Analysis of temporal and spatial expression of the CcaR regulatory elementin the cephamycin C biosynthetic pathway using green fluorescent protein

The DNA-binding capability of a key secondary metabolite regulatory element (CcaR) in the Streptomyces clavuligerus cephamycin C pathway was investiga ted by gel mobility retardation and DNase I footprinting analysis. These re sults revealed that CcaR specifically binds to the promoter region of the l ysine-epsilon -aminotransferase gene (lat). Green fluorescent protein (GFP) was subsequently used as a reporter to analyse in vivo expression of CcaR. The corresponding isogenic strain containing ccaR::gfp in the chromosome p roduced cephamycin C at levels similar to those of wild-type S. clavuligeru s. Confocal laser scanning microscopy revealed that expression of CcaR in l iquid culture was temporally dynamic and spatially heterogeneous in S. clav uligerus mycelia. The highly fluorescent seed culture mycelia quickly lost fluorescence upon inoculation into fresh culture medium. The characteristic green colour reappeared in a small portion of mycelia during mid-exponenti al growth phase. As the culture aged, the population expressing CcaR expand ed, and the expression level increased. This was followed by a reduction in the CcaR-expressing population towards the end of the culture period. Duri ng peak expression, CcaR was distributed uniformly in mycelia, but became l ocalized distal to the chromosome when the culture entered stationary phase . In solid phase analysis, abundant CcaR expression was evident in the subs trate mycelia, but was completely absent in aerial hyphae. These results sh ow regulatory linkage between ccaR and lat, whose expression profile showed a similar spatial decoupling between morphogenesis and antibiotic producti on. In addition, visualizing CcaR within S. clavuligerus mycelia demonstrat es a distinct pattern of localization over the course of physiological diff erentiation.