Polar accumulation of the metabolic sensory histidine kinases DcuS and CitA in Escherichia coli

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 Escherichia coli, 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 dcuS-yfp (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.

Oligomeric Sensor Kinase DcuS in the Membrane of Escherichia coli and in Proteoliposomes: Chemical Cross-linking and FRET Spectroscopy

“…3B, lanes 2 to 5) showed an approximately 8-to 10-fold increase in the content of DcuS (Fig. 3B) (37). But again, all samples with detectable DcuS contained DcuS of the 61-kDa, the 120-kDa, and the major 240-kDa form, and there was no overproportional increase for the high-M r forms of DcuS with increasing induction of DcuS.…”

“…The fusions were placed on different plasmids within E. coli cells. In the DcuS-YFP fusion (encoded by pMW407), the DcuS protein is still functional in sensing and signal transduction in vivo (Table 2), and the YFP protein shows normal fluorescence (37). The DcuS-CFP fusion protein (encoded by pMW408) was tested in a similar way for the functionality of DcuS.…”

“…3B). E. coli containing the low-copy-number DcuS CysϪ expression plasmid pMW967 was induced for increasing levels of DcuS CysϪ by increasing the levels of the inducer (0 to 333 M arabinose), as described earlier (37). The proteins from the cell homogenate were subjected to SDS-PAGE and analyzed for DcuS by immunoblotting.…”

“…The FRET studies were performed with DcuS fused to the CFP or YFP derivative of the GFP protein or with DcuS labeled chemically with fluorescent dyes. DcuS forms stable fusion proteins with CFP or YFP, as tested earlier by immunoblotting (37). For quantitative and reproducible analysis of FRET efficiency, a method described by Gordon et al (12) was extended.…”

“…The dcuS-YFP fusion (pMW407) was constructed and cloned into pBAD30 as described previously (37). The dcuS-CFP fusion was constructed in the same way, whereas the ECFP gene was amplified from plasmid pECFP (Clontech) and dcuS-CFP was finally cloned from pMW386 via XbaI into pBAD18-Kan.…”