Phosphorelay through the bifunctional phosphotransferase PhyT controls the general stress response in an alphaproteobacterium

Abstract

Two-component systems constitute phosphotransfer signaling pathways and enable adaptation to environmental changes, an essential feature for bacterial survival. The general stress response (GSR) in the plant-protecting alphaproteobacterium Sphingomonas melonis Fr1 involves a two-component system consisting of multiple stress-sensing histidine kinases (Paks) and the response regulator PhyR; PhyR in turn regulates the alternative sigma factor EcfG, which controls expression of the GSR regulon. While Paks had been shown to phosphorylate PhyR in vitro, it remained unclear if and under which conditions direct phosphorylation happens in the cell, as Paks also phosphorylate the single domain response regulator SdrG, an essential yet enigmatic component of the GSR signaling pathway. Here, we analyze the role of SdrG and investigate an alternative function of the membrane-bound PhyP (here re-designated PhyT), previously assumed to act as a PhyR phosphatase. In vitro assays show that PhyT transfers a phosphoryl group from SdrG to PhyR via phosphoryl transfer on a conserved His residue. This finding, as well as complementary GSR reporter assays, indicate the participation of SdrG and PhyT in a Pak-SdrG-PhyT-PhyR phosphorelay. Furthermore, we demonstrate complex formation between PhyT and PhyR. This finding is substantiated by PhyT-dependent membrane association of PhyR in unstressed cells, while the response regulator is released from the membrane upon stress induction. Our data support a model in which PhyT sequesters PhyR, thereby favoring Pak-dependent phosphorylation of SdrG. In addition, PhyT assumes the role of the SdrG-phosphotransferase to activate PhyR. Our results place SdrG into the GSR signaling cascade and uncover a dual role of PhyT in the GSR.

Two paralogous EcfG σ factors hierarchically orchestrate the activation of the General Stress Response in Sphingopyxis granuli TFA

“…Several GSR regulons have been defined in diverse α-proteobacterial species, including C. crescentus 15 , Sinorhizobium. meliloti 9 , M. extorquens 26 , Bradyrhizobium japonicum 27 , R. etli 28 and Sphingomonas melonis 13 , and a common feature in all of them is the high abundance of target genes with unknown function, a situation also described here for TFA (only 37% of the GSR-dependent genes have a predicted function). Among the genes directly regulated by the GSR, some obvious targets could be found, such as most of the regulatory elements of the response (the nepR2ecfG1 operon, phyR1 and phyR2, and the HWE family histidine kinase SGRAN_1165 located next to phyR2), as described in the literature 4 .…”

“…the genes with annotated function are the stress inducible csbD (SGRAN_3063), two katA catalases (SGRAN_1770 and SGRAN_2521) 4 , the ecnAB toxin-antitoxin system 29 (SGRAN_1922), an mscS mechanosensitive ion channel 30 (SGRAN_0895), an osmC peroxiredoxin 31 (SGRAN_3924), an RND efflux system component 15 (SGRAN_4134) and the ku-ligD non-homologous DNA repair system (SGRAN_4136 and SGRAN_4135) 32 , some of them recently found GSR-dependent in the sphingomonad S. melonis 13 . In addition, a large representation of envelope related elements also appear in this regulon (e.g., SGRAN_1221, SGRAN_1797, SGRAN_1798, SGRAN_2253, SGRAN_2364, SGRAN_2492 or SGRAN_2662) 4 .…”

Regulation of theErythrobacter litoralisDSM 8509 general stress response by visible light

“…However, the major effect of deleting or overexpressing genes encoding LOV-HWE kinases appears to be dysregulation of the general stress response (GSR) system (Foreman et al, 2012, Kim et al, 2014, which determines cell survival across a range of stress conditions . There is increasing evidence that multiple HWE/HisKA2-family kinases function as part of complex regulatory networks that regulate the GSR in Alphaproteobacteria by influencing the phosphorylation state of the anti-anti-σ factor, PhyR (Kaczmarczyk et al, 2014, Gottschlich et al, 2018, Lori et al, 2018, Correa et al, 2013. Phospho-PhyR activates an extracytoplasmic function (ECF) σ factor -EcfG -by binding and sequestering its anti-σ factor, NepR (Figure 1).…”

Regulation of the Erythrobacter litoralis DSM 8509 general stress response by visible light

“…However, the major effect of deleting or overexpressing genes encoding LOV‐HWE kinases appears to be dysregulation of the general stress response (GSR) system (Foreman et al , ; Kim et al , ), which determines cell survival across a range of stress conditions (Fiebig et al , ; Francez‐Charlot et al , ). There is increasing evidence that multiple HWE/HisKA2‐family kinases function as part of complex regulatory networks that regulate the GSR in Alphaproteobacteria by influencing the phosphorylation state of the anti‐anti‐σ factor, PhyR (Correa et al , ; Kaczmarczyk et al , ; Gottschlich et al , ; Lori et al , ). Phospho‐PhyR activates an extracytoplasmic function (ECF) σ factor – EcfG – by binding and sequestering its anti‐σ factor, NepR (Francez‐Charlot et al , ) (Fig.…”