Regulation of tylosin production and morphological differentiation in Streptomyces fradiae by TylP, a deduced g-butyrolactone receptor activator, adpA (Ohnishi et al., 1999). A variation on this theme is seen in Streptomyces virginiae, in which gbutyrolactones ('butanolides') bind to BarA and derepress virginiamycin biosynthesis with no concurrent effect on sporulation (Nakano et al., 1998). In the latter case, BarA controls the expression of various genes, including barB. Because the tylosin biosynthetic (tyl ) gene cluster of Streptomyces fradiae (Fig. 1) contains obvious orthologues of barA and barB (namely tylP and tylQ respectively; Bate et al., 1999), it seemed likely a priori that one or more g-butyrolactone(s) regulate tylosin production and that the TylP protein (deduced to be a g-butyrolactone receptor) might be a repressor involved in such regulation. The aim of the present work was to test that hypothesis and (if substantiated) to identify targets for TylP. In doing so, we were mindful that g-butyrolactones and their receptors typically operate at relatively 'high' levels in regulatory cascades that control antibiotic production in actinomycetes. That being so and given that the tyl cluster is remarkable in containing at least five regulatory genes (including tylR, tylS and tylT, in addition to tylP and tylQ; Bate et al., 1999;, we were also interested in the possibility of establishing a hierarchical order among the tyl regulators. Accordingly, gene expression analysis (involving reverse transcription-polymerase chain reaction, RT-PCR) was applied to the entire tyl cluster before and after the onset of tylosin production in S. fradiae wild type and, concurrently, in engineered strains in which tylP was constitutively overexpressed or had been specifically disrupted. Such studies were complemented by fermentation analysis and by characterization of tyl promoters transplanted into S. lividans.
Results
Gene expression analysisWhen transcript analysis was first applied to the tyl gene cluster , it became clear that the TylQ protein is a repressor that controls tylR (a global activator of the tyl cluster; Bate et al., 1999), and that tylQ must be repressed before tylosin production can begin. Thus, in S. fradiae wild type, although tylQ was expressed before the onset of tylosin biosynthesis (i.e. at 18 h; Fig. 2), it was the only silent gene in the cluster after tylosin production had commenced (i.e. at 40 h; Fig. 2). In Molecular Microbiology (2002) 45(3), [735][736][737][738][739][740][741][742][743][744] George Stratigopoulos, Atul R. Gandecha and Eric Cundliffe* Department of Biochemistry, University of Leicester, Leicester LE1 7RH, UK.
SummaryDuring promoter-probe analysis carried out in Streptomyces lividans, the TylP protein powerfully inhibited reporter gene expression from the tylP promoter, raising the likelihood that tylP is autoregulated in its native host, Streptomyces fradiae. Also in S. lividans, TylP negatively controlled the tylQ promoter, even though tylQ could still be switched off in ...