title: Capturing the target genes of BldD in Saccharopolyspora erythraea using improved genomic SELEX method.
abstract: BldD (SACE_2077), a key developmental regulator in actinomycetes, is the first identified transcriptional factor in Saccharopolyspora erythraea positively regulating erythromycin production and morphological differentiation. Although the BldD of S. erythraea binds to the promoters of erythromycin biosynthetic genes, the interaction affinities are relatively low, implying the existence of its other target genes in S. erythraea. Through the genomic systematic evolution of ligands by exponential enrichment (SELEX) method that we herein improved, four DNA sequences of S. erythraea A226, corresponding to the promoter regions of SACE_0306 (beta-galactosidase), SACE_0811 (50S ribosomal protein L25), SACE_3410 (fumarylacetoacetate hydrolase), and SACE_6014 (aldehyde dehydrogenase), were captured with all three BldD concentrations of 0.5, 1, and 2 μM, while the previously identified intergenic regions of eryBIV-eryAI and ermE-eryCI plus the promoter region of SACE_7115, the amfC homolog for aerial mycelium formation, could be captured only when the BldD's concentration reached 2 μM. Electrophoretic mobility shift assay (EMSA) analysis indicated that BldD specifically bound to above seven DNA sequences, and quantitative real-time PCR (qRT-PCR) assay showed that the transcriptional levels of the abovementioned target genes decreased when bldD was disrupted in A226. Furthermore, SACE_7115 and SACE_0306 in A226 were individually inactivated, showing that SACE_7115 was predominantly involved in aerial mycelium formation, while SACE_0306 mainly controlled erythromycin production. This study provides valuable information for better understanding of the pleiotropic regulator BldD in S. erythraea, and the improved method may be useful for uncovering regulatory networks of other transcriptional factors.
authors.affiliation: Institute of Health Sciences, School of Life Sciences, Anhui University, Hefei, 230601, China.
authors.forename: Hang
authors.initials: H
authors.lastname: Wu
authors.forename: Yongrong
authors.initials: Y
authors.lastname: Mao
authors.forename: Meng
authors.initials: M
authors.lastname: Chen
authors.forename: Hui
authors.initials: H
authors.lastname: Pan
authors.forename: Xunduan
authors.initials: X
authors.lastname: Huang
authors.forename: Min
authors.initials: M
authors.lastname: Ren
authors.forename: Hao
authors.initials: H
authors.lastname: Wu
authors.forename: Jiali
authors.initials: J
authors.lastname: Li
authors.forename: Zhongdong
authors.initials: Z
authors.lastname: Xu
authors.forename: Hualing
authors.initials: H
authors.lastname: Yuan
authors.forename: Ming
authors.initials: M
authors.lastname: Geng
authors.forename: David T
authors.initials: DT
authors.lastname: Weaver
authors.forename: Lixin
authors.initials: L
authors.lastname: Zhang
authors.forename: Buchang
authors.initials: B
authors.lastname: Zhang
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics*; D021901:DNA, Intergenic; D004917:Erythromycin / Q000096:biosynthesis; D005285:Fermentation; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D023281:Genomics; D011401:Promoter Regions, Genetic; D060888:Real-Time Polymerase Chain Reaction; D016930:Saccharopolyspora / Q000235:genetics*; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D021901:DNA, Intergenic; D014157:Transcription Factors; D004917:Erythromycin; D001616:beta-Galactosidase