Control of Chey-P Levels in the Chemotactic Sensory Transduction Cascade of Bacillus Subtilis
Szurmant, Hendrik
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https://hdl.handle.net/2142/84806
Description
Title
Control of Chey-P Levels in the Chemotactic Sensory Transduction Cascade of Bacillus Subtilis
Author(s)
Szurmant, Hendrik
Issue Date
2004
Doctoral Committee Chair(s)
Ordal, George W.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Language
eng
Abstract
Bacteria sense their environment and respond to it in order to enhance their chance for survival. Motility is one output created that enables bacteria to colonize in optimal conditions. Since environments are ever changing, the signal transduction mechanism controlling this behavior has to be able to respond on a time scale of seconds. This is achieved by phosphorylation of an intracellular signaling molecule CheY (to generate CheY-P) in response to changing environments. As important is the rapid dephosphorylation of this protein, to reset the system and prepare it for new signals. The flagella proteins FliM and FliY are the interaction target for CheY-P in Bacillus subtilis. Binding of CheY-P to FliM induces a counterclockwise rotation of the motility apparatus, the flagella. FliM in Escherichia coli was previously demonstrated to have the same function. FliY on the other hand is not found in E. coli. Phenotypes of a mutant incapable of binding CheY-P led to the hypothesis that FliY actively dephosphorylates CheY-P. This hypothesis was verified by incubating CheY with FliY in vitro and following the rate of CheY-P hydrolysis. CheC, which shares homology with the N-terminal domain of FliY, also shares its function to dephosphorylate CheY-P. With the knowledge that there are two proteins in the B. subtilis chemotaxis signal transduction cascade, an attempt was made to clarify the specific roles of these two proteins. Specific activities and per cell copy number were measured for both these proteins. FliY is expressed at much higher levels than CheC. The activity of CheC is relatively low compared to that of FliY, but this activity was shown to be enhanced 5-fold by the chemotaxis protein CheD. In vivo, a double mutant lacking cheC and the region of fliY that encodes the CheY-P binding domain is almost completely smooth swimming, implying that these cells contain very high levels of CheY-P. CheC appears to be primarily involved in restoring normal CheY-P levels following the addition of attractant, whereas FliY seems to act on CheY-P constitutively. A model for how FliY, CheC, and CheD work together to regulate CheY-P levels in the bacterium is proposed.
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