title: Identification of two novel regulatory genes involved in pristinamycin biosynthesis and elucidation of the mechanism for AtrA-p-mediated regulation in Streptomyces pristinaespiralis.
abstract: In this study, using a transposon-based strategy, two novel regulatory genes were identified as being involved in the biosynthesis of both pristinamycin I (PI) and II (PII) in Streptomyces pristinaespiralis, including a TetR-family regulatory gene atrA-p (SSDG_00466) and an orphan histidine kinase gene SSDG_02492. The mechanism by which AtrA-p exerted a positive role in pristinamycin production was elucidated. We showed that deletion of atrA-p resulted in a delayed production of both PI and PII as well as reduced PII production. Transcriptional analysis integrated with electrophoretic mobility shift assays (EMSAs) demonstrated that AtrA-p played a positive role in pristinamycin production by directly activating the transcription of two cluster-situated regulatory genes, spbR and papR5, which encode a γ-butyrolactone receptor protein and a TetR-family repressor, respectively. The precise AtrA-p-binding sites upstream of these two targets were determined, which allowed the identification of a relatively conserved binding motif comprising two 5-nt inverted repeats separated by a variable 5-nt sequence (5'-GGAAT-n5-ATTCC-3') possibly required for the regulation of AtrA-like regulators in Streptomyces. Base substitutions of the AtrA-p-binding sites on the genome caused similar decreases in spbR and papR5 transcription as those observed in ∆atrA-p. Taken together, herein, a novel mechanism for AtrA-dependent regulation of antibiotic biosynthesis was revealed in S. pristinaespiralis, which is distinct from those of its homologs, AtrA-c from Streptomyces coelicolor, AtrA-g from Streptomyces griseus, and AtrA from Streptomyces roseosporus that perform their effects in antibiotic biosynthesis directly via pathway-specific activator genes or the biosynthetic structural genes.
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, People's Republic of China.
authors.forename: Wenfang
authors.initials: W
authors.lastname: Wang
authors.forename: Jinzhong
authors.initials: J
authors.lastname: Tian
authors.forename: Lei
authors.initials: L
authors.lastname: Li
authors.forename: Mei
authors.initials: M
authors.lastname: Ge
authors.forename: Hong
authors.initials: H
authors.lastname: Zhu
authors.forename: Guosong
authors.initials: G
authors.lastname: Zheng
authors.forename: He
authors.initials: H
authors.lastname: Huang
authors.forename: Lijun
authors.initials: L
authors.lastname: Ruan
authors.forename: Weihong
authors.initials: W
authors.lastname: Jiang
authors.forename: Yinhua
authors.initials: Y
authors.lastname: Lu
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001665:Binding Sites; D004251:DNA Transposable Elements; D024202:Electrophoretic Mobility Shift Assay; D017353:Gene Deletion; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D053263:Gene Regulatory Networks; D005809:Genes, Regulator; D016254:Mutagenesis, Insertional; D025762:Pristinamycin / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D004251:DNA Transposable Elements; D025762:Pristinamycin