Using a fluorescent dual-reporter in streptomyces to study bldA-dependant gene expression

Stone, Jack William

Thesis

Using a fluorescent dual-reporter in streptomyces to study bldA-dependant gene expression

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Creator

Stone, Jack William

Rights statement

Strathclyde Thesis Copyright

Awarding institution

University of Strathclyde

Date of award

2026

Thesis identifier

T17615

Person Identifier (Local)

202162198

Qualification Level

Doctoral (Postgraduate)

Qualification Name

Doctor of Philosophy (PhD)

Department, School or Faculty

Strathclyde Institute of Pharmacy and Biomedical Sciences

Abstract

With rising antimicrobial resistance, new strategies are needed to discover and improve antibiotics. Improved genetic tools may accelerate antibiotic discovery and production. Many existing antimicrobial compounds are natural products of Streptomyces bacteria. In Streptomyces, the Leucine-tRNAUAA (encoded by bldA) decodes the rare TTA codon, and loss of bldA results in deficiencies in aerial hyphae, sporulation and antibiotic production. Expression of bldA is reported to be repressed by the global regulator BldD in Streptomyces coelicolor. Reporter assays, including fluorescent proteins and RNA aptamers, are widely used to monitor gene regulation, and their combination offers a promising genetic tool. This thesis describes the design, construction, and validation of the ‘Broccomyces ‘dualreporter system, which combines the Broccoli aptamer (transcriptional output) and a modified version of mCherry (TTA-mCherry, translational output). Broccomyces was tested in S. coelicolor using the bldA translational control system. TTA-mCherry levels varied with bldA availability, confirming translational reporter viability, however the Broccoli transcriptional reporter was non-functional in liquid media. Unexpectedly, TTA-mCherry was also expressed in bldA-deficient backgrounds, suggesting alternative mechanisms for decoding rare codons. The Broccomyces system was further tested under the ccaR promoter in S. clavuligerus and demonstrated utility for quantifying the translational effects of 5`UTR mutations relevant to antibiotic production. TTA-mCherry detection in a bldA-deficient background prompted whole genome sequencing of a historical bldA- strain, highlighting hundreds of previously undocumented mutations, with potential regulatory effects. An isogenic bldA deletion strain in S. coelicolor M145 was subsequently created and characterised through complementation, metabolite profiling, utilisation assays and transcriptomics. Analysis indicates the bldA tRNA impacts far more than morphological development and antibiotic production. This work establishes Broccomyces as a versatile translational reporter and demonstrates the wider regulatory role of bldA, with implications for engineering antibiotic production in Streptomyces.

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