts with all AAs except the acidic AAs aspartate and glutamate. Binding parameters are determined for aspartate, glutamate, phenylalanine and proline. In Chapter 5, five quaternary ammonium compounds (QACs) are quantified from the host and non-host seed exudates, which reveals distinctive QAC profiles. S. meliloti is found to display strong chemotaxis to all QACs, which is further shown to be mediated mostly by McpX. McpX is then established as a direct binder to all QACs as well as proline, with dissociation constants ranging from nanomolar to millimolar. These studies have increased our knowledge of how chemoreceptors sense attractants, and they have contributed to the bank of known attractant molecules for bacteria. Our new understandings of chemotaxis and how it relates to the Sinorhizobium-alfalfa model can allow for manipulations of the system to enhance chemotaxis to the host, thus propagating the symbiosis more efficiently, ultimately leading to greater crop yields. / Ph. D.

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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.