Understanding and exploiting general transcription factors in the antibiotic-producing Streptomyces

Most of our antibiotics derive from the Actinobacteria, a phylum of Gram positive bacteria that inhabit diverse terrestrial and aquatic habitats and includes the Streptomyces genus that produce, for example, chloramphenicol, neomycin and erythromycin. While the use of antibiotics in the second half of the 20th century transformed human health care, antimicrobial resistance in pathogenic bacteria has steadily increased and now presents itself as a major global threat to health and society. Researchers are therefore hunting for new antibiotics to replace those that are becoming obsolete and the Actinobacteria are being revisited as a major source. One reason is because recent analysis of genome sequences is revealing genes that might direct antibiotic production, but are not switched on under laboratory conditions. Scientists are therefore very interested in finding new ways to switch on such cryptic biosynthetic genes so that the products can be assayed. One approach is to generate mutations that affect the structure and function of RNA polymerase - the enzyme that transcribes (reads) DNA to generate an RNA copy that can then be translated into the respective protein. Certain mutations are thought to redistribute the RNA polymerase away genes involved in growth towards those involved in secondary metabolism including antibiotic production. These mutations might mimic the behavior of this enzyme when the bacteria run out of nutrients - the so called stringent response that ensures that RNA polymerase switches from transcribing genes involved in growth to those involved in surviving the stress. However, this process is poorly understood in Actinobacteria and recent findings have shown that RNA polymerase itself differs from well-studied model organism E. coli in containing two accessory protein factors, called RbpA and CarD, that are required for transcription. This project will investigate the stringent response and its relationship with these transcription factors. It will produce new knowledge that will help us understand the role of RbpA and CarD and generate new new ways to reactivate cryptic antibiotic biosynthetic gene clusters and engineer, using synthetic biology, new recombinant bacterial strains for the production of novel antimicrobials.

Antibiotics are usually produced towards the end of growth when genes involved in macromolecular biosynthesis (e.g. rRNA operons) are downregulated, leading to a redistribution of RNA polymerase (RNAP) towards genes involved in e.g. secondary metabolism. Where studied, rRNA promoters tend to have kinetic properties that confer unusual sensitivity to the concentration of initiating nucleotides (iNTP) and/or the bacterial alarmone ppGpp; they form unusually unstable binary open complexes with RNAP (RPo), which necessitates high [iNTP] and low [ppGpp] for activity. In the actinobacteria, initiation is also influenced by two newly discovered general transcription factors, RbpA and CarD, that each contact the upstream edge of -10 elements to stimulate/stabilize RPo complexes. Building on our prior studies we will: 1. Understand how stable RNA synthesis is stringently controlled in Streptomyces and how GTFs influence this response. We will generate strains in which the iNTP (GTP) levels can be adjusted then modulate the level and activities of the GTFs to determine the influence on stable RNA expression. We will also study the kinetics of transcription initiation in vitro using recombinant proteins. 2. Understand and exploit the overproduction of antibiotics by RbpA mutants. We will investigate whether the level and activities of RbpA and CarD are influenced by growth phase using Chip-seq, western, and LC-MS/MS analysis. Since certain RbpA mutants overproduce the antibiotic actinorhodin, we will also develop RbpA as a tool to reawaken cryptic antibiotic biosynthetic clusters. 3. understand how promoter architecture defines transcriptional output by RbpA and CarD. We will use in vitro NGS based techniques to determine promoter dependency on RbpA and CarD and how these factors influence each other. The results will provide key insights into transcription initiation and the stringent response in Actinobacteria with important implications for antibiotic discovery.

The beneficiaries of this research include: (1) Pharma and Biotech companies interested in generating new antibiotics. Such companies will benefit from the new knowledge generated concerning the expression of antibiotic biosynthesis genes and the reawakening of silent antibiotic biosynthetic clusters, helping to reveal the full biosynthetic capabilities of the actinobacteria. The outcomes will also provide key information about promoter structure and function, which will aid in the refactoring of pathways to provide sufficient material for analysis and clinical trials. Prior to publication of our data, we will protect any arising IP to enhance value and maximise opportunities for collaborative research or licensing. We will then pro-actively engage with such beneficiaries with the help of the University of Sussex Research and Enterprise Office. Research data will be published in peer-reviewed open access journals, to broaden the reach of the outcomes. (2) Members of the wider academic community. The outcomes will be of great interest to researchers studying the mechanism of transcription initiation and the stringent response in many different bacterial models. Outputs will be disseminated primarily by traditional routes of publication in peer-reviewed journals, presentation at international research meetings, and the development of new collaborations where appropriate. Data will also be made available on the Sussex Research Online repository, immediately upon acceptance for publication, and publicised through established social media routes with the help of the University of Sussex Press and Communications office. (3) Skills, training and the knowledge economy. The PDRA employed on the project will be trained in a wide variety of cutting edge biochemical, microbiological and genome-based techniques, and this project will therefore enhance UK competiveness in this area. The PDRA will be encouraged to engage with the University of Sussex Professional Development Unit to acquire and enhance skills such as public speaking. The University of Sussex takes the development of early-career researchers very seriously and retained a European Commission HR Excellence in Research award in 2015. The School of Life Sciences was awarded an Athena Swan Silver award in 2016 in recognition of its career development support, irrespective of gender. The development of the PDRA will be monitored through an annual appraisal scheme. As part of their development, the PDRA will also design short-term projects and be involved in the training of undergraduate and intern students that join the laboratory, thereby broadening the impact of this research. (4) The general public and other stakeholders. Ultimately this research could lead to the discovery of new antibiotics, desperately needed to tackle the global problem of antimicrobial resistance. The research therefore has important benefits for health and society with the associated cost savings for the NHS. We will engage with younger members of the public through talks and hands-on experiments at schools including developing activities related to antibiotic discovery as part of the Small World Initiative. We will also deliver public seminars on antibiotic awareness to interest groups such as U3A. The applicant has been actively engaged in such activities in recent years. (5) Public sector health professionals. Clinicians and senior health providers benefit from an improved understanding of where key medicines, such as antibiotics, come from, how they act, and approaches for their discovery. The PI will therefore contribute to local network groups including the Brighton and Sussex Infection and Immunology Research Network.