RESUMO
A chromosomal fragment of bacteria Photorhabdus luminescence Zm1, which contains the lux operon, was cloned into the vector pUC18. The hybrid clone containing plasmid pXen7 with the EcoRI fragment approximately 7-kb was shown to manifest a high level of bioluminescence. By subcloning and restriction analysis of the EcoRI fragment, the location of luxCDABE genes relative to restriction sites was determined. The nucleotide sequence of the DNA fragment containing the luxA and luxB genes encoding alpha- and beta-subunits of luciferase was determined. A comparison with the nucleotide sequences of luxAB genes in Hm and Hw strains of Ph. luminescence revealed 94.5 and 89.7% homology, respectively. The enterobacterial repetitive intergenic sequence (ERIC) of 126 bp typical for Hw strains was identified in the spacer between the luxD and luxA genes. The lux operon of Zm1 is assumed to emerge through recombination between Hm and Hw strains. Luciferase of Ph. luminescence was shown to possess a high thermal stability: its activity decreased by a factor of 10 at 44 degrees C for 30 min, whereas luciferases of marine bacteria Vibrio fischeri and Vibrio harveyi were inactivated by one order of magnitude at 44 degrees C for 1 and 6 min, respectively. The lux genes of Ph. luminescence are suggested for use in gene engineering and biotechnology.
Assuntos
Luciferases/genética , Óperon , Photorhabdus/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Vetores Genéticos , Luciferases/metabolismo , Dados de Sequência Molecular , Photorhabdus/enzimologia , Recombinação Genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Vibrio/enzimologiaAssuntos
Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Protease La , Regulon , Proteínas Repressoras/genética , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Transativadores/genética , Vibrio/genética , Proteases Dependentes de ATP , Escherichia coli/enzimologia , Hidrólise , Mutação , PlasmídeosRESUMO
The possibility of application of the bioluminescence method (Lux-test) for studying in vivo functional activity of Escherichia coli protease Lon and its mutants was demonstrated. This assay is based on the capacity of protease Lon and its mutant forms for specific degradation of the LuxR protein, a positive transcriptional activator of the right operon luxICDABE from the marine bacterium Vibrio fischeri, and thus to affect the level of AB luciferase in the cells. A correlation between in vitro activity of the protease Lon mutants and the intensity of bioluminescence measured by the Lux-test was revealed.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/enzimologia , Proteínas de Choque Térmico/metabolismo , Mutação , Protease La , Regulon , Proteínas Repressoras/genética , Serina Endopeptidases/metabolismo , Transativadores/genética , Vibrio/genética , Proteases Dependentes de ATP , Proteínas de Bactérias/genética , Proteínas de Choque Térmico/genética , Luciferases/genética , Medições Luminescentes , Óperon , Serina Endopeptidases/genéticaRESUMO
Bacterial luciferases are highly suitable test substrates for the analysis of refolding of misfolded proteins, as they are structurally labile and loose activity at 42 degrees C. Heat-denatured thermolabile Vibrio fischeri luciferase and thermostable Photorhabdus luminescens luciferase were used as substrates. We found that their reactivation requires the activity of the DnaK chaperone system. The DnaKJ chaperones were dispensable in vivo for de novo folding at 30 degrees C of the luciferase, but essential for refolding after a heat-shock. The rate and yield of DnaKJ refolding of the P. luminescens thermostable luciferase were to a marked degree lower as compared with the V. fischeri thermolabile luciferase. The refolding activity of the DnaKJ chaperones was examined at various temperatures. Between 30 and 37 degrees C, the refolding rates of the V. fischeri luciferase decreased and the reaction reached a complete arrest at temperatures above 40 degrees C. The rate of DnaKJ-mediated refolding of the thermostable luciferase at first increased between 30 and 37 degrees C and then decreased at the range of 37-44 degrees C. We observed that the rate of DnaKJ-mediated refolding of the heat-denatured P. luminescens thermostable luciferase, but not of the thermolabile V. fischeri luciferase, decreased during the prolonged incubation at a high (47 degrees C) temperature. The efficiency and reversibility of protein refolding arrest during and after heat-shock strongly depended on the stability of the DnaKJ-denatured luciferase complex. It is supposed that the thermostable luciferase is released during the heat-shock, whereas the thermolabile luciferase remained bound to the chaperone.
