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Kathryn Styles

Project title: Investigating signalling molecule and transcriptional repressor interactions in Streptomyces

Kathryn Styles, Vilmos Fulop and Christophe Corre

School of Life Sciences, University of Warwick, Coventry, United Kingdom

MmfR, is a TetR-family homodimeric protein consisting of an N-terminal DNA-binding domain and a C-terminal ligand-binding domain. MmfR specifically binds to the small signalling molecules MMFs (methylenomycin furans) and has been shown to control the production of the methylenomycin antibiotic in Streptomyces coelicolor A3(2). MmfR binds to three different intergenic regions, directly controlling the expression of five operons in the methylenomycin cluster.

With the aim of developing a novel inducible expression system, the project first investigates the molecular interactions between MmfR and the MMFs as well as different promoter regions. In silico, key residues in the ligand- and DNA-binding sites of MmfR have been identified through sequence and structural analysis and a comparison of motifs with orthologues such as GBL-binding (gamma-butyrolactone) proteins. These key residues have been selected for site directed mutagenesis. A luciferase reporter gene assay is also being developed in vivo, employing the MMFs and other potential analogous inducer molecules. Reporter genes (luxCDABE) replace methylenomycin biosynthetic cluster with luciferase genes, which produce a bioluminescent product. These lux genes are repressed by MmfR but their expression is induced by MMF signaling molecules.

Key references:

Cuthbertson, L. & Nodwell, J. R. (2013) The TetR Family of Regulators. Microbiology and Molecular Biology Reviews, 77 (3): 440-475.

Corre, C., Song, L., O'Rourke, S., Chater, K. F. & Challis, G. L. (2008) 2-Alkyl-4-hydroxymethylfuran-3-carboxylic acids, antibiotic production inducers discovered by Streptomyces coelicolor genome mining. Proceedings of the National Academy of Sciences of the United States of America, 105 (45): 17510-17515.

Craney, A., Hohenauer, T., Xu, Y., Navani, N. K., Li, Y. & Nodwell, J. (2007) A synthetic luxCDABE gene cluster optimized for expression in high-GC bacteria. Nucleic Acids Research, 35 (6): e46-Article No.: e46.

Corre group site:

http://www2.warwick.ac.uk/fac/sci/chemistry/research/corre/corregroup

MIBTP training year:

http://prezi.com/v3_qyiljzn1f/investigating-microbial-hormonestranscriptional-repressors/

Project worked on at WHO during professional internship:

http://www.who.int/medicines/areas/priority_medicines/en/

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Kathryn Styles

K dot Styles at warwick dot ac dot uk