Lokalisierung und Funktion der Chemotaxis- und Motorkomponenten von Sinorhizobium meliloti: Eine in vivo Untersuchung unter Einsatz der FRET-Technik
Final Report Abstract
Six of the transmembrane (McpT-X, McpZ) and both soluble receptors (McpY, IcpA) of Sinorhizobium meliloti localize to the cell pole. Only McpS, encoded from the symbiotic plasmid pSymA, is evenly distributed in the cell. While the synthesis of all polar localized receptors is confined to exponential growth correlating with the motility phase of cells, McpS is only weakly expressed throughout cell culture growth. Therefore, motile S. meliloti cells form one major Chemotaxis cluster that harbors all chemoreceptors except for McpS. The response regulator for the flagellar motor, CheY2-P, is inactivated via a phosphotransfer back to CheA, and then lo a second response regulator, CheY1, which acts as a phosphate sink. NMR fitration studies of CheY2 and CheY2-P with the isolated P2 domain of CheA revealed two overlapping sites in the P2 domain that selectively bind either CheY2 or CheY2-P. Using isolated CheA domains in phosphorylation experiments, we could show that the P2 domain is dispensable for the phosphorylation of CheY2, but that it is essenfial for the retrotransfer from CheY2-P to CheA. We also found that CheX, a new small Chemotaxis protein, binds to CheA, where it might serve as regulatory domain to control the phosphate flow between the kinase and the response regulators. One major goal of this proposal was the analysis of Chemotaxis and motor components using the FRET technique in vivo. However, we found several pitfalls during the course of our experiments. We were able to overcome the problem of expression levels and ratios between the two FRET components by constructing a tightly regulated vector. Another problem was the autofluorescence of the attractant proline due to the formation of poly-proline that obscured our results. For future experiments, we will need to purify the substrate before use. We documented a positive FRET response between FliM-CFP and CheY2-YFP once. Unfortimately, we were not able to reproduce this result. We are worried about the high concentrations of attractant that we had to apply before we were able lo observe a response. At this point, we cannot exclude that osmotic effects instead of true chemotactic responsed were causing the FRET effect. We believe that the FRET technique can be used to study the chemotactic response of S. meliloti. However, a more detailed understanding of the intracellular signalling process, in particular the adaptation mechanism is essential to successfully apply this sensitive technique.
Publications
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(2008) FliL is essential for swarming: motor rotation in absence of FliL fractures the flagellar rod in swarmer cells of Salmonella enterica. Molecular Microbiology 68:328-341
U. Attmannspacher, Scharf B.E. and Harshey R.M.
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(2008) Interaction of CheY2 and CheY2-P with the cognate CheA kinase in the chemosensory-signalling chain of Sinorhizobium meliloti. Molecular Microbiology 69, 1373-1384
H. Riepl, Maurer T., Kalbitzer H.R., Meier V.M., Schmitt R., Scharf B.
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(2009) Celiular localization of predicted transmembrane and soluble chemoreceptors in Sinorhizobium meliloti. Journal of Bacteriology 191, 5724-5733
V.M. Meier & Scharf B.E.
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(2009) Upward mobility and alternative lifestyles: a report from the 10th biennial meeting on Bacterial Locomotion and Signal Transduction. Molecular Microbiology 73, 5-19
B.E. Scharf, Aldridge P.D., Kirby, J.R., Crane, B.R.
