Characterisation of BldC a novel transcription factor required for development and antibiotic production in Streptomyces

The harmless soil bacteria called streptomycetes are vital to human welfare because they are the source of the vast majority of antibiotics used by doctors to cure infectious diseases, as well as providing us with numerous other medicines used, for example, to treat cancer, and to help organ transplant patients. We have identified a key regulator (called a 'transcription factor') that switches the genetic machinery of these useful bacteria to allow them to reproduce themselves and to make antibiotics. The aim of this work is to find out exactly how this regulator works at the molecular level, how its activity is controlled, to understand what it does in the cell (what genes it controls), and how this brings about antibiotic production and bacterial reproduction. We also know that similar regulators are present in other bacteria, including pathogenic bacteria that cause fatal diseases like tuberculosis and diphtheria. Thus, this work will also provide potential insights into the function of these regulators in other bacteria important to human health.

A major challenge in Streptomyces developmental biology is to connect the well-defined cell biological processes underlying morphogenesis to the master regulators identified by classical genetics, by dissecting the regulatory networks that link the two. We seek to understand the physiological function, post-translational regulation and mechanism of action of one such master regulator, BldC, a novel transcription factor required for differentiation and antibiotic production in Streptomyces, the bacteria that are the most abundant source of clinically important antibiotics and other bioactive molecules. To understand the physiological function of BldC, we will use ChIP-chip to identify the complete regulon of genes under BldC control, and to determine how the BldC regulon changes during development. In addition, we will use microarray transcriptional profiling to determine in vivo for each target gene, whether BldC functions as an activator or a repressor, and when. We have discovered that BldC is phosphorylated on Thr38 and Thr42. We will determine how Thr38/42 substitutions blocking phosphorylation or mimicking constitutive phosphorylation affect BldC activity in vivo and in vitro to determine how phosphorylation affects BldC function. BldC is 68 residues long and is related to the DNA-binding domain of the MerR family, but lacks the dimerisation helix and effector-recognition domain. Despite the absence of a dimerisation helix, purified BldC binds specifically to target promoters. These observations raise important questions about the mechanism of action of BldC. We will define the position of BldC binding in a range of target promoters (sites of activation and repression, if both occur) and, using in vivo and in vitro approaches, we will determine the mechanisms by which BldC controls transcription at these sites. We will determine the oligomeric state of BldC in solution and bound to its targets, and determine mechanistically and structurally how BldC binds DNA.

WHO WILL BENEFIT FROM THIS RESEARCH, AND HOW? BldC plays an important role in the control not only of sporulation, but also of antibiotic production. Streptomycetes and their actinomycete relatives produce 80% of the commercially important antibiotics, and are also a rich source of other types of bioactive molecules such as anticancer agents and immunosupressants, in total accounting for $40 billion of revenue in the pharmaceutical industry worldwide. The proposed research may therefore have implications for pharmaceutical companies manufacturing antibiotics and other medically important compounds of actinomycete origin. By analogy, another Bld regulator discussed in the grant, BldD, was recently shown to directly control production of the antibiotic erythromycin in the actinomycete Saccharopolyspora erythraea [Chng et al. (2008) Proc Natl Acad Sci U S A. 105:11346-51], suggesting an obvious route to increase erythromycin production levels by rational manipulation of BldD, a protein originally identified, like BldC, through the academic analysis of development in Streptomyces coelicolor. WHAT WILL BE DONE TO ENSURE THAT THEY HAVE THE OPPORTUNITY TO BENEFIT FROM THIS RESEARCH? Academic research at the John Innes Centre (JIC) with potential implications is patented through Plant Biosciences Ltd (PBL), a technology transfer company based at JIC that is jointly and equally owned by the Gatsby Charitable Foundation and the JIC. The purpose of Plant Bioscience Ltd is to bring the results of research in plant and microbial sciences at the Centre into public use for public benefit through commercial exploitation. PBL meets all patent filing, marketing and licensing expenses in respect of technologies it develops for JIC. Streptomyces research is prominent in PBL's portfolio. As an illustration, two spin-out companies have been established based on JIC Streptomyces group patents: Novacta Biosystems Ltd, founded at JIC in 2003 and now based at Welwyn Garden City Biopark, where it employs about 30 people; and Procarta Biosystems, founded at JIC in 2008. Thus, there are well established routes for delivery of IP arising from Streptomyces research at the John Innes Centre.