The general stress response regulatory network in the alphaproteobacterium sphingopyxis granuli tfa

Resum

Far from living in controlled laboratory conditions, bacteria continuously face challenging situations in natural environments. To improve their chances of survival, they adapt their physiology and metabolism, not only with specific responses, but also triggering the so-called General Stress Response (GSR). This is an unspecific response that prepares the cell to deal with a number of unrelated stresses. Usually, the alphaproteobacterial GSR is regulated by an ECF sigma factor named EcfG, a NepR anti-sigma factor and a PhyR response regulator, which sequesters NepR upon phosphorylation by stress-sensing histidine kinases. This titration of NepR releases EcfG to initiate the transcription of the GSR regulon. This work sheds light on the regulatory pathway that signals and activates the GSR in S. granuli, a species with the ability to use the organic solvent tetralin as carbon and energy source, and the first facultative anaerobe described within its genus. After inspecting the S. granuli genome, a catalogue of the sigma factors and the GSR regulators encoded in it was defined. Particularly, this species bears two paralogs of each of the GSR regulators. To address the specific role of each of them in the regulation and in the final transcriptional output of the response, a number of single and multiple mutants of these regulatory genes were constructed and their ability to activate the GSR and respond to stress were addressed. As a result, the sigma factor EcfG2 was identified as the main activator of the GSR. Also, a GSR direct regulon has been defined in this species by dRNA-seq, comprising 104 genes distributed in 79 operons. Furthermore, the two sigma factors involved in the transcriptional activation (EcfG1 and EcfG2) were purified and their relative contribution to the transcription of individual GSR target promoters was studied by setting up an in vitro transcription (IVT) system using the native S. granuli core RNA polymerase. Based on this IVT setup, and after the purification of the rest of GSR regulators (NepR1, NepR2, PhyR1 and PhyR2), the whole regulatory system could be reconstructed in vitro. The resulting data allowed to address the hierarchical role of these elements in the regulation and a specificity in the stress signaling by PhyR1 and PhyR2. Besides, this has provided evidence of the indirect negative feedback exerted by NepR2 by titrating PhyR1 and PhyR2 instead of the  factors, thus releasing NepR1 to perform the actual inhibition of EcfG1 and EcfG2, which supports a hypothesis that had only been speculated in previous literature. Apart from the study of the GSR signaling pathway, this work also provides evidence of the posttranscriptional control of this response, aimed at the main GSR regulator EcfG2, as an additional layer of regulation