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

Abstract

SummaryThe 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-1-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.

DNA-binding activity of LndI protein and temporal expression of the gene that upregulates landomycin E production in Streptomyces globisporus 1912

“…For EGFPproduction analysis, mycelia grown in TSB media were washed with deionized water and applied to glass slides. To study cell viability, propidium iodide (PI) was used as an indicator in a procedure described elsewhere (Khetan et al, 2000;Kyung et al, 2001). …”

“…EGFP expression levels can be quantified reliably (Kataoka et al, 1999), and its use does not cause any artifacts that would result from a highly stable reporter protein (Khetan et al, 2000;Kyung et al, 2001). To analyse the spatial and temporal expression pattern of the lndI gene the wC31-based plasmid pIJ8660H, carrying P lndI transcriptionally fused to the EGFP gene, was constructed.…”

Spatio-temporal expression of the pathway-specific regulatory gene redD in S. coelicolor

“…Method for Western blot analysis was performed as described by Kyung et al(2001), in which 30 µg of the total protein extract was separated by SDS-PAGE (10%) and the purified EYFP (27 kDa) was used as a positive control.…”

Transcriptional and translational analysis of the ccaR gene from Streptomyces clavuligerus

“…However, the detailed mechanism by which ccaR regulates clavulanic acid and cephamycin C production has been more difficult to determine at the molecular level, and studies to date have generated conflicting results (Kyung et al, 2001;Santamarta et al, 2002).…”