1887

Abstract

Signal transduction in prokaryotes is frequently accomplished by two-component regulatory systems in which a histidine protein kinase is the sensory component. Many of these sensory kinases control metabolic processes that do not show an obvious requirement for inhomogeneous distribution within bacterial cells. Here, the sensory kinases DcuS and CitA, two histidine kinases of , were investigated. Both are membrane-integral and involved in the regulation of carboxylate metabolism. The two-component sensors were fused with yellow fluorescent protein (YFP) and live images of immobilized cells were obtained by confocal laser fluorescence microscopy. The fluorescence of the fusion proteins was concentrated at the poles of the cells, indicating polar accumulation of the sensory kinases. For quantitative evaluation, line profiles of the imaged fluorescence intensities were generated; these revealed that the fluorescence intensity of the polar bright spots was 2.3–8.5 times higher than that of the cytoplasm. With respect to the cylindrical part of the membrane, the values were lower by about 40 %. The polar accumulation was comparable to that of methyl-accepting chemotaxis proteins (MCPs) and MCP-related proteins. The degree of DcuS–YFP localization was independent of the presence of MCP and the expression level of (or DcuS concentration). The presence of effector (fumarate or citrate, respectively) increased the polar accumulation by more than 20 %. Cell fractionation demonstrated that polar accumulation was not related to inclusion body formation. Therefore, sensory kinases DcuS and CitA, which regulate metabolic processes without obvious polar function, exhibit polar accumulation.

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2008-08-01
2019-11-19
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Supplements

The supplementary data file [ PDF] (354 kb) contains experimental results confirming the formation of fluorescent DcuS-YFP fusion protein by fluorescence spectroscopy, showing the intensity ratios versus z-position of the focus, and demonstrating that polar accumulation responds to the presence of effector. It includes the following figures: Fluorescence emission spectra of DcuS-YFP fusion protein. Dependence of the intensity ratio on the focus z-position. Influence of effectors fumarate and citrate on the polar accumulation of DcuS-YFP and CitA-YFP. Four QuickTime movie files are also available: 3D-reconstruction of an cell expressing DcuS-YFP (strain JM109/pMW407; in the presence of 20 mM fumarate; custom-built microscope) [ QuickTime file] (698 kb) 3D-reconstruction of an cell expressing DcuS-YFP (strain UU1250/pMW407; without fumarate; custom-built microscope). Movies 2a [ QuickTime file] (1571 kb) and 2b [ QuickTime file] (1300 kb) show the same cells but differ with respect to the thresholds. 3D-reconstruction of an cell expressing DcuS-YFP (strain JM109pMW407; without fumarate; commercial microscope) [ QuickTime file] (1393 kb) 3D-reconstruction of an cell expressing DcuS-YFP (strain JM109/pMW407; without fumarate; commercial microscope) [ QuickTime file] (2000 kb)

PDF

The supplementary data file [ PDF] (354 kb) contains experimental results confirming the formation of fluorescent DcuS-YFP fusion protein by fluorescence spectroscopy, showing the intensity ratios versus z-position of the focus, and demonstrating that polar accumulation responds to the presence of effector. It includes the following figures: Fluorescence emission spectra of DcuS-YFP fusion protein. Dependence of the intensity ratio on the focus z-position. Influence of effectors fumarate and citrate on the polar accumulation of DcuS-YFP and CitA-YFP. Four QuickTime movie files are also available: 3D-reconstruction of an cell expressing DcuS-YFP (strain JM109/pMW407; in the presence of 20 mM fumarate; custom-built microscope) [ QuickTime file] (698 kb) 3D-reconstruction of an cell expressing DcuS-YFP (strain UU1250/pMW407; without fumarate; custom-built microscope). Movies 2a [ QuickTime file] (1571 kb) and 2b [ QuickTime file] (1300 kb) show the same cells but differ with respect to the thresholds. 3D-reconstruction of an cell expressing DcuS-YFP (strain JM109pMW407; without fumarate; commercial microscope) [ QuickTime file] (1393 kb) 3D-reconstruction of an cell expressing DcuS-YFP (strain JM109/pMW407; without fumarate; commercial microscope) [ QuickTime file] (2000 kb)

MOVIE

The supplementary data file [ PDF] (354 kb) contains experimental results confirming the formation of fluorescent DcuS-YFP fusion protein by fluorescence spectroscopy, showing the intensity ratios versus z-position of the focus, and demonstrating that polar accumulation responds to the presence of effector. It includes the following figures: Fluorescence emission spectra of DcuS-YFP fusion protein. Dependence of the intensity ratio on the focus z-position. Influence of effectors fumarate and citrate on the polar accumulation of DcuS-YFP and CitA-YFP. Four QuickTime movie files are also available: 3D-reconstruction of an cell expressing DcuS-YFP (strain JM109/pMW407; in the presence of 20 mM fumarate; custom-built microscope) [ QuickTime file] (698 kb) 3D-reconstruction of an cell expressing DcuS-YFP (strain UU1250/pMW407; without fumarate; custom-built microscope). Movies 2a [ QuickTime file] (1571 kb) and 2b [ QuickTime file] (1300 kb) show the same cells but differ with respect to the thresholds. 3D-reconstruction of an cell expressing DcuS-YFP (strain JM109pMW407; without fumarate; commercial microscope) [ QuickTime file] (1393 kb) 3D-reconstruction of an cell expressing DcuS-YFP (strain JM109/pMW407; without fumarate; commercial microscope) [ QuickTime file] (2000 kb)

MOVIE

The supplementary data file [ PDF] (354 kb) contains experimental results confirming the formation of fluorescent DcuS-YFP fusion protein by fluorescence spectroscopy, showing the intensity ratios versus z-position of the focus, and demonstrating that polar accumulation responds to the presence of effector. It includes the following figures: Fluorescence emission spectra of DcuS-YFP fusion protein. Dependence of the intensity ratio on the focus z-position. Influence of effectors fumarate and citrate on the polar accumulation of DcuS-YFP and CitA-YFP. Four QuickTime movie files are also available: 3D-reconstruction of an cell expressing DcuS-YFP (strain JM109/pMW407; in the presence of 20 mM fumarate; custom-built microscope) [ QuickTime file] (698 kb) 3D-reconstruction of an cell expressing DcuS-YFP (strain UU1250/pMW407; without fumarate; custom-built microscope). Movies 2a [ QuickTime file] (1571 kb) and 2b [ QuickTime file] (1300 kb) show the same cells but differ with respect to the thresholds. 3D-reconstruction of an cell expressing DcuS-YFP (strain JM109pMW407; without fumarate; commercial microscope) [ QuickTime file] (1393 kb) 3D-reconstruction of an cell expressing DcuS-YFP (strain JM109/pMW407; without fumarate; commercial microscope) [ QuickTime file] (2000 kb)

MOVIE

The supplementary data file [ PDF] (354 kb) contains experimental results confirming the formation of fluorescent DcuS-YFP fusion protein by fluorescence spectroscopy, showing the intensity ratios versus z-position of the focus, and demonstrating that polar accumulation responds to the presence of effector. It includes the following figures: Fluorescence emission spectra of DcuS-YFP fusion protein. Dependence of the intensity ratio on the focus z-position. Influence of effectors fumarate and citrate on the polar accumulation of DcuS-YFP and CitA-YFP. Four QuickTime movie files are also available: 3D-reconstruction of an cell expressing DcuS-YFP (strain JM109/pMW407; in the presence of 20 mM fumarate; custom-built microscope) [ QuickTime file] (698 kb) 3D-reconstruction of an cell expressing DcuS-YFP (strain UU1250/pMW407; without fumarate; custom-built microscope). Movies 2a [ QuickTime file] (1571 kb) and 2b [ QuickTime file] (1300 kb) show the same cells but differ with respect to the thresholds. 3D-reconstruction of an cell expressing DcuS-YFP (strain JM109pMW407; without fumarate; commercial microscope) [ QuickTime file] (1393 kb) 3D-reconstruction of an cell expressing DcuS-YFP (strain JM109/pMW407; without fumarate; commercial microscope) [ QuickTime file] (2000 kb)

MOVIE

The supplementary data file [ PDF] (354 kb) contains experimental results confirming the formation of fluorescent DcuS-YFP fusion protein by fluorescence spectroscopy, showing the intensity ratios versus z-position of the focus, and demonstrating that polar accumulation responds to the presence of effector. It includes the following figures: Fluorescence emission spectra of DcuS-YFP fusion protein. Dependence of the intensity ratio on the focus z-position. Influence of effectors fumarate and citrate on the polar accumulation of DcuS-YFP and CitA-YFP. Four QuickTime movie files are also available: 3D-reconstruction of an cell expressing DcuS-YFP (strain JM109/pMW407; in the presence of 20 mM fumarate; custom-built microscope) [ QuickTime file] (698 kb) 3D-reconstruction of an cell expressing DcuS-YFP (strain UU1250/pMW407; without fumarate; custom-built microscope). Movies 2a [ QuickTime file] (1571 kb) and 2b [ QuickTime file] (1300 kb) show the same cells but differ with respect to the thresholds. 3D-reconstruction of an cell expressing DcuS-YFP (strain JM109pMW407; without fumarate; commercial microscope) [ QuickTime file] (1393 kb) 3D-reconstruction of an cell expressing DcuS-YFP (strain JM109/pMW407; without fumarate; commercial microscope) [ QuickTime file] (2000 kb)

MOVIE
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