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

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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.

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