Interaction of lipoprotein QseG with sensor kinase QseE in the periplasm controls the phosphorylation state of the two-component system QseE/QseF in Escherichia coli

Abstract

Histidine kinase QseE and response regulator QseF compose a two-component system in Enterobacteriaceae. In Escherichia coli K-12 QseF activates transcription of glmY and of rpoE from Sigma 54-dependent promoters by binding to upstream activating sequences. Small RNA GlmY and RpoE (Sigma 24) are important regulators of cell envelope homeostasis. In pathogenic Enterobacteriaceae QseE/QseF are required for virulence. In enterohemorrhagic E. coli QseE was reported to sense the host hormone epinephrine and to regulate virulence genes post-transcriptionally through employment of GlmY. The qseEGF operon contains a third gene, qseG, which encodes a lipoprotein attached to the inner leaflet of the outer membrane. Here, we show that QseG is essential and limiting for activity of QseE/QseF in E. coli K-12. Metabolic 32P-labelling followed by pull-down demonstrates that phosphorylation of the receiver domain of QseF in vivo requires QseE as well as QseG. Accordingly, QseG acts upstream and through QseE/QseF by stimulating activity of kinase QseE. 32P-labelling also reveals an additional phosphorylation in the QseF C-terminus of unknown origin, presumably at threonine/serine residue(s). Pulldown and two-hybrid assays demonstrate interaction of QseG with the periplasmic loop of QseE. A mutational screen identifies the Ser58Asn exchange in the periplasmic loop of QseE, which decreases interaction with QseG and concomitantly lowers QseE/QseF activity, indicating that QseG activates QseE by interaction. Finally, epinephrine is shown to have a moderate impact on QseE activity in E. coli K-12. Epinephrine slightly stimulates QseF phosphorylation and thereby glmY transcription, but exclusively during stationary growth and this requires both, QseE and QseG. Our data reveal a three-component signaling system, in which the phosphorylation state of QseE/QseF is governed by interaction with lipoprotein QseG in response to a signal likely derived from the cell envelope.

Small RNA ‐binding protein RapZ mediates cell envelope precursor sensing and signaling in Escherichia coli

“…Complementation of the rapZ + strain with the plasmid encoding wild‐type QseF resulted in intermediate glmY’‐lacZ levels, whereas ~2‐fold lower activities were measured in the presence of the QseF‐D56A variant, which mimics non‐phosphorylated QseF (Fig C). The latter activities reflect the residual ability of non‐phosphorylated QseF to activate glmY expression when overproduced (Göpel & Görke, ). High glmY expression levels were obtained in the presence of the QseF‐D56E variant, which mimics phosphorylated QseF (Fig C).…”

“…Nonetheless, both domains of RapZ and proper oligomerization are required for undisturbed binding of QseE/QseF suggesting that tetrameric RapZ activates this TCS (Fig EV1). Recently, we have shown that QseE/QseF employ the lipoprotein QseG as third essential component (Göpel & Görke, ). Kinase QseE requires interaction with QseG in the periplasm for activity suggesting that a quaternary signaling complex involving RapZ must form to obtain a fully activated TCS (Fig ).…”

“…The EMBO Journal 39: e103848 | 2020 ª 2020 The Authors presence of the QseF-D56A variant, which mimics non-phosphorylated QseF ( Fig 3C). The latter activities reflect the residual ability of non-phosphorylated QseF to activate glmY expression when overproduced (Göpel & Görke, 2018). High glmY expression levels were obtained in the presence of the QseF-D56E variant, which mimics phosphorylated QseF ( Fig 3C).…”

Omnipresent Maxwell's demons orchestrate information management in living cells

“…In this system, energy involvement is more complex than with standard EPBs, in that instead of being an ATP‐binding protein, QseF needs to be phosphorylated by QseE in complex with outer membrane sensor QseG to activate its cognate promoters. However, we witness also an energy‐dependent recycling process in that QseE must be dephosphorylated by a second activity of the QseE protein (phosphatase) to start another round of control (Gopel and Gorke, ).…”

Feedback regulation of small RNA processing by the cleavage product

“…This mechanism may be in particular important when GlmY levels are low, e.g. when the QseE/QseG/QseF three-component system, which controls glmY transcription, is in the OFF state [34,35]. Under these conditions, accumulation of GlmZ* may limit further processing of full-length GlmZ thereby maintaining a robust GlmS basal level.…”