Regulation of Iron Storage by CsrA Supports Exponential Growth of Escherichia coli

ABSTRACT The global regulatory protein CsrA coordinates gene expression in response to physiological cues reflecting cellular stress and nutrition. CsrA binding to the 5′ segments of mRNA targets affects their translation, RNA stability, and/or transcript elongation. Recent studies identified probable mRNA targets of CsrA that are involved in iron uptake and storage in Escherichia coli, suggesting an unexplored role for CsrA in regulating iron homeostasis. Here, we assessed the impact of CsrA on iron-related gene expression, cellular iron, and growth under various iron levels. We investigated five new targets of CsrA regulation, including the genes for 4 ferritin or ferritin-like iron storage proteins (ISPs) and the stress-inducible Fe-S repair protein, SufA. CsrA bound with high affinity and specificity to ftnB, bfr, and dps mRNAs and inhibited their translation, while it modestly activated ftnA expression. Furthermore, CsrA was found to regulate cellular iron levels and support growth by repressing the expression of genes for ISPs, most importantly, ferritin B (FtnB) and bacterioferritin (Bfr). Iron starvation did not substantially affect cellular levels of CsrA or its small RNA (sRNA) antagonists, CsrB and CsrC. csrA disruption led to increased resistance to the lethal effects of H2O2 during exponential growth, consistent with a regulatory role in oxidative stress resistance. We propose that during exponential growth and under minimal stress, CsrA represses the deleterious expression of the ISPs that function under oxidative stress and stationary-phase conditions (FtnB, Bfr, and Dps), thus ensuring that cellular iron is available to processes that are required for growth. IMPORTANCE Iron is an essential micronutrient for nearly all living organisms but is toxic in excess. Consequently, the maintenance of iron homeostasis is a critical biological process, and the genes involved in this function are tightly regulated. Here, we explored a new role for the bacterial RNA binding protein CsrA in the regulation of iron homeostasis. CsrA was shown to be a key regulator of iron storage genes in Escherichia coli, with consequential effects on cellular iron levels and growth. Our findings establish a model in which robust CsrA activity during the exponential phase of growth leads to repression of genes whose products sequester iron or divert it to unnecessary stress response processes. In so doing, CsrA supports E. coli growth under iron-limiting laboratory conditions and may promote fitness in the competitive iron-limited environment of the host large intestine.

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The Irr and RirA Proteins Participate in a Complex Regulatory Circuit and Act in Concert To Modulate Bacterioferritin Expression in Ensifer meliloti 1021

Applied and Environmental Microbiology ◽

10.1128/aem.00895-17 ◽

2017 ◽

Vol 83 (16) ◽

Cited By ~ 5

Author(s):

Daniela Costa ◽

Vanesa Amarelle ◽

Claudio Valverde ◽

Mark R. O'Brian ◽

Elena Fabiano

Keyword(s):

Gene Expression ◽

Small Rna ◽

Iron Homeostasis ◽

Sufficient Conditions ◽

Iron Storage ◽

Content Type ◽

Reading Frame ◽

Regulatory Circuit ◽

Ensifer Meliloti ◽

Rhizobial Species

ABSTRACT In this work we found that the bfr gene of the rhizobial species Ensifer meliloti, encoding a bacterioferritin iron storage protein, is involved in iron homeostasis and the oxidative stress response. This gene is located downstream of and overlapping the smc03787 open reading frame (ORF). No well-predicted RirA or Irr boxes were found in the region immediately upstream of the bfr gene although two presumptive RirA boxes and one presumptive Irr box were present in the putative promoter of smc03787. We demonstrate that bfr gene expression is enhanced under iron-sufficient conditions and that Irr and RirA modulate this expression. The pattern of bfr gene expression as well as the response to Irr and RirA is inversely correlated to that of smc03787. Moreover, our results suggest that the small RNA SmelC759 participates in RirA- and Irr-mediated regulation of bfr expression and that additional unknown factors are involved in iron-dependent regulation. IMPORTANCE E. meliloti belongs to the Alphaproteobacteria, a group of bacteria that includes several species able to associate with eukaryotic hosts, from mammals to plants, in a symbiotic or pathogenic manner. Regulation of iron homeostasis in this group of bacteria differs from that found in the well-studied Gammaproteobacteria. In this work we analyzed the effect of rirA and irr mutations on bfr gene expression. We demonstrate the effect of an irr mutation on iron homeostasis in this bacterial genus. Moreover, results obtained indicate a complex regulatory circuit where multiple regulators, including RirA, Irr, the small RNA SmelC759, and still unknown factors, act in concert to balance bfr gene expression.

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Cell Envelope Perturbation Induces Oxidative Stress and Changes in Iron Homeostasis in Vibrio cholerae

Journal of Bacteriology ◽

10.1128/jb.00092-09 ◽

2009 ◽

Vol 191 (17) ◽

pp. 5398-5408 ◽

Cited By ~ 33

Author(s):

Aleksandra E. Sikora ◽

Sinem Beyhan ◽

Michael Bagdasarian ◽

Fitnat H. Yildiz ◽

Maria Sandkvist

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Outer Membrane ◽

Vibrio Cholerae ◽

Iron Homeostasis ◽

Cell Envelope ◽

Membrane Integrity ◽

Genetic Alterations ◽

Iron Storage ◽

Study Gene Expression

ABSTRACT The Vibrio cholerae type II secretion (T2S) machinery is a multiprotein complex that spans the cell envelope. When the T2S system is inactivated, cholera toxin and other exoproteins accumulate in the periplasmic compartment. Additionally, loss of secretion via the T2S system leads to a reduced growth rate, compromised outer membrane integrity, and induction of the extracytoplasmic stress factor RpoE (A. E. Sikora, S. R. Lybarger, and M. Sandkvist, J. Bacteriol. 189:8484-8495, 2007). In this study, gene expression profiling reveals that inactivation of the T2S system alters the expression of genes encoding cell envelope components and proteins involved in central metabolism, chemotaxis, motility, oxidative stress, and iron storage and acquisition. Consistent with the gene expression data, molecular and biochemical analyses indicate that the T2S mutants suffer from internal oxidative stress and increased levels of intracellular ferrous iron. By using a tolA mutant of V. cholerae that shares a similar compromised membrane phenotype but maintains a functional T2S machinery, we show that the formation of radical oxygen species, induction of oxidative stress, and changes in iron physiology are likely general responses to cell envelope damage and are not unique to T2S mutants. Finally, we demonstrate that disruption of the V. cholerae cell envelope by chemical treatment with polymyxin B similarly results in induction of the RpoE-mediated stress response, increased sensitivity to oxidants, and a change in iron metabolism. We propose that many types of extracytoplasmic stresses, caused either by genetic alterations of outer membrane constituents or by chemical or physical damage to the cell envelope, induce common signaling pathways that ultimately lead to internal oxidative stress and misregulation of iron homeostasis.

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Overexpression offetA(ybbL) andfetB(ybbM), Encoding an Iron Exporter, Enhances Resistance to Oxidative Stress in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.02322-13 ◽

2013 ◽

Vol 79 (23) ◽

pp. 7210-7219 ◽

Cited By ~ 20

Author(s):

Sergios A. Nicolaou ◽

Alan G. Fast ◽

Eiko Nakamaru-Ogiso ◽

Eleftherios T. Papoutsakis

Keyword(s):

Oxidative Stress ◽

Escherichia Coli ◽

Iron Overload ◽

Parent Strain ◽

Iron Homeostasis ◽

Intracellular Iron ◽

Paramagnetic Resonance ◽

Content Type ◽

Genomic Libraries ◽

Metal Transporter

ABSTRACTReactive oxygen species are generated by redox reactions and the Fenton reaction of H2O2and iron that generates the hydroxyl radical that causes severe DNA, protein, and lipid damage. We screenedEscherichia coligenomic libraries to identify a fragment, containingcueR,ybbJ,qmcA,ybbL, andybbM, which enhanced resistance to H2O2stress. We report that the ΔybbLand ΔybbMstrains are more susceptible to H2O2stress than the parent strain and thatybbLandybbMoverexpression overcomes H2O2sensitivity. TheybbLandybbMgenes are predicted to code for an ATP-binding cassette metal transporter, and we demonstrate that YbbM is a membrane protein. We investigated various metals to identify iron as the likely substrate of this transporter. We propose the gene namesfetAandfetB(for Fe transport) and the gene product names FetA and FetB. FetAB allows for increased resistance to oxidative stress in the presence of iron, revealing a role in iron homeostasis. We show that iron overload coupled with H2O2stress is abrogated byfetAandfetBoverexpression in the parent strain and in the Δfurstrain, where iron uptake is deregulated. Furthermore, we utilized whole-cell electron paramagnetic resonance to show that intracellular iron levels in the Δfurstrain are decreased by 37% byfetAandfetBoverexpression. Combined, these findings show thatfetAandfetBencode an iron exporter that has a role in enhancing resistance to H2O2-mediated oxidative stress and can minimize oxidative stress under conditions of iron overload and suggest that FetAB facilitates iron homeostasis to decrease oxidative stress.

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Assessment of Insertion Sequence Mobilization as an Adaptive Response to Oxidative Stress in Acinetobacter baumannii Using IS-seq

Journal of Bacteriology ◽

10.1128/jb.00833-16 ◽

2017 ◽

Vol 199 (9) ◽

Cited By ~ 11

Author(s):

Meredith S. Wright ◽

Stephanie Mountain ◽

Karen Beeri ◽

Mark D. Adams

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Hydrogen Peroxide ◽

Acinetobacter Baumannii ◽

Insertion Sequence ◽

Iron Homeostasis ◽

Bacterial Species ◽

Growth Conditions ◽

Content Type ◽

Is Elements

ABSTRACT Insertion sequence (IS) elements are found throughout bacterial genomes and contribute to genome variation by interrupting genes or altering gene expression. Few of the more than 30 IS elements described in Acinetobacter baumannii have been characterized for transposition activity or expression effects. A targeted sequencing method, IS-seq, was developed to efficiently map the locations of new insertion events in A. baumannii genomes and was used to identify novel IS sites following growth in the presence of hydrogen peroxide, which causes oxidative stress. Serial subculture in the presence of subinhibitory concentrations of hydrogen peroxide led to rapid selection of cells carrying an ISAba1 element upstream of the catalase-peroxidase gene katG. Several additional sites for the elements ISAba1, ISAba13, ISAba25, ISAba26, and ISAba125 were found at low abundance after serial subculture, indicating that each element is active and contributes to genetic variation that may be subject to selection. Following hydrogen peroxide exposure, rapid changes in gene expression were observed in genes related to iron homeostasis. The IS insertions adjacent to katG resulted in more than 20-fold overexpression of the gene and increased hydrogen peroxide tolerance. IMPORTANCE Insertion sequences (IS) contribute to genomic and phenotypic variation in many bacterial species, but little is known about how transposition rates vary among elements or how selective pressure influences this process. A new method for identifying new insertion locations that arise under experimental growth conditions in the genome, termed IS-seq, was developed and tested with cells grown in the presence of hydrogen peroxide, which causes oxidative stress. Gene expression changes in response to hydrogen peroxide exposure are similar to those observed in other species and include genes that control free iron concentrations. New IS insertions adjacent to a gene encoding a catalase enzyme confirm that IS elements can rapidly contribute to adaptive variation in the presence of selection.

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IscR Regulates Synthesis of Colonization Factor Antigen I Fimbriae in Response to Iron Starvation in Enterotoxigenic Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00214-15 ◽

2015 ◽

Vol 197 (18) ◽

pp. 2896-2907 ◽

Cited By ~ 23

Author(s):

Sara Haines ◽

Nadège Arnaud-Barbe ◽

David Poncet ◽

Sylvie Reverchon ◽

Julien Wawrzyniak ◽

...

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Virulence Factors ◽

Promoter Region ◽

Promoter Activity ◽

Enterotoxigenic Escherichia Coli ◽

Iron Availability ◽

Iron Starvation ◽

Content Type ◽

The Impact

ABSTRACTIron availability functions as an environmental cue for enteropathogenic bacteria, signaling arrival within the human host. As enterotoxigenicEscherichia coli(ETEC) is a major cause of human diarrhea, the effect of iron on ETEC virulence factors was evaluated here. ETEC pathogenicity is directly linked to production of fimbrial colonization factors and secretion of heat-labile enterotoxin (LT) and/or heat-stable enterotoxin (ST). Efficient colonization of the small intestine further requires at least the flagellin binding adhesin EtpA. Under iron starvation, production of the CFA/I fimbriae was increased in the ETEC H10407 prototype strain. In contrast, LT secretion was inhibited. Furthermore, under iron starvation, gene expression of thecfa(CFA/I) andetp(EtpBAC) operons was induced, whereas transcription of toxin genes was either unchanged or repressed. Transcriptional reporter fusion experiments focusing on thecfaoperon further showed that iron starvation stimulatedcfaApromoter activity in ETEC, indicating that the impact of iron on CFA/I production was mediated by transcriptional regulation. Evaluation ofcfaApromoter activity in heterologousE. colisingle mutant knockout strains identified IscR as the regulator responsible for inducingcfafimbrial gene expression in response to iron starvation, and this was confirmed in an ETEC ΔiscRstrain. The global iron response regulator, Fur, was not implicated. IscR binding sites were identifiedin silicowithin thecfaApromoter and fixation confirmed by DNase I footprinting, indicating that IscR directly binds the promoter region to induce CFA/I.IMPORTANCEPathogenic enterobacteria modulate expression of virulence genes in response to iron availability. Although the Fur transcription factor represents the global regulator of iron homeostasis inEscherichia coli, we show that several ETEC virulence factors are modulated by iron, with expression of the major fimbriae under the control of the iron-sulfur cluster regulator, IscR. Furthermore, we demonstrate that the apo form of IscR, lacking an Fe-S cluster, is able to directly fix the corresponding promoter region. These results provide further evidence implicating IscR in bacterial virulence and suggest that IscR may represent a more general regulator mediating the iron response in enteropathogens.

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Impact of Nutritional Factors on the Proteome of Intestinal Escherichia coli: Induction of OxyR-Dependent Proteins AhpF and Dps by a Lactose-Rich Diet

Applied and Environmental Microbiology ◽

10.1128/aem.00244-12 ◽

2012 ◽

Vol 78 (10) ◽

pp. 3580-3591 ◽

Cited By ~ 15

Author(s):

Monique Rothe ◽

Carl Alpert ◽

Wolfram Engst ◽

Stephanie Musiol ◽

Gunnar Loh ◽

...

Keyword(s):

Oxidative Stress ◽

Escherichia Coli ◽

Osmotic Stress ◽

Stress Responses ◽

Luciferase Reporter ◽

Nutritional Factors ◽

Content Type ◽

E Coli ◽

K 12 ◽

The Impact

ABSTRACTTo study the impact of nutritional factors on protein expression of intestinal bacteria, gnotobiotic mice monoassociated withEscherichia coliK-12 were fed three different diets: a diet rich in starch, a diet rich in nondigestible lactose, and a diet rich in casein. Two-dimensional gel electrophoresis and electrospray-tandem mass spectrometry were used to identify differentially expressed proteins of bacteria recovered from small intestine and cecum. Oxidative stress response proteins such as AhpF, Dps, and Fur, all of which belong to the oxyR regulon, were upregulated inE. coliisolates from mice fed the lactose-rich diet. Luciferase reporter gene assays demonstrated that osmotic stress caused by carbohydrates led to the expression ofahpCFanddps, which was not observed in anE. coliΔoxyRmutant. Growth ofahpCFandoxyRdeletion mutants was strongly impaired when nondigestible sucrose was present in the medium. The wild-type phenotype could be restored by complementation of the deletions with plasmids containing the corresponding genes and promoters. The results indicate that some OxyR-dependent proteins play a major role in the adaptation ofE. colito osmotic stress. We conclude that there is an overlap of osmotic and oxidative stress responses. Mice fed the lactose-rich diet possibly had a higher intestinal osmolality, leading to the upregulation of OxyR-dependent proteins, which enable intestinalE. colito better cope with diet-induced osmotic stress.

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The Small RNA RyhB Contributes to Siderophore Production and Virulence of Uropathogenic Escherichia coli

Infection and Immunity ◽

10.1128/iai.02287-14 ◽

2014 ◽

Vol 82 (12) ◽

pp. 5056-5068 ◽

Cited By ~ 34

Author(s):

Gaëlle Porcheron ◽

Rima Habib ◽

Sébastien Houle ◽

Mélissa Caza ◽

François Lépine ◽

...

Keyword(s):

Oxidative Stress ◽

Escherichia Coli ◽

Urinary Tract ◽

Type Strain ◽

Iron Homeostasis ◽

Wild Type Strain ◽

Iron Acquisition ◽

Wild Type ◽

Content Type ◽

E Coli

ABSTRACTInEscherichia coli, the small regulatory noncoding RNA (sRNA) RyhB and the global ferric uptake regulator (Fur) mediate iron acquisition and storage control. Iron is both essential and potentially toxic for most living organisms, making the precise maintenance of iron homeostasis necessary for survival. While the roles of these regulators in iron homeostasis have been well studied in a nonpathogenicE. colistrain, their impact on the production of virulence-associated factors is still unknown for a pathogenicE. colistrain. We thus investigated the roles of RyhB and Fur in iron homeostasis and virulence of the uropathogenicE. coli(UPEC) strain CFT073. In a murine model of urinary tract infection (UTI), deletion offuralone did not attenuate virulence, whereas a ΔryhBmutant and a ΔfurΔryhBdouble mutant showed significantly reduced bladder colonization. The Δfurmutant was more sensitive to oxidative stress and produced more of the siderophores enterobactin, salmochelins, and aerobactin than the wild-type strain. In contrast, while RyhB was not implicated in oxidative stress resistance, the ΔryhBmutant produced lower levels of siderophores. This decrease was correlated with the downregulation ofshiA(encoding a transporter of shikimate, a precursor of enterobactin and salmochelin biosynthesis) andiucD(involved in aerobactin biosynthesis) in this mutant grown in minimal medium or in human urine.iucDwas also downregulated in bladders infected with the ΔryhBmutant compared to those infected with the wild-type strain. Our results thus demonstrate that the sRNA RyhB is involved in production of iron acquisition systems and colonization of the urinary tract by pathogenicE. coli.

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Death by Cystine: an Adverse Emergent Property from a Beneficial Series of Reactions

Journal of Bacteriology ◽

10.1128/jb.00546-15 ◽

2015 ◽

Vol 197 (23) ◽

pp. 3626-3628 ◽

Cited By ~ 4

Author(s):

Larry Reitzer

Keyword(s):

Oxidative Stress ◽

Escherichia Coli ◽

Sulfur Metabolism ◽

Emergent Property ◽

Futile Cycle ◽

Content Type ◽

Link Type

In this issue of theJournal of Bacteriology, Chonoles Imlay et al. (K. R. Chonoles Imlay, S. Korshunov, and J. A. Imlay, J Bacteriol 197:3629–3644, 2015,http://dx.doi.org/10.1128/JB.00277-15) show that oxidative stress kills sulfur-restrictedEscherichia coligrown with sublethal H2O2when challenged with cystine. Killing requires rapid and seemingly unregulated cystine transport and equally rapid cystine reduction to cysteine. Cysteine export completes an energy-depleting futile cycle. Each reaction of the cycle could be beneficial. Together, a cystine-mediated vulnerability emerges during the transition from a sulfur-restricted to a sulfur-replete environment, perhaps because of complexities of sulfur metabolism.

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Pathovar Transcriptomes

mSystems ◽

10.1128/msystems.00049-17 ◽

2017 ◽

Vol 2 (4) ◽

Cited By ~ 1

Author(s):

Amy Platenkamp ◽

Jay L. Mellies

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Regulatory Networks ◽

Virulence Gene ◽

Model System ◽

Genomic Sequences ◽

Bacterial Strains ◽

Content Type ◽

Virulence Gene Expression ◽

Link Type

ABSTRACT Archetypal pathogenic bacterial strains are often used to elucidate regulatory networks of an entire pathovar, which encompasses multiple lineages and phylogroups. With enteropathogenic Escherichia coli (EPEC) as a model system, Hazen and colleagues (mSystems 6:e00024-17, 2017, https://doi.org/10.1128/mSystems.00024-17 ) used 9 isolates representing 8 lineages and 3 phylogroups to find that isolates with similar genomic sequences exhibit similarities in global transcriptomes under conditions of growth in medium that induces virulence gene expression, and they found variation among individual isolates. Archetypal pathogenic bacterial strains are often used to elucidate regulatory networks of an entire pathovar, which encompasses multiple lineages and phylogroups. With enteropathogenic Escherichia coli (EPEC) as a model system, Hazen and colleagues (mSystems 6:e00024-17, 2017, https://doi.org/10.1128/mSystems.00024-17 ) used 9 isolates representing 8 lineages and 3 phylogroups to find that isolates with similar genomic sequences exhibit similarities in global transcriptomes under conditions of growth in medium that induces virulence gene expression. They also found variation among individual isolates. Their work illustrates the importance of moving beyond observing regulatory phenomena of a limited number of regulons in a few archetypal strains, with the possibility of correlating clinical symptoms to key transcriptional pathways across lineages and phylogroups.

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