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Biochemical investigations of TetR-family regulators that control antibiotic biosynthesis in Streptomyces bacteria
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Fullwood, Alexander James (2019) Biochemical investigations of TetR-family regulators that control antibiotic biosynthesis in Streptomyces bacteria. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3453473~S15
Abstract
Many gene clusters encoding secondary metabolites biosynthesis in Streptomyces bacteria are controlled by complex regulatory networks, producing silent phenotypes when Streptomyces are grown under standard laboratory conditions. 2-alkyl-4-hydroxymethylfuran-3-carboylic acids (AHFCAs) induce secondary metabolite production in Streptomyces spp. By dissociating ArpA-subfamily transcriptional repressors from pseudo-palindromic autoregulator responsive elements (AREs).
In Streptomyces coelicolor A3(2) the AHFCA receptor MmfR and the pseudo-AHFCA receptor MmyR regulate the methylenomycin (mmy) gene cluster in tandem by controlling the expression of the mmy transcriptional activator mmyB as well as a 5-gene AHFCA regulatory cassette encoding mmfR, mmyR and the three AHFCA biosynthetic enzymes mmfLHP. Orthologous regulatory cassettes can be found within gene clusters of novel antibacterial interest across many Streptomyces spp., including Streptomyces venezuelae, Streptomyces avermitilis and Streptomyces sclerotialus.
The aim of this project was to characterise in vitro both the DNA-binding mechanisms and AHFCA structure-activity relations (SAR) of MmfR/MmyR orthologues found in S. avermitilis (AvaL1/AvaL2) and S. sclerotialus (SclM1/SclM4). All orthologues excluding SclM4 were solubilised in vitro, and crystallisation trials yielded cruystals of AvaL1, which remain to be diffracted. Gel shift assays demonstrated AvaL1/Sc1M1 binding to three/four AREs within their respective gene clusters, matching the 24-bp MmfR-type ARE consensus sequence 5’-AAnATACCTTCG|CGAAGGTATnTT-3’.
Surface plasmon resonance (SPR) established that AvaL1 bound to the three S. avermitilis AREs as a pair of homodimers with a mean KD of 123.7 nM ± 11.2 nM at 25ﹾC. Kinetic analysis inferred a two state conformation-dependent. DNA-binding mechanism likely associated with the stoichiometry of bound dimers. AvaL1 binding to DNA was more significant impaired in the presence of endogenous S. avermitilis AHFCAs (mean IC50 = 0.48 μM ± 0.06 μM), rationalised in silico by enlargement of the AvaL1 ligand-binding pocket. The pseudo-AHFCA receptor AvaL2 was also shown not bind to the shame AREs as AvaL1, and its effector remains unknown.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QD Chemistry Q Science > QH Natural history > QH426 Genetics Q Science > QR Microbiology |
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Library of Congress Subject Headings (LCSH): | Streptomyces, Antibiotics -- Synthesis, Genetic regulation | ||||
Official Date: | June 2019 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | School of Life Sciences | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Corre, Christophe ; Napier, R. (Richard) ; Fulop, Vilmos | ||||
Format of File: | |||||
Extent: | xix, 240 leaves : illustrations, charts | ||||
Language: | eng |
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