Quantification and functional characterization of Sinorhizobium meliloti chemotaxis proteins
Arapov, Timofey Dmitryevich
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The flagellated soil-dwelling bacterium Sinorhizobium meliloti is known for its symbiotic relationship with several leguminous genera. The symbiosis between the bacterium and its host plants facilitates fixation of atmospheric nitrogen and ultimately replenishment of nitrogen to the soil. However, before nitrogen fixation can occur, the bacterial cells must actively travel to the plant's roots and successfully induce formation of a plant organ called a root nodule. To initiate the nodulation process, the bacterium needs to be in direct contact with the root hairs. This requires free living bacterial cells within the soil to sense the presence of their host plant and travel to its roots. S. meliloti is able to do this through a process called chemotaxis. Chemotaxis is the ability to respond to chemical gradients within the environment by directed movement. It is facilitated by Methyl-accepting Chemotaxis Proteins (MCPs) as part of a two-component signal transduction system. These receptor proteins are able to bind ligands and influence the state of the signal transduction system, ultimately controlling flagellar behavior. The chemotaxis system of Escherichia coli has been well characterized and serves as a useful point of comparison to that of S. melilot throughout this work. Within this work we have determined the stoichiometry of all chemotaxis proteins of S. meliloti by means of quantitative immunoblotting. Chapter 2 addresses the stoichiometry of MCPs and the histidine kinase CheA. The eight MCPs were grouped by total abundance within the cell, in high abundance (McpV), low abundance (IcpA, McpU, McpX, and McpW), and very low abundance (McpY, McpZ and McpT). The approximate cellular ratio of these three receptor groups is 300:30:1. The chemoreceptor-to-CheA ratio is 22.3:1, highly similar to the 23:1 ratio known for Bacilius subtiltis. Chapter 3 continues the investigation of the protein stoichiometry, expanding to all chemotaxis proteins. We compare ratios of S. meliloti chemotaxis proteins to those of E. coli and B. subtilis. We address the possible reasons for the high ratio of CheR / CheB to the total amount of receptors. Proteins again can be grouped by abundance: CheD, CheY1, and CheY2 are the most abundant. CheR and CheB appear in lower amounts, CheS and CheT appear to be auxiliary proteins, and finally CheW1 and CheW2, which directly interact with the receptors and CheA. Chapter 4 focuses on altered receptor abundance in S. meliloti due to the fusion of common epitope tags to the C-terminus. The fusion of these tags promotes greater cellular abundance of many receptors including McpU. The fusion of charged residues to the C-terminus promotes a greater increase in McpU abundance thanthe addition of single amino acid residues. Truncations of McpU were made to investigate the presence of a protease recognition site near the C-terminus. These truncations resulted in an increase in abundance similar to those resulting from epitope tag fusions. As epitope tags are widely used in protein studies to help determine protein stoichiometry, this study obviates a potential stumbling block for future experimenters. The function of CheT, a small protein (13.4 kDa) encoded by the last gene in S. meliloti's major chemotaxis operon, is the subject of chapter 5. A cheT deletion strain is chemotactically deficient compared to the S. meliloti wild-type strain. Through two separate experiments (a glutaraldehyde cross-linking assay and co-purification) we demonstrated that CheT interacts with the methyltransferase CheR. We also investigate its possible role in CheR's methylation activity through a series of methylation assays. This work contributes to our understanding of Sinorhizobium meliloti's chemotaxis signal transduction system. We have discovered evidence for new a protein-protein interaction within our system and have revealed the abundance of all chemotaxis proteins within the cell. We also showed that fusions of epitope tags to various chemotaxis proteins can dramatically influence their abundance. We shed light on the possible function of a previously uncharacterized protein, although more work is required to determine its exact role.
General Audience Abstract
Sinorhizboium meliloti is a bacterium that lives in the soil and forms a symbiotic relationship with many plants including alfalfa, a commonly grown cover crop. This symbiotic relationship is important because it allows for nitrogen to be replenished into the soil without the use of artificial fertilizer. However, to form this relationship the bacterial cells in the soil must be able to colonize the plant roots. The soil is a complex environment with many different kinds of chemical molecules and sources of nutrients. Like many other types of bacteria, S. meliloti uses flagella (long helical structures that rotate much like a propeller) to move through the soil. Control of the flagella falls to what is known as a chemotaxis signal transduction system, which can be thought of as a navigation system for each bacterial cell. The system has proteins that act as receptors to sense different chemical molecules. The bacterial cells can sense signals for the plant and move towards their host. This work shows the abundance of each type of receptor and other components within the cell. It also examines the function of a previously unknown protein, CheT, within the chemotaxis system.
- Doctoral Dissertations