David A. Hodgson

Systems biology approaches to study metabolic switching in Streptomyces coelicolor A3(2) depend on cultivation conditions ensuring high reproducibility and distinct phases of culture growth and secondary metabolite production. In addition, biomass concentrations must be sufficiently high to allow for extensive time-series sampling before occurrence of a given nutrient depletion for transition triggering. The present study describes for the first time the development of a dedicated optimized submerged batch fermentation strategy as the basis for highly time-resolved systems biology studies of metabolic switching in S. coelicolor A3(2).

Background: The transition from exponential to stationary phase in Streptomyces coelicolor is accompanied by a major metabolic switch and results in a strong activation of secondary metabolism. Here we have explored the underlying reorganization of the metabolome by combining computational predictions based on constraint-based modeling and detailed transcriptomics time course observations. Results: We reconstructed the stoichiometric matrix of S. coelicolor, including the major antibiotic biosynthesis pathways, and performed flux balance analysis to predict flux changes that occur when the cell switches from biomass to antibiotic production. We defined the model input based on observed fermenter culture data and used a dynamically varying objective function to represent the metabolic switch. The predicted fluxes of many genes show highly significant correlation to the time series of the corresponding gene expression data. Individual mispredictions identify novel links between antibiotic production and primary metabolism. Conclusion: Our results show the usefulness of constraint-based modeling for providing a detailed interpretation of time course gene expression data.

During the lifetime of a fermenter culture, the soil bacterium S. coelicolor undergoes a major metabolic switch from exponential growth to antibiotic production. We have studied gene expression patterns during this switch, using a specifically designed Affymetrix genechip and a high-resolution time-series of fermenter-grown samples. Surprisingly, we find that the metabolic switch actually consists of multiple finely orchestrated switching events. Strongly coherent clusters of genes show drastic changes in gene expression already many hours before the classically defined transition phase where the switch from primary to secondary metabolism was expected. The main switch in gene expression takes only 2 hours, and changes in antibiotic biosynthesis genes are delayed relative to the metabolic rearrangements. Furthermore, global variation in morphogenesis genes indicates an involvement of cell differentiation pathways in the decision phase leading up to the commitment to antibiotic biosynthesis. Our study provides the first detailed insights into the complex sequence of early regulatory events during and preceding the major metabolic switch in S. coelicolor, which will form the starting point for future attempts at engineering antibiotic production in a biotechnological setting.

InMyxococcus xanthusphotoprotective carotenoids are produced in response to illumination due to regulated expression of carotenoid biosynthesis genes at two loci. Induction of the carotenogenesis regulon is dependent on expression of thecarQRSoperon. The first gene product of the operon, CarQ, is a sigma factor belonging to the ECF family and is responsible for light-dependent initiation of transcription at thecarQRSpromoter. We defined the minimalcarQRSpromoter as a 145-bp fragment of DNA upstream of thecarQRStranscriptional start site, which includes the promoter for a divergent gene,gufA. In order to elucidate regions with the promoter required for activity, point mutations were introduced into thecarQRSpromoter between positions −151 and 6. While most sequence changes abolishedcarQRSpromoter activity, two changes enhanced promoter activity and two changes caused the mutant promoter to become constitutive and independent of CarQ. The promoter-null point mutations and 6-bp deletion mutations implied that thecarQRSpromoter requires a functionalgufApromoter for transcriptional activity and vice versa. By mapping the extent of the promoter region, identifying sequences important for promoter activity, and highlighting potential topological effects, we provide a foundation for further analysis of thecarQRSpromoter.