Two paralogous EcfG σ factors hierarchically orchestrate the activation of the General Stress Response in Sphingopyxis granuli TFA

Abstract

two paralogous ecfG σ factors hierarchically orchestrate the activation of the General Stress Response in Sphingopyxis granuli tfA Rubén de Dios, elena Rivas-Marin, eduardo Santero & francisca Reyes-Ramírez *Under ever-changing environmental conditions, the General Stress Response (GSR) represents a lifesaver for bacteria in order to withstand hostile situations. in α-proteobacteria, the ecfG-type extracytoplasmic function (ecf) σ factors are the key activators of this response at the transcriptional level. in this work, we address the hierarchical function of the ecf σ factor paralogs EcfG1 and EcfG2 in triggering the GSR in Sphingopyxis granuli TFA and describe the role of EcfG2 as global switch of this response. In addition, we define a GSR regulon for TFA and use in vitro transcription analysis to study the relative contribution of each ecfG paralog to the expression of selected genes. We show that the features of each promoter ultimately dictate this contribution, though EcfG2 always produced more transcripts than EcfG1 regardless of the promoter. These first steps in the characterisation of the GSR in tfA suggest a tight regulation to orchestrate an adequate protective response in order to survive in conditions otherwise lethal.In an environment where conditions are constantly changing, bacteria need to adapt their physiology in order to increase their chances of survival. These adaptations are not only mediated by particular responses to certain stimuli, but also by global changes in the transcriptional profile. These changes allow bacteria to thrive in conditions that otherwise would be considered hostile habitats for them. Global transcriptional adaptations can be exerted by a number of mechanisms, all of them ending up in a regulator that allows or prevents the expression of certain genes 1 . One direct way to alter the transcription is the swapping of σ factors, small switchable subunits of the RNA polymerase (RNAP) whose main functions are the promoter recognition and the double strand unwinding to form the transcription bubble 2 . The general classification of σ factors comprises two main groups: the σ 70 and the σ 54 families.

Identification of two fnr genes and characterisation of their role in the anaerobic switch in Sphingopyxis granuli strain TFA

“…An obvious paralelism is observed in the Global Stress Response of this bacterium, controlled by two extracytoplasmic function sigma factors EcfGs, in which ecfG1 , whose expression is activated by the constitutively produced EcfG2, just plays an auxiliary role in modulating the global stress response 43 . In this case, a similar auxiliary role of FixK in the global Fnr-mediated activation control could be exerted by competing for Fnr target sites or by forming FnrN-FixK inefficient heterodimers, which would result in a baroque but functional way of auto-modulating Fnr activity under anaerobic conditions.…”

“…Those belonging to category 3 appear to be genes responding to an energy limitation stress due to anaerobic respiration in the WT strain, which is intensified in the absence of respiration in the WT in the absence of nitrate or in the mutants unable to respire nitrate. However, this response appears to be specific of anaerobiosis because these genes are not induced under other energy limiting conditions such as carbon starvation in aerobiosis, which induces the General Stress Response of this bacterium 43 . One possibility is that they respond to a high of NADH/NAD + ratio but the nature of the signal or the response mechanism remains uncharacterised.…”

Extracytoplasmic Function σ Factors as Tools for Coordinating Stress Responses

“…In most instances, with a few exceptions such as the FeclR-like systems and some others [12], they are autoregulated by a positive feedback loop dependent on the regulated ECF itself. An intermediate situation of this type of transcriptional regulation is found in the general stress response (GSR, a global response mediated by ECFs in Alphaproteobacteria that is discussed in detail below) regulatory system described for Sphingopyxis granuli TFA [74]. Here, upon activation of the GSR signaling pathway, the constitutively expressed ECF EcfG2 drives the transcription of the paralogous EcfG1, which is also able to control its own transcription.…”

“…The presence of EcfG paralogs has been studied in a few organisms [74,76,116,117], and much effort has been made trying to establish possible regulatory differences among them, either by transcriptomic studies or at the level of gene expression phenotype. In spite of this, there is no general consensus about possible specific functions so far.…”

“…However, transcriptomic analyses suggest that the majority of the GSR regulon in M. extorquens (490 genes) is transcribed by an additive contribution of different combinations of the six EcfG paralogs encoded in this species (EcfG1-6). In S. granuli TFA, with EcfG1 and EcfG2, differential functions between the two paralogs have been detected by in vitro transcription assays, showing that although they seem to share the same target genes, EcfG2 produces higher levels of transcription than EcfG1, regardless of the promoter, and seems more important to induce GSR [74]. However, ecfG1 transcription is strongly induced by EcfG2 under stress conditions, reaching sufficient protein levels to activate target genes by itself.…”