Copper plays essential roles in biology, but abnormal interactions are damaging. Reliable quantification of copper-protein interactions will underpin the molecular understanding of copper nutrition and toxicity. We have previously established two high affinity probes, Bathocuproine disulfonate (Bcs) and Bicinchoninate (Bca) anions, that are capable of in vitro quantification of Cu(i) binding with affinities from pico- to atto-molar concentrations. Quantitative probes are required for Cu(i) binding of lower affinity for proteins and peptides typically associated with neurodegenerative diseases. The present work evaluates two classic Fe(ii) ligands Ferene S (Fs) and Ferrozine (Fz) as quantitative probes for Cu(i). Both react with Cu(i) quantitatively to yield well-defined complex anions [Cu(I)(Fs)2](3-) (λmax = 484 nm, ε = 6700 cm(-1) M(-1)) and [Cu(I)(Fz)2](3-) (λmax = 470 nm, ε = 4320 cm(-1) M(-1)). These complexes are sensitive to aerial oxidation (E1/2∼ +0.36 V vs. SHE) and to substitution by other ligands (e.g., Cl(-), MeCN). However, they can be protected effectively under anaerobic conditions by suitable reductants and an excess of the free probe ligands. Formation constants β2 were determined by two approaches: direct metal ion titration and ligand competition. They provided estimates which differed by ∼3 orders of magnitude. The sources of these differences were examined carefully to consolidate the affinities of the two probes to a unified standard (10(15.1) M(-2) for Fz and 10(13.7) M(-2) for Fs). It is apparent that application of direct metal ion titrations to quantification of Cu(i) binding affinities is problematical and should be avoided. The four ligands Bcs, Bca, Fz and Fs in combination form a set of versatile probes for ligand competition experiments and are capable of detecting and differentiating an extended spectrum of Cu(i) binding affinities from nano- to atto-molar concentrations. Selected examples of quantification of weaker Cu(i) binding in proteins and peptides are provided, including that of an amyloid-β peptide.

Two-component systems are major signal transduction pathways, which consist of histidine kinases and response regulators that communicate through phosphorylation. Here, we highlight a distinct class of single-domain response regulators containing the PFXFATG[G/Y] motif that are activated by a mechanism distinct from the Y-T coupling described for prototypical receiver domains. We first solved the structures of inactive and active SdrG, a representative of the FAT GUY family, and then biochemically and genetically characterized variants in which residues in this motif were mutated. Our results support a model of activation mainly driven by a conserved lysine and reveal that the rotation of the threonine induces the reorganization of several aromatic residues in and around the PFXFATG[G/Y] motif to generate intermediates resembling those occurring during classical Y-T coupling. Overall, this helps define a new subfamily of response regulators that emerge as important players in physiological adaptation.

The general stress response (GSR) represents an important trait to survive in the environment by leading to multiple stress resistance. In alphaproteobacteria, the GSR is under the transcriptional control of the alternative sigma factor EcfG. Here we performed transcriptome analyses to investigate the genes controlled by EcfG of Sphingomonas melonis Fr1 and the plasticity of this regulation under stress conditions. We found that EcfG regulates genes for proteins that are typically associated with stress responses. Moreover, EcfG controls regulatory proteins, which likely fine-tune the GSR. Among these, we identified a novel negative GSR feedback regulator, termed NepR2, on the basis of gene reporter assays, phenotypic analyses, and biochemical assays. Transcriptional profiling of signaling components upstream of EcfG under complex stress conditions showed an overall congruence with EcfG-regulated genes. Interestingly however, we found that the GSR is transcriptionally linked to the regulation of motility and biofilm formation via the single domain response regulator SdrG and GSR-activating histidine kinases. Altogether, our findings indicate that the GSR in S. melonis Fr1 underlies a complex regulation to optimize resource allocation and resilience in stressful and changing environments.

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.