scholarly journals Dual Repression by Fe2+-Fur and Mn2+-MntR of the mntH Gene, Encoding an NRAMP-Like Mn2+ Transporter in Escherichia coli

2001 ◽  
Vol 183 (16) ◽  
pp. 4806-4813 ◽  
Author(s):  
Silke I. Patzer ◽  
Klaus Hantke
Keyword(s):  
Escherichia Coli ◽  
Consensus Sequence ◽  
Transcriptional Regulator ◽  
Binding Region ◽  
Gene Encoding ◽  
Lower Affinity ◽  
E Coli ◽  
Metal Transporter ◽  
Dnase I Footprinting

ABSTRACT The uptake of Mn2+, a cofactor for several enzymes inEscherichia coli, is mediated by MntH, a proton-dependent metal transporter, which also recognizes Fe2+ with lower affinity. MntH belongs to the NRAMP family of eukaryotic Fe2+ and Mn2+ transporters. In E. coli strains with chromosomal mntH-lacZ fusions,mntH was partially repressed by both Mn2+ and Fe2+. Inactivation of fur resulted in the loss of Fe2+-dependent repression of mntHtranscription, demonstrating that Fe2+ repression depends on the global iron regulator Fur. However, these furmutants still showed Mn2+-dependent repression ofmntH. The Mn2+-responsive transcriptional regulator of mntH was identified as the gene product ofo155 (renamed MntR). mntR mutants were impaired in Mn2+ but not Fe2+ repression ofmntH transcription. Binding of purified MntR to themntH operator was manganese dependent. The binding region was localized by DNase I footprinting analysis and covers a nearly perfect palindrome. The Fur binding site, localized within 22 nucleotides of the mntH operator by in vivo operator titration assays, resembles the Fur-box consensus sequence.

A novel peroxiredoxin of the plant Sedum lineare is a homologue of Escherichia coli bacterioferritin co-migratory protein (Bcp)

2000 ◽  
Vol 351 (1) ◽  
pp. 107-114 ◽  
Author(s):  
Wei KONG ◽  
Susumu SHIOTA ◽  
Yixin SHI ◽  
Hiroaki NAKAYAMA ◽  
Koji NAKAYAMA
Keyword(s):  
Escherichia Coli ◽  
Peroxidase Activity ◽  
Discrete Group ◽  
Sequence Data ◽  
Accession Number ◽  
Gene Encoding ◽  
Reaction Cycle ◽  
Mutant Proteins ◽  
E Coli

We cloned a gene encoding a 17-kDa protein from a cDNA library of the plant Sedum lineare and found that its deduced amino acid sequence showed similarities to those of Escherichia coli bacterioferritin co-migratory protein (Bcp) and its homologues, which comprise a discrete group associated with the peroxiredoxin (Prx) family. Studies of the recombinant 17-kDa protein produced in E. coli cells revealed that it actually had a thioredoxin-dependent peroxidase activity, the hallmark of the Prx family. PrxQ, as we now designate the 17-kDa protein, had two cysteine residues (Cys-44 and Cys-49) well conserved among proteins of the Bcp group. These two cysteines were demonstrated to be essential for the thioredoxin-dependent peroxidase activity by analysis of mutant proteins, suggesting that these residues are involved in the formation of an intramolecular disulphide bond as an intermediate in the reaction cycle. Expression of PrxQ suppressed the hypersensitivity of an E. coli bcp mutant to peroxides, indicating that it might exert an antioxidant activity in vivo. The sequence data presented have been deposited in the GenBank/EMBL/DDBJ nucleotide sequence databases under the accession number AB037598.

scholarly journals In Vivo Transduction with Shiga Toxin 1-Encoding Phage

1998 ◽  
Vol 66 (9) ◽  
pp. 4496-4498 ◽  
Author(s):  
David W. K. Acheson ◽  
Joachim Reidl ◽  
Xiaoping Zhang ◽  
Gerald T. Keusch ◽  
John J. Mekalanos ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gastrointestinal Tract ◽  
Shiga Toxin ◽  
Recipient Strain ◽  
Gene Encoding ◽  
E Coli ◽  
A Subunit

ABSTRACT To facilitate the study of intestinal transmission of the Shiga toxin 1 (Stx1)-converting phage H-19B, Tn10d-blamutagenesis of an Escherichia coli H-19B lysogen was undertaken. Two mutants containing insertions in the gene encoding the A subunit of Stx1 were isolated. The resultant ampicillin-resistantE. coli strains lysogenic for these phages produced infectious H-19B particles but not active toxin. These lysogens were capable of transducing an E. coli recipient strain in the murine gastrointestinal tract, thereby demonstrating that lysogens of Shiga toxin-converting phages give rise to infectious virions within the host gastrointestinal tract.

scholarly journals Evidence for an Arginine Exporter Encoded by yggA (argO) That Is Regulated by the LysR-Type Transcriptional Regulator ArgP in Escherichia coli

2004 ◽  
Vol 186 (11) ◽  
pp. 3539-3546 ◽  
Author(s):  
Madhusudan R. Nandineni ◽  
J. Gowrishankar
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Control ◽  
Transcriptional Regulator ◽  
Basic Amino Acid ◽  
Missense Mutations ◽  
Reading Frame ◽  
E Coli ◽  
Regulator Protein ◽  
Outward Transport

ABSTRACT The anonymous open reading frame yggA of Escherichia coli was identified in this study as a gene that is under the transcriptional control of argP (previously called iciA), which encodes a LysR-type transcriptional regulator protein. Strains with null mutations in either yggA or argP were supersensitive to the arginine analog canavanine, and yggA-lac expression in vivo exhibited argP+ -dependent induction by arginine. Lysine supplementation phenocopied the argP null mutation in that it virtually abolished yggA expression, even in the argP+ strain. The dipeptides arginylalanine and lysylalanine behaved much like arginine and lysine, respectively, to induce and to turn off yggA transcription. Dominant missense mutations in argP (argPd ) that conferred canavanine resistance and rendered yggA-lac expression constitutive were obtained. The protein deduced to be encoded by yggA shares similarity with a basic amino acid exporter (LysE) of Corynebacterium glutamicum, and we obtained evidence for increased arginine efflux from E. coli strains with either the argPd mutation or multicopy yggA+ . The null yggA mutation abolished the increased arginine efflux from the argPd strain. Our results suggest that yggA encodes an ArgP-regulated arginine exporter, and we have accordingly renamed it argO (for “arginine outward transport”). We propose that the physiological function of argO may be either to prevent the accumulation to toxic levels of canavanine (which is a plant-derived antimetabolite) or arginine or to maintain an appropriate balance between the intracellular lysine and arginine concentrations.

scholarly journals Growth Phase and Growth Rate Regulation of therapA Gene, Encoding the RNA Polymerase-Associated Protein RapA in Escherichia coli

2001 ◽  
Vol 183 (20) ◽  
pp. 6126-6134 ◽  
Author(s):  
Julio E. Cabrera ◽  
Ding Jun Jin
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Rna Polymerase ◽  
Growth Phase ◽  
Gene Encoding ◽  
Rate Regulation ◽  
E Coli ◽  
Growth Rate Control

ABSTRACT The Escherichia coli rapA gene encodes the RNA polymerase (RNAP)-associated protein RapA, which is a bacterial member of the SWI/SNF helicase-like protein family. We have studied therapA promoter and its regulation in vivo and determined the interaction between RNAP and the promoter in vitro. We have found that the expression of rapA is growth phase dependent, peaking at the early log phase. The growth phase control ofrapA is determined at least by one particular feature of the promoter: it uses CTP as the transcription-initiating nucleotide instead of a purine, which is used for most E. colipromoters. We also found that the rapA promoter is subject to growth rate regulation in vivo and that it forms intrinsic unstable initiation complexes with RNAP in vitro. Furthermore, we have shown that a GC-rich or discriminator sequence between the −10 and +1 positions of the rapA promoter is responsible for its growth rate control and the instability of its initiation complexes with RNAP.

scholarly journals The soxRS response of Escherichia coli can be induced in the absence of oxidative stress and oxygen by modulation of NADPH content

Microbiology ◽  
2011 ◽  
Vol 157 (4) ◽  
pp. 957-965 ◽  
Author(s):  
Adriana R. Krapp ◽  
María Victoria Humbert ◽  
Néstor Carrillo
Keyword(s):  
Oxidative Stress ◽  
Escherichia Coli ◽  
Methyl Viologen ◽  
Transcriptional Regulator ◽  
E Coli ◽  
Enhanced Sensitivity ◽  
Genes Encoding ◽  
Cellular Signal ◽  
Redox Shuttle

The soxRS regulon protects Escherichia coli cells against superoxide and nitric oxide. Oxidation of the SoxR sensor, a [2Fe–2S]-containing transcriptional regulator, triggers the response, but the nature of the cellular signal sensed by SoxR is still a matter of debate. In vivo, the sensor is maintained in a reduced, inactive state by the activities of SoxR reductases, which employ NADPH as an electron donor. The hypothesis that NADPH levels affect deployment of the soxRS response was tested by transforming E. coli cells with genes encoding enzymes and proteins that lead to either build-up or depletion of the cellular NADPH pool. Introduction of NADP+-reducing enzymes, such as wheat non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase or E. coli malic enzyme, led to NADPH accumulation, inhibition of the soxRS regulon and enhanced sensitivity to the superoxide propagator methyl viologen (MV). Conversely, expression of pea ferredoxin (Fd), a redox shuttle that can oxidize NADPH via ferredoxin-NADP(H) reductase, resulted in execution of the soxRS response in the absence of oxidative stress, and in higher tolerance to MV. Processes that caused NADPH decline, including oxidative stress and Fd activity, correlated with an increase in total (NADP++NADPH) stocks. SoxS expression can be induced by Fd expression or by MV in anaerobiosis, under conditions in which NADPH is oxidized but no superoxide can be formed. The results indicate that activation of the soxRS regulon in E. coli cells exposed to superoxide-propagating compounds can be triggered by depletion of the NADPH stock rather than accumulation of superoxide itself. They also suggest that bacteria need to finely regulate homeostasis of the NADP(H) pool to enable proper deployment of this defensive response.

scholarly journals The transcription factor DNR from Pseudomonas aeruginosa specifically requires nitric oxide and haem for the activation of a target promoter in Escherichia coli

Microbiology ◽  
2009 ◽  
Vol 155 (9) ◽  
pp. 2838-2844 ◽  
Author(s):  
Nicoletta Castiglione ◽  
Serena Rinaldo ◽  
Giorgio Giardina ◽  
Francesca Cutruzzolà
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Consensus Sequence ◽  
Signal Molecules ◽  
Reporter System ◽  
Nucleotide Substitutions ◽  
E Coli

Pseudomonas aeruginosa is a well-known pathogen in chronic respiratory diseases such as cystic fibrosis. Infectivity of P. aeruginosa is related to the ability to grow under oxygen-limited conditions using the anaerobic metabolism of denitrification, in which nitrate is reduced to dinitrogen via nitric oxide (NO). Denitrification is activated by a cascade of redox-sensitive transcription factors, among which is the DNR regulator, sensitive to nitrogen oxides. To gain further insight into the mechanism of NO-sensing by DNR, we have developed an Escherichia coli-based reporter system to investigate different aspects of DNR activity. In E. coli DNR responds to NO, as shown by its ability to transactivate the P. aeruginosa norCB promoter. The direct binding of DNR to the target DNA is required, since mutations in the helix–turn–helix domain of DNR and specific nucleotide substitutions in the consensus sequence of the norCB promoter abolish the transcriptional activity. Using an E. coli strain deficient in haem biosynthesis, we have also confirmed that haem is required in vivo for the NO-dependent DNR activity, in agreement with the property of DNR to bind haem in vitro. Finally, we have shown, we believe for the first time, that DNR is able to discriminate in vivo between different diatomic signal molecules, NO and CO, both ligands of the reduced haem iron in vitro, suggesting that DNR responds specifically to NO.

scholarly journals Escherichia coli RNA Polymerase Recognition of a σ70-Dependent Promoter Requiring a −35 DNA Element and an Extended −10 TGn Motif

2006 ◽  
Vol 188 (24) ◽  
pp. 8352-8359 ◽  
Author(s):  
India Hook-Barnard ◽  
Xanthia B. Johnson ◽  
Deborah M. Hinton
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Consensus Sequence ◽  
Perfect Match ◽  
Start Site ◽  
E Coli ◽  
Functional Differences ◽  
Extension Analysis

ABSTRACT Escherichia coli σ70-dependent promoters have typically been characterized as either −10/−35 promoters, which have good matches to both the canonical −10 and the −35 sequences or as extended −10 promoters (TGn/−10 promoters), which have the TGn motif and an excellent match to the −10 consensus sequence. We report here an investigation of a promoter, Pminor, that has a nearly perfect match to the −35 sequence and has the TGn motif. However, Pminor contains an extremely poor σ70 −10 element. We demonstrate that Pminor is active both in vivo and in vitro and that mutations in either the −35 or the TGn motif eliminate its activity. Mutation of the TGn motif can be compensated for by mutations that make the −10 element more canonical, thus converting the −35/TGn promoter to a −35/−10 promoter. Potassium permanganate footprinting on the nontemplate and template strands indicates that when polymerase is in a stable (open) complex with Pminor, the DNA is single stranded from positions −11 to +4. We also demonstrate that transcription from Pminor incorporates nontemplated ribonucleoside triphosphates at the 5′ end of the Pminor transcript, which results in an anomalous assignment for the start site when primer extension analysis is used. Pminor represents one of the few −35/TGn promoters that have been characterized and serves as a model for investigating functional differences between these promoters and the better-characterized −10/−35 and extended −10 promoters used by E. coli RNA polymerase.

scholarly journals Characterization and in Vivo Cloning of prlC, a Suppressor of Signal Sequence Mutations in Escherichia coli K12

Genetics ◽  
1987 ◽  
Vol 116 (4) ◽  
pp. 513-521
Author(s):  
Nancy J Trun ◽  
Thomas J Silhavy
Keyword(s):  
Escherichia Coli ◽  
Genetic Mapping ◽  
Signal Sequence ◽  
Illegitimate Recombination ◽  
Mutant Phenotype ◽  
E Coli ◽  
Physical Location ◽  
Sequence Deletion ◽  
New Alleles

ABSTRACT The prlC gene of E. coli was originally identified as an allele, prlC1, which suppresses certain signal sequence mutations in the genes for several exported proteins. We have isolated six new alleles of prlC that also confer this phenotype. These mutations can be placed into three classes based on the degree to which they suppress the lamBsignal sequence deletion, lamBs78. Genetic mapping reveals that the physical location of the mutations in prlC correlates with the strength of the suppression, suggesting that different regions of the gene can be altered to yield a suppressor phenotype. We also describe an in vivo cloning procedure using λplacMu9H. The procedure relies on transposition and illegitimate recombination to generate a specialized transducing phage that carries prlC1. This method should be applicable to any gene for which there is a mutant phenotype.

scholarly journals Mechanistic insights into synergy between nalidixic acid and tetracycline against clinical isolates of Acinetobacter baumannii and Escherichia coli

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Amit Gaurav ◽  
Varsha Gupta ◽  
Sandeep K. Shrivastava ◽  
Ranjana Pathania
Keyword(s):  
Escherichia Coli ◽  
Acinetobacter Baumannii ◽  
Nalidixic Acid ◽  
Bacterial Infections ◽  
Clinical Isolates ◽  
Hospital Environment ◽  
Infection Model ◽  
Drug Resistant ◽  
E Coli

AbstractThe increasing prevalence of antimicrobial resistance has become a global health problem. Acinetobacter baumannii is an important nosocomial pathogen due to its capacity to persist in the hospital environment. It has a high mortality rate and few treatment options. Antibiotic combinations can help to fight multi-drug resistant (MDR) bacterial infections, but they are rarely used in the clinics and mostly unexplored. The interaction between bacteriostatic and bactericidal antibiotics are mostly reported as antagonism based on the results obtained in the susceptible model laboratory strain Escherichia coli. However, in the present study, we report a synergistic interaction between nalidixic acid and tetracycline against clinical multi-drug resistant A. baumannii and E. coli. Here we provide mechanistic insight into this dichotomy. The synergistic combination was studied by checkerboard assay and time-kill curve analysis. We also elucidate the mechanism behind this synergy using several techniques such as fluorescence spectroscopy, flow cytometry, fluorescence microscopy, morphometric analysis, and real-time polymerase chain reaction. Nalidixic acid and tetracycline combination displayed synergy against most of the MDR clinical isolates of A. baumannii and E. coli but not against susceptible isolates. Finally, we demonstrate that this combination is also effective in vivo in an A. baumannii/Caenorhabditis elegans infection model (p < 0.001)

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

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