• Journal of Microbiology and Biotechnology
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• 제32권9호
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• pp.1134-1145
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• 2022

SCO6993 (606 amino acids) in Streptomyces coelicolor belongs to the large ATP-binding regulators of the LuxR family regulators having one DNA-binding motif. Our previous findings predicted that SCO6993 may suppress the production of pigmented antibiotics, actinorhodin, and undecylprodigiosin, in S. coelicolor, resulting in the characterization of its properties at the molecular level. SCO6993-disruptant, S. coelicolor ΔSCO6993 produced excess pigments in R2YE plates as early as the third day of culture and showed 9.0-fold and 1.8-fold increased production of actinorhodin and undecylprodigiosin in R2YE broth, respectively, compared with that by the wild strain and S. coelicolor ΔSCO6993/SCO6993⁺. Real-time polymerase chain reaction analysis showed that the transcription of actA and actII-ORF4 in the actinorhodin biosynthetic gene cluster and that of redD and redQ in the undecylprodigiosin biosynthetic gene cluster were significantly increased by SCO6993-disruptant. Electrophoretic mobility shift assay and DNase footprinting analysis confirmed that SCO6993 protein could bind only to the promoters of pathway-specific transcriptional activator genes, actII-ORF4 and redD, and a specific palindromic sequence is essential for SCO6993 binding. Moreover, SCO6993 bound to two palindromic sequences on its promoter region. These results indicate that SCO6993 suppresses the expression of other biosynthetic genes in the cluster by repressing the transcription of actII-ORF4 and redD and consequently negatively regulating antibiotic production.

• Journal of Microbiology and Biotechnology
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• 제31권11호
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• pp.1591-1600
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• 2021

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 β-glucuronidase activity in an API ZYM assay and toward two artificial substrates, p-nitrophenyl-β-D-glucuronide and AS-BI-β-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 β-glucuronidase activity, that has a distinct primary structure and physiological role from those of previously reported β-glucuronidases.