Streptomyces coelicolor, a multicellular bacterium with complex cell cycle is a model organism for bacterial development and antibiotic production (Flardh and Buttner, 2009). Its life cycle begins with filamentous growth producing multigenomic and branching hyphae. Later, the aerial parts of the long, hyphal filaments undergo synchronous septation producing a chain of unigenomic compartments, the pre-spores. Concomitant with aerial hyphae formation, biologically active secondary metabolites are produced and the fate of the non-sporulating vegetative hyphae is cell lysis. Our preliminary data suggest that this highly regulated life cycle involves communication between the different cellular compartments within S.coelicolor.  We have identified a putative oligopeptide (OP) for cross-talk between the spore chains and the vegetative mycelium.

The aim of the proposed project is to characterise the role of the oligopeptide signalling during the development and antibiotic production of S.coelicolor.  First, knockout mutation in the gene encoding the oligopeptide will be generated and the mutant phenotype will be analysed using microscopy techniques. Our preliminary data has shown that expression of this oligopeptide is dependent on one of the RNA polymerase sigma factors, SigN, and also on a yet unidentified transcriptional activator. We have identified the binding site for this activator and we will purify and identify this activator using affinity purification with the target DNA immobilised to magnetic beads (Dynal). The DNA binding protein will be identified using MALDI-TOF (JIC). We will monitor expression of the oligopeptide using transcriptional mapping in the wild-type and in different developmental mutants and we will also localise the oligopeptide using fluorescently labelled antibodies. In order to monitor the transcription within the specific compartments during OP signalling, we will perform transcriptomics/RNAseq using RNA isolated from the wild-type and several mutants of sigN(1) the genes encoding the oligopeptide (2) and the activator protein (3). The oligopeptide will be overexpressed and purified and its role in controlling gene expression within the vegetative mycelium will be shown by monitoring egfp fusions to genes that are dependent on the oligopeptide signalling.

Flardh, K. & M. J. Buttner, (2009) Streptomyces morphogenetics: dissecting differentiation in a filamentous bacterium. Nat Rev Microbiol 7: 36-49.