scholarly journals Regulation of the Cobalt/Nickel Efflux OperondmeRFin Agrobacterium tumefaciens and a Link between the Iron-Sensing Regulator RirA and Cobalt/Nickel Resistance

2016 ◽  
Vol 82 (15) ◽  
pp. 4732-4742 ◽  
Author(s):  
Thanittra Dokpikul ◽  
Paweena Chaoprasid ◽  
Kritsakorn Saninjuk ◽  
Sirin Sirirakphaisarn ◽  
Jaruwan Johnrod ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Iron Homeostasis ◽  
Regulatory Protein ◽  
Nickel Resistance ◽  
Mobility Shift ◽  
Potent Inducer ◽  
Content Type ◽  
Cobalt Nickel ◽  
Metal Efflux ◽  
Cobalt And Nickel

ABSTRACTTheAgrobacterium tumefaciensC58 genome harbors an operon containing thedmeR(Atu0890) anddmeF(Atu0891) genes, which encode a transcriptional regulatory protein belonging to the RcnR/CsoR family and a metal efflux protein belonging to the cation diffusion facilitator (CDF) family, respectively. ThedmeRFoperon is specifically induced by cobalt and nickel, with cobalt being the more potent inducer. Promoter-lacZtranscriptional fusion, an electrophoretic mobility shift assay, and DNase I footprinting assays revealed that DmeR repressesdmeRFtranscription through direct binding to the promoter region upstream ofdmeR. A strain lackingdmeFshowed increased accumulation of intracellular cobalt and nickel and exhibited hypersensitivity to these metals; however, this strain displayed full virulence, comparable to that of the wild-type strain, when infecting aNicotiana benthamianaplant model under the tested conditions. Cobalt, but not nickel, increased the expression of many iron-responsive genes and reduced the induction of the SoxR-regulated genesodBII. Furthermore, control of iron homeostasis via RirA is important for the ability ofA. tumefaciensto cope with cobalt and nickel toxicity.IMPORTANCEThe molecular mechanism of the regulation ofdmeRFtranscription by DmeR was demonstrated. This work provides evidence of a direct interaction of apo-DmeR with the corresponding DNA operator siteinvitro. The recognition site for apo-DmeR consists of 10-bp AT-rich inverted repeats separated by six C bases (5′-ATATAGTATACCCCCCTATAGTATAT-3′). Cobalt and nickel cause DmeR to dissociate from thedmeRFpromoter, which leads to expression of the metal efflux genedmeF. This work also revealed a connection between iron homeostasis and cobalt/nickel resistance inA. tumefaciens.

scholarly journals Involvement of PatE, a Prophage-Encoded AraC-Like Regulator, in the Transcriptional Activation of Acid Resistance Pathways of Enterohemorrhagic Escherichia coli Strain EDL933

2012 ◽  
Vol 78 (15) ◽  
pp. 5083-5092 ◽  
Author(s):  
Jennifer K. Bender ◽  
Judyta Praszkier ◽  
Matthew J. Wakefield ◽  
Kathryn Holt ◽  
Marija Tauschek ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Activation ◽  
Regulatory Protein ◽  
Acid Resistance ◽  
Enterohemorrhagic Escherichia Coli ◽  
Transcriptional Analysis ◽  
Mobility Shift ◽  
Content Type ◽  
Infectious Dose ◽  
Genes Encoding

ABSTRACTEnterohemorrhagicEscherichia coli(EHEC) O157:H7 is a lethal human intestinal pathogen that causes hemorrhagic colitis and the hemolytic-uremic syndrome. EHEC is transmitted by the fecal-oral route and has a lower infectious dose than most other enteric bacterial pathogens in that fewer than 100 CFU are able to cause disease. This low infectious dose has been attributed to the ability of EHEC to survive in the acidic environment of the human stomach.In silicoanalysis of the genome of EHEC O157:H7 strain EDL933 revealed a gene,patE, for a putative AraC-like regulatory protein within the prophage island, CP-933H. Transcriptional analysis inE. colishowed that the expression ofpatEis induced during stationary phase. Data from microarray assays demonstrated that PatE activates the transcription of genes encoding proteins of acid resistance pathways. In addition, PatE downregulated the expression of a number of genes encoding heat shock proteins and the type III secretion pathway of EDL933. Transcriptional analysis and electrophoretic mobility shift assays suggested that PatE also activates the transcription of the gene for the acid stress chaperonehdeAby binding to its promoter region. Finally, assays of acid tolerance showed that increasing the expression of PatE in EHEC greatly enhanced the ability of the bacteria to survive in different acidic environments. Together, these findings indicate that EHEC strain EDL933 carries a prophage-encoded regulatory system that contributes to acid resistance.

scholarly journals Transcriptome-Based Analysis of the Pantoea stewartii Quorum-Sensing Regulon and Identification of EsaR Direct Targets

2014 ◽  
Vol 80 (18) ◽  
pp. 5790-5800 ◽  
Author(s):  
Revathy Ramachandran ◽  
Alison Kernell Burke ◽  
Guy Cormier ◽  
Roderick V. Jensen ◽  
Ann M. Stevens
Keyword(s):  
Quorum Sensing ◽  
Cell Envelope ◽  
Regulatory Protein ◽  
Homoserine Lactone ◽  
Sequencing Analysis ◽  
Mobility Shift ◽  
Content Type ◽  
Proteomic Approach ◽  
Stewart's Wilt ◽  
Cell Densities

ABSTRACTPantoeastewartiisubsp.stewartiiis a proteobacterium that causes Stewart's wilt disease in corn plants. The bacteria form a biofilm in the xylem of infected plants and produce capsule that blocks water transport, eventually causing wilt. At low cell densities, the quorum-sensing (QS) regulatory protein EsaR is known to directly repress expression ofesaRitself as well as the genes for the capsular synthesis operon transcription regulator,rcsA, and a 2,5-diketogluconate reductase,dkgA. It simultaneously directly activates expression of genes for a putative small RNA,esaS, the glycerol utilization operon,glpFKX, and another transcriptional regulator,lrhA. At high bacterial cell densities, all of this regulation is relieved when EsaR binds an acylated homoserine lactone signal, which is synthesized constitutively over growth. QS-dependent gene expression is critical for the establishment of disease in the plant. However, the identity of the full set of genes controlled by EsaR/QS is unknown. A proteomic approach previously identified around 30 proteins in the QS regulon. In this study, a whole-transcriptome, next-generation sequencing analysis of rRNA-depleted RNA from QS-proficient and -deficientP. stewartiistrains was performed to identify additional targets of EsaR. EsaR-dependent transcriptional regulation of a subset of differentially expressed genes was confirmed by quantitative reverse transcription-PCR (qRT-PCR). Electrophoretic mobility shift assays demonstrated that EsaR directly bound 10 newly identified target promoters. Overall, the QS regulon ofP. stewartiiorchestrates three major physiological responses: capsule and cell envelope biosynthesis, surface motility and adhesion, and stress response.

scholarly journals GouR, a TetR Family Transcriptional Regulator, Coordinates the Biosynthesis and Export of Gougerotin in Streptomyces graminearus

2013 ◽  
Vol 80 (2) ◽  
pp. 714-722 ◽  
Author(s):  
Junhong Wei ◽  
Yuqing Tian ◽  
Guoqing Niu ◽  
Huarong Tan
Keyword(s):  
Biological Activities ◽  
Regulatory Protein ◽  
Specific Inhibitor ◽  
Biosynthetic Gene Cluster ◽  
Binding Activity ◽  
Major Facilitator Superfamily ◽  
Transcriptional Analysis ◽  
Mobility Shift ◽  
Content Type ◽  
Inhibitor Of Protein Synthesis

ABSTRACTGougerotin is a peptidyl nucleoside antibiotic. It functions as a specific inhibitor of protein synthesis by binding ribosomal peptidyl transferase and exhibits a broad spectrum of biological activities.gouR, situated in the gougerotin biosynthetic gene cluster, encodes a TetR family transcriptional regulatory protein. Gene disruption and genetic complementation revealed thatgouRplays an important role in the biosynthesis of gougerotin. Transcriptional analysis suggested that GouR represses the transcription of thegouL-to-gouBoperon consisting of 11 structural genes and activates the transcription of the major facilitator superfamily (MFS) transporter gene (gouM). Electrophoresis mobility shift assays (EMSAs) and DNase I footprinting experiments showed that GouR has specific DNA-binding activity for the promoter regions ofgouL,gouM, andgouR. Our data suggested that GouR modulates gougerotin production by coordinating its biosynthesis and export inStreptomyces graminearus.

scholarly journals Iron-Response Element Binding to Iron Regulatory Protein (IRP) 1 and 2 Are Indispensable for Adipose Tissue Browning

2020 ◽  
Vol 4 (Supplement_2) ◽  
pp. 1283-1283
Author(s):  
Mikyoung You ◽  
Soonkyu Chung
Keyword(s):  
Adipose Tissue ◽  
Iron Metabolism ◽  
Knockout Mice ◽  
Iron Homeostasis ◽  
Regulatory Protein ◽  
Mrna Translation ◽  
Intracellular Iron ◽  
Mobility Shift ◽  
Iron Regulatory Proteins ◽  
Tissue Browning

Abstract Objectives Intracellular iron homeostasis is tightly regulated in posttranscriptional levels via iron regulatory proteins (IRPs). IRPs bind to the iron-responsive elements (IREs), leading to either mRNA translation or stability. Our recent study demonstrated that iron metabolism is intimately linked with adipose tissue browning and thermogenic activation. However, the role of IRP/IRE interactions in the adipose tissue is poorly understood. We aim to characterize the IRP/IRE interactions in the adipose tissue in terms of depot-specificity and thermogenic potential. Methods To induce adipocyte browning, mice were administrated with beta-3 adrenoceptor agonist CL316243 (CL) for 5 days, and different depots of adipose tissue of epididymal (eWAT), inguinal (iWAT), brown (BAT), and liver were collected. Iron metabolism and thermogenesis were evaluated. To investigate the IRP/IRE binding, electrophoretic mobility shift assay (EMSA) was performed in the cytosolic using the fluorescence-labeled IRE (IR-IRE). To distinguish the IRE binding with IRP1 and 2, the cytosolic fraction from IRP1 and 2 knockout mice were used as positive controls. Results In a normal temperature, the constitutive IRP/IRE binding was found in the BAT, but not in the eWAT and iWAT. In response to CL treatment, iron content and transferrin receptor levels significantly increased in the WAT. Accordingly, the IRE/IRPs binding significantly increased in the CL-treated iWAT. Genetic deletion of IRP1 or 2 poses a marginal impact on constitutively active BAT development, suggesting IRP1 and 2 plays a compensatory role. Unlikely to BAT, the deletion of either IRP1 or 2 failed to induce WAT browning in the IRP1 and 2 knockout mice with CL stimulation. Consistently, both IRE binding to IRP1 and 2 were manifest in the CL treated iWAT, implicating that IRP1 and 2 plays a separate and synergistic function for WAT browning. Conclusions Our study defined the depot-specific iron regulatory metabolism in the adipose tissue using an innovative EMSA method. We demonstrated that, for the first time in our knowledge, IRE binding to both IRP1 and IRP2 is indispensable for the thermogenic activation of WAT, which is distinct from the iron regulatory mechanism found in the BAT. We propose that iron metabolism in the WAT is a novel determinant for WAT browning and thermogenic energy expenditure. Funding Sources None.

scholarly journals Mutations within themepAOperator Affect Binding of the MepR Regulatory Protein and Its Induction by MepA Substrates in Staphylococcus aureus

2015 ◽  
Vol 197 (6) ◽  
pp. 1104-1114 ◽  
Author(s):  
Bryan D. Schindler ◽  
Susan M. Seo ◽  
Ivan Birukou ◽  
Richard G. Brennan ◽  
Glenn W. Kaatz
Keyword(s):  
Staphylococcus Aureus ◽  
Binding Sites ◽  
Regulatory Protein ◽  
Titration Calorimetry ◽  
Multidrug Efflux ◽  
Mobility Shift ◽  
Content Type ◽  
Operator Mutant ◽  
Current Sequence

The expression ofmepA, encoding theStaphylococcus aureusMepA multidrug efflux protein, is repressed by the MarR homologue MepR. Repression occurs through binding of two MepR dimers to an operator with two homologous and closely approximated pseudopalindromic binding sites (site 1 [S1] and site 2 [S2]). MepR binding is impeded in the presence of pentamidine, a MepA substrate. The effects of variousmepAoperator mutations on MepR binding were determined using electrophoretic mobility shift assays and isothermal titration calorimetry, and anin vivoconfirmation of the effects observed was established for a fully palindromic operator mutant. Altering the S1-S2 spacing by 1 to 4 bp severely impaired S2 binding, likely due to a physical collision between adjacent MepR dimers. Extension of the spacing to 9 bp eliminated the S1 binding-mediated DNA allostery required for efficient S2 binding, consistent with positive cooperative binding of MepR dimers. Binding of a single dimer to S1 was maintained when S2 was disrupted, whereas disruption of S1 eliminated any significant binding to S2, also consistent with positive cooperativity. Palindromization of binding sites, especially S2, enhanced MepR affinity for themepAoperator and reduced MepA substrate-mediated MepR induction. As a result, the on-off equilibrium between MepR and its binding sites was shifted toward the on state, resulting in less free MepR being available for interaction with inducing ligand. The selective pressure(s) under whichmepAexpression is advantageous likely contributed to the accumulation of mutations in themepAoperator, resulting in the current sequence from which MepR is readily induced by MepA substrates.

High-Level Resistance to Cobalt and Nickel in Cuban Serratia marcescens Strains Isolated from Serpentine Deposits

2007 ◽  
Vol 20-21 ◽  
pp. 521-525
Author(s):  
Jeannette Marrero ◽  
Georg Auling ◽  
Orquidea Coto ◽  
Dietrich Nies
Keyword(s):  
Serratia Marcescens ◽  
Molecular Mechanisms ◽  
Regulatory Protein ◽  
Metal Resistance ◽  
Uptake System ◽  
Nickel Resistance ◽  
16S Rrna Analysis ◽  
E Coli ◽  
Flow Equilibrium ◽  
Cobalt And Nickel

A collection of highly nickel and cobalt-resistant enterobacteria were isolated from the Punta Gorda serpentine deposit (Moa, Cuba). The most nickel and cobalt resistant strain (termed C- 1) was assigned to Serratia marcescens by 16S rRNA analysis and DNA/DNA hybridization and the molecular mechanisms underlying its inducible cobalt and nickel resistance was investigated. Genes involved in metal resistance were identified by transposon mutagenesis followed by selection for Co- and Ni-sensitive derivatives. The transposon insertion causing the highest decrease in metal resistance was located in the ncrABC determinant. The three ORFs (ncrA, ncrB and ncrC) were cloned in E. coli. The predicted NcrA product was an NreB ortholog of the major facilitator protein superfamily and was central for Co/Ni resistance in S. marcescens strain C-1. NcrA also mediated metal resistance in E. coli and caused decreased accumulation of Co and Ni in this heterologous host. NcrB may be a regulatory protein. NcrC was a protein of the Ni–Co transport (NiCoT) protein family and necessary for full metal resistance in E. coli, but only when NcrA was also present. Without NcrA, NcrC caused a slight decrease in metal resistance and mediated increased accumulation of Ni and Co. As the cytoplasmic metal concentration can be assumed to be the result of a flow equilibrium of uptake and efflux processes, this interplay between metal uptake system NcrC and metal efflux system NcrA may contribute to nickel and cobalt resistance in this bacterium.

scholarly journals RegR Virulence Regulon of Rabbit-Specific Enteropathogenic Escherichia coli Strain E22

2013 ◽  
Vol 81 (4) ◽  
pp. 1078-1089 ◽  
Author(s):  
Yogitha N. Srikhanta ◽  
Dianna M. Hocking ◽  
Judyta Praszkier ◽  
Matthew J. Wakefield ◽  
Roy M. Robins-Browne ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Regulatory Protein ◽  
Bioinformatic Analysis ◽  
Coli Strain ◽  
Mobility Shift ◽  
Gene Encoding ◽  
Gene Target ◽  
Content Type ◽  
E Coli ◽  
Genes Encoding

ABSTRACTAraC-like regulators play a key role in the expression of virulence factors in enteric pathogens, such as enteropathogenicEscherichia coli(EPEC), enterotoxigenicE. coli, enteroaggregativeE. coli, andCitrobacter rodentium. Bioinformatic analysis of the genome of rabbit-specific EPEC (REPEC) strain E22 (O103:H2) revealed the presence of a gene encoding an AraC-like regulatory protein, RegR, which shares 71% identity to the global virulence regulator, RegA, ofC. rodentium. Microarray analysis demonstrated that RegR exerts 25- to 400-fold activation on transcription of several genes encoding putative virulence-associated factors, including a fimbrial operon (SEF14), a serine protease, and an autotransporter adhesin. These observations were confirmed by proteomic analysis of secreted and heat-extracted surface-associated proteins. The mechanism of RegR-mediated activation was investigated by using its most highly upregulated gene target,sefA. Transcriptional analyses and electrophoretic mobility shift assays showed that RegR activates the expression ofsefAby binding to a region upstream of thesefApromoter, thereby relieving gene silencing by the global regulatory protein H-NS. Moreover, RegR was found to contribute significantly to virulence in a rabbit infection experiment. Taken together, our findings indicate that RegR controls the expression of a series of accessory adhesins that significantly enhance the virulence of REPEC strain E22.

scholarly journals Chlamydia trachomatisAlters Iron-Regulatory Protein-1 Binding Capacity and Modulates Cellular Iron Homeostasis in HeLa-229 Cells

2009 ◽  
Vol 2009 ◽  
pp. 1-7 ◽  
Author(s):  
Harsh Vardhan ◽  
Apurb R. Bhengraj ◽  
Rajneesh Jha ◽  
Aruna Singh Mittal
Keyword(s):  
Iron Homeostasis ◽  
Binding Capacity ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Iron Regulatory Protein ◽  
Mobility Shift ◽  
Cellular Iron ◽  
Infected Cells ◽  
Mobility Shift Assay ◽  
Cellular Iron Homeostasis

Chlamydia trachomatis(CT) is the leading cause of diseases related to reproductive health and iron plays important role in chlamydial pathogenesis. Iron homeostasis in chlamydia-infected cells is not clear thus far. This study shows that expression of the transferrin receptor (TfR) is downregulated, whereas expression of the ferritin heavy chain is upregulated in CT-infected HeLa-229 cells. Expression of iron-regulatory protein (IRP)-1 predominates over IRP-2 in infected cells. In infected cells, attenuated binding activity of IRP-iron responsive elements (IREs) is observed using the electrophoretic mobility-shift assay. These results suggest that iron homeostasis is modulated in CT-infected HeLa cells at the interface of acquisition and commensal use of iron.

scholarly journals Diverse Genetic Regulon of the Virulence-Associated Transcriptional Regulator MucR in Brucella abortus 2308

2013 ◽  
Vol 81 (4) ◽  
pp. 1040-1051 ◽  
Author(s):  
Clayton C. Caswell ◽  
Ahmed E. M. Elhassanny ◽  
Emilie E. Planchin ◽  
Christelle M. Roux ◽  
Jenni N. Weeks-Gorospe ◽  
...  
Keyword(s):  
Brucella Abortus ◽  
Iron Homeostasis ◽  
Brucella Melitensis ◽  
Cell Envelope ◽  
Transcriptional Regulator ◽  
Growth Defect ◽  
Mobility Shift ◽  
Content Type ◽  
Polysaccharide Biosynthesis

ABSTRACTThe Ros-type regulator MucR is one of the few transcriptional regulators that have been linked to virulence inBrucella. Here, we show that aBrucella abortusin-framemucRdeletion strain exhibits a pronounced growth defect duringin vitrocultivation and, more importantly, that themucRmutant is attenuated in cultured macrophages and in mice. The genetic basis for the attenuation ofBrucella mucRmutants has not been defined previously, but in the present study the genes regulated by MucR inB. abortushave been elucidated using microarray analysis and real-time reverse transcription-PCR (RT-PCR). InB. abortus2308, MucR regulates a wide variety of genes whose products may function in establishing and maintaining cell envelope integrity, polysaccharide biosynthesis, iron homeostasis, genome plasticity, and transcriptional regulation. Particularly notable among the MucR-regulated genes identified isarsR6(nolR), which encodes a transcriptional regulator previously linked to virulence inBrucella melitensis16 M. Importantly, electrophoretic mobility shift assays (EMSAs) determined that a recombinant MucR protein binds directly to the promoter regions of several genes repressed by MucR (includingarsR6[nolR]), and inBrucella, as in other alphaproteobacteria, MucR binds to its own promoter to repress expression of the gene that encodes it. Overall, these studies have uncovered the diverse genetic regulon of MucR inBrucella, and in doing so this work has begun to define the MucR-controlled genetic circuitry whose misregulation contributes to the virulence defect ofBrucella mucRmutants.

scholarly journals Vibrio choleraeCsrA Directly RegulatesvarATo Increase Expression of the Three Nonredundant Csr Small RNAs

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Heidi A. Butz ◽  
Alexandra R. Mey ◽  
Ashley L. Ciosek ◽  
Shelley M. Payne
Keyword(s):  
Gene Expression ◽  
Vibrio Cholerae ◽  
Small Rnas ◽  
Feedback Loop ◽  
Rna Binding ◽  
Regulatory Protein ◽  
Fine Tuning ◽  
Mobility Shift ◽  
Content Type

ABSTRACTCsrA, an RNA-binding global regulator, is an essential protein inVibrio cholerae.V. choleraeCsrA is regulated by three small RNAs (sRNAs), namely, CsrB, CsrC, and CsrD, which act to sequester and antagonize the activity of CsrA. Although the sRNAs were considered to be largely redundant, we found that they differ in expression, half-life, and the ability to regulate CsrA. Further, we identified a feedback loop in the Csr system in which CsrA increases the synthesis of these antagonistic sRNAs. Because the Csr sRNAs are positively regulated by VarA, we determined the effects of CsrA on VarA levels. The level of VarA was reduced in acsrAmutant, and we found that CsrA directly bound tovarAmRNA in an electrophoretic mobility shift assayin vitroand in an CsrA-RNA immunoprecipitation assayin vivo. Thus,varAmRNA is anin vivo-verified direct target of CsrA inV. cholerae, and this is the first demonstration of CsrA directly binding to avarA/uvrY/gacAhomolog. Additionally, we demonstrated that avarAtranslational fusion was less active in acsrAmutant than in wild-typeV. cholerae, suggesting that CsrA enhancesvarAtranslation. We propose that this autoregulatory feedback loop, in which CsrA increases the production of the nonredundant Csr sRNAs by regulating the amount of VarA, provides a mechanism for fine-tuning the availability of CsrA and, thus, of its downstream targets.IMPORTANCEVibrio choleraeis a major human pathogen, causing epidemics and pandemics of cholera.V. choleraepersists in the aquatic environment, providing a constant source for human infection. Success in transitioning from the environment to the human host and back requires the bacterium to rapidly respond and to adjust its gene expression and metabolism to these two very different habitats. Our findings show that CsrA, an RNA-binding regulatory protein, plays a central role in regulating these transitions. CsrA activity is controlled by the antagonistic sRNAs CsrB, CsrC, and CsrD, and these sRNAs respond to changes in the availability of nutrients. CsrA autoregulates its own activity by controlling these sRNAs via their primary regulator VarA. Thus, the change in CsrA availability in response to nutrient availability allowsV. choleraeto alter gene expression in response to environmental cues.

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