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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.

SUBMITTER: Gottschlich L

PROVIDER: S-EPMC5898713 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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

Gottschlich Lisa L Bortfeld-Miller Miriam M Gäbelein Christoph C Dintner Sebastian S Vorholt Julia A JA

PLoS genetics 20180413 4

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. Whi ...[more]

PMID: 29652885

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Project description:BACKGROUND: Dinoroseobacter shibae, a member of the Roseobacter clade abundant in marine environments, maintains morphological heterogeneity throughout growth, with small cells dividing by binary fission and large cells dividing by budding from one or both cell poles. This morphological heterogeneity is lost if the quorum sensing (QS) system is silenced, concurrent with a decreased expression of the CtrA phosphorelay, a regulatory system conserved in Alphaproteobacteria and the master regulator of the Caulobacter crescentus cell cycle. It consists of the sensor histidine kinase CckA, the phosphotransferase ChpT and the transcriptional regulator CtrA. Here we tested if the QS induced differentiation of D. shibae is mediated by the CtrA phosphorelay. RESULTS: Mutants for ctrA, chpT and cckA showed almost homogeneous cell morphology and divided by binary fission. For ctrA and chpT, expression in trans on a plasmid caused the fraction of cells containing more than two chromosome equivalents to increase above wild-type level, indicating that gene copy number directly controls chromosome number. Transcriptome analysis revealed that CtrA is a master regulator for flagellar biosynthesis and has a great influence on the transition to stationary phase. Interestingly, the expression of the autoinducer synthase genes luxI2 and luxI3 was strongly reduced in all three mutants, resulting in loss of biosynthesis of acylated homoserine-lactones with C14 side-chain, but could be restored by expressing these genes in trans. Several phylogenetic clusters of Alphaproteobacteria revealed a CtrA binding site in the promoters of QS genes, including Roseobacters and Rhizobia. CONCLUSIONS: The CtrA phosphorelay induces differentiation of a marine Roseobacter strain that is strikingly different from that of C. crescentus. Instead of a tightly regulated cell cycle and a switch between two morphotypes, the morphology and cell division of Dinoroseobacter shibae are highly heterogeneous. We discovered for the first time that the CtrA phosphorelay controls the biosynthesis of signaling molecules. Thus cell-cell communication and differentiation are interlinked in this organism. This may be a common strategy, since we found a similar genetic set-up in other species in the ecologically relevant group of Alphaproteobacteria. D. shibae will be a valuable model organism to study bacterial differentiation into pleomorphic cells.

Dataset Information

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

Publications

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

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