Functional Analysis ofrelAandrshA, TworelA/spoTHomologues ofStreptomyces coelicolorA3(2)

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Abstract
Deletion of the (p)ppGpp synthetase gene,relA, ofStreptomyces coelicolorA3(2) results in loss of production of the antibiotics actinorhodin (Act) and undecylprodigiosin (Red) and delayed morphological differentiation when the mutant is grown under conditions of nitrogen limitation. To analyze the role of (p)ppGpp as an intracellular signaling molecule for the initiation of antibiotic production, several C-terminally deleted derivatives ofS. coelicolor relAthat could potentially function in the absence of ribosome activation were placed under the control of the thiostrepton-inducibletipApromoter. While 0.82- and 1.28-kb N-terminal segments failed to restore (p)ppGpp and antibiotic production upon induction in arelAnull mutant, 1.46- and 2.07-kb segments did. Under conditions of phosphate limitation, deletion ofrelAhad little or no effect on Act or Red synthesis, potentially reflecting an alternative mechanism for ppGpp synthesis. A secondS. coelicolorRelA homologue (RshA, with 42% identity toS. coelicolorRelA) was identified in the genome sequence. However, deletion ofrshAhad no effect on the ability of therelAmutant to make Act and Red when grown under conditions of phosphate limitation. While high-level induction oftipAp::rshAin therelAmutant resulted in growth inhibition, low-level induction restored antibiotic production and sporulation. In neither case, nor in therelAmutant that was grown under phosphate limitation and producing Act and Red, could (p)ppGpp synthesis be detected. Thus, a ppGpp-independent mechanism exists to activate antibiotic production under conditions of phosphate limitation that can be mimicked by overexpression ofrshA.