Eduardo A. Groisman
Published: May 11, 2020
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AbstractTo survive an environmental stress, organisms must detect the stress and mount an appropriate response. One way that bacteria do so is by phosphorelay systems that respond to a stress by activating a regulator that modifies gene expression. To ensure an appropriate response, a given regulator is typically activated solely by its cognate phosphorelay protein(s). However, we now report that the regulator RcsB is activated by both cognate and non-cognate phosphorelay proteins depending on the condition experienced by the bacterium Salmonella enterica serovar Typhimurium. The RcsC and RcsD proteins form a phosphorelay that activates their cognate regulator RcsB in response to outer membrane stress and cell wall perturbations, conditions Salmonella experiences during infection. Surprisingly, the non-cognate phosphorelay protein BarA activates RcsB during logarithmic growth in Luria-Bertani medium in three ways. That is, BarA’s cognate regulator SirA promotes transcription of the rcsDB operon; the SirA-dependent regulatory RNAs CsrB and CsrC further increase RcsB-activated gene transcription; and BarA activates RcsB independently of the RcsC, RcsD, and SirA proteins. Activation of a regulator by multiple sensors broadens the spectrum of environments in which a set of genes is expressed without evolving binding sites for different regulators at each of these genes.
The phosphorelay is a form of signal transduction used by organisms in all three domains of life. Typically, a phosphorelay consists of sensor proteins that respond to specific signals by activating a cognate regulatory protein that alters gene expression. Phosphorelays exhibit specificity towards their cognate regulators, thereby ensuring that any changes in gene expression help an organism cope with the experienced stress (and not to an unrelated stress). However, we now report that the regulator RcsB is activated by both cognate and non-cognate phosphorelay proteins in the bacterium Salmonella enterica serovar Typhimurium. The phosphorelay proteins RcsC and RcsD activate RcsB upon cell envelope perturbations whereas the non-cognate phosphorelay protein BarA activates RcsB during rapid growth in Luria-Bertani medium. Our findings establish that BarA controls gene expression via both its cognate regulator SirA and the non-cognate regulator RcsB. In addition, they demonstrate that RcsB controls gene expression in response to multiple signals detected by the RcsC, RcsD and BarA proteins.
Citation: Salvail H, Groisman EA (2020) The phosphorelay BarA/SirA activates the non-cognate regulator RcsB in Salmonella enterica. PLoS Genet 16(5):
https://doi.org/10.1371/journal.pgen.1008722Editor: Sean Crosson, Michigan State University, UNITED STATESReceived: February 12, 2020; Accepted: March 18, 2020; Published: May 11, 2020Copyright: © 2020 Salvail, Groisman. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Data Availability: All relevant data are within the manuscript and its Supporting Information files.Funding: This research was supported, in part, by National Institutes of Health grant AI120558 to E.A.G. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing interests: The authors have declared that no competing interests exist.
IntroductionSurvival in a hostile environment requires gene products that protect an organism from the particular stresses present in that environment. Phosphorelay systems allow bacteria to respond to environmental insults by activating a regulatory protein that alters the expression of a subset of genes [1, 2]. To ensure a specific response to a given stress condition, phosphorelay systems usually restrict activation to their respective cognate regulatory proteins [3–5]. However, we now report that a regulatory protein is activated both by cognate and non-cognate phosphorelay proteins depending on the stress experienced by the bacterium Salmonella enterica serovar Typhimurium.
Bacteria and cell wall-containing eukaryotes utilize two-component systems to alter behavior in response to changes in environmental or cellular conditions [6, 7]. The prototypical two-component system consists of a sensor that responds to a specific signal(s) by modifying the activity of a cognate regulator through phosphorylation. Phosphorelays are versions of two-component systems in which there is a three-step phosphotransfer, whereby the phosphoryl group is shuttled from the sensor kinase to the response regulator via an intermediary phosphotransferase protein or domain, as opposed to the single phosphotransfer event from a sensor to a regulator in classical two-component systems [8, 9].
One of the best-studied phosphorelays in the family Enterobacteriacae is Rcs, which consists of the RcsC, RcsD and RcsB proteins [10, 11] (Fig 1). RcsC and RcsD are inner membrane proteins, whereas RcsB is a cytoplasmic DNA binding regulator that alters expression of its target genes when bacteria experience outer membrane stress or cell wall perturbations. These stresses are believed to promote RcsC autophosphorylation at a histidine residue followed by phosphotransfer to an aspartate residue within RcsC, followed by phosphotransfer to a histidine residue in RcsD, and ending in phosphotransfer to an aspartate residue on RcsB. Phosphorylated RcsB activates or represses a subset of genes involved in numerous cellular processes, including motility, biofilm formation, and the general stress response [3, 12–14]. Surprisingly, full expression of RcsB-activated genes has been observed in mutants lacking either the rcsC or rcsD genes [15, 16], raising the possibility of RcsB being activated by signaling proteins other than RcsC and RcsD.
Fig 1. The phosphorelay BarA/SirA activates the non-cognate regulator RcsB.Upon polymyxin B-induced outer membrane damage or mecillinam-induced cell wall damage (left panel), the sensor kinase RcsC and the phosphotransferase RcsD activate their cognate response regulator RcsB, which then alters transcription of its target genes (e.g., rprA). The single-headed arrows for the RcsC/RcsD/RcsB phosphotransfer cascade indicate that upon outer membrane and cell wall damages, the phosphate flow is directed toward RcsB. Under these conditions, BarA is dispensable for RcsB activation. Upon rapid growth in LB media (right panel), BarA uses multiple mechanisms to activate RcsB. That is, BarA’s cognate regulator SirA promotes transcription of the rcsDB operon, increasing the amounts of the RcsD and RcsB proteins; the SirA-dependent regulatory RNAs CsrB and CsrC reduce RcsC expression, probably through titration of the CsrA protein, thus limiting RcsB dephosphorylation by RcsC and RcsD. BarA also activates RcsB independently of the RcsC, RcsD, and SirA proteins. The latter mechanism may entail direct phosphotransfer from BarA to RcsB and is depicted by a dashed arrow and a question mark. The double-headed arrows for the RcsC/RcsD/RcsB phosphotransfer cascade indicate that upon rapid growth in LB media, the phosphate flow may be reversed, resulting in the phosphate being taken away from RcsB by RcsD and RcsC. RcsF is an outer membrane lipoprotein that senses envelope stress. IgaA is a negative regulator of the RcsC/RcsD/RcsB phosphorelay.
https://doi.org/10.1371/journal.pgen.1008722.g001We now report that the sensor phosphorelay protein BarA activates the non-cognate regulator RcsB in a RcsC- and RcsD-independent manner. We establish that BarA utilizes several mechanisms to increase the amounts of active RcsB protein. And we identify conditions in which RcsB activation requires RcsC and RcsD but not BarA. Our findings indicate that a given regulatory protein can be activated by non-cognate phosphorelay proteins. This activation expands the spectrum of environments in which a given set of genes is expressed without an organism needing to evolve binding sites for different regulatory proteins at the regulatory region of each target gene. Because the proteins that constitute a given phosphorelay are often encoded by independently transcribed genes, they may be more prone to establish physiological interactions with “non-cognate” partners.
A condition that activates the regulator RcsB independently of the RcsC, RcsD and RcsF proteins
We hypothesized that RcsB is activated by a non-cognate sensor because RcsB-dependent genes are fully expressed in the absence of either the rcsC or rcsD genes [15, 16], and also because the possibility of RcsC phosphorylating RcsB in the absence of RcsD, or of RcsD phosphorylating RcsB in the absence of RcsC  seemed unlikely .
To test this hypothesis, we examined the fluorescence of isogenic Salmonella strains with mutations in various rcs genes and harboring a medium copy number plasmid with a transcriptional fusion between the RcsB-activated rprA promoter and a promoterless gfp gene. Although the RcsB protein can form heterodimers with several proteins [10, 17, 18], rprA transcription is carried out by RcsB homodimers , which are favored upon RcsB phosphorylation . Bacteria were grown in regular (i.e., NaCl-containing) Luria-Bertani (LB) media and in LB without NaCl as a means to decrease osmolarity.
Wild-type Salmonella fluoresced when incubated on LB agar plates lacking NaCl at 37˚C for 24 h (Fig 2). Fluorescence was 4-fold higher on regular (i.e., NaCl-containing) LB plates than in LB plates lacking NaCl. These results are in agreement with the notion that increased osmolarity activates the Rcs system [19, 20] (Fig 2). The rcsB mutant did not fluoresce on either plate (Fig 2), demonstrating that rprA transcription is fully dependent on RcsB under the two conditions [14, 15]. By contrast, the rcsC and rcsD single mutants exhibited wild-type fluorescence on NaCl-lacking LB plates (Fig 2). While displaying half of the wild-type strain fluorescence on LB plates, the fluorescence of the rcsC and rcsD single mutants was higher than that of the rcsB mutant (Fig 2). These results indicated that the RcsB-dependent rprA gene is still expressed in the absence of either RcsC or RcsD [15, 16].
Fig 2. The response regulator RcsB can be activated independently of the RcsC, RcsD and RcsF proteins.Fluorescence from wild-type (14028s), rcsB (EG12925), rcsC (HS1350), rcsD (HS1382), rcsC rcsD (HS1383) and rcsF (HS1326) Salmonella harboring plasmid pRprA-GFP (rprA-gfp) following 24 h of growth on LB solid medium without (-NaCl) or with (+NaCl) NaCl. Data are representative of three independent experiments, which gave similar results. Quantification of the fluorescence is provided on the right panel of the figure. Values derived from three independent experiments (mean ± standard deviation) were statistically analyzed by Prism 8 using two-tailed unpaired t test. Statistical significance is indicated by * P