1. Corynebacterium glutamicum CrtR and Its Orthologs in Actinobacteria : Conserved Function and Application as Genetically Encoded Biosensor for Detection of Geranylgeranyl Pyrophosphate.
- Author
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Henke NA, Austermeier S, Grothaus IL, Götker S, Persicke M, Peters-Wendisch P, and Wendisch VF
- Subjects
- Actinobacteria genetics, Binding Sites, Corynebacterium glutamicum genetics, Gene Expression Regulation, Bacterial, Promoter Regions, Genetic, Actinobacteria metabolism, Bacterial Proteins physiology, Biosensing Techniques, Carotenoids metabolism, Corynebacterium glutamicum metabolism, Polyisoprenyl Phosphates analysis, Transcription Factors physiology
- Abstract
Carotenoid biosynthesis in Corynebacterium glutamicum is controlled by the MarR-type regulator CrtR, which represses transcription of the promoter of the crt operon (P crtE ). Geranylgeranyl pyrophosphate (GGPP), and to a lesser extent other isoprenoid pyrophosphates, interfere with the binding of CrtR to its target DNA in vitro, suggesting they act as inducers of carotenoid biosynthesis. CrtR homologs are encoded in the genomes of many other actinobacteria. In order to determine if and to what extent the function of CrtR, as a metabolite-dependent transcriptional repressor of carotenoid biosynthesis genes responding to GGPP, is conserved among crtR ). Geranylgeranyl pyrophosphate (GGPP), and to a lesser extent other isoprenoid pyrophosphates, interfere with the binding of CrtR to its target DNA in vitro, suggesting they act as inducers of carotenoid biosynthesis. CrtR homologs are encoded in the genomes of many other actinobacteria. In order to determine if and to what extent the function of CrtR, as a metabolite-dependent transcriptional repressor of carotenoid biosynthesis genes responding to GGPP, is conserved among actinobacteria , five CrtR orthologs were characterized in more detail. EMSA assays showed that the CrtR orthologs from Corynebacterium callunae , Acidipropionibacterium jensenii , Paenarthrobacter nicotinovorans , Micrococcus luteus and Pseudarthrobacter chlorophenolicus bound to the intergenic region between their own gene and the divergently oriented gene, and that GGPP inhibited these interactions. In turn, the CrtR protein from C. glutamicum bound to DNA regions upstream of the orthologous crtR genes that contained a 15 bp DNA sequence motif conserved between the tested bacteria. Moreover, the CrtR orthologs functioned in C. glutamicum in vivo at least partially, as they complemented the defects in the pigmentation and expression of a P crtE _ gfp uv transcriptional fusion that were observed in a crtR deletion mutant to varying degrees. Subsequently, the utility of the P crtE _ gfp uv transcriptional fusion and chromosomally encoded CrtR from C. glutamicum as genetically encoded biosensor for GGPP was studied. Combined FACS and LC-MS analysis demonstrated a correlation between the sensor fluorescent signal and the intracellular GGPP concentration, and allowed us to monitor intracellular GGPP concentrations during growth and differentiate between strains engineered to accumulate GGPP at different concentrations.
- Published
- 2020
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