The Streptomyces master regulator BldD binds c-di-GMP sequentially to create a functional BldD2-(c-di-GMP)4 complex.

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Streptomyces are ubiquitous soil bacteria that undergo a complex developmental transition coinciding with their production of antibiotics. This transition is controlled by binding of a novel tetrameric form of the second messenger, 3?-5? cyclic diguanylic acid (c-di-GMP) to the master repressor, BldD. In all domains of life, nucleotide-based second messengers allow a rapid integration of external and internal signals into regulatory pathways that control cellular responses to changing conditions. c-di-GMP can assume alternative oligomeric states to effect different functions, binding to effector proteins as monomers, intercalated dimers or, uniquely in the case of BldD, as a tetramer. However, at physiological concentrations c-di-GMP is a monomer and little is known about how higher oligomeric complexes assemble on effector proteins and if intermediates in assembly pathways have regulatory significance. Here, we show that c-di-GMP binds BldD using an ordered, sequential mechanism and that BldD function necessitates the assembly of the BldD2-(c-di-GMP)4 complex.