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Title: AtrA-mediated transcriptional regulation in Streptomyces secondary metabolite production and development
Author: Towle, Jane Elizabeth
ISNI:       0000 0001 3535 689X
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2007
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Production of the tilue-pigmented polyketide antibiotic actinorhodin by Streptomyces co~licolor is controlled by transcriptional regulationof the pathway-specific gene actlIORF4. Previous work in our laboratory identified a member of the TetR-family of' transcriptional regulators, designated AtrA @ctinorhodin !ranscriptional regulator A), that can bind to two regions of the actlI-ORF4 promoter. Work in this thesis shows that disruption of atrA results in reduced levels of actinorhodin production. Furthermore, , QPCR analysis reveals the reduction is associated with diminished levels of actlI-ORF4 mRNA. These results suggest AtrA works as an activator of transcription of actIl- ORF4. Here it is shown that atrA is a limiting factor in the production of actinorhodin, as constitutive expression of atrA from an integrative plasmid results in early and enhanced levels of actinorhodin. QPCR analysis also indicates that atrA is regulated at the level of transcription. Additional experiments involving the constitutive expression of atrA reveal a possible role in aerial hyphae formation. Within this thesis, mutation of a single amino acid (R71A) is shown to significaIitlyreduce the DNA-binding activity of AtrA. Constitutive expression of this mutant from an integrative plasmid 1:?locks actinorhodin production in wild-type cells, showing it has a dominant-negative phenotype. This mutant could provide a powerful tool for assessing the requirement for atrA in strains showing enhanced actinorhodin production or disparate species, without the need to delete atrA from the chromosome. The atrA deletion strain and the plasmid expressing wild-type atrA were used to investigate the role of atrA in afsR- and relAmediated regulation of actinorhodin production. Results described here insinuate that under certain conditions afsR and atrA are ·both required for the biosynthesis of actinorhodin, and that relA and atrA may work synergistically to initiate actinorhodin production. Moreover, it is shown that the stimulation of actinorhodin production by the addition of exogenous y-butyrolactones requires atrA.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available