Listeria monocytogenes Requires the RsbX Protein To Prevent SigB Activation under Nonstressed Conditions

Abstract

The survival of microbial cells under changing environmental conditions requires an efficient reprogramming of transcription, often mediated by alternative sigma factors. The Gram-positive human pathogen Listeria monocytogenes senses and responds to environmental stress mainly through the alternative sigma factor σ B (SigB), which controls expression of the general stress response regulon. SigB activation is achieved through a complex series of phosphorylation/dephosphorylation events culminating in the release of SigB from its anti-sigma factor RsbW. At the top of the signal transduction pathway lies a large multi-protein complex known as the stressosome that is believed to act as a sensory hub for stresses. Following signal detection, stressosome proteins become phosphorylated. Resetting of the stressosome is hypothesized to be exerted by a putative phosphatase, RsbX, which presumably removes phosphate groups from stressosome proteins post-stress. We addressed the role of the RsbX protein in modulating the activity of the stressosome and consequently regulating SigB activity in L. monocytogenes . We show that RsbX is required to reduce SigB activation/levels under non-stress conditions and that it is required for appropriate SigB mediated stress-adaptation. A strain lacking RsbX displayed impaired motility and biofilm formation, but also an increased survival at low pH. Our results could suggest that absence of RsbX alter the multi-protein composition of the stressosome without dramatically affecting its phosphorylation status. Overall the data show that RsbX plays a critical role in modulating the signal transduction pathway by blocking SigB activation under non-stressed conditions. Importance Pathogenic bacteria need to sense and respond to stresses to survive harsh environments but also be able to turn off the response when no longer facing stress. Activity of the stress-sigma factor SigB in the human pathogen Listeria monocytogenes is controlled by a hierarchic system having a large stress-sensing multiprotein complex known as the stressosome at the top. Following stress exposure, proteins in the stressosome become phosphorylated leading to SigB activation. We have studied the role of a putative phosphatase, RsbX, which is hypothesized to dephosphorylate stressosome proteins. RsbX is not only critical to switch off the stress response post-stress but also to keep the activity of SigB low at non-stressed conditions to prevent unnecessary gene-expression and save energy.

Acid stress signals are integrated into the σB-dependent general stress response pathway via the stressosome in the food-borne pathogen Listeria monocytogenes

“…Further structural and genetic studies will be needed to fully elucidate the role phosphorylation in signal transduction. The role of the putative phosphatase RsbX in resetting the sensing–ready state of the stressosome has recently been suggested [ 56 ] but further work will be needed to elucidate the details of precisely how it interacts with the stressosome.…”

“…The RsbT kinase is responsible for phosphorylation of all residues in the RsbR1 and RsbS and the inactivation or deletion of this protein results on the impairment of the σ B activation in both L. monocytogenes and B. subtilis [41,42,[52][53][54]. Once the stress has dissipated or the bacterium has adequately responded to it, the stressosome is restored to the ground state by dephosphorylation of the core proteins, through the action of the serine phosphatase RsbX [52,[55][56][57][58].…”

The Vibrio vulnificus stressosome is dispensable in nutrient-rich media

“…In this work, we focused on the phenotypic characterization of a V. vulnificus stressosome mutant lacking the upstream module of the stressosome genetic locus. This includes the genes encoding the three proteins forming the stressosome (VvRsbR, VvRsbS and VvRsbT) and the phosphatase VvRsbX [51,52]. We decided to perform this characterization in the rich medium LBN, which is one of the most commonly used media in studies of the physiology of V. vulnificus.…”

In vitroevolution ofListeria monocytogenesreveals selective pressure for loss of SigB and AgrA function at different incubation temperatures

“…The RsbV-RsbW interaction favours the association of σ B with the RNA polymerase, to form the holoenzyme E σ B and consequently transcription of the GSR regulon. Once the stress conditions abate, or the cell has adequately responded to them, the stressosome is reset to a sensing-competent state through the action of the RsbX phosphatase, which re-establishes the appropriate phosphorylation state of RsbR and RsbS (Oliveira et al, 2021; Xia et al, 2016). All the proteins involved in this signal cascade are encoded by the sigB operon, which comprises rsbRSTUVW - sigB - rsbX (Ferreira et al, 2004).…”