Abstract
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 core GSR regulators (the response regulator PhyR, the anti-σ factor NepR and the σ factor EcfG) are duplicated in TFA, being PhyR1 and PhyR2, NepR1 and NepR2 and EcfG1 and EcfG2. Based on multiple genetic, phenotypical and biochemical evidences including in vitro transcription assays, we have assigned distinct functional features to each paralogue 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 σ factors upon activation of the GSR and it would modulate the GSR activity indirectly by titrating the PhyR regulators.