RESUMEN
Streptomyces coelicolor is a filamentous soil bacterium producing several kinds of antibiotics. S. coelicolor abs8752 is an abs (antibiotic synthesis deficient)-type mutation at the absR locus; it is characterized by an incapacity to produce any of the four antibiotics synthesized by its parental strain J1501. A chromosomal DNA fragment from S. coelicolor J1501, capable of complementing the abs- phenotype of the abs8752 mutant, was cloned and analyzed. DNA sequencing revealed that two complete ORFs (SCO6992 and SCO6993) were present in opposite directions in the clone. Introduction of SCO6992 in the mutant strain resulted in a remarkable increase in the production of two pigmented antibiotics, actinorhodin and undecylprodigiosin, in S. coelicolor J1501 and abs8752. However, introduction of SCO6993 did not show any significant difference compared to the control, suggesting that SCO6992 is primarily involved in stimulating the biosynthesis of antibiotics in S. coelicolor. In silico analysis of SCO6992 (359 aa, 39.5 kDa) revealed that sequences homologous to SCO6992 were all annotated as hypothetical proteins. Although a metalloprotease domain with a conserved metal-binding motif was found in SCO6992, the recombinant rSCO6992 did not show any protease activity. Instead, it showed very strong ß-glucuronidase activity in an API ZYM assay and toward two artificial substrates, p-nitrophenyl-ß-D-glucuronide and AS-BI-ß-D-glucuronide. The binding between rSCO6992 and Zn2+ was confirmed by circular dichroism spectroscopy. We report for the first time that SCO6992 is a novel protein with ß-glucuronidase activity, that has a distinct primary structure and physiological role from those of previously reported ß-glucuronidases.
Asunto(s)
Antibacterianos/biosíntesis , Proteínas Bacterianas/genética , Glucuronidasa/genética , Streptomyces coelicolor/genética , Secuencia de Aminoácidos , Antraquinonas/metabolismo , Proteínas Bacterianas/metabolismo , Dosificación de Gen , Regulación Bacteriana de la Expresión Génica , Prueba de Complementación Genética , Glucuronidasa/metabolismo , Mutación , Prodigiosina/análogos & derivados , Prodigiosina/biosíntesis , Alineación de Secuencia , Streptomyces coelicolor/enzimologíaRESUMEN
The methylation of myo-inositol forms O-methyl inositol (D-ononitol) when plants are under abiotic stress in a reaction catalyzed by myo-inositol methyltransferase (IMT). D-Ononitol can serve as an osmoprotectant that prevents water loss in plants. We isolated the IMT cDNA from Glycine max and found by RT-PCR analysis that GmIMT transcripts are induced by drought and salinity stress treatments in the leaves of soybean seedlings. We confirmed the protein product of GmIMT and its substrate using a recombinant system in E. coli. Transgenic Arabidopsis plants over-expressing GmIMT displayed improved tolerance to dehydration stress treatment and to a lesser extent high salinity stress treatment. These results indicate that GmIMT is functional in heterologous Arabidopsis plants.