Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
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Item Developing Tools for Investigating Chemotaxis Signal Clusters in Bacillus subtilis(2012) Rogers, James Allen; Stewart, Richard C; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Many bacteria make use of a set of dedicated chemoreceptor proteins to control a His-Asp signaling system; this control converts environmental sensory information into instructions that regulate flagellar rotation, enabling chemotaxis. This thesis summarizes my investigations of some of the chemotaxis signaling proteins in Bacillus subtilis, particularly coupling proteins CheW and CheV. Proteins CheA, CheW, CheV, CheY, and FliM were each expressed in B. subtilis as translational fusions with either YFP or CFP. These fusion proteins were then shown to fluoresce in living bacterial cells. Motility experiments were conducted to compare the function of these fusion proteins to their wild type counterparts. This thesis proposes a series of experiments that would use these fluorescent fusion proteins to further explore the idea that these chemotaxis proteins change position when B. subtilis encounters chemostimuli.Item Binding Interactions in the Bacterial Chemotaxis Signal Transduction Pathway(2008-12-08) Eaton, Anna Kolesar; Stewart, Richard C; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The investigation of signal transduction pathways is critical to the basic understanding of cellular processes as these pathways function to regulate diverse processes in both eukaryotes and prokaryotes. This dissertation focuses on understanding some of the biochemical events that take place in the chemotaxis signal transduction pathway of bacteria. In this system, cell-surface receptor proteins regulate a histidine protein kinase, CheA, that autophosphorylates and then transfers its phosphate to an effector protein, CheY. Phospho-CheY, in turn, influences the direction of flagellar rotation. This sequence of biochemical events establishes a chain of communication that ultimately allows the chemotaxis receptor proteins to regulate the swimming pattern of the bacterial cell when it encounters gradients of attractant and repellent chemicals in its environment. The three projects presented in this dissertation sought to fill basic gaps in our current understanding of CheA and CheY function. In the first project, I examined the nucleotide binding reaction of CheA using the fluorescent nucleotide analogue, TNP-ATP [2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate]. TNP-ATP is an effective inhibitor for CheA. By monitoring the fluorescence of TNP-ATP when it bound to CheA, I examined the affinity of the binding interaction and discovered that the two ATP binding sites of each CheA dimer exhibited negative cooperativity in their interactions with TNP-ATP. This is the first evidence of cooperativity in the histidine protein kinase superfamily. In the second project, I focused on elucidating the binding mechanism that underlies formation of the CheA:TNP-ATP complex. My results indicated a three-step mechanism, including rapid formation of a low-affinity complex, followed by two steps during which conformational changes give rise to the final high-affinity complex. This same basic mechanism applied to CheA from Escherichia coli and from Thermotoga maritima. In the third project, I turned my attention to studying the CheY phosphorylation and binding reactions using fluorescently labeled versions of CheY. The results of this final study indicated that CheY proteins labeled with the fluorophore Badan [6-bromoacetyl-2-(dimethylamino)naphthalene] could be useful tools for investigating CheY biochemistry. However my results also brought to light some of the limitations and difficulties of this approach.Item The Role of CheV in S. typhimurium Chemotaxis(2006-12-11) Dougherty, Megan; Stewart, Richard C; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The chemotaxis systems of Escherichia coli and Salmonella typhimurium are thought to be virtually identical. However, recently a putative chemotaxis gene, cheV, was found to be present in S. typhimurium but not in E. coli. Sequence analysis shows that the CheV protein shares sequence similarity to both CheW and CheY. My thesis research investigated whether cheV does play a role in S. typhimurium chemotaxis. My results show that disruption of the cheV gene had no effect on S. typhimurium's swarming ability and only a minor effect on the ability of S. typhimurium to sense/respond to serine and its ability to accomplish surface motility. My results also indicate that overexpression of the cheV gene disrupts S. typhimurium's swarming ability, as well as, S. typhimurium's ability to sense/respond to serine and S. typhimurium's ability to accomplish surface motility. Overall, these results suggest that CheV may be involved in S. typhimurium chemotaxis.