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Applied Microbiology and Biotechnology

, Volume 99, Issue 6, pp 2683–2692 | Cite as

Capturing the target genes of BldD in Saccharopolyspora erythraea using improved genomic SELEX method

  • Hang Wu
  • Yongrong Mao
  • Meng Chen
  • Hui Pan
  • Xunduan Huang
  • Min Ren
  • Hao Wu
  • Jiali Li
  • Zhongdong Xu
  • Hualing Yuan
  • Ming Geng
  • David T. WeaverEmail author
  • Lixin ZhangEmail author
  • Buchang ZhangEmail author
Applied genetics and molecular biotechnology

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.

Keywords

Saccharopolyspora erythraea Improved genomic SELEX method BldD amfC SACE_0306 

Notes

Acknowledgments

This work was supported by the National Program on Key Basic Research Project (973 program, 2013CB734000), Open Funding Project of the State Key Laboratory of Bioreactor Engineering (2013), The National Natural Science Foundation of China (31300081, 30870069), The Natural Science Foundation of Anhui Province (1208085MC46), The Initial Foundation of Doctoral Scientific Research in Anhui University (01001904), and National Innovation experiment program for University Students (201410357024). LZ is an Awardee for National Distinguished Young Scholar Program in China.

Supplementary material

253_2014_6255_MOESM1_ESM.pdf (281 kb)
ESM 1 (PDF 280 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Hang Wu
    • 1
  • Yongrong Mao
    • 1
  • Meng Chen
    • 1
  • Hui Pan
    • 1
  • Xunduan Huang
    • 1
  • Min Ren
    • 1
  • Hao Wu
    • 1
  • Jiali Li
    • 1
  • Zhongdong Xu
    • 3
  • Hualing Yuan
    • 3
  • Ming Geng
    • 3
  • David T. Weaver
    • 1
    Email author
  • Lixin Zhang
    • 1
    • 2
    Email author
  • Buchang Zhang
    • 1
    Email author
  1. 1.Institute of Health Sciences, School of Life SciencesAnhui UniversityHefeiChina
  2. 2.CAS Key Laboratory of Pathogenic Microbiology & Immunology, Institute of MicrobiologyChinese Academy of SciencesBeijingChina
  3. 3.School of Life SciencesHefei Normal UniversityHefeiChina

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