scholarly journals The General Stress Response Is Conserved in Long-Term Soil-Persistent Strains of Escherichia coli

2016 ◽  
Vol 82 (15) ◽  
pp. 4628-4640 ◽  
Author(s):  
Yinka Somorin ◽  
Florence Abram ◽  
Fiona Brennan ◽  
Conor O'Byrne
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Stress Resistance ◽  
Sigma Factor ◽  
Content Type ◽  
General Stress Response ◽  
E Coli ◽  
General Stress ◽  
Stress Protection

ABSTRACTAlthoughEscherichia coliis generally considered to be predominantly a commensal of the gastrointestinal tract, a number of recent studies suggest that it is also capable of long-term survival and growth in environments outside the host. As the extraintestinal physical and chemical conditions are often different from those within the host, it is possible that distinct genetic adaptations may be required to enable this transition. Several studies have shown a trade-off between growth and stress resistance in nutrient-poor environments, with lesions in therpoSlocus, which encodes the stress sigma factor RpoS (σS). In this study, we investigated a unique collection of long-term soil-persistentE. coliisolates to determine whether the RpoS-controlled general stress response is altered during adaptation to a nutrient-poor extraintestinal environment. The sequence of therpoSlocus was found to be highly conserved in these isolates, and no nonsense or frameshift mutations were detected. Known RpoS-dependent phenotypes, including glycogen synthesis and γ-aminobutyrate production, were found to be conserved in all strains. All strains expressed the full-length RpoS protein, which was fully functional using the RpoS-dependent promoter reporter fusion PgadX::gfp. RpoS was shown to be essential for long-term soil survival ofE. coli, since mutants lackingrpoSlost viability rapidly in soil survival assays. Thus, despite some phenotypic heterogeneity, the soil-persistent strains all retained a fully functional RpoS-regulated general stress response, which we interpret to indicate that the stresses encountered in soil provide a strong selective pressure for maintaining stress resistance, despite limited nutrient availability.IMPORTANCEEscherichia colihas been, and continues to be, used as an important indicator species reflecting potential fecal contamination events in the environment. However, recent studies have questioned the validity of this, sinceE. colihas been found to be capable of long-term colonization of soils. This study investigated whether long-term soil-persistentE. colistrains have evolved altered stress resistance characteristics. In particular, the study investigated whether the main regulator of genes involved in stress protection, the sigma factor RpoS, has been altered in the soil-persistent strains. The results show that RpoS stress protection is fully conserved in soil-persistent strains ofE. coli. They also show that loss of therpoSgene dramatically reduces the ability of this organism to survive in a soil environment. Overall, the results indicate that soil represents a stressful environment forE. coli, and their survival in it requires that they deploy a full stress protection response.

scholarly journals A Mutant RNA Polymerase Activates the General Stress Response, Enabling Escherichia coli Adaptation to Late Prolonged Stationary Phase

mSphere ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Pabitra Nandy ◽  
Savita Chib ◽  
Aswin Seshasayee
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Stationary Phase ◽  
Sigma Factor ◽  
Slow Growth ◽  
Content Type ◽  
General Stress Response ◽  
E Coli ◽  
General Stress ◽  
Small Colony

ABSTRACT Escherichia coli populations undergo repeated replacement of parental genotypes with fitter variants deep in stationary phase. We isolated one such variant, which emerged after 3 weeks of maintaining an E. coli K-12 population in stationary phase. This variant displayed a small colony phenotype and slow growth and was able to outcompete its ancestor over a narrow time window in stationary phase. The variant also shows tolerance to beta-lactam antibiotics, though not previously exposed to the antibiotic. We show that an RpoC(A494V) mutation confers the slow growth and small colony phenotype on this variant. The ability of this mutation to confer a growth advantage in stationary phase depends on the availability of the stationary-phase sigma factor σS. The RpoC(A494V) mutation upregulates the σS regulon. As shown over 20 years ago, early in prolonged stationary phase, σS attenuation, but not complete loss of activity, confers a fitness advantage. Our study shows that later mutations enhance σS activity, either by mutating the gene for σS directly or via mutations such as RpoC(A494V). The balance between the activities of the housekeeping major sigma factor and σS sets up a trade-off between growth and stress tolerance, which is tuned repeatedly during prolonged stationary phase. IMPORTANCE An important general mechanism of a bacterium’s adaptation to its environment involves adjusting the balance between growing fast and tolerating stresses. One paradigm where this plays out is in prolonged stationary phase: early studies showed that attenuation, but not complete elimination, of the general stress response enables early adaptation of the bacterium E. coli to the conditions established about 10 days into stationary phase. We show here that this balance is not static and that it is tilted back in favor of the general stress response about 2 weeks later. This can be established by direct mutations in the master regulator of the general stress response or by mutations in the core RNA polymerase enzyme itself. These conditions can support the development of antibiotic tolerance although the bacterium is not exposed to the antibiotic. Further exploration of the growth-stress balance over the course of stationary phase will necessarily require a deeper understanding of the events in the extracellular milieu.

scholarly journals Fitness, Stress Resistance, and Extraintestinal Virulence in Escherichia coli

2013 ◽  
Vol 81 (8) ◽  
pp. 2733-2742 ◽  
Author(s):  
Alexandre Bleibtreu ◽  
Pierre-Alexis Gros ◽  
Cédric Laouénan ◽  
Olivier Clermont ◽  
Hervé Le Nagard ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Stress Response ◽  
Mouse Model ◽  
Stress Resistance ◽  
Maximum Growth ◽  
Competitive Fitness ◽  
Content Type ◽  
General Stress Response ◽  
E Coli

ABSTRACTThe extraintestinal virulence ofEscherichia coliis dependent on numerous virulence genes. However, there is growing evidence for a role of the metabolic properties and stress responses of strains in pathogenesis. We assessed the respective roles of these factors in strain virulence by developing phenotypic assays for measuringin vitroindividual and competitive fitness and the general stress response, which we applied to 82 commensal and extraintestinal pathogenicE. colistrains previously tested in a mouse model of sepsis. Individual fitness properties, in terms of maximum growth rates in various media (Luria-Bertani broth with and without iron chelator, minimal medium supplemented with gluconate, and human urine) and competitive fitness properties, estimated as the mean relative growth rate per generation in mixed cultures with a reference fluorescentE. colistrain, were highly diverse between strains. The activity of the main general stress response regulator, RpoS, as determined by iodine staining of the colonies, H2O2resistance, andrpoSsequencing, was also highly variable. No correlation between strain fitness and stress resistance and virulence in the mouse model was found, except that the maximum growth rate in urine was higher for virulent strains. Multivariate analysis showed that the number of virulence factors was the only independent factor explaining the virulence in mice. At the species level, growth capacity and stress resistance are heterogeneous properties that do not contribute significantly to the intrinsic virulence of the strains.

scholarly journals IraL Is an RssB Anti-adaptor That Stabilizes RpoS during Logarithmic Phase Growth in Escherichia coli and Shigella

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Andrew J. Hryckowian ◽  
Aurelia Battesti ◽  
Justin J. Lemke ◽  
Zachary C. Meyer ◽  
Rodney A. Welch
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Stress Response ◽  
Sigma Factor ◽  
Logarithmic Phase ◽  
Phase Growth ◽  
Content Type ◽  
General Stress Response ◽  
E Coli ◽  
K 12

ABSTRACTRpoS (σS), the general stress response sigma factor, directs the expression of genes under a variety of stressful conditions. Control of the cellular σSconcentration is critical for appropriately scaled σS-dependent gene expression. One way to maintain appropriate levels of σSis to regulate its stability. Indeed, σSdegradation is catalyzed by the ClpXP protease and the recognition of σSby ClpXP depends on the adaptor protein RssB. Three anti-adaptors (IraD, IraM, and IraP) exist inEscherichia coliK-12; each interacts with RssB andinhibitsRssBactivity under different stress conditions, thereby stabilizing σS. Unlike K-12, someE. coliisolates, including uropathogenicE. colistrain CFT073, show comparable cellular levels of σSduring the logarithmic and stationary growth phases, suggesting that there are differences in the regulation of σSlevels amongE. colistrains. Here, we describe IraL, an RssB anti-adaptor that stabilizes σSduring logarithmic phase growth in CFT073 and otherE. coliandShigellastrains. By immunoblot analyses, we show that IraL affects the levels and stability of σSduring logarithmic phase growth. By computational and PCR-based analyses, we reveal thatiraLis found in manyE. colipathotypes but not in laboratory-adapted strains. Finally, by bacterial two-hybrid and copurification analyses, we demonstrate that IraL interacts with RssB by a mechanism distinct from that used by other characterized anti-adaptors. We introduce a fourth RssB anti-adaptor found inE. colispecies and suggest that differences in the regulation of σSlevels may contribute to host and niche specificity in pathogenic and nonpathogenicE. colistrains.IMPORTANCEBacteria must cope with a variety of environmental conditions in order to survive. RpoS (σS), the general stress response sigma factor, directs the expression of many genes under stressful conditions in both pathogenic and nonpathogenicEscherichia colistrains. The regulation of σSlevels and activity allows appropriately scaled σS-dependent gene expression. Here, we describe IraL, an RssB anti-adaptor that, unlike previously described anti-adaptors, stabilizes σSduring the logarithmic growth phase in the absence of additional stress. We also demonstrate thatiraLis found in a large number ofE. coliandShigellaisolates. These data suggest that strains containingiraLare able to initiate σS-dependent gene expression under conditions under which strains withoutiraLcannot. Therefore, IraL-mediated σSstabilization may contribute to host and niche specificity inE. coli.

scholarly journals The Lumen of Human Intestinal Organoids Poses Greater Stress to Bacteria Compared to the Germ-Free Mouse Intestine: Escherichia coli Deficient in RpoS as a Colonization Probe

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Madeline R. Barron ◽  
Roberto J. Cieza ◽  
David R. Hill ◽  
Sha Huang ◽  
Veda K. Yadagiri ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Content Type ◽  
General Stress Response ◽  
E Coli ◽  
Germ Free ◽  
General Stress ◽  
Microbe Interactions ◽  
Mouse Intestine ◽  
Host Microbe Interactions

ABSTRACT Pluripotent stem-cell-derived human intestinal organoids (HIOs) are three-dimensional, multicellular structures that model a naive intestinal epithelium in an in vitro system. Several published reports have investigated the use of HIOs to study host-microbe interactions. We recently demonstrated that microinjection of the nonpathogenic 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 further characterize the environment present in the HIO lumen. We generated an isogenic mutant in the general stress response sigma factor RpoS and employed this mutant to compare challenges faced by a bacterium during colonization of the HIO lumen relative to the germ-free mouse intestine. We demonstrate that the loss of RpoS significantly decreases the ability of ECOR2 to colonize HIOs, although it does not prevent colonization of germ-free mice. These results indicate that the HIO lumen is a more restrictive environment to E. coli than the germ-free mouse intestine, thus increasing our understanding of the HIO model system as it pertains to studying the establishment of intestinal host-microbe symbioses. IMPORTANCE Technological advancements have driven and will continue to drive the adoption of organotypic 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 is more restrictive than 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.

scholarly journals Global Analysis of the General Stress Response ofBacillus subtilis

2001 ◽  
Vol 183 (19) ◽  
pp. 5617-5631 ◽  
Author(s):  
Anja Petersohn ◽  
Matthias Brigulla ◽  
Stefan Haas ◽  
Jörg D. Hoheisel ◽  
Uwe Völker ◽  
...  
Keyword(s):  
Stress Response ◽  
Stress Resistance ◽  
Sigma Factor ◽  
Global Analysis ◽  
Transcriptional Analysis ◽  
Ethanol Stress ◽  
General Stress Response ◽  
Multiple Stress ◽  
Stress Genes ◽  
General Stress

ABSTRACT Gene arrays containing all currently known open reading frames ofBacillus subtilis were used to examine the general stress response of Bacillus. By proteomics, transcriptional analysis, transposon mutagenesis, and consensus promoter-based screening, 75 genes had previously been described as ςB-dependent general stress genes. The present gene array-based analysis confirmed 62 of these already known general stress genes and detected 63 additional genes subject to control by the stress sigma factor ςB. At least 24 of these 125 ςB-dependent genes seemed to be subject to a second, ςB-independent stress induction mechanism. Therefore, this transcriptional profiling revealed almost four times as many regulon members as the proteomic approach, but failure of confirmation of all known members of the ςB regulon indicates that even this approach has not yet elucidated the entire regulon. Most of the ςB-dependent general stress proteins are probably located in the cytoplasm, but 25 contain at least one membrane-spanning domain, and at least 6 proteins appear to be secreted. The functions of most of the newly described genes are still unknown. However, their classification as ςB-dependent stress genes argues that their products most likely perform functions in stress management and help to provide the nongrowing cell with multiple stress resistance. A comprehensive screening program analyzing the multiple stress resistance of mutants with mutations in single stress genes is in progress. The first results of this program, showing the diminished salt resistance of yjbC and yjbD mutants compared to that of the wild type, are presented. Only a few new ςB-dependent proteins with already known functions were found, among them SodA, encoding a superoxide dismutase. In addition to analysis of the ςB-dependent general stress regulon, a comprehensive list of genes induced by heat, salt, or ethanol stress in a ςB-independent manner is presented. Perhaps the most interesting of the ςB-independent stress phenomena was the induction of the extracytoplasmic function sigma factor ςW and its entire regulon by salt shock.

scholarly journals Genome-Wide Analysis of the General Stress Response Network in Escherichia coli: σS-Dependent Genes, Promoters, and Sigma Factor Selectivity

2005 ◽  
Vol 187 (5) ◽  
pp. 1591-1603 ◽  
Author(s):  
Harald Weber ◽  
Tino Polen ◽  
Johanna Heuveling ◽  
Volker F. Wendisch ◽  
Regine Hengge
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Regulatory Network ◽  
Cell Physiology ◽  
Data Set ◽  
General Stress Response ◽  
General Stress ◽  
Genome Wide

ABSTRACT The σS (or RpoS) subunit of RNA polymerase is the master regulator of the general stress response in Escherichia coli. While nearly absent in rapidly growing cells, σS is strongly induced during entry into stationary phase and/or many other stress conditions and is essential for the expression of multiple stress resistances. Genome-wide expression profiling data presented here indicate that up to 10% of the E. coli genes are under direct or indirect control of σS and that σS should be considered a second vegetative sigma factor with a major impact not only on stress tolerance but on the entire cell physiology under nonoptimal growth conditions. This large data set allowed us to unequivocally identify a σS consensus promoter in silico. Moreover, our results suggest that σS-dependent genes represent a regulatory network with complex internal control (as exemplified by the acid resistance genes). This network also exhibits extensive regulatory overlaps with other global regulons (e.g., the cyclic AMP receptor protein regulon). In addition, the global regulatory protein Lrp was found to affect σS and/or σ70 selectivity of many promoters. These observations indicate that certain modules of the σS-dependent general stress response can be temporarily recruited by stress-specific regulons, which are controlled by other stress-responsive regulators that act together with σ70 RNA polymerase. Thus, not only the expression of genes within a regulatory network but also the architecture of the network itself can be subject to regulation.

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

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.

scholarly journals A mutant RNA polymerase activates the general stress response, enabling E. coli adaptation to late prolonged stationary phase

2020 ◽  
Author(s):  
Pabitra Nandy ◽  
Savita Chib ◽  
Aswin Seshasayee
Keyword(s):  
Stress Response ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Sigma Factor ◽  
Slow Growth ◽  
General Mechanism ◽  
General Stress Response ◽  
E Coli ◽  
General Stress ◽  
Small Colony

AbstractEscherichia coli populations undergo repeated replacement of parental genotypes with fitter variants deep in stationary phase. We isolated one such variant, which emerged after three weeks o of maintaining an E. coli K12 population in stationary phase. This variant displayed a small colony phenotype, slow growth and was able to outcompete its ancestor over a narrow time window in stationary phase. The variant also shows tolerance to beta-lactam antibiotics, though not previously exposed to the antibiotic. We show that an RpoC (A494V) mutation confers the slow growth and small colony phenotype to this variant. The ability of this mutation to confer a growth advantage in stationary phase depends on the availability of the stationary phase sigma factor σS. The RpoC (A494V) mutation up-regulates the σS regulon. As shown over 20 years ago, early in prolonged stationary phase, σS attenuation, but not complete loss of activity, confers a fitness advantage. Our study shows that later mutations enhance σS activity, either by mutating the gene for σS directly, or via mutations such as RpoC (A494V). The balance between the activities of the housekeeping major sigma factor and σS sets up a trade-off between growth and stress tolerance, which is tuned repeatedly during prolonged stationary phase.ImportanceAn important general mechanism of bacterial adaptation to its environment involves adjusting the balance between growing fast, and tolerating stresses. One paradigm where this plays out is in prolonged stationary phase: early studies showed that attenuation, but not complete elimination, of the general stress response enables early adaptation of the bacterium E. coli to the conditions established about 10 days into stationary phase. We show here that this balance is not static and that it is tilted back in favour of the general stress response about two weeks later. This can be established by direct mutations in the master regulator of the general stress response, or by mutations in the core RNA polymerase enzyme itself. These conditions can support the development of antibiotic tolerance though the bacterium is not exposed to the antibiotic. Further exploration of the growth-stress balance over the course of stationary phase will necessarily require a deeper understanding of the events in the extracellular milieu.

scholarly journals Proteorhodopsin Overproduction Enhances the Long-Term Viability of Escherichia coli

2019 ◽  
Vol 86 (1) ◽  
Author(s):  
Yizhi Song ◽  
Michaël L. Cartron ◽  
Philip J. Jackson ◽  
Paul A. Davison ◽  
Mark J. Dickman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Raman Spectroscopy ◽  
Cell Membrane ◽  
Single Cell ◽  
Marine Bacteria ◽  
Membrane Area ◽  
Content Type ◽  
E Coli ◽  
Wide Range

ABSTRACT Genes encoding the photoreactive protein proteorhodopsin (PR) have been found in a wide range of marine bacterial species, reflecting the significant contribution that PR makes to energy flux and carbon cycling in ocean ecosystems. PR can also confer advantages to enhance the ability of marine bacteria to survive periods of starvation. Here, we investigate the effect of heterologously produced PR on the viability of Escherichia coli. Quantitative mass spectrometry shows that E. coli, exogenously supplied with the retinal cofactor, assembles as many as 187,000 holo-PR molecules per cell, accounting for approximately 47% of the membrane area; even cells with no retinal synthesize ∼148,000 apo-PR molecules per cell. We show that populations of E. coli cells containing PR exhibit significantly extended viability over many weeks, and we use single-cell Raman spectroscopy (SCRS) to detect holo-PR in 9-month-old cells. SCRS shows that such cells, even incubated in the dark and therefore with inactive PR, maintain cellular levels of DNA and RNA and avoid deterioration of the cytoplasmic membrane, a likely basis for extended viability. The substantial proportion of the E. coli membrane required to accommodate high levels of PR likely fosters extensive intermolecular contacts, suggested to physically stabilize the cell membrane and impart a long-term benefit manifested as extended viability in the dark. We propose that marine bacteria could benefit similarly from a high PR content, with a stabilized cell membrane extending survival when those bacteria experience periods of severe nutrient or light limitation in the oceans. IMPORTANCE Proteorhodopsin (PR) is part of a diverse, abundant, and widespread superfamily of photoreactive proteins, the microbial rhodopsins. PR, a light-driven proton pump, enhances the ability of the marine bacterium Vibrio strain AND4 to survive and recover from periods of starvation, and heterologously produced PR extends the viability of nutrient-limited Shewanella oneidensis. We show that heterologously produced PR enhances the viability of E. coli cultures over long periods of several weeks and use single-cell Raman spectroscopy (SCRS) to detect PR in 9-month-old cells. We identify a densely packed and consequently stabilized cell membrane as the likely basis for extended viability. Similar considerations are suggested to apply to marine bacteria, for which high PR levels represent a significant investment in scarce metabolic resources. PR-stabilized cell membranes in marine bacteria are proposed to keep a population viable during extended periods of light or nutrient limitation, until conditions improve.

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