The functional differences between paralogous regulators define the control of the General Stress Response in Sphingopyxis granuli TFA

Mapping Intimacies ◽

10.1101/2021.10.15.464624 ◽

2021 ◽

Author(s):

Rubén de Dios ◽

Eduardo Santero ◽

Francisca Reyes-Ramírez

Keyword(s):

Stress Response ◽

In Vitro Transcription ◽

Integrative Model ◽

General Stress Response ◽

Adverse Conditions ◽

General Stress ◽

Functional Differences ◽

Functional Features ◽

Stress Signals

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 GSR triggered by TFA is driven by two extracytoplasmic function σ factors (ECFs), EcfG1 and EcfG2, whose functional differences have been addressed previously, being EcfG2 the main activator. Upstream in this cascade, NepR anti-s factors directly inhibit EcfG activity under non-stress conditions, whereas PhyR response regulators sequester the NepR elements upon stress sensing to relieve EcfG inhibition. These elements, which are essential mediators of the GSR regulation, are duplicated in TFA, being NepR1 and NepR2, and PhyR1 and PhyR2. Here, based on multiple genetic, phenotypical and biochemical evidences including in vitro transcription assays, we have assigned distinct functional features to each of these paralogs 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 s factors upon activation of the GSR and it would modulate the GSR activity indirectly by titrating the PhyR regulators.

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WrpA Is an Atypical Flavodoxin Family Protein under Regulatory Control of the Brucella abortus General Stress Response System

Journal of Bacteriology ◽

10.1128/jb.00982-15 ◽

2016 ◽

Vol 198 (8) ◽

pp. 1281-1293 ◽

Cited By ~ 11

Author(s):

Julien Herrou ◽

Daniel M. Czyż ◽

Jonathan W. Willett ◽

Hye-Sook Kim ◽

Gekleng Chhor ◽

...

Keyword(s):

Stress Response ◽

Mouse Model ◽

Binding Site ◽

Brucella Abortus ◽

Chronic Infection ◽

Content Type ◽

General Stress Response ◽

General Stress

ABSTRACTThe general stress response (GSR) system of the intracellular pathogenBrucella abortuscontrols the transcription of approximately 100 genes in response to a range of stress cues. The core genetic regulatory components of the GSR are required forB. abortussurvival under nonoptimal growth conditionsin vitroand for maintenance of chronic infection in anin vivomouse model. The functions of the majority of the genes in the GSR transcriptional regulon remain undefined.bab1_1070is among the most highly regulated genes in this regulon: its transcription is activated 20- to 30-fold by the GSR system under oxidative conditionsin vitro. We have solved crystal structures of Bab1_1070 and demonstrate that it forms a homotetrameric complex that resembles those of WrbA-type NADH:quinone oxidoreductases, which are members of the flavodoxin protein family. However,B. abortusWrbA-relatedprotein (WrpA) does not bind flavin cofactors with a high affinity and does not function as an NADH:quinone oxidoreductasein vitro. Soaking crystals with flavin mononucleotide (FMN) revealed a likely low-affinity binding site adjacent to the canonical WrbA flavin binding site. Deletion ofwrpA(ΔwrpA) does not compromise cell survival under acute oxidative stressin vitroor attenuate infection in cell-based or mouse models. However, a ΔwrpAstrain does elicit increased splenomegaly in a mouse model, suggesting that WrpA modulatesB. abortusinteraction with its mammalian host. Despite high structural homology with canonical WrbA proteins, we propose thatB. abortusWrpA represents a functionally distinct member of the diverse flavodoxin family.IMPORTANCEBrucella abortusis an etiological agent of brucellosis, which is among the most common zoonotic diseases worldwide. The general stress response (GSR) regulatory system ofB. abortuscontrols the transcription of approximately 100 genes and is required for maintenance of chronic infection in a murine model; the majority of GSR-regulated genes remain uncharacterized. We presentin vitroandin vivofunctional and structural analyses of WrpA, whose expression is strongly induced by GSR under oxidative conditions. Though WrpA is structurally related to NADH:quinone oxidoreductases, it does not bind redox cofactors in solution, nor does it exhibit oxidoreductase activityin vitro. However, WrpA does affect spleen inflammation in a murine infection model. Our data provide evidence that WrpA forms a new functional class of WrbA/flavodoxin family proteins.

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Loss of RpoS results in attenuated Escherichia coli colonization of human intestinal organoids and a competitive disadvantage within the germ-free mouse intestine

10.1101/2020.07.30.230003 ◽

2020 ◽

Author(s):

Madeline R. Barron ◽

Roberto J. Cieza ◽

David R. Hill ◽

Sha Huang ◽

Veda K. Yadagiri ◽

...

Keyword(s):

Escherichia Coli ◽

Stress Response ◽

General Stress Response ◽

E Coli ◽

Germ Free ◽

General Stress ◽

Intestinal Organoids ◽

Mouse Intestine

AbstractPluripotent stem-cell-derived human intestinal organoids (HIOs) are three-dimensional, multicellular structures that model a previously uncolonized, naïve intestinal epithelium in an in vitro system. We recently demonstrated that microinjection of the non-pathogenic Escherichia coli strain, ECOR2, into HIOs induced morphological and functional maturation of the HIO epithelium, including increased secretion of mucins and cationic antimicrobial peptides. In the current work, we use ECOR2 as a biological probe to investigate the bacterial response to colonization of the HIO lumen. In E. coli and other Gram-negative bacteria, adaptation to environmental stress is regulated by the general stress response sigma factor, RpoS. We generated an isogenic ∆rpoS ECOR2 mutant to compare challenges faced by a bacterium during colonization of the HIO lumen relative to the germ-free mouse intestine, which is currently the best available system for studying the initial establishment of bacterial populations within the gut. We demonstrate that loss of RpoS significantly decreases the ability of ECOR2 to colonize HIOs, though it does not prevent colonization of germ-free mice. Rather, the ∆rpoS ECOR2 exhibits a fitness defect in the germ-free mouse intestine only in the context of microbial competition. These results indicate that HIOs pose a differentially restrictive luminal environment to E. coli during colonization, thus increasing our understanding of the HIO model system as it pertains to studying the establishment of intestinal host-microbe symbioses.ImportanceTechnological advancements have and will continue to drive the adoption of organoid-based systems for investigating host-microbe interactions within the human intestinal ecosystem. Using E. coli deficient in the RpoS-mediated general stress response, we demonstrate that the type or severity of microbial stressors within the HIO lumen differ from those of the in vivo environment of the germ-free mouse gut. This study provides important insight into the nature of the HIO microenvironment from a microbiological standpoint.

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An Extracytoplasmic Function Sigma Factor Acts as a General Stress Response Regulator in Sinorhizobium meliloti

Journal of Bacteriology ◽

10.1128/jb.00175-07 ◽

2007 ◽

Vol 189 (11) ◽

pp. 4204-4216 ◽

Cited By ~ 96

Author(s):

Laurent Sauviac ◽

Heinui Philippe ◽

Kounthéa Phok ◽

Claude Bruand

Keyword(s):

Stress Response ◽

Stationary Phase ◽

Stress Responses ◽

Sigma Factor ◽

Sinorhizobium Meliloti ◽

In Planta ◽

General Stress Response ◽

Stress Response Genes ◽

General Stress

ABSTRACT Sinorhizobium meliloti genes transcriptionally up-regulated after heat stress, as well as upon entry into stationary phase, were identified by microarray analyses. Sixty stress response genes were thus found to be up-regulated under both conditions. One of them, rpoE2 (smc01506), encodes a putative extracytoplasmic function (ECF) sigma factor. We showed that this sigma factor controls its own transcription and is activated by various stress conditions, including heat and salt, as well as entry into stationary phase after either carbon or nitrogen starvation. We also present evidence that the product of the gene cotranscribed with rpoE2 negatively regulates RpoE2 activity, and we therefore propose that it plays the function of anti-sigma factor. By combining transcriptomic, bioinformatic, and quantitative reverse transcription-PCR analyses, we identified 44 RpoE2-controlled genes and predicted the number of RpoE2 targets to be higher. Strikingly, more than one-third of the 60 stress response genes identified in this study are RpoE2 targets. Interestingly, two genes encoding proteins with known functions in stress responses, namely, katC and rpoH2, as well as a second ECF-encoding gene, rpoE5, were found to be RpoE2 regulated. Altogether, these data suggest that RpoE2 is a major global regulator of the general stress response in S. meliloti. Despite these observations, and although this sigma factor is well conserved among alphaproteobacteria, no in vitro nor in planta phenotypic difference from the wild-type strain could be detected for rpoE2 mutants. This therefore suggests that other important actors in the general stress response have still to be identified in S. meliloti.

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