Mass spectrometric screening of transcriptional regulators involved in antibiotic biosynthesis in Streptomyces coelicolor A3(2).
DNA-affinity capture assay (DACA) coupled with liquid chromatography-tandem mass spectrometry analysis was applied to identify the transcriptional regulators involved in the biosynthesis of actinorhodin (Act) and undecylprodigiosin (Red) in Streptomyces coelicolor. The aim of this analysis was to determine the specific transcriptional regulators binding to the promoter region of actII-ORF4 or redD. The results of the DACA, as the first screening tool, identified eight proteins, including AdpA, as candidate regulators binding to those promoter regions. To show the direct physical relationship between the regulators and promoters, we purified four regulators over-expressed in soluble form in Escherichia coli and subjected these to an electrophoretic mobility shift assay (EMSA). The results of the EMSA appeared to be compatible with the DACA results for those regulators. A null mutant was also constructed for one of these regulators, SCO6008, which showed early Red production and quite delayed Act production in R5(-) medium. These observations suggest that DACA can be widely used to find new regulators and that the regulator SCO6008 may be involved in antibiotic production through its binding to the redD promoter.
Park
Sung-Soo
SS
School of Chemical and Biological Engineering, Institute of Bioengineering, Seoul National University, Kwanak-gu, Seoul, Korea.
Yang
Yung-Hun
YH
Song
Eunjung
E
Kim
Eun-Jung
EJ
Kim
Woo Seong
WS
Sohng
Jae Kyung
JK
Lee
Hei Chan
HC
Liou
Kwang Kyoung
KK
Kim
Byung-Gee
BG
D000880:Anthraquinones / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000302:isolation & purification*; D002853:Chromatography, Liquid / Q000379:methods; D004269:DNA, Bacterial / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D055786:Gene Knockout Techniques; D013058:Mass Spectrometry / Q000379:methods; D011353:Prodigiosin / Q000031:analogs & derivatives / Q000096:biosynthesis; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D048372:Streptomyces coelicolor / Q000737:chemistry* / Q000502:physiology*; D014157:Transcription Factors / Q000302:isolation & purification*; D000095622:Benzoisochromanequinones
D000880:Anthraquinones; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011353:Prodigiosin; D014157:Transcription Factors; C047347:undecylprodigiosin; C013390:actinorhodin; D000095622:Benzoisochromanequinones