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

• Published in: Molecular microbiology Vol. 40; no. 3; pp. 530 - 541
• Main Authors: Kyung, Yun Seung, Hu, Wei‐Shou, Sherman, David H.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.05.2001; Blackwell Publishing Ltd
• Subjects: Alcohol Oxidoreductases - biosynthesis; Alcohol Oxidoreductases - genetics; Alcohol Oxidoreductases - metabolism; Base Sequence; CcaR protein; cephamycin C; Cephamycins - metabolism; Chromosomes, Bacterial; Culture Media; DNA-Binding Proteins - biosynthesis; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Gene Expression; Green Fluorescent Proteins; L-Lysine 6-Transaminase; lat gene; Luminescent Proteins - genetics; lysine-^e-aminotransferase; Microscopy, Confocal; Molecular Sequence Data; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Streptomyces - genetics; Streptomyces - growth & development; Streptomyces - metabolism; Streptomyces clavuligerus; Time Factors; Transaminases - genetics
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1046/j.1365-2958.2001.02386.x

The DNA‐binding capability of a key secondary metabolite regulatory element (CcaR) in the Streptomyces clavuligerus cephamycin C pathway was investigated by gel mobility retardation and DNase I footprinting analysis. These results revealed that CcaR specifically binds to the promoter region of the lysine‐ε‐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 produced cephamycin C at levels similar to those of wild‐type S. clavuligerus. Confocal laser scanning microscopy revealed that expression of CcaR in liquid culture was temporally dynamic and spatially heterogeneous in S. clavuligerus 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‐exponential growth phase. As the culture aged, the population expressing CcaR expanded, and the expression level increased. This was followed by a reduction in the CcaR‐expressing population towards the end of the culture period. During peak expression, CcaR was distributed uniformly in mycelia, but became localized distal to the chromosome when the culture entered stationary phase. In solid phase analysis, abundant CcaR expression was evident in the substrate mycelia, but was completely absent in aerial hyphae. These results show regulatory linkage between ccaR and lat, whose expression profile showed a similar spatial decoupling between morphogenesis and antibiotic production. In addition, visualizing CcaR within S. clavuligerus mycelia demonstrates a distinct pattern of localization over the course of physiological differentiation.