Streptomyces bacteria represent an unusual group of Gram-positive bacteria that possess high GC content genomes (typically 74% G+C). They are of considerable importance to society as they are responsible for producing most of the antibiotics that are used in human and veterinary medicine. They remain a focus internationally for the identification of new antibiotics to combat worldwide antimicrobial resistance and as a bacterial platform for enhancing production of existing antibiotics through synthetic biology approaches.
It has recently been discovered in studies on the model bacterium, Escherichia coli, that ‘cold-shock’ proteins (CSPs), a universally conserved protein family, fulfil important roles in regulating messenger RNA secondary structure when the organism is subjected to low temperatures. CSPs bind the mRNA to facilitate its unfolding, making it available for translation by ribosomes. We have recently revealed that translation of the homologous CSP family in Streptomyces bacteria is enhanced following exposure to heat-shock. We hypothesise that the CSPs in Streptomyces, which are highly abundant proteins, have evolved to fulfil an essential function at all temperatures to maintain mRNAs in a translatable state; this is because the high %G+C content of the streptomycete genes generates considerable secondary structure in the encoded mRNAs, hindering the ability of ribosomes to translate them.
This interdisciplinary project brings together our complementary expertise in genetic manipulation and genome wide analysis of translation in Streptomyces, cutting-edge expertise in mass spectrometry-based proteomics (Brighton) and in cutting edge in vitro array-based screening of transcription and translation (Portsmouth) to understand the role of CSPs in Streptomyces and to exploit this information to modulate gene expression in synthetic biology platforms, both in vivo and in vitro. The student will investigate the function of each member of the CSP family in vivo through deletion, depletion and over-expression of individual CSP genes. This will involve analysis of the ‘translatome’ using the cutting edge ‘Ribosome Profiling’ technique and MS-based proteomics. Each CSP will also be analysed in a novel array-based in vitro transcription-translation system to examine the respective influences of the E. coli and Streptomyces CSPs on translation of fusion reporters at different CSP concentrations and at different temperatures. This biochemical work will also evaluate the functional targets of the CSPs. The outputs of the project could find applications in industrial biotechnology and in in vitro synthetic biology platforms.
The student will develop a broad range of transferable skills in experimental genomics, mass spectrometry-based proteomics, molecular genetics and in vitro nucleic acid and protein biochemistry, coupled with a practical grounding in bioinformatic analysis of genomic and proteomic data. They will be primarily based at the Moulsecoomb campus in Brighton (Brighton Genomics and mass spectrometry facility) but will also undertake array-based experiments on a regular basis at the neighbouring University of Portsmouth, in the laboratory of Prof Anastasia Callaghan. Portsmouth and Brighton universities are both members of the UK’s University Alliance.
Selected relevant references
- Bucca G, Pothi R, Hesketh A, Moller-Levet C, Hodgson DA, Laing EE, et al. Translational control plays an important role in the adaptive heat-shock response of Streptomyces coelicolor. Nucleic Acids Res. 2018;46(11):5692-703.
- Jeong Y, Kim JN, Kim MW, Bucca G, Cho S, Yoon YJ, et al. The dynamic transcriptional and translational landscape of the model antibiotic producer Streptomyces coelicolor A3(2). Nat Commun. 2016;7:11605.
- Norouzi M, Pickford AR, Butt LE, Vincent HA, Callaghan AJ. (2019) Application of mRNA Arrays for the Production of mCherry Reporter-Protein Arrays for Quantitative Gene Expression Analysis. ACS Synthetic Biology 8:207-215
- Phillips JO, Butt LE, Henderson CA, Devonshire M, Healy J, Conway SJ, et al. High-density functional-RNA arrays as a versatile platform for studying RNA-based interactions. Nucleic Acids Res. 2018;46(14):e86.
- Zhang Y, Burkhardt DH, Rouskin S, Li GW, Weissman JS, Gross CA. A Stress Response that Monitors and Regulates mRNA Structure Is Central to Cold Shock Adaptation. Mol Cell. 2018;70(2):274-86 e7.