title: Beta-glucuronidase as a sensitive and versatile reporter in actinomycetes.
abstract: Here we describe a versatile and sensitive reporter system for actinomycetes that is based on gusA, which encodes the β-glucuronidase enzyme. A series of gusA-containing transcriptional and translational fusion vectors were constructed and utilized to study the regulatory cascade of the phenalinolactone biosynthetic gene cluster. Furthermore, these vectors were used to study the efficiency of translation initiation at the ATG, GTG, TTG, and CTG start codons. Surprisingly, constructs using a TTG start codon showed the best activity, whereas those using ATG or GTG were approximately one-half or one-third as active, respectively. The CTG fusion showed only 5% of the activity of the TTG fusion. A suicide vector, pKGLP2, carrying gusA in its backbone was used to visually detect merodiploid formation and resolution, making gene targeting in actinomycetes much faster and easier. Three regulatory genes, plaR1, plaR2, and plaR3, involved in phenalinolactone biosynthesis were efficiently replaced with an apramycin resistance marker using this system. Finally, we expanded the genetic code of actinomycetes by introducing the nonproteinogenic amino acid N-epsilon-cyclopentyloxycarbonyl-l-lysine with the GusA protein as a reporter.
authors.affiliation: Helmholtz Institute for Pharmaceutical Research, Saarland Campus, Building C2.3, 66123 Saarbrucken, Germany.
authors.forename: Maksym
authors.initials: M
authors.lastname: Myronovskyi
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Welle
authors.forename: Viktor
authors.initials: V
authors.lastname: Fedorenko
authors.forename: Andriy
authors.initials: A
authors.lastname: Luzhetskyy
mesh_terms: D039903:Actinobacteria / Q000201:enzymology* / Q000235:genetics; D018387:Codon, Initiator / Q000235:genetics; D004224:Diterpenes; D017930:Genes, Reporter / Q000235:genetics*; D005822:Genetic Vectors; D005966:Glucuronidase / Q000235:genetics*; D006027:Glycosides / Q000096:biosynthesis / Q000235:genetics; D008239:Lysine / Q000031:analogs & derivatives; D009328:Nebramycin / Q000031:analogs & derivatives / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D018387:Codon, Initiator; D004224:Diterpenes; D006027:Glycosides; D011993:Recombinant Fusion Proteins; C558342:phenalinolactone A; D009328:Nebramycin; C011666:apramycin; D005966:Glucuronidase; D008239:Lysine