title: Complex two-component signaling regulates the general stress response in Alphaproteobacteria.
abstract: The general stress response (GSR) in Alphaproteobacteria was recently shown to be controlled by a partner-switching mechanism that is triggered by phosphorylation of the response regulator PhyR. Activation of PhyR ultimately results in release of the alternative extracytoplasmic function sigma factor σ(EcfG), which redirects transcription toward the GSR. Little is known about the signal transduction pathway(s) controlling PhyR phosphorylation. Here, we identified the single-domain response regulator (SDRR) SdrG and seven histidine kinases, PakA to PakG, belonging to the HWE/HisKA2 family as positive modulators of the GSR in Sphingomonas melonis Fr1. Phenotypic analyses, epistasis experiments, and in vitro phosphorylation assays indicate that Paks directly phosphorylate PhyR and SdrG, and that SdrG acts upstream of or in concert with PhyR, modulating its activity in a nonlinear pathway. Furthermore, we found that additional SDRRs negatively affect the GSR in a way that strictly requires PhyR and SdrG. Finally, analysis of GSR activation by thermal, osmotic, and oxidative stress indicates that Paks display different degrees of redundancy and that a specific kinase can sense multiple stresses, suggesting that the GSR senses a particular condition as a combination of, rather than individual, molecular cues. This study thus establishes the alphaproteobacterial GSR as a complex and interlinked network of two-component systems, in which multiple histidine kinases converge to PhyR, the phosphorylation of which is, in addition, subject to regulation by several SDRRs. Our finding that most HWE/HisKA2 kinases contribute to the GSR in S. melonis Fr1 opens the possibility that this notion might also be true for other Alphaproteobacteria.
authors.affiliation: Department of Biology, Institute of Microbiology, Eidgenössiche Technische Hochschule Zurich, 8093 Zurich, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Kaczmarczyk
authors.affiliation: Department of Biology, Institute of Microbiology, Eidgenössiche Technische Hochschule Zurich, 8093 Zurich, Switzerland.
authors.forename: Ramon
authors.initials: R
authors.lastname: Hochstrasser
authors.affiliation: Department of Biology, Institute of Microbiology, Eidgenössiche Technische Hochschule Zurich, 8093 Zurich, Switzerland.
authors.forename: Julia A
authors.initials: JA
authors.lastname: Vorholt
authors.affiliation: Department of Biology, Institute of Microbiology, Eidgenössiche Technische Hochschule Zurich, 8093 Zurich, Switzerland fanne@micro.biol.ethz.ch.
authors.forename: Anne
authors.initials: A
authors.lastname: Francez-Charlot
mesh_terms: D020561:Alphaproteobacteria / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D000431:Ethanol / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D000071677:Histidine Kinase; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics*; D012965:Sodium Chloride / Q000494:pharmacology; D020578:Sphingomonas / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D013312:Stress, Physiological / Q000235:genetics*; D013696:Temperature; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D007527:Isoenzymes; D012808:Sigma Factor; D000431:Ethanol; D012965:Sodium Chloride; D020122:tert-Butylhydroperoxide; D011494:Protein Kinases; D000071677:Histidine Kinase
keywords: ECF sigma factor; general stress response; histidine kinase; response regulator; two-component system