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Transcription of the 74 kb Pseudomonas fluorescens mupirocin [pseudomonic acid (PA)] biosynthesis cluster depends on quorum sensing-dependent regulation via the LuxI/LuxR homologues MupI/MupR. To facilitate analysis of novel PAs from pathway mutants, we investigated factors that affect mup gene expression. First, the signal produced by MupI was identified as N-(3-oxodecanoyl)homoserine lactone, but exogenous addition of this molecule did not activate mupirocin production prematurely nor did expression of mupI in trans increase metabolite production. Second, we confirmed that mupX, encoding an amidase/hydrolase that can degrade N-acylhomoserine lactones, is also required for efficient expression, consistent with its occurrence in a regulatory module linked to unrelated genes in P. fluorescens. Third, and most significantly, mupR expression in trans to wild type and mutants can increase production of antibiotic and novel intermediates up to 17-fold.