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김민정,TAKUYA NIHIRA,최선욱 한국응용생명화학회 2012 Applied Biological Chemistry (Appl Biol Chem) Vol.55 No.5
Global regulator for secondary metabolism, AfsR, is phosphorylated by the serine/threonine kinase AfsK. Phosphorylation of AfsR activates the transcription of afsS, resulting in the increased production of secondary metabolites in Streptomyces strains. We isolated an afsR homologue regulatory gene from Streptomyces acidiscabies ATCC 49003, which produces thaxtomin A and WS5995B as secondary metabolites. To examine the function of afsR in the production of secondary metabolites in S. acidiscabies, an intact 2,976 bp open reading frame of afsR was identified and characterized. In S. lividans TK 24 strain, the exconjugant harboring afsR high expression vector, began to generate actinorhodin at 36 h of culture, and the amount of accumulated actinorhodin became 10-fold higher than that of the exconjugant harboring the vector lacking the afsR gene. To clarify the in vivo function of afsR, an afsR-disrupted mutant was constructed and analyzed. No morphological difference was observed between the wild-type strain and the afsR disruptant, but production of thaxtomin A and WS5995B of the afsR disruptant were significantly decreased compared to those of the wild-type strains. Specially, WS5995B production was almost abolished by the disruption of only the afsR gene. These changes were restored to the original wild-type phenotype by the introduction of the intact afsR gene into the afsR disruptant, suggesting that the afsR gene participates in the production of secondary metabolites of S. acidiscabies.
Kim, Min-Jeong,Nihira, Takuya,Choi, Sun-Uk The Korean Society for Applied Biological Chemistr 2012 Applied Biological Chemistry (Appl Biol Chem) Vol.55 No.5
Global regulator for secondary metabolism, AfsR, is phosphorylated by the serine/threonine kinase AfsK. Phosphorylation of AfsR activates the transcription of afsS, resulting in the increased production of secondary metabolites in Streptomyces strains. We isolated an afsR homologue regulatory gene from Streptomyces acidiscabies ATCC 49003, which produces thaxtomin A and WS5995B as secondary metabolites. To examine the function of afsR in the production of secondary metabolites in S. acidiscabies, an intact 2,976 bp open reading frame of afsR was identified and characterized. In S. lividans TK 24 strain, the exconjugant harboring afsR high expression vector, began to generate actinorhodin at 36 h of culture, and the amount of accumulated actinorhodin became 10-fold higher than that of the exconjugant harboring the vector lacking the afsR gene. To clarify the in vivo function of afsR, an afsR-disrupted mutant was constructed and analyzed. No morphological difference was observed between the wild-type strain and the afsR disruptant, but production of thaxtomin A and WS5995B of the afsR disruptant were significantly decreased compared to those of the wild-type strains. Specially, WS5995B production was almost abolished by the disruption of only the afsR gene. These changes were restored to the original wild-type phenotype by the introduction of the intact afsR gene into the afsR disruptant, suggesting that the afsR gene participates in the production of secondary metabolites of S. acidiscabies.
Conjugal Transfer of Plasmid DNA from Escherichia coli to Streptomyces lavendulae FRI-5
KITANI, SHIGERU,BIBB, MERVYN J.,NIHIRA, TAKUYA,YAMADA, YASUHIRO 한국미생물 · 생명공학회 2000 Journal of microbiology and biotechnology Vol.10 No.4
Streptomyces lavendulae FRI-5 produces the γ-butyrolactone autoregulator IM-2, which is required for nucleoside antibiotic production. We have developed a system for introducing DNA into S. lavendulae FRI-5 via conjugal transfer from Escherichia coli. Conditions were established for conjugation of the oriT- and attP-containing plasmid pSET152 from E. coli ET12567 (pUZ8002) to FRI-5. Conjugation resulted in integration of the plasmid at the chromosomal øC31 attB site. The frequency of intergeneric conjugation varied with the medium used. The highest frequency (1.6×l0^-5 per recipient) was obtained on ISP medium 2 containing 10mM MgCl_2. Southern blot and phenotypic analyses of exconjugants revealed that S. lavendulae FRI-5 contains a unique øC31 attB site, and that integration of heterologous DNA into the attB site did not interfere with morphological differentiation or IM-2-dependent signal transduction, including the production of a blue pigment. This system will now enable detailed genetic analysis of the regulation of antibiotic production in S. lavendulae FRI-5.