The functional differences between paralogous regulators define the control of the General Stress Response in Sphingopyxis granuli TFA

Sphingopyxis granuli TFA is a contaminant degrading alphaproteobacterium that responds to adverse conditions by inducing the General Stress Response (GSR), an adaptive response that controls the transcription of a variety of genes to overcome adverse conditions. The GSR triggered by TFA is driven by two extracytoplasmic function {sigma} factors (ECFs), EcfG1 and EcfG2, whose functional differences have been addressed previously, being EcfG2 the main activator. Upstream in this cascade, NepR anti-{sigma} factors directly inhibit EcfG activity under non-stress conditions, whereas PhyR response regulators sequester the NepR elements upon stress sensing to relieve EcfG inhibition. These elements, which are essential mediators of the GSR regulation, are duplicated in TFA, being NepR1 and NepR2, and PhyR1 and PhyR2. Here, based on multiple genetic, phenotypical and biochemical evidences including in vitro transcription assays, we have assigned distinct functional features to each of these paralogs and assessed their contribution to the GSR regulation, dictating its timing and the intensity. We show that different stress signals are differentially integrated into the GSR by PhyR1 and PhyR2, therefore producing different levels of GSR activation. We demonstrate in vitro that both NepR1 and NepR2 bind EcfG1 and EcfG2, although NepR1 produces a more stable interaction than NepR2. Conversely, NepR2 interacts with phosphorylated PhyR1 and PhyR2 more efficiently than NepR1. We propose an integrative model where NepR2 would play a dual negative role: it would directly inhibit the {sigma} factors upon activation of the GSR and it would modulate the GSR activity indirectly by titrating the PhyR regulators. IMPORTANCEIn Alphaproteobacteria, the General Stress Response (GSR) aims at protecting against a variety of stresses. Needing to integrate different signals, its modulation is capital to produce a proportionate response according to the environmental conditions. Individual alphaproteobacterial species have evolved distinct GSR cascades in which the information flow is usually straightforward to ascertain due to the presence of a single copy of at least one of its main regulators (PhyR, NepR and EcfG), restricting the regulatory possibilities. However, Sphingopyxis granuli TFA encodes two paralogs of each regulator, multiplying the possible regulatory interplays. We demonstrate that functional differences between paralogous GSR regulators allow an intrinsic feedback regulation in this pathway. We provide evidence of a NepR anti-{sigma} factor that exerts a dual negative feedback regulation on the GSR by interacting with the EcfG {sigma} factors and with the PhyR regulators. This would attune its output to the actual needs of the cell.