Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.14.3 (luciferase)
 38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Expression of the Vibrio fischeri luminescence genes (luxR and luxICDABEG) in Escherichia coli requires autoinducer (N-3-oxohexanoyl homoserine lactone) and LuxR protein, which activate transcription of luxICDABEG (genes for autoinducer synthase and the luminescence enzymes), and cyclic AMP (cAMP) and cAMP receptor protein (CRP), which activate transcription of the divergently expressed luxR gene. In E. coli and in V. fischeri, the autoinducer-LuxR protein-dependent induction of luxICDABEG transcription (called autoinduction) is delayed by glucose, whereas it is promoted by iron restriction, but the mechanisms for these effects are not clear. To examine in V. fischeri control of lux gene expression by autoinducer, cAMP, glucose, and iron, lux::Mu dI(lacZ) and lux deletion mutants of V. fischeri were constructed by conjugation and gene replacement procedures. beta-Galactosidase synthesis in a luxC::lacZ mutant exhibited autoinduction. In a luxR::lacZ mutant, complementation by the luxR gene was necessary for luminescence, and addition of cAMP increased beta-galactosidase activity four- to sixfold. Furthermore, a luxI::lacZ mutant produced no detectable autoinducer but responded to its addition with induced synthesis of beta-galactosidase. These results confirm in V. fischeri key features of lux gene regulation derived from studies with E. coli. However, beta-galactosidase specific activity in the luxI::lacZ mutant, without added autoinducer, exhibited an eight- to tenfold decrease and rise back during growth, as did beta-galactosidase and luciferase specific activities in the luxR::lacZ mutant and luciferase specific activity in a delta(luxR luxICD) mutant. The presence of glucose delayed the rise back in beta-galactosidase and luciferase specific activities in these strains, whereas iron restriction promoted it. Thus, in addition to transcriptional control by autoinducer and LuxR protein, the V. fischeri lux system exhibits a cell density-dependent modulation of expression that does not require autoinducer, LuxR protein, or known lux regulatory sites. The response of autoinducer-LuxR protein-independent modulation to glucose and iron may account for how these environmental factors control lux gene expressions.
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PMID:Cell density-dependent modulation of the Vibrio fischeri luminescence system in the absence of autoinducer and LuxR protein. 131 12

Iron controls luminescence in Vibrio fischeri by an indirect but undefined mechanism. To gain insight into that mechanism, the involvement of cyclic AMP (cAMP) and cAMP receptor protein (CRP) and of modulation of DNA levels in iron control of luminescence were examined in V. fischeri and in Escherichia coli containing the cloned V. fischeri lux genes on plasmids. For V. fischeri and E. coli adenylate cyclase (cya) and CRP (crp) mutants containing intact lux genes (luxR luxICDABEG), presence of the iron chelator ethylenediamine-di(o-hydroxyphenyl acetic acid) (EDDHA) increased expression of the luminescence system like in the parent strains only in the cya mutants in the presence of added cAMP. In the E. coli strains containing a plasmid with a Mu dl(lacZ) fusion in luxR, levels of beta-galactosidase activity (expression from the luxR promoter) and luciferase activity (expression from the lux operon promoter) were both 2-3-fold higher in the presence of EDDHA in the parent strain, and for the mutants this response to EDDHA was observed only in the cya mutant in the presence of added cAMP. Therefore, cAMP and CRP are required for the iron restriction effect on luminescence, and their involvement in iron control apparently is distinct from the known differential control of transcription from the luxR and luxICDABEG promoters by cAMP-CRP. Furthermore, plasmid and chromosomal DNA levels were higher in E. coli and V. fischeri in the presence of EDDHA. The higher DNA levels correlated with an increase in expression of chromosomally encoded beta-galactosidase in E. coli and with a higher level of autoinducer in cultures of V. fischeri. These results implicate cAMP-CRP and modulation of DNA levels in the mechanism of iron control of the V. fischeri luminescence system.
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PMID:Mechanism for iron control of the Vibrio fischeri luminescence system: involvement of cyclic AMP and cyclic AMP receptor protein and modulation of DNA level. 132 97

Luciferase-based biosensors are becoming increasingly used for environmental monitoring. A transcriptional fusion of the Vibrio harveyi luxAB genes (encoding bacterial luciferase) to the fliC gene of Escherichia coli was constructed and luminescence shown to be induced (in liquid media) in the presence of 1-10 micrograms/ml aluminum, but not copper, iron or nickel. Moreover, luminescence is markedly increased at pH 5.5, where aluminum is more soluble than at pH 7.0. However, aluminum also stimulated luciferase activity when the luxAB genes were located in the xyl operon. This suggests that aluminum stimulates luciferase enzyme activity in vivo. These results are specific to E. coli, as no such aluminum stimulation was observed in the luminescent bacterium V. harveyi. These results have important implications in the generalized use of these clones for environmental monitoring, where aluminum can be present at elevated concentrations.
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PMID:Characterization of the effects of aluminum on luciferase biosensors for the detection of ecotoxicity. 147 Dec 23

Iron influences luminescence in Vibrio fischeri; cultures iron-restricted for growth rate induce luminescence at a lower optical density (OD) than faster growing, iron-replete cultures. An iron restriction effect analogous to that in V. fischeri (slower growth, induction of luminescence at a lower OD) was established using Escherichia coli tonB and tonB+ strains transformed with recombinant plasmids containing the V. fischeri lux genes (luxR luxICDABEG) and grown in the presence and absence of the iron chelator ethylenediamine-di(o-hydroxylphenyl acetic acid) (EDDHA). This permitted the mechanism of iron control of luminescence to be examined. A fur mutant and its parent strain containing the intact lux genes exhibited no difference in the OD at induction of luminescence. Therefore, an iron-binding repressor protein apparently is not involved in iron control of luminescence. Furthermore, in the tonB and in tonB+ strains containing lux plasmids with Mu dI(lacZ) fusions in luxR, levels of beta-galactosidase activity (expression from the luxR promoter) and luciferase activity (expression from the luxICDABEG promoter) both increased by a similar amount (8-9 fold each for tonB, 2-3 fold each for tonB+) in the presence of EDDHA. Similar results were obtained with the luxR gene present on a complementing plasmid. The previously identified regulatory factors that control the lux system (autoinducer-LuxR protein, cyclic AMP-cAMP receptor protein) differentially control expression from the luxR and luxICDABEG promoters, increasing expression from one while decreasing expression from the other. Consequently, these results suggest that the effect of iron on the V. fischeri luminescence system is indirect.
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PMID:Iron control of the Vibrio fischeri luminescence system in Escherichia coli. 151 May 56

luxAB gene fusions in the Escherichia coli genome were used to screen for clones displaying transcriptional changes in the presence of aluminum. One clone was found that contained a luciferase gene fusion in which transcription was increased in the presence of aluminum and which was subsequently shown to be induced by copper, iron, and nickel. Cloning of the metal-regulated gene, hybridization to the ordered phage lambda bank of the E. coli chromosome, and sequencing of DNA adjacent to the luxAB fusion revealed that the insertion occurred within the fliC (hag) gene of E. coli. This gene encodes flagellin, the filament subunit of the bacterial motility organ, and is under the control of several regulatory cascades. These results suggest that environmental metals may play a role in the regulation of the motility potential of E. coli and that this bioluminescent gene fusion clone (or derivatives thereof) may be used to prepare a biosensor for the rapid detection of metal contamination in water samples.
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PMID:Transcription of the Escherichia coli fliC gene is regulated by metal ions. 176 97

We have developed a high-efficiency nucleic acid delivery system that uses receptor-mediated endocytosis to carry DNA macromolecules into cells. We accomplished this by conjugating the iron-transport protein transferrin to polycations that bind nucleic acids. Human transferrin, as well as the chicken homologue conalbumin, has been covalently linked to the small DNA-binding protein protamine or to polylysines of various sizes through a disulfide linkage. These modified transferrin molecules maintain their ability to bind their cognate receptor and to mediate efficient iron transport into the cell. The transferrin-polycation molecules form electrophoretically stable complexes with double-stranded DNA, single-stranded DNA, and modified RNA molecules independent of nucleic acid size (from short oligonucleotides to DNA of 21 kilobase pairs). When complexes of transferrin-polycation and a bacterial plasmid DNA containing the gene for Photinus pyralis luciferase are supplied to eukaryotic cells, high-level expression of the luciferase gene occurs, demonstrating transferrin receptor-mediated endocytosis and expression of the imported DNA. We refer to this delivery system as "transferrinfection."
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PMID:Transferrin-polycation conjugates as carriers for DNA uptake into cells. 233 90

Xenorhabdus luminescens, a newly isolated luminous bacterium collected from a human wound, was characterized. The effects of ionic strength, temperature, oxygen, and iron on growth and development of the bioluminescent system were studied. The bacteria grew and emitted light best at 33 degrees C in a medium with low salt, and the medium after growth of cells to a high density was found to have antibiotic activity. The emission spectrum peaked at 482 nm in vivo and at 490 nm in vitro. Both growth and the development of luminescence in X. luminescens required oxygen and iron. The isolated luciferase itself exhibited a temperature optimum at about 40 degrees C; after purification by affinity chromatography, it showed two bands (52 and 41 kilodaltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicative of an alpha and beta subunit structure. Reduced flavin mononucleotide (Km of 1.4 microM) and tetradecanal (Km of 2.1 microM) were the best substrates for the luciferase, and the first-order decay constant under these conditions at 37 degrees C was 0.79 s-1.
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PMID:Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound. 260 98

Genes necessary for luminescence (lux genes) in the marine bacterium Photobacterium leiognathi, strain PL721, were isolated and expressed in Escherichia coli. A 15-kb fragment obtained from a partial digestion of PL721 DNA with HindIII was cloned into the plasmid pACYC184, resulting in the hybrid plasmid pSD721. When pSD721 was transformed into E. coli ED8654, the resulting transformants were luminous with no additions to the cells, indicating that it contained the structural genes coding for the alpha and beta subunits of luciferase (luxA and luxB), and for components involved in aldehyde biosynthesis. Hybridization analysis with luxA and luxB 32P probes confirmed the location of these two genes on the 15-kb insert. When pSD721 was transformed into four different strains of E. coli, luminescence expression varied widely in amount and in pattern. In some strains, luminescence developed like an autoinducible system, and at maximum induction was very bright, even with no addition of aldehyde, while in others, luminescence was 100-fold less, and no induction was seen. In no case was luminescence affected by shifts in temperature, osmolarity, or iron concentration. These results indicate that, while the complete lux regulon is apparently contained on the 15-kb cloned fragment, the regulation of the lux regulon in pSD721 is subject to host controls by E. coli, controls which vary widely among different E. coli strains.
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PMID:Isolation of the lux genes from Photobacterium leiognathi and expression in Escherichia coli. 330 37

The changes of energy metabolism on vasogenic edema have been largely examined using biochemical quantitative assay. However, the relationship between the sequential changes and blood-brain barrier (BBB) breakdown is not well understood. In the present study, the sequential changes of energy metabolism and potassium in relation to BBB breakdown following the cold-induced brain edema were investigated histochemically. Adult male Wistar rats, weighing 200-250g, were anesthesized with pentobarbital and a burr hole was made in the left parietal region. For evaluating the breakdown of BBB, 2.5% Evans blue (EB) was injected 30 min. before injury, except in the 5 min. model in which it was injected at the time of cold injury. An iron-bar precooled in liquid N2 was placed over the surface for 30 seconds and they were frozen in situ in liquid N2 at 5 min., 2 hrs., 6 hrs., 12 hrs., and 24 hrs., after producing the lesion. The frozen brain was sectioned using a precooled saw in the coronal plane. The brain section was placed in liquid N2 bath and illuminated with 366 nm light (UV) from a 200 watt mercury lamp and Corning filter 5840. NADH fluorescence was recorded photographically through Corning filter 3387 and 5562. Regional ATP and potassium content were investigated histochemically in thin sections with luciferine-luciferase method and Macallum's technique, respectively. At 5 min. after cold injury, leakage of EB was limited within the lesion. Potassium and ATP were decreased in the lesion. NADH fluorescence was increased slightly in the cortex around the lesion.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[The sequential changes of energy metabolism following cold-induced brain edema--a histochemical study]. 362 Feb 15

Synthesis of luciferase (an autoinducible enzyme) is repressed by iron in the symbiotic bioluminescent bacterium Vibrio fischeri. Possible mechanisms of iron regulation of luciferase synthesis were tested with V. fischeri and with Escherichia coli clones containing plasmids carrying V. fischeri luminescence genes. Experiments were conducted in complete medium with and without the synthetic iron chelator ethylenediamine-di(o-hydroxyphenyl acetic acid). Comparison of the effect of ethylenediamine-di(o-hydroxyphenyl acetic acid) and another growth inhibitor, (2-n-heptyl-4-hydroxyquinoline-N-oxide), showed that iron repression is not due to inhibition of growth. A quantitative bioassay for autoinducer was developed with E. coli HB101 containing pJE411, a plasmid carrying V. fischeri luminescence genes with a transcriptional fusion between luxI and E. coli lacZ. Bioassay experiments showed no effect of iron on either autoinducer activity or production (before induction) or transcription of the lux operon. Ethylenediamine-di(o-hydroxyphenyl acetic acid) did not affect luciferase induction in E. coli strains with wild-type iron assimilation (ED8654) or impaired iron assimilation (RW193) bearing pJE202 (a plasmid with functional V. fischeri lux genes), suggesting that the genes responsible for the iron effect are missing or substituted in these clones. Two models are consistent with the data: (i) iron represses autoinducer transport, and (ii) iron acts through an autoinduction-independent regulatory system (e.g., an iron repressor).
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PMID:Mechanisms of iron regulation of luminescence in Vibrio fischeri. 392 Feb 2


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