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.

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.